CN119306700A

CN119306700A - DNMT1-HDAC dual-target inhibitor and preparation method and application thereof

Info

Publication number: CN119306700A
Application number: CN202411190504.4A
Authority: CN
Inventors: 方浩; 孔祥谦; 侯旭奔; 常英杰; 郭铧慧
Original assignee: Shandong University; Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Shandong University; Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2024-08-28
Filing date: 2024-08-28
Publication date: 2025-01-14

Abstract

本发明涉及一种如式(I)所示DNMT1‑HDAC双靶点抑制剂及其制备方法和应用，本发明化合物不仅表现出良好的DNMT1和HDAC酶抑制活性，而且对实体瘤和血液瘤均有显著抗肿瘤活性。该类化合物作为首次报道的基于DNMT1和HDACs的双靶点抑制剂，在癌症治疗方面具有很大前景； The present invention relates to a DNMT1-HDAC dual-target inhibitor as shown in formula (I), and a preparation method and application thereof. The compound of the present invention not only exhibits good DNMT1 and HDAC enzyme inhibitory activity, but also has significant anti-tumor activity against both solid tumors and hematological tumors. As the first reported dual-target inhibitor based on DNMT1 and HDACs, this type of compound has great prospects in cancer treatment;

Description

DNMT1-HDAC double-target inhibitor and preparation method and application thereof

Technical Field

The invention relates to a DNMT1-HDAC double-target inhibitor, and a preparation method and application thereof, and belongs to the technical field of medicines.

Background

Cancer is a disease characterized by genetic abnormalities and is characterized by resistance to treatment and high mortality. However, epigenetic science has gradually been considered as a factor responsible for cancer behavior, which cannot be explained by genetic alterations alone. In general, the heterogeneity of tumor cells in growth potential, invasiveness, malignant transformation capacity, and resistance to therapeutic intervention can be attributed to the interference of epigenetic regulation, with cancer cells causing extensive and profound changes in the epigenetic genome compared to normal cells, including DNA methylation, histone acetylation, histone methylation, and chromatin remodeling. Among them, abnormal histone tail lysine acetylation and CpG island hypermethylation in the promoter region of tumor suppressor gene are driving factors and biomarkers for tumor progression and prognosis failure.

DNA methylation is regulated by DNA methyltransferases (DNMT). The human DNMT family includes 5 members, which can be further divided into two groups, including typical members (DNMT 1, DNMT3A and DNMT 3B), which can add an s-adenosyl-L-methionine (SAM) -derived methyl group to the C-5 position of CpG island cytosine, and atypical members (DNMT 2 and DNMT 3L), which are not catalytically active. Unlike DNMT3A and DNMT3B, which establish a methylation pattern from scratch, DNMT1 is responsible for maintaining DNA methylation and transferring the methylation pattern from pattern to daughter strand. Furthermore, compared to other DNMTs, the levels of DNMT1 are dominant in both normal tissues and metastatic cancer tissues. Currently, DNMT inhibitors are classified into covalent nucleoside inhibitors and non-nucleoside reversible inhibitors. The former is represented by azacytidine (AZA), decitabine (DAC), and sibirin, and the latter is represented by RG108 and SGI-1027.

Histone Deacetylases (HDACs) can scavenge lysine acetyl groups from histone tails or non-histone substrates. Humans have found 18 HDACs and classified into four classes according to catalytic mechanism and intracellular localization, including zinc dependent class I (HDAC 1,2, 3, 8), class IIa (HDAC 4, 5, 7, 9), class IIb (HDAC 6, 10), class IV (HDAC 11) and class III (Sirtuin 1-7) that is NAD ⁺ dependent. To date, HDAC inhibitors are commonly used to treat hematological malignancies, with some inhibitors having been FDA approved, including vorinostat (SAHA), belinostat (PDX 101), panobinostat (LBH 589), sitadaniline (CS 055), and romidepsin (FK 228).

The combination of DNMT inhibitors and HDAC inhibitors is useful in the treatment of a variety of cancers, such as Acute Myelogenous Leukemia (AML), multiple Myeloma (MM), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma (TCL), and myelodysplastic syndrome, among others. For example, AML patients receive a combination of azacytidine and panobinostat for which the observed clinical efficacy is superior to panobinostat single drug therapy. While the combination of DNMT and HDAC inhibitors has certain advantages in the treatment of AML and tumor immunotherapy, the drug-drug interactions disadvantageously limit the achievement of synergistic effects, such as complex dosing regimens and negative effects on pharmacokinetics. In contrast, designing DNMT/HDAC dual target inhibitors may circumvent these potential drawbacks. However, the DNMT/HDAC dual target inhibitors reported to date do not have good inhibition of DNMT or HDAC and lack subtype selectivity for DNMT1, resulting in no apparent therapeutic advantage compared to single drug treatment.

Therefore, the development of an effective DNMT1/HDAC dual inhibitor has important clinical significance.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a DNMT1-HDAC double-target inhibitor, and a preparation method and application thereof.

The technical scheme of the invention is as follows:

a DNMT1-HDAC dual-target inhibitor, wherein the structural formula of the inhibitor is shown in formula (I) or a pharmaceutically acceptable salt of formula (I):

wherein X is an aromatic heterocycle or one of the following linking groups:

A is one of the following structures:

R ₁ is hydroxy or 2-aminophenyl;

R ₂ is one of the following structures

R ₃ is hydrogen atom, which is one of the following structures:

The DNMT1-HDAC double-target 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 -hydroxysuccinimide (11 a);

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

N ¹ - (1- (6- ((2-amino-2-oxo-1-phenylethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) -N ⁶ -hydroxy hexanediamide (11 c);

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

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

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

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-toluenediamide (11 g);

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 (11 h);

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) butanediamide (11 i);

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) gluta-namide (11 j);

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) hexanediamide (11 k);

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) butanediamide (11 l);

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 (11 m);

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-oxoprop-1-en-1-yl) phenyl) butanediamide (11N);

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

(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 (11 p);

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 (11 q);

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

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

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 (11 t);

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 (11 u);

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 (11 v);

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 (15 a)

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 (15 b);

(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 (19 a);

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

(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 (23 a);

(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) -23 a);

(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) -23 a);

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 (23 b);

(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);

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 (30 a);

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 (30 b);

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 (30 c);

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 (30 d);

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 (30 e);

N ¹ - (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) ethyl) -N ⁴ -hydroxysuccinamide (40 a);

n ¹ - (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) ethyl) -N ⁵ -hydroxyglutaramide (40 b);

N ¹ - (3- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) propyl) -N ⁴ -hydroxysuccinamide (40 c);

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

N ¹ - (1- (3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) piperidin-4-yl) -N ⁵ -hydroxyglutaramide (44 a);

(E) - (1- (3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) piperidin-4-yl) -N ⁵ -hydroxyglutaramide (44 b);

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

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

The DNMT1-HDAC double-target inhibitor is selected from one of the following structures:

A second object of the present invention is to provide a method for preparing a DNMT1-HDAC dual-target inhibitor, the method comprising any one of the following methods:

the preparation method of the compounds 11a-11V is as follows:

Reagents and conditions (a) p-toluenesulfonyl chloride, N, N-diisopropylethylamine, 4-dimethylaminopyridine, acetonitrile, 80 ℃ for 6 hours, (b) N-propanal, N-methylmorpholine, anhydrous ethanol, room temperature for 30 hours, (C) N, N-diisopropylethylamine, N, N-dimethylformamide, 85 ℃ for 5 hours, (d) isopentyl nitrite, copper chloride, acetonitrile, 80 ℃ for 2.5 hours, (e) aminopiperidine derivatives, potassium carbonate, dichloromethane, 4 hours, (f) 4M hydrochloric acid/ethyl acetate, room temperature for 12 hours, (g) 1) 9a-N, isobutyl chloroformate, N-methylmorpholine, dichloromethane/tetrahydrofuran (1:1), room temperature for 6 hours, (2) 9o-t, sodium triacetoxyborohydride, acetic acid, room temperature for 12 hours; 3) 9u, potassium carbonate, acetonitrile, 80 ℃ for 6 hours, (h) 4M hydrochloric acid/ethyl acetate, room temperature for 12 hours;

