A kind of p-amidobenzenesulfonyl compound and its application

Abstract

The present invention provides a p-amidobenzenesulfonyl compound and its application, wherein the p-amidobenzenesulfonyl compound has a structure shown in the following formula I. The p-amidobenzenesulfonyl compound provided by the present invention exhibits excellent inhibitory activity on PHGDH, and has strong proliferation inhibitory activity on cell lines with PHGDH gene amplification or high PHGDH expression; and can be used as a PHGDH inhibitor for preparing anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a p-amidobenzenesulfonyl compound and application thereof. Background Art

[0002] The difficulty in curing tumors is largely due to the complexity of gene mutations and metabolic pathway changes in tumor cells. Exploring the biological characteristics that are common in tumor cells but different from normal cells and specifically intervening in these characteristics are the key to improving the effectiveness of tumor treatment. As early as the 1920s, the metabolic changes of tumor cells attracted the attention of German physiologist Otto Warburg, the famous "Warburg effect", also known as "aerobic glycolysis".

[0003] With the development of modern molecular biology technology, changes in many metabolic pathways such as fatty acid metabolism, glutamine metabolism, serine metabolism, one-carbon unit metabolism, and choline metabolism have also been summarized as the "Warburg effect". More and more evidence shows that tumor-related genes (activation of oncogenes or loss and inactivation of tumor suppressor genes), cellular microenvironment (hypoxia), transcription factors, non-coding RNA, etc., as well as mutations in metabolic enzymes themselves or changes in the activity of metabolic regulatory proteins can all lead to metabolic reprogramming of tumor cells, giving tumor cells a characteristic metabolic pattern. In clinical practice, the 18F-FDG PET / CT technology, which was developed based on the high glucose uptake capacity of tumor cells, can detect most primary and metastatic epithelial tumors, and has become an efficient and specific means of tumor diagnosis, staging, and efficacy monitoring. Therefore, regulating tumor metabolism is of great significance for the early diagnosis, intervention, and treatment of tumors.

[0004] Aerobic glycolysis of tumor cells is one of the important signs of metabolic abnormalities. The intermediates produced can be integrated into various metabolic pathways to produce nucleotides, lipids, amino acids and NADPH, etc., providing important raw materials for the synthesis of life substances. Among them, the serine synthetic pathway (SSP), as an important branch of the glycolysis pathway, controls the flux of glycolysis intermediates into serine and its downstream substances. Under certain pressures, tumor cells have to use this pathway to provide raw materials and energy for proliferation. This pathway mainly includes three consecutive enzyme-catalyzed reactions, specifically: under the synergistic action of the cofactor NAD+, phosphoglycerate dehydrogenase (PHGDH) oxidizes about 10% of the glycolysis intermediate 3-phosphoglycerate (3-PG) to 3-phosphopyruvate (3P-pyruvate), and then converts it into 3-phosphoserine (3P-serine) under the action of phosphoserine aminotransferase 1 (PSAT1), and finally converts it into serine (Serine) through phosphoserine phosphatase (PSPH). Serine can be further transformed to provide precursor materials for a variety of biosynthetic pathways. For example, serine can be converted into glycine by serine hydroxymethyltransferase (SHMT), which is beneficial to the synthesis of nucleotides and glutathione. The accumulation of serine can allosterically activate pyruvate kinase isozyme M2 (PKM2), thereby accelerating glycolytic flux and promoting the occurrence and development of tumors.

[0005] Phosphoglycerate dehydrogenase (PHGDH) is the key rate-limiting enzyme in the first step of the serine synthesis pathway, controlling the de novo synthesis pathway of serine. Many studies have shown that in some tumor cells (especially breast cancer cells, non-small cell lung cancer cells and melanoma cells lacking estrogen receptors), some intermediates of the glycolysis pathway enter the serine synthesis pathway, PHGDH enzyme activity is enhanced, the gene is amplified and the protein is highly expressed, participating in the regulation of tumor cell proliferation, apoptosis and invasion. After silencing the PHGDH gene or inhibiting the activity of the PHGDH enzyme, the flow of the serine de novo synthesis pathway is reduced, which can significantly reduce the proliferation of these cancer cells in vivo and in vitro, promote cell apoptosis, and inhibit tumor cell invasion, while it has no effect on tumor cells that normally express PHGDH. Studies have also shown that PHGDH plays an important role in the formation of secondary (recurrent or metastatic) tumors by controlling the redox homeostasis of mitochondria in cells, and has potential significance for the targeted treatment of advanced cancer. In addition, the use of inhibitors or siRNA to interfere with PHGDH can overcome the drug resistance caused by erlotinib in the treatment of lung cancer. This is mainly because the inhibition of PHGDH can cause a significant increase in the DNA damage marker γH2AX in resistant cells (PC9 and HCC827), and reduce the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), thus revealing that PHGDH is a potential target for overcoming the drug resistance of lung cancer EGFR-TKIs. Therefore, as tumor metabolism has become a research hotspot again, PHGDH is an important new target in anti-tumor research and development strategies. Its inhibitors can not only treat tumors through a new mechanism of action, but also have the potential to solve the drug resistance problem caused by current tumor treatment, which has important research value.

[0006] Currently, no PHGDH inhibitor has entered clinical research. The main reasons are as follows: (1) Although Professor Snell and others found in the 1980s that the PHGDH enzyme activity in rat liver cancer cells was 1.7-10.6 times that in normal liver tissue, it has not attracted much attention. It was not until 2011 that a number of functional genomics articles in Nature and Cell magazines brought PHGDH back into people's attention. Its small molecule inhibitor was first reported in 2014. Therefore, the inhibitors of this target have a short history and are relatively small in number and structure type; (2) The reported inhibitors have poor in vitro activity, and the enzyme and cell activities are both at the micromolar level; (3) The pharmacokinetic properties of some compounds are poor, which affects the in vivo biological research of the compounds.

[0007] Therefore, finding PHGDH inhibitors with novel structure, strong activity and good pharmacokinetic properties is a key scientific issue that needs to be solved urgently and has important research value. Summary of the invention

[0008] In view of the shortcomings of the prior art, the object of the present invention is to provide a p-amidobenzenesulfonyl compound and its application. The p-amidobenzenesulfonyl compound provided by the present invention can be used to prepare a PHGDH inhibitor.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a para-amidobenzenesulfonyl compound, wherein the para-amidobenzenesulfonyl compound has a structure as shown in the following formula I:

[0011]

[0012] Among them, R 1selected from halogen, substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl, substituted or unsubstituted Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched alkyl, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarboxyl, substituted or unsubstituted C3-C20 ( For example, it can be C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) cycloalkyl, substituted or unsubstituted C2-C20 (for example, it can be C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heterocycloalkyl, substituted or unsubstituted C2-C2 any one of C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) alkylaryl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) alkoxyaryl;

[0013] R 2selected from substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl, any one of a C7, C8, C9, C10, etc.) straight or branched alkyl, a substituted or unsubstituted C3-C20 (for example, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) cycloalkyl, and a substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heterocycloalkyl;

[0014] n is selected from 0 or 1.

[0015] In the present invention, the substituted substituents are each independently selected from halogen, carboxyl, hydroxyl, amide, unsubstituted or R a Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched alkyl, unsubstituted or R a Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, unsubstituted or R a Substituted C1-C10 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylhydroxyl, unsubstituted or R a Substituted C1-C10 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarbonyl, unsubstituted or R a substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylamide, unsubstituted or Ra-substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxyamide, NR N1 R N2 , unsubstituted or R aSubstituted C1-C10 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarboxyl, unsubstituted or R a Substituted C3-C20 (for example, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) cycloalkyl, unsubstituted or R a Substituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heterocycloalkyl, unsubstituted or R a Substituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, unsubstituted or R a Any one of substituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl;

[0016] R N1 , R N2 Each is independently selected from any one of hydrogen, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched chain alkyl;

[0017] R a Any one selected from halogen, hydroxyl, nitro, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight or branched alkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy or C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) haloalkyl.

[0018] In the present invention, the para-amidobenzenesulfonyl compound is selected from any one of the structures shown in the following formula II to formula VI:

[0019]

[0020] Among them, R 11Any one selected from halogen, substituted or unsubstituted C4-C20 (for example, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) nitrogen-containing heterocycloalkyl;

[0021] R 12 selected from substituted or unsubstituted C3-C20 (for example, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) cycloalkyl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heterocycloalkyl, substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight or branched alkyl, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, substituted or unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarboxyl;

[0022] R 2 Any one selected from substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl.

[0023] p is an integer selected from 0-5 (for example, 0, 1, 2, 3, 4, 5); X is selected from CH 2 , O or NH; L 1Any one selected from a single bond, a substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, or a substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl;

[0024] q is selected from an integer of 0-5 (for example, 0, 1, 2, 3, 4, 5); Y is selected from CH 2 , carbonyl, O, NH, Any one of L 2 Any one selected from a single bond, a substituted or unsubstituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl, or a substituted or unsubstituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl; Indicates the attachment site of a group.

[0025] In the present invention, the R 2 Any one selected from substituted C2-C20 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) heteroaryl;

[0026] The substituents of the substituted C2-C20 heteroaryl are selected from unsubstituted or R b Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched alkyl, unsubstituted or R b Any one of substituted C6-C20 (for example, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, etc.) aryl groups;

[0027] The R bAny one selected from halogen, hydroxyl, nitro, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight or branched alkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy or C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) haloalkyl.

[0028] In the present invention, the R 2 Any one selected from substituted thiazolyl, substituted thienyl, and substituted triazole;

[0029] The substituted substituents are each independently selected from unsubstituted or R c Substituted phenyl, unsubstituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched chain alkyl;

[0030] The R c Any one selected from halogen, hydroxyl, nitro, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight or branched alkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy or C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) haloalkyl.

[0031] In the present invention, the R 11 is selected from halogen, unsubstituted or R d Any one of substituted C4-C12 (for example, C4, C5, C6, C7, C8, C9, C10, C11, C12, etc.) nitrogen-containing heterocycloalkyl;

[0032] The R d Any one selected from carboxyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched chain alkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy.

[0033] In the present invention, the R 12 Selected from unsubstituted or R e Substituted C2-C12 (e.g., C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, etc.) heterocycloalkyl, unsubstituted or R eSubstituted C3-C12 (e.g., C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, etc.) cycloalkyl, unsubstituted or R e Substituted C6-C12 (for example, C6, C7, C8, C9, C10, C11, C12, etc.) aryl, unsubstituted or R e Substituted C2-C12 (e.g., C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, etc.) heteroaryl, unsubstituted or R e Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched alkyl, unsubstituted or R e Substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, unsubstituted or R e Any one of substituted C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarboxyl groups;

[0034] The R e is selected from hydroxyl, carboxyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) straight chain or branched alkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkoxy, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylhydroxyl, C2-C12 (for example, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) , C6, C7, C8, C9, C10, C11, C12, etc.) heterocycloalkyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarbonyl, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylamide, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.) alkylcarboxyl, NR N3 R N4 Any of the following:

[0035] The R N3 , R N4 Each is independently selected from hydrogen, C1-C10 (for example, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, etc.), any one of a straight chain or branched chain alkyl group.

[0036] In the present invention, the L 1 , L 2 Each is independently selected from a single bond, a substituted or unsubstituted C6-C12 (for example, C6, C7, C8, C9, C10, C11, C12, etc.) aryl group, or a substituted or unsubstituted C5-C12 (for example, C5, C6, C7, C8, C9, C10, C11, C12, etc.) heteroaryl group.