The preparation method of the compounds 15a, 15b, 19a and 19b is as follows:

reagents and conditions (a) aminopiperidine derivative, potassium carbonate, methylene chloride, 4 hours, (b) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (C) 9u, potassium carbonate, acetonitrile, 80 ℃,6 hours, (d) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (e) piperazine-1-t-butyl carboxylate, potassium carbonate, methylene chloride, 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 ℃,6 hours, (h) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours;

The preparation of compounds 23a, (R) -23a, (S) -23a, 23b, 27, 30a-e is as follows:

Reagents and conditions, (a) piperidine-4-aminomethyl tert-butyl formate, potassium carbonate, dichloromethane, 4 hours, (b) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (C) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours, (2) 9u, potassium carbonate, acetonitrile, 80 ℃,6 hours, (d) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (e) tert-butyl (2- (methylamino) ethyl) carbamate, potassium carbonate, dichloromethane, 4 hours, (f) 4M hydrochloric acid/ethyl acetate, 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, (d) 28d, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours, 3 e, 28e, potassium carbonate, 6 j) ethyl acetate, 6 hours, 12 hours;

the preparation of compounds 40a-d was as follows:

Reagents and conditions, (a) methanesulfonyl chloride, triethylamine, dichloromethane, room temperature for 4 hours, (b) diisobutylaluminum hydride, tetrahydrofuran, -20 ℃,6 hours, (C) p-toluenesulfonyl chloride, triethylamine, DMAP, acetonitrile, 80 ℃,4 hours, (d) 4, triethylamine, DMF,85 ℃,5 hours, (e) isoamyl nitrite, cupric chloride, acetonitrile, 80 ℃,2.5 hours, (f) 2-methylamino derivative, potassium carbonate, dichloromethane, 4 hours, (g) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (h) 1) 9a or 9b, isobutyl chloroformate, N-methylmorpholine, dichloromethane/tetrahydrofuran (1:1), room temperature, 6 hours, 2) 9u, potassium carbonate, acetonitrile, 80 ℃,6 hours, (i) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours;

the preparation of compounds 44a-c, 48 was as follows:

Reagents and conditions (a) t-butyl 4-carbamate, potassium carbonate, methylene chloride, 4 hours, (b) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (C) 1) 9b, isobutyl chloroformate, N-methylmorpholine, methylene chloride/tetrahydrofuran (1:1), room temperature, 6 hours; 2) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 3) 9u, potassium carbonate, acetonitrile, 80 ℃,6 hours, (d) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (e) piperazine-1-t-butyl carboxylate, potassium carbonate, methylene chloride, 4 hours, (f) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours, (g) 9u, potassium carbonate, acetonitrile, 80 ℃,6 hours, (h) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours.

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

The DNMT1-HDAC double-target inhibitor is applied to the preparation of medicaments for treating tumor diseases related to targets.

The tumor is 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 cancer, leukemia or myeloma.

The invention has the technical characteristics and advantages that:

1. The compound not only shows good DNMT1 and HDAC enzyme inhibition activity, but also has stronger in-vitro and in-vivo anti-tumor activity, and can obviously delay tumor growth.

2. The compound of the invention has further development and research value as a double-target anti-tumor drug based on DNMT1 and HDAC reported for the first time.

Drawings

FIG. 1 is a graph showing the results of antitumor activity of compound (R) -23a, combination (GSK 5032 and SAHA) on human acute monocytic leukemia cells MV4-11 in vivo, wherein A is tumor-bearing mouse weight change, and B is tumor volume growth curve of tumor-bearing mouse.

FIG. 2 is a graph showing the results of the anti-tumor activity of the compound (R) -23a, the combination of GSK5032 and SAHA, on MC38 of colon cancer cells of mice, wherein A is the change of body weight of tumor-bearing mice, and B is the tumor volume growth curve of tumor-bearing mice.

Detailed Description

The features of the present invention are further described below by way of examples, but the present invention is not limited to the following examples.

Reagents, starting materials, or intermediates for the examples are commercially available or may be prepared by literature procedures. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

Example 1 preparation of intermediate 6

2-Amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate (2)

2-Hydroxy-2-phenylacetamide (10 g,66.15 mmol), N-diisopropylethylamine (DMAP, 25.65g,198.46 mmol) and 4-dimethylaminopyridine (1.21 g,9.92 mmol) were added to acetonitrile (100 mL) 4-methylbenzenesulfonyl chloride (18.92 g,99.23 mmol), stirred at room temperature for 6h, and the solvent was removed by rotary evaporation. The resulting residue was washed with 100mL of water, the mixture was extracted with ethyl acetate (30 mL. Times.3), and the separated organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product, which was purified by silica gel column chromatography to give Compound 2, the synthetic methods of Compound (R) -2, (S) -2 and 34 were the same as in this procedure.

White solid, yield 77.2％,¹H 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).

(R) -2-amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate ((R) -2)

White solid, yield 69.1％,¹H 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).

(S) -2-amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate ((S) -2)

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).

4-Methylmorpholine-4-ammonium salt-6-amino-3, 5-dicyano-4-ethylpyridine-2-thioate (4)

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

Brown solid, yield 30.4％,¹H 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).

2- ((6-Amino-3, 5-dicyano-4-ethylpyridin-2-yl) thio) -2-phenylacetamide (5)

Compound 4 (661mg, 1.96 mmol) and N, N-diisopropylethylamine (760 mg,5.88 mmol) were dissolved in DMF (5 mL) and compound 2 (598 mg,1.96 mmol) was added thereto, heated to 85℃for 5 hours, and then the solvent was removed by rotary evaporation. The resulting residue was washed with 50mL of water, the mixture was extracted with ethyl acetate (15 mL. Times.3), and the separated organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product, which was purified by silica gel column chromatography to give Compound 5, the synthetic methods of Compound (R) -5, (S) -5 and 35 were the same as the procedure. Pale yellow solid, yield 68.1％,¹H 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).

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

Yellow solid, yield 40.7％.¹H 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).

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

Yellow solid, yield 45.1％.¹H 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).

2- ((6-Chloro-3, 5-dicyano-4-ethylpyridin-2-yl) thio) -2-phenylacetamide (6)

Compound 5a (402 mg,1.19 mmol) and copper chloride (797 mg,5.93 mmol) were dissolved in acetonitrile (20 mL), and isoamyl nitrite (278 mg,2.37 mmol) was added thereto, and the temperature was raised to 80℃for 2.5 hours, followed by rotary evaporation to remove the solvent. The resulting residue was washed with 100mL of water, the mixture was extracted with ethyl acetate (30 mL. Times.3), and the separated organic phase was dried over anhydrous sodium sulfate and concentrated to give a crude product, which was purified by silica gel column chromatography to give Compound 6, the synthetic methods of Compound (R) -6, (S) -6 and 36 were the same as in this procedure. Pale yellow solid, yield 31.3％.¹H 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).

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

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＝8.2,6.3Hz,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).

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

Pale yellow solid, yield 30.1％,.¹H 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).

EXAMPLE 2 preparation of intermediate 7a

To a solution of compound 6 (200 mg,0.56 mmol) and potassium carbonate (232 mg,1.68 mmol) in CH ₂Cl₂ (10 mL) was added tert-butyl piperidine-4-carbamate (224 mg,1.12 mmol), and the mixture was stirred at room temperature for 4 hours and then washed with 100mL of water. The organic phase was then separated, dried over anhydrous sodium sulfate and concentrated to give the crude product. The latter was purified by silica gel column chromatography to give 7a, and the synthetic methods of the compounds 7b, 12a, 12b, 16, 20, (R) -20, (S) -20, 24, 37a-c, 41 and 45 were the same as those of the procedure.

Tert-butyl (1-6 ((2-amino-2-oxo-1-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin 2-yl) piperidin-4-yl) carbamate (7 a)

White solid, yield 68.5％.¹H 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),4.45(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).