[0037] In the present invention, the p-amidobenzenesulfonyl compound has any one of the structures shown in A2, B1-B25, C1-C6, D1-D23, E1-E5, and F1-F7:

[0038]

[0039]

[0040]

[0041] In a second aspect, the present invention provides a use of the p-amidobenzenesulfonyl compound according to the first aspect in the preparation of a phosphoglycerate dehydrogenase inhibitor.

[0042] In a third aspect, the present invention provides a use of the para-amidobenzenesulfonyl compound according to the first aspect in the preparation of anti-tumor drugs.

[0043] The enzyme inhibition activity IC of the preferred compound D8 of the present invention on PHGDH 50 The IC 50 The values ​​ranged from 3.5μM to 21.6μM. MST experiments showed that D8 had a strong binding affinity with PHGDH protein, with a Kd value of 2.76μM; D8 could inhibit the de novo synthesis of serine in MDA-MB-468 cells; flow cytometry experiments showed that compound D8 could block the G1 phase of the cell cycle; molecular dynamics experiments and amino acid mutation experiments jointly revealed that D8 had a strong binding affinity with PHGDH NAD + The action mode of the binding pocket; pharmacokinetic experiments showed that compound D8 had excellent oral bioavailability (F% = 82.0%); in vivo antitumor experiments showed that compound D8 had good antitumor activity in the PC9 transplanted tumor mouse model.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The invention provides a para-amidobenzenesulfonyl compound, which exhibits excellent inhibitory activity on PHGDH and has strong proliferation inhibitory activity on cell lines with PHGDH gene amplification or high PHGDH expression; and can be used as a PHGDH inhibitor for preparing anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 For D8 and NAD + Lineweaver-Burk plot of the competition test.

[0047] Figure 2 Lineweaver-Burk plot for the competition test between D8 and 3-PG.

[0048] Figure 3 Diagram of the binding mode of D8 in the cis-PHGDH model.

[0049] Figure 4 Diagram of the binding mode of D8 in the trans-PHGDH model.

[0050] Figure 5 D8 inhibits the expression of 13 C6-Glucose Synthesis 13 C3-serine test results graph.

[0051] Figure 6 This is a graph showing the anti-proliferative activity test results of compound D8 on cancer cell lines.

[0052] Figure 7 The drug concentration distribution in mouse plasma after intravenous injection and oral administration of D8.

[0053] Figure 8 The graph shows the changes in tumor volume.

[0054] Fig. 9 The weight of the tumor was measured on day 31 of treatment.

[0055] Fig.10 This is the weight growth curve of mice. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0057] The explanations of the English abbreviations in the following embodiments and test examples are as follows:

[0058] PHGDH, phosphoglycerate dehydrogenase; SSP, serine biosynthesis pathway; 3-PG, 3-phosphoglycerate; 3-PPyr, 3-phosphohydroxypyruvate; NAD, nicotinamide adenine dinucleotide; PK, pharmacokinetics; MST, microthermophoresis; TGI, tumor growth inhibition; TNBC, triple-negative breast cancer; AUC, mean area under the curve; MD, molecular dynamics.

[0059] (I) Compound A1, Compounds B1-B25 and Synthesis

[0060] Commercially available 4-(chlorosulfonyl)benzoic acid (Compound 1) was reacted with 4-phenylthiazol-2-amine (Compound 2) in the presence of triethylamine to generate Compound A1. Using triethylamine as a base at room temperature, different amine derivatives were reacted with A1 to generate Compounds B1-B3, B5-B12, B14-B21, B24-B25 and Compound 3-6. Compound 3-6 was reacted with 1M NaOH aqueous solution and CH 3 OH solution to generate B4, B13, B22 and B23. For the synthesis of compound B16, the key intermediate 9 was obtained by two consecutive reactions. First, tetrahydrofuran was used as a solvent, and in the presence of pyridine, commercially available 4,6-dimethylpyrimidine-2-amine (compound 7) and methyl 4-(chlorosulfonyl)benzoate (compound 8) were reacted to generate the corresponding ester; then the ester was hydrolyzed in a sodium hydroxide aqueous solution to generate an intermediate (compound 9), and the intermediate (compound 9) reacted with the corresponding amine to generate compound B16.

[0061] The synthetic reaction formula is as follows:

[0062] Synthesis of compounds A1, B1-B3, B5-B12, B14-B21, B24 and B25:

[0063]

[0064]

[0065] Synthesis of compounds B4, B13, B22 and B23:

[0066]

[0067] Synthesis of compound B16:

[0068]

[0069] Reaction conditions: (a) Et 3 N,THF,reflux,4h;(b)Et 3 N,THF,rt,2h; (c)1M NaOH(aq.),CH3 OH,rt,2h;(d)Pyridine,THF,rt,4h;(e)1M NaOH(aq.),CH 3 OH,rt,2h;(f)HATU,DIPEA,THF,rt,6h.

[0070] (II) Synthesis of Compounds C1-C6 and Compounds D1-D23

[0071] The synthesis steps of compounds C1-C6 and compounds D1-D23 are the same as the synthesis of compound B16. The synthesis reaction formula is shown below:

[0072]

[0073] Reaction conditions: (a) Et 3 N,THF,reflux,4h; (b))1M NaOH(aq.),CH 3 OH,rt,2h; (c)HATU,DIPEA,THF,rt,4h.

[0074] Preparation Example 1

[0075] Synthesis of compound 9: To a solution of 4,6-dimethylpyrimidin-2-amine (compound 7, 123 mg, 1.0 mmol) in THF (8 mL) was added methyl 4-(chlorosulfonyl)benzoate (compound 8, 235 mg, 1.0 mmol) and pyridine (243 μL, 3.0 mmol) at room temperature, and after stirring for about 4 hours, the solvent was removed under reduced pressure. The resulting residue was dissolved in CH 3 OH (3 mL) and NaOH (1 mol / L) aqueous solution (3 mL), and stirred for another 2 hours. Then the solvent was removed under reduced pressure, and the residue was acidified to pH = 2 or lower with HCl (1 mmol / L) aqueous solution. The solution was extracted with EtOAc (3×20 mL), and the combined organic matter was washed with anhydrous Na 2 SO 4 Dry and concentrate in vacuo. Chromatography on silica gel (DCM / CH 3 The residue was purified by 4% paraformaldehyde (2-(4-(4-hydroxy-1-hydroxy-2 ...

[0076] 1 H NMR (600 MHz, DMSO-d 6 )δ8.07(m,4H),6.75(s,1H),2.25(s,6H).ESI-MS:calcdfor[M+H] + m / z 308.1, found: 308.2.

[0077] Preparation Example 2

[0078] The synthesis steps of compound 11 are the same as those of compound 9, and compound 8 and methyl-3-aminoacetophenone (compound 10) are used to prepare the compound 11, which is a white solid with a yield of 72%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.75(s,1H),8.08(d,J=8.4Hz,2H),7.89(d,J=8.4Hz,2H),7.68(d,J=7.7Hz,1H),7.65(d, J=1.7Hz,1H),7.42(t,J=7.9Hz,1H),7.37(dd,J=8.1,1.2Hz,1H),2.51(s,3H).ESI-MS:calcd for[MH] - m / z318.0, found:318.1.

[0079] Example 1

[0080] Synthesis of Compound A1: Using THF (20 mL) as solvent, 4-(chlorosulfonyl)benzoic acid (Compound 1, 1.14 g, 5.16 mmol), 4-phenylthiazol-2-amine (Compound 2, 1.0 g, 5.67 mmol) and Et 3 N (786 μL, 5.67 mmol) and refluxed for 4 h. After the solvent was removed by concentration under reduced pressure, EtOAc (100 mL) and water (20 mL) were added. The liquid was separated, the aqueous layer was extracted with EtOAc (3×20 mL), the organic layers were combined, and anhydrous Na 2 SO 4 The mixture was dried and concentrated in vacuo. The crude compound was purified by silica gel column chromatography (PE / EA=10 / 1, v / v) to give A1 (879 mg, 45% yield) as a pale yellow solid.

[0081] 1 H NMR (600 MHz, DMSO-d 6 )δ12.84(s,1H),8.07(d,J=8.3Hz,2H),7.97(d,J=7.3Hz,2H),7.74(d,J=8.3Hz,2H),7.71(s,1H),7.46(t,J=7.7Hz,2H),7.34(t,J=7.3Hz,1H). 13 CNMR (150MHz, DMSO-d 6)δ165.47,158.96,152.41,149.58,134.82,132.37,129.22,128.44,128.29,126.25,126.10,109.05.

[0082] Example 2

[0083] Synthesis of Compound B1: Compound A1 (379 mg, 1.0 mmol) and Et 3 N (278 μL, 2.0 mmol). The mixture was stirred for 4 hours and monitored by TLC. After the solvent was removed by concentration under reduced pressure, EtOAc (100 mL) and water (20 mL) were added. The organic layer was washed with anhydrous Na 2 SO 4 Dry and concentrate in vacuo.The crude compound was purified by silica gel column chromatography (PE / EA=4 / 1, v / v) to give compound B1 (365 mg, 85% yield) as a white solid.

[0084] 1 H NMR (600 MHz, DMSO-d 6 )δ13.09(s,1H),8.36(d,J=8.4Hz,2H),7.97(d,J=7.2Hz,2H),7.91(d,J=8.4Hz,2H),7.76( s,1H),7.46(t,J=7.7Hz,2H),7.35(t,J=7.3Hz,1H),3.68–3.62(m,4H),2.97–2.91(m,4H). 13 C NMR (150 MHz, DMSO-d 6 )δ169.75,163.52,157.60,148.69,137.25,135.67,133.65,128.76,128.19,127.31,127.22,125.20,108.32,64.69,45.29.HRMS(ESI):[M+H] + Calculate for C 20 H 20 N 3 O 4 S 2 + 430.0890,found 430.0894.HPLCpurity,99%.

[0085] Example 3

[0086] The synthesis steps of compound B2 are the same as those of compound B1, and it is prepared from compound A1 and thiomorpholine. It is a white solid with a yield of 90%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.07(s,1H),8.34(d,J=8.4Hz,2H),7.97(d,J=7.4Hz,2H),7.92(d,J=8.4Hz,2H),7.76( s,1H),7.46(t,J=7.7Hz,2H),7.35(t,J=7.3Hz,1H),3.30-3.25(m,4H),2.71–2.67(m,4H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.59,158.68,149.76,140.03,136.60,134.72,129.90,129.25,128.38,127.86,126.27,109.38,48.27,26.89.HRMS(ESI):[M+Na] + Calculate for C 20 H 19 N 3 O 3 S 3 Na + 468.0481,found 468.0476.HPLCpurity,99%.

[0087] Example 4

[0088] The synthesis steps of compound B3 are the same as those of compound B1. It is prepared from compound A1 and thiomorpholine-1,1-dioxide as a white solid with a yield of 87%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.08(s,1H),8.36(d,J=8.3Hz,2H),8.00(d,J=8.3Hz,2H),7.96(d,J=7.7Hz,2H),7.76( s,1H),7.46(t,J=7.6Hz,2H),7.35(t,J=7.4Hz,1H),3.56-3.48(s,4H),3.33–3.28(m,4H). 13 C NMR (150 MHz, DMSO-d 6)δ164.55,158.67,149.76,139.79,137.01,134.71,130.08,129.26,128.39,127.92,126.27,109.41,50.63,45.57.HRMS(ESI):[M+H] + Calculate for C 20 H 20 N 3 O 5 S 3 + 478.0560, found 478.0557. HPLC purity, 99%.