Tert-butyl (1-6 ((2-amino-2-oxo-1-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin 2-yl) piperidin-4-yl) (methyl) carbamate (7 b)

White solid, yield 69.5％,¹H 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),4.45(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).

EXAMPLE 3 preparation of intermediate 8a

1- (6- ((2-Amino-2-oxo-1-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-ammonium salt (8 a)

The synthesis of compounds 8b, 13a, 13b, 17, 21, (R) -21, (S) -21, 25, 38a-c, 42 and 46 was followed by addition of compound 7a (292 mg,0.56 mmol) to saturated ethyl hydrogen acetate (10 mL), stirring at room temperature for 12 hours, filtering, and recrystallization of the purified cake from ethyl acetate (20 mL) to give 8 a. White solid, yield 85.8％.¹H 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).

1- (6- ((2-Amino-2-oxo-1-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) -N-methylpiperidin-4-ammonium salt (8 b)

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).

EXAMPLE 4 preparation of intermediates 9a-f

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 15mL CH ₂Cl₂ and the reaction stirred at room temperature for 3 hours. The organic phase was then washed with saturated NaCl solution (30 ml), the organic phase was separated, dried over anhydrous Na ₂SO₄, and concentrated to give the crude product, which was purified by silica gel column chromatography to give compounds 9a-f.

4-Oxo-4- (((tetrahydro-2H-pyran-2-yl) oxo) amino) butanoic acid (9 a)

White solid, yield 53％.¹H 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).

5-Oxo-5- (((tetrahydro-2H-pyran-2-yl) oxo) amino) pentanoic acid (9 b)

White solid, yield 45.7％.¹H 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).

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

White solid, yield 48.3％.¹H 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).

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

Colorless oily liquid, yield 48.1％.¹H 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).

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

White solid, yield 46％.¹H 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).

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

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).

EXAMPLE 5 preparation of intermediate 9g-9u

4- ((Tetrahydro-2H-pyran-2-yl) carbamoyl) benzoic acid (9 g)

The corresponding carboxylic acid (5.31 mmol), EDCI (1.23 g,6.4 mmol), DMAP (843 mg,6.9 mmol), and pyranhydroxylamine (6272 mg,5.31 mmol) were added to 15mL CH ₂Cl₂ and reacted at room temperature for 8 hours. After completion of the reaction, the reaction mixture was washed with 1M aqueous hydrochloric acid (100 mL) and saturated aqueous sodium chloride (100 mL) in this order. The organic phase was then dried over anhydrous Na ₂SO₄ and concentrated. Purification by silica gel column chromatography gave 9g, and the procedure was the same as for the synthesis of compound 9 h-u. Colorless oily liquid, yield 70.8％.¹H 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).

4- ((4- (((Tetrahydro-2H-pyran-2-yl) oxo) carbamoyl) benzyl) oxo) benzoic acid (9H)

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).

4-Oxo-4- ((4- (((tetrahydro-2H-pyran-2-yl) oxo) carbamoyl) phenyl) amino) butanoic acid (9 i)

White solid, yield 44.5％.¹H 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).

5-Oxo-5- ((4- (((tetrahydro-2H-pyran-2-yl) oxo) carbamoyl) phenyl) amino) pentanoic acid (9 j)

White solid, yield 27.9％.¹H 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).

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

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).

4-Oxo-4- ((4- (((tetrahydro-2H-pyran-2-yl) carbamoyl) benzyl) amino) butanoic acid (9 l)

White solid, yield 54.2％.¹H 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).

5-Oxo-5- ((4- (((tetrahydro-2H-pyran-2-yl) carbamoyl) benzyl) amino) pentanoic acid (9 m)

White solid, yield 48.1％.¹H 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,1H),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).

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

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).

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

White solid, yield 57.2％.¹H 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).

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

White solid, yield 39.1％.¹H 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).

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

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).

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

White solid, yield 47.7％.¹H 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＝7.7Hz,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).

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

White solid, yield 52.8％.¹H 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).

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

White solid, yield 62.9％.¹H 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,1H),3.52–3.45(m,1H),2.01–1.97(m,2H),1.73(p,J＝6.7Hz,3H),1.68–1.60(m,3H),1.51(q,J＝7.6Hz,6H),1.43–1.37(m,2H),1.31(q,J＝4.5Hz,2H).

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

White solid, yield 29.4％.¹H 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).

Example 6 preparation of intermediates 10a-10n

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) butanediamide (10 a)

9A (190 mg,0.875 mmol) and NMM (110 mg,1.09 mmol) were added to THF (10 mL), isobutyl chloroformate (120 mg,0.875 mmol) was added, and stirred at 0deg.C for 0.5h. The resulting filtrate was added dropwise to CH ₂Cl₂ (10 mL) containing 8a (200 mg,0.437 mmol) and N-methylmorpholine (44 mg,0.437 mmol) at 0 ℃. The mixture was stirred at room temperature for 6 hours, then the solvent was removed by rotary evaporation, and the resulting residue was washed with 100mL of water and extracted with ethyl acetate (30 mL. Times.3). The organic phase was washed successively with 1M aqueous hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate (100 mL), and saturated aqueous sodium chloride (100 mL). The organic phase was then dried over anhydrous Na ₂SO₄ and concentrated. Purification by silica gel column chromatography gave 10a, and the procedure was the same for the synthesis of compounds 10b-n, 29a-c, 39a-c and 43 a. White solid, yield 44.1％.¹H 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,3H),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＝12.7Hz,2H),1.64(s,3H),1.50(s,3H),1.43(t,J＝10.3Hz,2H),1.20(t,J＝7.6Hz,3H).

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 (10 b)

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).

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) hexanediamide (10 c)

White solid, yield 60.4％.¹1H 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).

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) pimelic acid amide (10 d)

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).

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) octanediamide (10 e)

White solid, yield 31.1％.¹H 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).

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) octanediamide (10 f)

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＝7.4Hz,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).

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 (10 g)

White solid, yield 48.2％.¹H 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).

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)

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).

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) butanamide (10 i)

White solid, yield 38.6％.¹H 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).

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) gluta-mide (10 j)

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＝7.6Hz,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).

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) hexanediamide (10 k)

White solid, yield 41.4％.¹H 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,4H),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),4.10–4.01(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,2H),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).

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) butanamide (10 l)

White solid, yield 42.5％.¹H 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,2H),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).

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) gluta-namide (10 m)

White solid, yield 41.3％.¹H 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,2H),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).

(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) butanediamide (10N)

White solid, yield 35.9％.¹H 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).

EXAMPLE 7 preparation of intermediate 10o-10t

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 (10 o)

To a mixture of MeOH/CH ₂Cl₂ (1:1, 10 mL) was added 8a (350 mg,0.766 mmol) and 9o (287 mg,1.15 mmol), and after 9 hours at room temperature, sodium triacetoxyborohydride (649 mg,3.06 mmol) was added and stirring was continued at room temperature for 3 hours. After the solvent was removed by rotary evaporation, the resulting residue was washed with 50mL of water, and the mixture was extracted with ethyl acetate (30 mL. Times.3). The separated organic phase was washed with 1M aqueous hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate (100 mL), and saturated aqueous sodium chloride (100 mL) in this order. The organic phase was then dried over anhydrous Na ₂SO₄ and concentrated. Purification by silica gel column chromatography gave 10o, and the synthesis of compounds 10p-t, 18a, 22, (R) -22, (S) -22, 26, 29d and 43b was carried out in the same manner as in this procedure. White solid, yield 41.2％.¹H 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,2H),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).

(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 (10 p)

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＝12.5Hz,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).

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 (10 q)

White solid, yield 38.2％.¹H 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,2H),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)

(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 (10 r)

White solid, yield 35.1％.¹H 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),2.03–1.95(m,2H),1.70(s,3H),1.53(d,J＝5.0Hz,3H),1.42(s,2H),1.20(t,J＝7.6Hz,3H).

(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 (10 s)

White solid, yield 33.8％.¹H 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).

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 (10 t)

White solid, yield 32.1％.¹H 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).