[0089] Example 5

[0090] The synthesis steps of compound B5 are the same as those of compound B1. It is prepared from compound A1 and 4-piperidineethanol as a white solid with a yield of 75%. 1 H NMR (600 MHz, CDCl 3 )δ10.90(s,1H),7.95(d,J=8.3Hz,2H),7.73(t,J=8.7Hz,4H),7.35(t,J=7.6Hz,2H),7.28(d,J=7.4Hz,1H),7.24(s,1H),3.78(d,J=11. 7Hz,2H),3.67(t,J=6.3Hz,2H),2.27-2.20(m,2H),1.76(d,J=12.7Hz,2H),1.50(q,J=6.4Hz,2H),1.44–1.37(m,1H),1.36–1.28(m,2H). 13 C NMR (150 MHz, CDCl 3 )δ163.67,158.46,150.33,140.26,135.64,133.87,128.85,128.31,128.09, 128.02,126.06,108.55,60.02,46.38,38.61,31.66,31.40.HRMS(ESI):[M+H] + Calculate for C 23 H 26 N 3 O 4 S 2 + 472.1359,found472.1360.HPLC purity,99%.

[0091] Example 6

[0092] The synthesis steps of compound B6 are the same as those of compound B1. It is prepared from compound A1 and cyclohexylamine as a white solid with a yield of 83%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.02(s,1H),8.28(d,J=8.4Hz,2H),7.99–7.95(m,4H),7.88(d,J=7.4Hz,1H),7.74(s,1H),7.46(t,J=7.7Hz,2H),7.35 (t,J=7.3Hz,1H),3.07–2.95(m,1H),1.58(d,J=8.2Hz,4H),1.45(d,J=12.1Hz,1H),1.20–1.10(m,4H),1.07-0.98(m,1H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.78,158.71,149.72,146.14,135.67,134.74,129.62,129.25,128. 36,126.82,126.25,109.31,52.67,33.73,25.28,24.81.HRMS(ESI):[M+H] + Calculate for C 22 H 24 N 3 O 3 S 2 + 442.1254,found 442.1261.HPLC purity,97%.

[0093] Example 7

[0094] The synthesis steps of compound B7 are the same as those of compound B1. It is prepared from compound A1 and 4-aminotetrahydropyran. It is a white solid with a yield of 30%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.01(s,1H),8.28(d,J=6.7Hz,2H),8.08–7.91(m,5H),7.75(s,1H),7.45(t,J=5.6Hz,2H),7.35(t, J=8.2Hz,1H),3.72(d,J=9.2Hz,2H),3.29–3.18(m,3H),1.53(d,J=10.4Hz,2H),1.37(d,J=9.3Hz,2H). 13 C NMR (150 MHz, DMSO-d 6)δ164.75,158.70,149.74,145.95,135.82,134.74,129.69,129.26,128.38,126.87,126.26,109.34,65.99,49.86,33.80.HRMS(ESI):[M+H] + Calculate for C 21 H 22 N 3 O 4 S 2 + 444.1046,found 444.1049.HPLC purity,96%.

[0095] Example 8

[0096] The synthesis steps of compound B8 are the same as those of compound B1. It is prepared from compound A1 and 1-methylpiperidin-4-amine as a white solid with a yield of 75%. 1 H NMR (600 MHz, DMSO-d 6 )δ8.29(d,J=8.4Hz,2H),8.01(d,J=7.0Hz,1H),7.97(dd,J=7.6,6.3Hz,4H),7.74(s,1H),7.46(t,J=7.7Hz,2H),7.35(t,J=7.4 Hz,1H),3.09–3.01(m,1H),2.74(d,J=10.2Hz,2H),2.20(s,3H),2.14–2.02(m,2H),1.59(d,J=10.8Hz,2H),1.51–1.41(m,2H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.89,159.09,149.63,145.76,136.03,134.77,129.65,129.24,128.33,126.85,126.25,109.22,54.13,46.03,32.63.HRMS(ESI):[M+H] + Calculate for C 22 H 25 N 4 O 3 S 2 + 457.1363,found 457.1360.HPLCpurity,98%.

[0097] Example 9

[0098] The synthesis steps of compound B9 are the same as those of compound B1. It is prepared from compound A1 and 1-acetylpiperidin-4-amine as a white solid with a yield of 73%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.02(s,1H),8.29(d,J=8.4Hz,2H),8.04(d,J=7.3Hz,1H),7.98(dd,J=13.8,7.9Hz ,4H),7.75(s,1H),7.46(t,J=7.7Hz,2H),7.35(t,J=7.3Hz,1H),4.07(d,J=13.2Hz,1H) ,3.65(d,J=13.9Hz,1H),3.32–3.25(m,1H),3.08–2.99(m,1H),2.71–2.62(m,1H),1.94 (s,3H),1.67–1.53(m,2H),1.29(td,J=14.5,4.0Hz,1H),1.19(td,J=14.8,4.2Hz,1H). 13 C NMR (150 MHz, DMSO-d 6 )δ168.37,164.74,158.73,149.73,145.80,135.85,134.74,129.71,129.25,128.37, 126.87,126.26,109.33,50.58,44.51,40.52,33.36,32.51,21.65.HRMS(ESI):[M+H] + Calculate for C 23 H 25 N 4 O 4 S 2 + 485.1312,found 485.1314.HPLC purity,99%.

[0099] Example 10

[0100] The synthesis steps of compound B10 are the same as those of compound B1. It is prepared from compound A1 and 4-methyl-1-piperazineethylamine as a white solid with a yield of 90%. 1 H NMR (600 MHz, CDCl 3)δ7.98(d,J=7.4Hz,2H),7.89(d,J=7.6Hz,2H),7.72(d,J=7.0Hz,2H),7.35(t,J=6.8Hz,2H), 7.29(d,J=7.0Hz,1H),7.23(s,1H),3.51–3.49(m,1H),3.02–2.97(m,2H),2.45–2.25(m,12H). 13 C NMR (150 MHz, DMSO-d 6 )δ163.74,157.91,148.57,143.41,134.87,133.67,128.51,128.17,127.26, 125.99,125.18,108.19,56.22,53.82,51.73,44.92,39.55.HRMS(ESI):[M+H] + Calculate for C 23 H 28 N 5 O 3 S 2 + 486.1628, found 486.1642. HPLC purity, 98%.