Example 8 preparation of intermediate 10u

To acetonitrile (10 mL) was added 8a (356 mg,0.78 mmol), 9u (242 mg,0.94 mmol) and K ₂CO₃ (162 mg,1.17 mmol), and reacted at 80℃for 6 hours. The reaction mixture was diluted with 50mL of water and extracted with ethyl acetate (20 mL. Times.3). The separated organic phase was dried sequentially over anhydrous Na ₂SO₄ and concentrated to give the crude product. The latter was purified by silica gel column chromatography to give 10u, and the synthesis of compounds 10v, 14a, 14b, 18b, 22b, 29e, 39d, 43c and 47 was carried out in the same manner as in this procedure.

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 (10 u)

White solid, yield 29.6％.¹H 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).

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 (10 u)

White solid, yield 31.1％.¹H NMR(400MHz,DMSO)δ11.54(s,1H),8.71(s,2H),7.92(s,1H),7.52(d,J＝7.3Hz,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).

EXAMPLE 9 preparation of the target Compound

The procedure was followed except for dissolving compound 10a (234 mg,0.378 mmol) prepared in example 6 in saturated ethyl hydrogen acetate (10 mL), stirring at room temperature for 12h, and recrystallizing the purified cake with EA or MeOH (20 mL) to give 11a, 11b-v, 15a, 15b, 19a, 19b, 23a, (R) -23a, (S) -23a, 23b, 27, 30a-e, 40a-d, 44a-c, and 48.

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

White solid, yield 44.1％,mp170-171℃.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),3.41–3.30(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).¹³C 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 C₂₆H₂₈N₇O₄S[M–H]^-534.1929,found 534.1932.HPLC t_R＝6.19min(99.89％purity).

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

White solid, yield 55.8％,mp171-173℃.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).¹³C NMR(101MHz,DMSO)δ174.66,171.46,170.00,164.79,164.40,158.45,136.69,129.11,128.66,128.61,116.43,115.15,93.77,87.56,53.63,46.96,45.79,45.59,34.97,33.48,33.22,32.06,32.00,27.58,21.19,14.01,8.90.HRMS-ESI(m/z)calc.for C₂₇H₃₀N₇O₄S[M-H]⁺548.2085,found 548.2088.HPLC t_R＝6.65min(97.33％purity).

N ¹ - (1- (6- ((2-amino-2-oxo-1-phenylethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) -N ⁶ -hydroxy hexanediamide (11 c)

White solid, yield 34.5％,mp173-175℃.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).¹³C 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 C₂₈H₃₂N₇O₄S[M-H]⁺562.2242,found 562.2246.HPLC t_R＝6.49min(98.83％purity).

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

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).¹³C 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 C₂₉H₃₅N₇O₄S[M+Na]⁺600.2375,found 600.2379.HPLC t_R＝12.41min(98.19％purity).

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

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＝7.2Hz,2H),1.87(d,J＝12.6Hz,2H),1.47 1.61–1.35(m,6H),1.23(dd,J＝12.8,8.4Hz,7H).¹³C 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 C₃₀H₃₇N₇O₄S[M+H]⁺592.2706,found 592.2708.HPLC t_R＝6.89min(99.98％purity).

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

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).¹³C 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 C₃₁H₃₉N₇O₄S[M+Na]⁺628.2682,found 628.2681.HPLC t_R＝11.64min(99.9％purity).

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-toluenediamide (11 g)

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)).¹³C 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 C₃₀H₂₉N₇O₄S[M+Na]⁺606.1905,found 606.1908.HPLC t_R＝6.73min(97.35％purity).

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 (11 h)

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).¹³C NMR(101MHz,DMSO)δ170.05,165.53,164.89,164.39,160.84,158.50,140.34,136.80,132.73,129.71,129.06,128.70,128.57,127.87,127.57,127.49,116.46,115.18,114.72,93.78,87.59,69.20,60.24,53.62,47.49,47.32,46.58,32.04,27.59,21.25,14.56,14.03.HRMS-ESI(m/z)calc.for C₃₇H₃₄N₇O₅S[M-H]^-688.2348,found 688.2348.HPLC t_R＝4.86min(99.92％purity).

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) butanediamide (11 i)

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).¹³C NMR(101MHz,DMSO)δ170.03,164.76,164.36,163.09,158.45,138.23,136.73,135.03,130.31,129.10,128.68,128.60,127.95,119.84,116.46,115.16,93.68,87.58,53.64,48.02,30.29,27.56,14.00.HRMS-ESI(m/z)calc.for C₃₃H₃₃N₈O₅S[M-H]⁺653.2300,found653.2302.HPLC t_R＝8.39min(99.06％purity).

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) gluta-namide (11 j)

White solid, yield 49.3％,mp214-216℃.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).¹³C 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 C₃₄H₃₆N₈O₅S[M+H]⁺669.2612,found 669.2615.HPLC t_R＝10.52min(97.52％purity).

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) hexanediamide (11 k)

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),7.74–7.64(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).¹³C NMR(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.for C₃₅H₃₇N₈O₅S[M-H]^-681.2613,found681.2617.HPLC t_R＝6.4min(97.15％purity).

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) butanediamide (11 l)

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),7.70(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),2.46–2.31(m,4H),1.88(d,J＝12.7Hz,2H),1.51–1.36(m,2H),1.21(t,J＝7.6Hz,3H).¹³C 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 C₃₄H₃₅N₈O₅S[M-H]^-667.2466,found 667.2467.HPLC t_R＝11.36min(99.30％purity).

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) gluta-namide (11 m)

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).¹³CNMR(101MHz,DMSO)δ172.34,171.56,169.99,164.79,164.54,164.40,158.47,143.47,136.65,131.68,129.85,129.13,128.66,127.47,127.37,116.44,115.14,93.78,87.59,53.65,46.94,45.59,42.20,35.36,35.19,32.09,32.02,27.58,22.05,14.02.HRMS-ESI(m/z)calc.for C₃₅H₃₇N₈O₅S[M-H]^-681.2613,found 681.2618.HPLC t_R＝6.21min(99.91％purity).

(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-oxoprop-1-en-1-yl) phenyl) butanediamide (11N)

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).¹³C 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 C₃₅H₃₅N₈O₅S[M-H]⁺679.2457,found 679.2462.HPLC t_R＝6.44min(97.25％purity).

4- (((1- (6- ((2-Amino-2-oxo-1-phenylethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) amino) methyl) -N-hydroxybenzoamide (11 o)

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,2H),2.86(s,1H),2.75(q,J＝7.6Hz,2H),1.44(s,2H),1.20(t,J＝7.6Hz,3H).¹³C NMR(101MHz,DMSO)δ170.05,164.94,164.40,164.05,158.62,136.67,135.58,133.57,130.72,130.55,129.94,129.15,128.70,128.64,127.56,116.32,115.00,94.43,88.17,54.16,53.64,47.05,46.39,46.27,45.90,28.41,27.59,13.98,8.93.HRMS-ESI(m/z)calc.for C₃₀H₃₂N₇O₃S[M+H]⁺570.2287,found 570.2297.HPLC t_R＝9.58min(97.78％purity).

(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 (11 p)

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＝12.9Hz,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).¹³C 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 C₃₂H₃₃N₇O₃S[M+H]⁺596.2438,found 596.2440.HPLC t_R＝8.11min(98.52％purity).

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 (11 q)

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),4.66(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＝12.8Hz,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).¹³C NMR(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 C₃₈H₃₉N₈O₄S[M+H]⁺703.2816,found 703.2821.HPLC t_R＝4.04min(96.86％purity).

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

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).¹³CNMR(101MHz,DMSO)δ170.07,166.15,164.92,164.40,163.20,158.57,141.58,138.45,136.65,135.74,134.96,133.90,130.54,129.16,128.70,128.66,128.25,127.98,127.96,119.19,116.34,115.03,94.34,88.11,54.07,53.60,46.97,46.38,46.26,42.92,28.43,27.61,14.01.HRMS-ESI(m/z)calc.for C₄₀H₄₁N₈O₄S[M+H]⁺729.2975,found 729.2979.HPLC t_R＝4.02min(97.99％purity).

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

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).¹³C NMR(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 C₄₀H₄₁N₈O₄S[M+Na]⁺724.2682,found 724.2676.HPLC t_R＝6.45min(97.29％purity).

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 (11 t)

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＝6.5Hz,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).¹³C NMR(101MHz,DMSO)δ170.07,169.58,164.92,164.41,159.57,158.58,136.60,132.12,129.17,128.69,124.16,116.33,115.02,114.98,94.34,88.10,67.94,53.71,53.62,47.00,46.37,46.26,32.66,28.95,28.78,28.45,27.60,25.69,25.52,14.01.HRMS-ESI(m/z)calc.for C₃₆H₄₃N₇O₄S[M+H]⁺670.3170,found 670.3177.HPLC t_R＝7.85min(98.24％purity)

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 (11 u)

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).¹³C NMR(101MHz,DMSO)δ170.04,164.80,164.48,162.35,158.42,136.51,129.17,128.68,116.45,115.39,115.15,93.77,87.55,53.63,47.76,46.99,31.77,27.58,14.00.HRMS-ESI(m/z)calc.for C₂₈H₂₉N₉O₃S[M+Na]⁺580.1811,found 580.1810.HPLC t_R＝5.74min(97.25％purity).

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 (11 v)

White solid, yield 36.1％,mp 185-187℃.¹H 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)δ170.02,164.80,164.40,161.99,158.37,157.48,136.73,129.11,128.66,116.51,115.17,114.92,93.84,87.63,53.64,52.34,47.62,29.49,29.06,27.59,14.01.HRMS-ESI(m/z)calc.for C₂₈H₂₉N₉O₃S[M+Na]⁺597.1965,found 594.1967.HPLC t_R＝4.52min(95.29％purity)

Tert-butyl-4- ((6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) (methyl) amino) piperidine-1-carbamic acid ester (12 a)

White solid, yield 38.3％.¹H 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).

Tert-butyl-4- ((6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) methyl) (methyl) amino) piperidine-1-carbamate (12 b)

White solid, yield 42.8％.¹H 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).

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

White solid, yield 56.2％.¹H 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).

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

White solid, yield 41.7％.¹H 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).

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 (14 a)

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,3H),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).

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 (14 b)

White solid, yield 45.3％.¹H 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),2.95–2.83(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).

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).¹³C 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 C₂₈H₂₉N₉O₃S[M+Na]⁺594.2007,found594.2002.HPLC t_R＝4.67min(96.98％purity).

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 (15 b)

White solid, yield 32.0％,mp 173-175℃.¹H 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 C₂₈H₂₉N₉O₃S[M+Na]⁺608.2160,found 608.2155.HPLC t_R＝4.55min(95.28％purity).

Tert-butyl-4- ((6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperazine-1-carbamate (16)

White solid, yield 81.2％.¹H 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).

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

White solid, yield 90.2％.¹H 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).

(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 (18 a)

White solid, yield 48.8％.¹H 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).

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 (18 b)

White solid, yield 50.2％.¹H 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).

(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 (19 a)

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).¹³C NMR(101MHz,DMSO)δ169.96,164.79,164.44,158.51,136.55,129.97,129.13,128.64,127.91,119.34,116.37,115.07,94.07,87.85,61.74,53.63,52.86,47.93,27.55,13.98.HRMS-ESI(m/z)calc.for C₃₁H₃₁N₇O₃S[M+Na]⁺604.2058,found 604.2059.HPLC t_R＝5.16min(95.27％purity).

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

White solid, yield 31.2％,mp 211-213℃.¹H 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)δ170.02,164.91,164.39,162.19,161.68,160.15,158.62,157.62,136.42,129.21,128.73,128.70,116.37,115.52,115.06,94.14,87.93,53.71,47.31,43.54,27.57,14.01.HRMS-ESI(m/z)calc.for C₂₆H₂₅N₉O₃S[M+Na]⁺566.1659,found 566.1655.HPLC t_R＝4.51min(96.89％purity).

Tert-butyl-4- ((6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) methyl) -1-carbamic acid ester (20)

White solid, yield 77.8％.¹H 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,2H),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).

Tert-butyl- (R) - ((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) methyl) -1-carbamic acid ester ((R) -20)

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).

Tert-butyl- (S) - ((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) methyl) -1-carbamic acid ester ((S) -20)

White solid, yield 66.2％.¹H 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).

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

White solid, yield 88.2％.¹H 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＝13.2Hz,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).

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

White solid, yield 67.6％.¹H 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).

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

White solid, yield 58.5％.¹H 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).

(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 (22 a)

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).

(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) -22 a)

White solid, yield 33.0％.White solid,yield 33.0％,¹H 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＝7.5Hz,2H),1.87(d,J＝12.2Hz,2H),1.70(s,3H),1.54(s,3H),1.19(t,J＝7.7Hz,3H).

(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) -22 a)

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＝7.3Hz,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).

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 (22 b)

White solid, yield 48.3％.¹H 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,1H),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).

(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-oxoprop-1-en-1-yl) benzyl) methylammonium salt (23 a)

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＝7.6Hz,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).¹³C 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 C₃₃H₃₅N₇O₃S[M+H]⁺610.2570,found 610.2552.HPLC t_R＝5.76min(97.46％purity).

(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-oxoprop-1-en-1-yl) benzyl) methylammonium salt ((R) -23 a)

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).¹³C NMR(101MHz,DMSO)δ170.03,164.83,164.30,162.96,158.50,137.93,136.77,135.84,133.40,131.36,131.31,129.07,128.73,128.68,128.56,127.99,120.53,116.42,115.14,93.77,87.73,56.49,53.64,51.48,50.41,47.90,47.73,45.78,32.81,30.05,27.55,19.03,13.99,8.88.HRMS-ESI(m/z)calc.for C₃₃H₃₅N₇O₃S[M+H]⁺610.2570,found 610.2572.HPLC t_R＝5.78min(98.02％purity).

(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-oxoprop-1-en-1-yl) benzyl) methylammonium salt ((S) -23 a)

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).¹³C NMR(101MHz,DMSO)δ170.03,164.83,164.31,162.95,158.50,137.95,136.75,135.83,133.40,131.35,131.30,129.08,128.75,128.68,128.57,128.00,120.50,116.43,115.15,93.76,87.73,56.49,53.63,51.49,50.41,47.89,47.73,32.81,30.06,30.03,27.55,14.00.HRMS-ESI(m/z)calc.for C₃₃H₃₅N₇O₃S[2M+H]⁺1219.5122,found 1219.5129.HPLC t_R＝5.82min(99.24％purity).

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 (23 b)

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).¹³C 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 C₂₈H₂₉N₉O₃S[M+H]⁺572.2155,found 572.2150.HPLC t_R＝11.25min(99.40％purity).

Tert-butyl (2- ((6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) (methyl) amino) ethyl) carbamate (24)

White solid, yield 85.2％.¹H 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).

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

White solid, yield 91.2％.¹H 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).

(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)

White solid, yield 51.5％.¹H 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).

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

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).¹³C NMR(101MHz,DMSO)δ170.55,164.48,163.01,158.18,138.04,136.44,135.92,133.44,131.14,129.17,128.79,128.71,128.12,120.48,116.82,115.15,93.97,87.12,52.94,50.58,48.06,44.46,27.62,13.96.HRMS-ESI(m/z)calc.for C₂₈H₂₉N₉O₃S[M+H]⁺570.2282,found 570.2284.HPLC t_R＝5.18min(96.49％purity).

EXAMPLE 10 preparation of target Compounds 30a-e

5- ((2- ((Tert-Butoxycarbamoyl) amino) phenyl) amino) -5-oxopentanoic acid (28 a)

The corresponding carboxylic acid (2.66 mmol), EDCI (616 mg,3.2 mmol), DMAP (422 mg,3.45 mmol), and tert-butyl 2-aminophenyl formate (5535 mg,2.66 mmol) were added to 15mL CH ₂Cl₂ and reacted at room temperature for 8 hours. After completion of the reaction, the reaction mixture was washed with 1M aqueous hydrochloric acid (100 mL) and saturated aqueous sodium chloride (100 mL) in this order. The organic phase was then dried over anhydrous Na ₂SO₄ and concentrated. Purification by silica gel column chromatography gave 28a, and the procedure was the same for the synthesis of compounds 28 d-e. White solid, yield 25.8％.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,1H),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).