[0101] Embodiment 11

[0102] The preparation steps of compound B11 are the same as those of compound B1. It is prepared from compound A1 and 4-(2-aminoethyl)morpholine as a white solid with a yield of 79%. 1 H NMR (600 MHz, CDCl 3 )δ10.57(s,1H),8.01(d,J=7.8Hz,2H),7.93(d,J=7.6Hz,2H),7.75(d,J=7.1Hz,2H),7.37(t,J=7.0Hz,2H),7.30( t,J=6.9Hz,1H),7.24(s,1H),3.65–3.58(m,4H),3.04(t,J=5.2Hz,2H),2.42(t,J=5.2Hz,2H),2.33–2.25(m,4H). 13 CNMR (150MHz, CDCl 3 )δ163.32,158.07,150.34,143.67,135.70,133.92,128.85,128.31,128. 21,127.57,126.07,108.55,66.75,56.26,52.96,38.94.HRMS(ESI):[M+H] +Calculate for C 22 H 25 N 4 O 4 S 2 + 473.1312,found 473.1314.HPLC purity,99%.

[0103] Example 12

[0104] The synthesis steps of compound B12 are the same as those of compound B1. It is prepared from compound A1 and 2-methoxyethylamine as a white solid with a yield of 90%. 1 H NMR (600 MHz, CDCl 3 )δ11.22(s,1H),7.91(d,J=7.4Hz,2H),7.81(d,J=7.5Hz,2H),7.68(d,J=6.9Hz,2H),7.31(t,J=6 .8Hz,2H),7.26(d,J=7.8Hz,2H),5.17(s,1H),3.42–3.37(m,2H),3.25(s,3H),3.15–3.10(m,2H). 13 C NMR (150 MHz, CDCl 3 )δ163.77,158.75,150.32,143.66,135.63,133.85,128.81,128.29,128.26,127.38,126.10,108.59,70.41,58.81,42.97.HRMS(ESI):[M+H] + Calculate for C 19 H 20 N 3 O 4 S 2 + 418.0890, found 418.0893. HPLC purity, 99%.

[0105] Embodiment 13

[0106] The synthesis steps of compound B14 are the same as those of compound B1. It is prepared from compound A1 and N,N-dimethylethylenediamine as a white solid with a yield of 79%. 1 H NMR (600 MHz, DMSO-d 6)δ12.95(s,1H),8.29(d,J=8.4Hz,2H),7.96(dd,J=8.1,2.5Hz,4H),7.74(s,1H),7.46(t,J= 7.7Hz,2H),7.35(t,J=7.3Hz,1H),2.90(t,J=6.8Hz,2H),2.27(t,J=6.7Hz,2H),2.07(s,6H). 13 C NMR (150 MHz, DMSO-d 6 )δ163.75,157.88,148.57,143.41,134.87,133.68,128.50,128.16,127.26,125.98,125.18,108.19,57.49,44.37,40.05.HRMS(ESI):[M+H] + Calculate for C 20 H 23 N 4 O 3 S 2 + 431.1206, found 431.1212. HPLC purity, 98%.

[0107] Embodiment 14

[0108] The synthesis steps of compound B15 are the same as those of compound B1. It is prepared from compound A1 and aniline as a white solid with a yield of 80%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(s,1H),10.48(s,1H),8.22(d,J=8.4Hz,2H),7.95(d,J=7.4Hz,2H),7.90(d,J=8.5Hz,2H),7.74(s,1H), 7.45(t,J=7.7Hz,2H),7.34(t,J=7.3Hz,1H),7.26(t,J=7.9Hz,2H),7.12(d,J=7.7Hz,2H),7.06(t,J=7.4Hz,1H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.61,158.64,149.71,143.17,137.77,136.40,134.71,129.76,129.68 ,129.24,128.36,127.29,126.24,124.91,120.80,109.33.HRMS(ESI):[M+H] + calcdfor C22 H 18 N 3 O 3 S 2 + 436.0784,found 436.0792.HPLC purity,97%.

[0109] Embodiment 15

[0110] The synthesis steps of compound B17 are the same as those of compound B1. It is prepared from compound A1 and 2H-pyrazol-3-ylamine as a white solid with a yield of 73%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.05(s,1H),8.28(d,J=8.4Hz,2H),8.08(d,J=2.9Hz,1H),7.98(d,J=8.4Hz,2H),7.95(d,J =7.6Hz,2H),7.75(s,1H),7.45(t,J=7.6Hz,2H),7.35(t,J=7.3Hz,1H),5.92(d,J=2.9Hz,1H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.40,161.53,158.58,149.75,140.21,137.55,135.29,134.69,129 .86,129.25,128.38,127.89,126.25,109.42,102.94.HRMS(ESI):[M+H] + Calculate for C 19 H 16 N 5 O 3 S 2 + 426.0689, found 426.0698. HPLC purity, 97%.

[0111] Example 16

[0112] The synthesis steps of compound B18 are the same as those of compound B1. It is prepared from compound A1 and 3-aminobenzyl alcohol as a white solid with a yield of 85%. 1 H NMR (600 MHz, DMSO-d 6)δ12.99(s,1H),10.47(s,1H),8.23(d,J=8.4Hz,2H),7.95(d,J=7.5Hz,2H),7.92(d,J=8.4Hz,2H),7.73(s,1H),7.45(t,J=7.7Hz ,2H),7.34(t,J=7.3Hz,1H),7.20(t,J=7.8Hz,1H),7.13(s,1H),7.03–6.97(m,2H),5.22(t,J=5.7Hz,1H),4.41(d,J=5.6Hz,2H). 13 C NMR (150 MHz, DMSO-d 6 )δ164.64,158.64,149.71,144.41,143.30,137.70,136.36,134.70,129.67,129.38,12 9.24,128.36,127.28,126.25,122.80,118.81,118.61,109.32,62.94.HRMS(ESI):[M+H] + Calculate for C 23 H 20 N 3 O 4 S 2 + 466.0890, found 466.0890. HPLC purity, 97%.