4- ((2- ((Tert-Butoxycarbamoyl) amino) phenyl) carbamoyl) benzoic acid (28 b)

White solid, yield 78.1％.¹H 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).

2- (4- ((2- ((Tert-Butoxycarbamoyl) amino) phenyl) carbamoyl) phenyl) acetic acid (28 c)

White solid, yield 96.4％.¹H 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).

Tert-butyl (2- (4-formylbenzamide) phenyl) carbamate (28 d)

Yellow solid, yield 43.9％.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),7.58–7.50(m,2H),7.25–7.11(m,2H),1.44(s,9H).

Tert-butyl (2- (2-chloropyrimidine-5-carboxamide) phenyl) carbamate (28 e)

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).

Tert-butyl (2- (5- ((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) amino) -5-oxopentylamino) phenyl) carbamate (29 a)

White solid, yield 84.3％.¹H 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),4.52–4.39(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).

Tert-butyl (2- (4- ((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) carbamoyl) benzamide) phenyl) carbamate (29 b)

White solid, yield 82.2％.¹H 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).

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) benzamide) phenyl) carbamate (29 c)

White solid, yield 75.5％.¹H 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＝11.2Hz,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).

Tert-butyl (2- (4- (((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) piperidin-4-yl) amino) methyl) benzamide) phenyl) carbamate (29 d)

White solid, yield 50.2％.¹H 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).

Tert-butyl (2- (2- ((1- (6- ((2-amino-2-oxo-phenethyl) thio) -3, 5-dicyano-4-ethylpyridin-2-yl) (methyl) amino) pyrimidine-carboxamide) phenyl) carbamate (29 e)

White solid, yield 46.0％.¹H 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),3.06(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).

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 (30 a)

White solid, yield 74.2％,mp 174-176℃.¹H NMR(400MHz,DMSO)δ9.09(s,1H),7.89(dd,J＝12.0,4.8Hz,2H),7.58–7.43(m,3H),7.41–7.28(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),5.53(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),2.34(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)δ171.62,171.51,171.27,169.98,164.79,164.41,158.50,155.11,142.37,136.64,129.13,128.66,126.18,125.82,123.98,121.63,116.60,116.42,116.32,115.11,93.85,87.63,53.69,46.96,45.62,35.62,35.32,32.07,28.47,27.57,24.13,22.03,13.99.HRMS-ESI(m/z)calc.for C₃₃H₃₆N₈O₃S[M+Na]⁺647.2529,found 647.2529.HPLC t_R＝10.24min(99.93％purity).

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 (30 b)

White solid, yield 76.5％,mp 247-249℃.¹H 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 C₃₆H₃₄N₈O₃S[M+Na]⁺681.2372,found 681.2374.HPLC t_R＝11.46min(99.31％purity).

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 (30 c)

White solid, yield 79.2％,mp 245-247℃.¹H NMR(400MHz,DMSO)δ10.41(d,J＝11.4Hz,1H),8.34(t,J＝7.0Hz,1H),8.04(td,J＝9.7,4.1Hz,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,1H),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 C₃₆H₃₄N₈O₃S[M–H]^-671.2558,found 671.2559.HPLC t_R＝5.18min(98.52％purity).

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 (30 d)

White solid, yield 70.2％,mp 221-223℃.¹H 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 C₃₆H₃₆N₈O₂S[M+Na]⁺645.2755,found 645.2756.HPLC t_R＝7.35min(97.74％purity).

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 (30 e)

White solid, yield 70.2％,mp 217-219℃.¹H 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＝7.9Hz,1H),6.59(t,J＝7.5Hz,1H),5.57(s,1H),4.95(s,2H),4.76(d,J＝13.3Hz,2H),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 C₃₄H₃₄N₁₀O₂S[M-H]^-645.2489,found 645.2494.HPLC t_R＝5.21min(96.94％purity).

EXAMPLE 11 preparation of target Compounds 40a-d, 44a-c, 48

Methyl 4- (N-methyl methanesulfonamido) benzoate (32)

Methanesulfonyl chloride (1.04 g,9.08 mmol) was slowly added to CH ₂Cl₂ (10 mL) containing 31 (1 g,6.05 mmol) and triethylamine (1.22 g,12.10 mmol) at 0℃and reacted at room temperature for 1.5 hours. The reaction solution was diluted with 50mL of CH ₂Cl₂, and the organic phase was washed successively with 1M aqueous hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate (100 mL), and saturated aqueous sodium chloride (100 mL). The organic phase was then dried over anhydrous Na ₂SO₄ and concentrated. Purification by silica gel column chromatography gave 32. Pale yellow solid, yield 83.5%. ¹ H NMR (400 MHz, DMSO). Delta.8.02-7.94 (m, 2H), 7.59-7.51 (m, 2H), 3.86 (s, 3H), 3.30 (s, 3H), 3.01 (s, 3H).

N- (4- (hydroxymethyl) phenyl) -N-methyl methanesulfonamide (33)

DIBAL (8.22 mmol) was added to a solution of 32 (1 g,4.11 mmol) in THF, stirred at-20℃for 6 hours, and after completion of the reaction quenched with 30mL of 10% NaOH solution. The mixture was then extracted with ethyl acetate (10 mL. Times.3) and the organic phase was concentrated to give the crude product, which was purified by column chromatography to give 33. Grey solid, yield 68.9％.¹H 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).

4- (N-Methylmethylsulfonyl) benzyl-4-methylbenzenesulfonate (34)

Pale yellow solid, yield 45.8%. MS-ESI (m/z) calc.for C ₁₆H₁₉N₁O₅S₂[M+H]⁺ 370.1.1.

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

Brown yellow solid, yield 71.6％.¹H 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).

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

Brown yellow solid, yield 31.3％.¹H 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).

Tert-butyl (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) (methyl) amino) ethyl) carbamate (37 a)

Yellow solid, yield 79.7％.¹H 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＝6.1Hz,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).

Tert-butyl (3- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) (methyl) amino) propyl) carbamate (37 b)

Yellow solid, yield 77.2％.¹H 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).

Tert-butyl (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) (methyl) amino) ethyl) (methyl) carbamate (37 c)

Yellow solid, yield 74.0％.¹H 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).

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

Pale yellow solid, yield 90.1％.¹H 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,2H),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).

3- ((3, 5-Dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) (methyl) amino) propyl-1-ammonium salt (38 b)

Pale yellow solid, yield 84.1％.¹H 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,2H),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).

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

Pale yellow solid, yield 85.0％.¹H 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).

N ¹ -2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonyl) benzyl) thio) pyridin-2-yl) (methyl) amino) ethyl) -N ⁴ - ((tetrahydro-2H-pyran-2-yl) oxo) butanediamide (39 a)

Pale yellow solid, yield 31.8％.¹H 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).

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

Pale yellow solid, yield 44.2％.¹H 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).

N ¹ -2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonyl) benzyl) thio) pyridin-2-yl) (methyl) amino) propyl) -N ⁴ - ((tetrahydro-2H-pyran-2-yl) oxo) butanediamide (39 c)

Pale yellow solid, yield 39.5％.¹H 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,2H),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).

2- ((2- ((3, 5-Dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin 2-yl) (methyl) amino) ethyl) (methyl) amino) -N- ((tetrahydro-2H-pyran-2-yl) oxo) pyrimidine-5-carboxamide (39 d)

Pale yellow solid, yield 39.2％.¹H 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,2H),3.52(d,J＝11.4Hz,1H),3.35(s,3H),3.21(s,3H),3.08(s,3H),2.92(s,3H),2.72(d,J＝7.6Hz,10H),1.71(s,3H),1.54(s,3H),1.17(t,J＝7.6Hz,3H).

N ¹ - (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) ethyl) -N ⁴ -hydroxysuccinamide (40 a)

Pale yellow solid, yield 21.2％,mp 152-154℃.¹H 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).¹³C 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 C₃₄H₃₄N₁₀O₂S[M+Na]⁺596.1726,found 596.1728.HPLC t_R＝3.98min(96.78％purity).

N ¹ - (2- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) ethyl) -N ⁵ -hydroxyglutaramide (40 b)

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＝8.2Hz,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).¹³C 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.for C₂₆H₃₃N₇O₅S₂[M+Na]⁺610.1836,found586.1831.HPLC t_R＝4.09min(95.64％purity).

N ¹ - (3- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) propyl) -N ⁴ -hydroxysuccinamide (40 c)

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).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 C₂₆H₃₃N₇O₅S₂[M-H]^-586.1887,found586.1883.HPLC t_R＝7.85min(98.24％purity).

2- ((2- ((3, 5-Dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) (methyl) amino) ethyl) (methyl) amino) -N-hydroxypyrimidine-5-carboxamide (40 d)

Orange solid, yield 27％,mp 155-157℃.¹H 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),4.12–4.05(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 C₂₇H₃₁N₉O₄S₂[M-H]^-608.1845,found 608.1640.HPLC t_R＝9.90min(98.58％purity).

Tert-butyl (1- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) piperidin-4-yl) carbamate (41)

White solid, yield 73.5％.¹H 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),3.27(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).

1- ((3, 5-Dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) piperidin-4-ammonium salt (42)

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＝17.7Hz,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).

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

White solid, yield 37％.¹H 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),4.80(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).

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

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).

2- ((1- (3, 5-Dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) piperidin-4-yl) amino) -N- ((tetrahydro-2H-pyran-2-yl) oxo) pyrimidine-5-carboxamide (43 c)

White solid, yield 40.9％.¹H 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).

N ¹ - (1- (3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) piperidin-4-yl) -N ⁵ -hydroxyglutaramide (44 a)

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).¹³C 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＝9.90min(98.58％purity).

(E) - (1- (3, 5-dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) phenyl) thio) pyridin-2-yl) piperidin-4-yl) -N ⁵ -hydroxyglutaramide (44 b)

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).¹³C NMR(101MHz,DMSO)δ165.04,164.52,163.17,158.44,141.25,138.45,136.14,129.81,127.91,126.83,116.47,115.19,94.33,87.74,53.23,46.47,38.20,35.48,33.49,27.60,14.02.HRMS-ESI(m/z)calc.for C₃₃H₃₇N₇O₄S₂[M+H]⁺660.2368,found 660.2371.HPLC t_R＝5.47min(96.01％purity).

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

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＝7.7Hz,2H),1.99(d,J＝12.3Hz,2H),1.57(d,J＝11.7Hz,2H),1.22(t,J＝7.5Hz,3H).¹³C 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 C₂₈H₃₁N₉O₄S₂[M-H]^-620.1840,found 620.1838.HPLC t_R＝2.27min(99.37％purity).

Tert-butyl 4- ((3, 5-dicyano-4-ethyl-6- ((4- (N-methylmethanesulfonamido) benzyl) thio) pyridin-2-yl) piperazine-1-carbamate (45)

White solid, yield 81.1％.¹H 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).

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

White solid, yield 86.1％.¹H 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).

2- (4- (3, 5-Dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) benzyl) thio) pyridin-2-yl) piperazin-1-yl) -N- ((tetrahydro-2-H-pyran-2-yl) oxo) pyrimidine-5-carboxamide (47)

White solid, yield 49.2％.¹H 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,4H),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).

2- (4- (3, 5-Dicyano-4-ethyl-6- ((4- (N-methylsulfonylamino) benzyl) thio) pyridin-2-yl) piperazin-1-yl) -N-hydroxypyrimidine-5-carboxamide (48)

White solid, yield 33％,mp 170-171℃.¹H 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 C₂₇H₂₉N₉O₄S₂[M-H]^-606.1688,found 606.1683.HPLC t_R＝9.16min(99.65％purity).

Experimental example 1 target Compound has inhibitory Activity against HDACs

1. Materials:

HDACs enzyme (HeLa cell nucleus extract), boc-Lys (Ac) -AMC substrate, pancreatin, EDTA, LBH-589 (10 mM in dimethyl sulfoxide), 96 Kong Quanhei ELISA plate, glycerol, ultrapure water

2. The method comprises the following steps:

Preparing a Buffer:

the formulation was 15mM Tris-HCl (pH=8.0), 250. Mu.M EDTA,250mM NaCl,10% glycerol.

Preparation of Trypsin solution:

the formulation was 10mg/mL pancreatin, buffer,2μ MLBH589.

Preparing a substrate solution:

The substrate was dissolved in DMSO to prepare a 30mM stock solution, which was then diluted to 300. Mu.M with buffer to give

The DMSO content was about 1%.

Dilution of enzyme solution:

the enzyme solution was diluted with buffer in a ratio of 1:80.

Preparation of compound solution:

Diluting the compound (test compound and positive control SAHA) to a final concentration of 5 Xwith buffer

Formulation and determination of 100% and blank:

50. Mu.L of buffer was mixed with 10. Mu.L of enzyme solution, after 5 minutes, 40. Mu.L of substrate was added to react at 37℃for 0.5 hours, then 100. Mu. L Trypsin solution was added to terminate the above reaction, and after 20 minutes at 37℃the fluorescence intensity was measured at (390 nm/460 nm) to give 100% absorbance, 60. Mu.L of buffer was added to 40. Mu.L of substrate, after 37℃for 0.5 hours, 100. Mu.L of Trypsin solution was added, and after 20 minutes at 37℃the fluorescence intensity was measured at (390 nm/460 nm) to give the blank absorbance.

Determination of HDACs inhibition by compounds:

50. Mu.L of buffer containing the drug was mixed with 10. Mu.L of enzyme solution and incubated for 5 minutes in advance, 40. Mu.L of substrate was added and reacted at 37℃for 0.5 hours, then 100. Mu.L of Trypsin solution was added to terminate the above reaction, and after 20 minutes of reaction at 37℃the fluorescence intensity was measured at (390 nm/460 nm) and the inhibition ratio and IC ₅₀ value were calculated using GRAPHPAD PRISM software and a formula. (Table 1)

TABLE 1 HDACs inhibitory Activity of target Compounds

^aA,IC₅₀ Greater than 5. Mu.M, B with IC ₅₀ between 1. Mu.M and 5. Mu.M, C with IC ₅₀ between 0.2. Mu.M and 1. Mu.M, D with IC ₅₀ below 0.2. Mu.M

^b Not tested

Experimental example 2 target Compound has DNMT1 inhibitory Activity

1. Materials:

MT1hypo-SFRP1-Luc cells, 96 Kong Quanhei ELISA plates, RPMI1640 medium, 10% fetal bovine serum (Hyclone Co., USA), PBS buffer; Luciferase detection reagent, positive control SAHA and GSK5032.

2. The method comprises the following steps:

inhibition of DNMT1 restores expression of the hypermethylated SFRP1 gene. Since SFRP1 has inserted the luciferase gene, the protein expression level can be determined by detecting the fluorescence intensity. MT1hypo-SFRP1-Luc cells (5400 per well) were seeded in black well plates and incubated in an incubator (37 ℃ C., 5% carbon dioxide) for 16 hours. Then adding compound dilutions prepared from corresponding cell culture media at different concentrations, and continuously placing the mixture into a constant temperature incubator for culturing for 72 hours after the addition. Finally, a luciferase detection reagent is added to each well and the corresponding fluorescence intensity is detected by an enzyme-labeled instrument. Inhibition activity was expressed as fold change (fluorescence intensity of compound group/fluorescence intensity of blank group) (table 2).