[0113] Embodiment 17

[0114] The synthesis steps of compound B19 are the same as those of compound B1. It is prepared from compound A1 and 2-(3-aminophenyl)ethanol as a white solid with a yield of 45%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.99(s,1H),10.43(s,1H),8.22(d,J=8.4Hz,2H),7.95(d,J=7.5Hz,2H),7.91(d,J=8.4Hz,2H),7.73(s,1H),7.45(t,J=7.7Hz,2H),7.3 5(t,J=7.3Hz,1H),7.15(t,J=7.8Hz,1H),6.99(s,1H),6.96-6.90(m,2H),4.67(t,J=5.2Hz,1H),3.53-3.49(m,2H),2.62(t,J=7.0Hz,2H). 13 C NMR (150 MHz, DMSO-d 6)δ164.63,158.63,149.71,143.23,141.27,137.59,136.33,134.69,129.63,129.46,129.2 5,128.38,127.33,126.24,125.50,121.21,118.24,109.32,62.39,39.34.HRMS(ESI):[M+H] + Calculate for C 24 H 22 N 3 O 4 S 2 + 480.1046,found 480.1054.HPLC purity,95%.

[0115] Embodiment 18

[0116] The synthesis steps of compound B20 are the same as those of compound B1. It is prepared from compound A1 and 3-(4-morpholinyl)aniline as a white solid with a yield of 70%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.97(s,1H),10.33(s,1H),8.22(d,J=8.5Hz,2H),7.95(d,J=7.2Hz,2H ),7.91(d,J=8.5Hz,2H),7.73(s,1H),7.45(t,J=7.7Hz,2H),7.34(t,J=7.4 Hz,1H),7.07(t,J=8.1Hz,1H),6.66(d,J=1.9Hz,1H),6.64(dd,J=8.3,2.0 Hz,1H),6.56(dd,J=7.9,1.2Hz,1H),3.72–3.68(m,4H),3.02–2.98(m,4H). 13 C NMR (150 MHz, DMSO-d 6 )δ151.09,148.62,142.19,137.50,135.29,133.63,129.05,128.57,128.16,127.2 8,126.28,125.16,110.62,110.16,108.25,105.97,65.34,47.48.HRMS(ESI):[M+H] + Calculate for C 26 H 25 N 4 O 4 S 2 +521.1312,found 521.1319.HPLC purity,97%.

[0117] Embodiment 19

[0118] The synthesis steps of compound B21 are the same as those of compound B1. It is prepared from compound A1 and 3-(4-methylpiperazine-1-yl)aniline as a white solid with a yield of 85%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.95(s,1H),10.32(s,1H),8.23(d,J=8.4Hz,2H),7.95(d,J=7.5Hz, 2H),7.91(d,J=8.4Hz,2H),7.73(s,1H),7.45(t,J=7.7Hz,2H),7.34(t,J =7.3Hz,1H),7.05(t,J=8.1Hz,1H),6.65(s,1H),6.63(d,J=8.3Hz,1H),6 .53(d,J=7.8Hz,1H),3.07–2.99(m,4H),2.44–2.38(m,4H),2.20(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ163.61,157.73,150.96,148.56,142.22,137.47,135.35,133.64,128.99,128.54,128.15,1 27.26,126.27,125.17,110.90,109.91,108.20,106.37,53.75,47.11,45.02.HRMS(ESI):[M+H] + Calculate for C 27 H 28 N 5 O 3 S 2 + 534.1628,found 534.1635.HPLCpurity,99%.

[0119] Embodiment 20

[0120] The synthesis steps of compound B24 are the same as those of compound B1. It is prepared from compound A1 and 3-aminoacetophenone as a white solid with a yield of 65%. 1 H NMR (600 MHz, DMSO-d 6)δ12.99(s,1H),10.75(s,1H),8.23(d,J=8.4Hz,2H),7.94(t,J=9.1Hz,4H),7.73(s,1H),7.7 0–7.66(m,2H),7.48-7.42(m,3H),7.39(d,J=8.3Hz,1H),7.34(t,J=7.3Hz,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.74,164.56,158.60,149.72,142.90,138.31,138.17,136.57,134.71,130.33,129.80 ,129.24,128.37,127.30,126.24,125.11,124.93,119.34,109.35,27.19.HRMS(ESI):[M+H] + calcdfor C 24 H 20 N 3 O 4 S 2 + 478.0890,found 478.0896.HPLC purity,98%.

[0121] Embodiment 21

[0122] The synthesis steps of compound B25 are the same as those of compound B1. It is prepared from compound A1 and 3-amino-N-methylbenzamide as a white solid with a yield of 80%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(s,1H),10.64(s,1H),8.44–8.40(m,1H),8.23(d,J=8.4Hz,2H),7.95(d,J=7.5Hz,2H),7.91(d,J=8.4Hz,2H),7.73(s, 1H),7.63(s,1H),7.50(d,J=7.7Hz,1H),7.45(t,J=7.7Hz,2H),7.34(t,J=7.9Hz,2H),7.28–7.24(m,1H),2.74(d,J=4.5Hz,3H). 13 C NMR (150 MHz, DMSO-d 6)δ166.43,164.57,158.61,149.71,143.03,138.00,136.48,136.21,134.71,129.74,12 9.71,129.24,128.36,127.27,126.24,123.09,120.01,109.34,26.74.HRMS(ESI):[M+H] + Calculate for C 24 H 21 N 4 O 4 S 2 + 493.0999, found 493.1008. HPLC purity, 97%.

[0123] Embodiment 22

[0124] Synthesis of compound B4: Compound A1 (379 mg, 1.0 mmol) and Et 3 N (278 μL, 2.0 mmol), stirred for about 4 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CH 3 OH (3 mL) and NaOH (1 mol / L) aqueous solution (3 mL) and stirred for another 2 hours. The solvent was then removed under reduced pressure and the residue was acidified to pH = 2 or lower with HCl (1 mol / L) aqueous solution. The solution was extracted with EtOAc (3×20 mL) and the combined organic matter was washed with anhydrous Na 2 SO 4 Dry and concentrate in vacuo. Chromatography on silica gel (DCM / CH 3 The obtained residue was purified by 4% paraformaldehyde (4% HCl, 0.1% NaOH=10 / 1, v / v) to give the desired compound B4 (396 mg, yield 84.0%) as a white solid.