TABLE 2 DNMT1 inhibitory Activity of target Compounds

^a A, fold change less than 100;B,fold change is between 100 and 200, C, fold change is between 200 and 300, D, fold change is greater than 300

Experimental example 3 antiproliferative Activity of target Compounds

1. Materials:

MV4-11 (human acute myelogenous leukemia cells), KG-1 (human acute myelogenous leukemia cells), THP-1 (human monocytic leukemia cells), jurkat (human T lymphocytic leukemia cells), K562 (human chronic myelogenous leukemia cells), MC38 (mouse colon cancer cells), 10% fetal bovine serum (Hyclone Co., USA), 2.5g.L ^-1 trypsin (Gibco Co., USA), CCK-8, modified RPMI1640 medium, DMEM medium (Hyclone Co., USA), positive controls SAHA and GSK5032,96 well plates.

2. The method comprises the following steps:

The cells were cultured conventionally, and the cells grown in logarithmic growth phase were collected for experiments, and solid tumor cells in logarithmic growth phase were diluted to 4X 10 ³. Mu.mL ^-1 (blood tumor cells were diluted to 1X 10 ⁴. ML ^-1) with a medium containing fetal bovine serum 10% RPMI1640 or DMEM, inoculated in 96-well plates (100. Mu.L per well) without adding cells as blank wells, then placed in an incubator (37 ℃ C., 5% carbon dioxide) for 8 hours, and then added with a target compound solution and a positive drug solution prepared with the medium as 100% wells without adding drugs, and after culturing in an incubator (37 ℃ C., 5% carbon dioxide) for 72 hours, 20. Mu.L of CCK-8 was added, and after three hours, absorbance values per well were measured with an ELISA reader at a wavelength of 450nm, and the inhibition rate and IC ₅₀ values were calculated (Table 3).

TABLE 3 antiproliferative activity of the compounds

^a A,IC₅₀ Greater than 20. Mu.M, B with IC ₅₀ between 4. Mu.M and 20. Mu.M, C with IC ₅₀ between 0.4. Mu.M and 4. Mu.M, D with IC ₅₀ below 0.4. Mu.M

Experimental example 4 therapeutic Effect of Compounds on human acute myelogenous leukemia

1. Materials:

cell line human acute myelogenous leukemia cell (MV 4-11).

The experimental animals were six-week-old BALB/c female nude mice purchased from Beijing vitamin Toril Hua laboratory animal technologies Co.

2. The method comprises the following steps:

approximately 2X 10 ⁷ MV4-11 cells were resuspended in 50. Mu.L of serum-free high-sugar medium and 50. Mu.L of matrigel and subcutaneously implanted in the right armpit area of BALB/c nude mice. When the tumor volume reached about 100mm ³, oral administration was started for 28 consecutive days. The dosage of the treatment group is 30mg/kg/d, the combined administration group is 30mg/kg/d+30mg/kg/d, and the control group is given with the same amount of physiological saline. Tumor size and mouse body weight were measured daily during the treatment period. Tumor volume calculation formula V (mm ³) =length (mm) ×width ² (mm)/2, tumor growth inhibition value (TGI) calculation formula tgi= (1-treatment group tumor average volume/control group tumor average volume) ×100%.

3. Experimental results:

As shown in the test result in FIG. 1, the tumor inhibition rate (TGI) of the target compound (R) -23a is preferably 98%, and the tumor inhibition effect is obviously better than that of the combined treatment group of the positive control medicine GSK5032 and SAHA.

Experimental example 5 therapeutic Effect of Compounds on colon cancer in mice

1. Materials:

Cell line mouse colon cancer cells (MC 38).

Experimental animals six weeks old BALB/c female nude mice were purchased from Beijing vitamin Toril Hua laboratory animal technologies Co.

2. The method comprises the following steps:

approximately 1.2X10 ⁶ MC38 cells were resuspended in 100. Mu.L of serum-free high-sugar medium and subcutaneously implanted in the right armpit area of BALB/c nude mice. When the tumor volume reached about 100mm ³, oral administration was started for 14 consecutive days. The dosage of the treatment group is 30mg/kg/d, the combined administration group is 30mg/kg/d+30mg/kg/d, and the control group is given with the same amount of physiological saline. Tumor size and mouse body weight were measured daily during the treatment period. Tumor volume calculation formula V (mm ³) =length (mm) ×width ² (mm)/2, tumor growth inhibition value (TGI) calculation formula tgi= (1-treatment group tumor average volume/control group tumor average volume) ×100%.

3. Experimental results:

The test results are shown in fig. 2, the tumor inhibition rate (TGI) of the target compound (R) -23a in the mouse colon cancer model is 82%, and the tumor inhibition effect is superior to that of the positive control medicine GSK5032 and the combination treatment group of the positive control medicine GSK5032 and SAHA.

Claims

1. A DNMT1-HDAC dual-target inhibitor, wherein the structural formula of the inhibitor is as shown in formula (I) or a pharmaceutically acceptable salt of formula (I):

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

A is one of the following structures:

_R1 is hydroxy or 2-aminophenyl;

_R2 is one of the following structures

_R3 is a hydrogen atom and is one of the following structures:

2. The DNMT1-HDAC dual-target inhibitor according to claim 1, 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 -hydroxysuccinamide (11a);

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);

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

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

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

N ¹ -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N ⁹ -hydroxyazelazamide (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-toluenediamide (11 g);

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)succinamide (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)glutaramide (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)adipamide (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)succinamide (111);

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)succinamide (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)piperidinium 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)piperidin-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)oxy)benzyl)piperidin-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-oxoheptyl)oxy)benzyl)piperidin-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)-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);

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);

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 -hydroxysuccinamide (40a);

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

N ¹ -(3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)propyl)-N ⁴ -hydroxysuccinamide (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 -hydroxyglutaramide (44a);

(E)-(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N ⁵ -hydroxyglutaramide (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).

3. The method for preparing the DNMT1-HDAC dual-target inhibitor according to claim 2, wherein the method comprises any one of the following methods:

The preparation methods of compounds 11a-11V are 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:

4. The preparation method according to claim 3, characterized in that in the preparation method of compounds 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-propionaldehyde, N-methylmorpholine, anhydrous ethanol, room temperature, 30 hours; (c) N,N-diisopropylethylamine, N,N-dimethylformamide, 85°C, 5 hours; (d) isoamyl nitrite, cupric chloride, acetonitrile, 80°C, 2.5 hours; (e) aminopiperidine derivatives, carbon 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.

5. The preparation method according to claim 3, characterized in that in the preparation method 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.

6. The preparation method according to claim 3, characterized in that in the preparation method of compounds 23a, (R)-23a, (S)-23a, 23b, 27, 30a-e,

The reagents and conditions are as follows: (a) piperidine-4-aminomethyl tert-butyl carbamate, potassium carbonate, dichloromethane, 4 hours; (b) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours; (c) 1) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 2) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (d) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours; (e) tert-butyl (2-(methylamino)ethyl)carbamate, potassium carbonate, dichloromethane, 4 hours; (f) 4M hydrochloric acid/ethyl acetate ethyl acetate, 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.

7. The preparation method according to claim 3, 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 hours; (b) diisobutylaluminum hydride, tetrahydrofuran, -20°C, 6 hours; (c) p-toluenesulfonyl chloride, triethylamine, DMAP, acetonitrile, 80°C, 4 hours; (d) 4, triethylamine, DMF, 85°C, 5 hours; (e) isoamyl nitrite, cupric chloride, acetonitrile, 80°C, 2.5 hours; (f) 2-methylamino derivative, potassium carbonate, dichloromethane, 4 hours; (g) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours; (h) 1) 9a or 9b, isobutyl chloroformate, N-methylmorpholine, dichloromethane/tetrahydrofuran (1:1), room temperature, 6 hours; 2) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (i) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours.

8. The preparation method according to claim 3, characterized in that in the preparation method of compounds 44a-c and 48,

Reagents and conditions are as follows: (a) tert-butyl 4-carbamate, potassium carbonate, dichloromethane, 4 hours; (b) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours; (c) 1) 9b, isobutyl chloroformate, N-methylmorpholine, dichloromethane/tetrahydrofuran (1:1), room temperature, 6 hours; 2) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 3) 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) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (h) 4M hydrochloric acid/ethyl acetate, room temperature, 12 hours.

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

10. Use of the DNMT1-HDAC dual-target inhibitor according to claim 1 in the preparation of a drug for treating tumor diseases related to the target;