[0125] 1 H NMR (600 MHz, DMSO-d 6 )δ13.08(s,1H),12.37(s,1H),8.35(d,J=8.4Hz,2H),7.97(d,J=7.5Hz,2H),7.91(d,J=8.3Hz,2H),7.76(s,1H),7.46(t,J=7.7 Hz,2H),7.35(t,J=7.3Hz,1H),3.56-3.50(m,2H),2.51-2.47(m,2H),2.36–2.27(m,1H),1.94–1.85(m,2H),1.61-1.52(m,2H).13 C NMR (150 MHz, DMSO-d 6 )δ175.63,164.63,139.44,136.47,134.69,129.79,129.26,128.38,128.07,126.27,109.38,45.66,39.17,27.63.HRMS(ESI):[M+H] + Calculate for C 22 H 22 N 3 O 5 S 2 + 472.0995, found 472.0995. HPLC purity, 97%.

[0126] Embodiment 23

[0127] The synthesis steps of compound B13 are the same as those of compound B4, and compound A1 and glycine methyl ester hydrochloride are used to prepare the compound B13, which is a white solid with a yield of 75%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.03(s,1H),12.76(s,1H),8.28(d,J=8.4Hz,3H),7.96(t,J=8.7Hz,4H),7. 75(s,1H),7.46(t,J=7.7Hz,2H),7.35(t,J=7.3Hz,1H),3.66(d,J=3.6Hz,2H). 13 C NMR (150 MHz, DMSO-d 6 )δ170.68,164.72,158.80,149.67,144.59,135.79,134.72,129.48,129.25,128.37,127.12,126.26,109.32,44.39.HRMS(ESI):[M+H] + Calculate for C 18 H 16 N 3 O 5 S 2 + 418.0526,found 418.0522.HPLCpurity,98%.

[0128] Embodiment 24

[0129] The synthesis steps of compound B22 are the same as those of compound B4, and compound A1 and methyl 3-aminobenzoate are used to prepare the compound B22 as a white solid with a yield of 95%.1 H NMR (600 MHz, DMSO-d 6 )δ13.11(s,1H),12.99(s,1H),10.75(s,1H),8.23(d,J=8.4Hz,2H),7.95(d,J=7.4Hz,2H),7.91(d,J=8.4Hz,2H ),7.73(d,J=8.6Hz,2H),7.63(d,J=6.6Hz,1H),7.45(t,J=7.7Hz,2H),7.42–7.38(m,2H),7.35(t,J=7.3Hz,1H). 13 CNMR (150MHz, DMSO-d 6 )δ167.14,142.89,138.14,132.33,130.16,129.79,129.25,128.37,127.25,126.24,125.66,124.75,121.21,109.34.HRMS(ESI):[M+H] + calcdfor C 23 H 18 N 3 O 5 S 2 + 480.0682,found 480.0689.HPLC purity,97%.

[0130] Embodiment 25

[0131] The synthesis steps of compound B23 are the same as those of compound B4, and compound A1 and methyl 3-aminophenylacetate are used to prepare the compound B23 as a white solid with a yield of 78%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.97(s,1H),10.51(s,1H),8.21(d,J=8.4Hz,2H),7.95(d,J=7.4Hz,2H),7.92(d,J=8.4Hz,2H),7.73(s,1H),7.45(t,J=7. 7Hz,2H),7.35(t,J=7.3Hz,1H),7.19(t,J=7.9Hz,1H),7.08(s,1H),7.00(d,J=8.2Hz,1H),6.95(d,J=7.6Hz,1H),3.49(s,2H). 13 C NMR (150 MHz, DMSO-d 6)δ172.85,164.67,158.69,149.64,143.15,137.72,136.65,134.65,129.65,129.60,12 9.26,128.38,127.32,126.24,125.95,121.47,118.81,109.31,41.06.HRMS(ESI):[M+H] + Calculate for C 24 H 20 N 3 O 5 S 2 + 494.0839,found 494.0844.HPLC purity,97%.

[0132] Embodiment 26

[0133] Synthesis of compound B16: Compound 9 (368 mg, 1.2 mmol) was added to a solution of HATU (684 mg, 1.8 mmol) and DIPEA (523 μL, 3.0 mmol) in THF (1 mL) at room temperature. Then 2-amino-4-phenylthiazole (176 mg, 1.0 mmol) was added and stirred for about 6 hours. The solvent was removed, and the residue was dissolved in ethyl acetate and washed with water and saturated aqueous ammonium chloride. The organic phase was washed with anhydrous Na 2 SO 4 Dry, concentrate under reduced pressure, and purify by flash column chromatography to obtain compound B16 (158 mg, yield 34%) as a white solid.

[0134] 1 H NMR (600 MHz, CDCl 3 )δ8.22(d,J=7.9Hz,2H),7.99(d,J=7.9Hz,2H),7.75(d,J=7.1Hz,2H),7.36(t ,J=7.0Hz,2H),7.29(d,J=7.0Hz,1H),7.21(s,1H),6.65(s,1H),2.34(s,6H). 13 C NMR (150 MHz, CDCl 3 )δ168.36,163.44,158.00,155.67,150.33,143.45,135.98,133.99,129.44 ,128.81,128.25,127.45,126.08,115.19,108.48,23.57.HRMS(ESI):[M+H] + Calculate for C 22H 20 N 5 O 3 S 2 + 466.1002,found 466.1004.HPLC purity,98%.

[0135] Embodiment 27

[0136] The synthesis steps of compound C1 are the same as those of compound B16, and it is prepared from compound 11 and 4-phenylthiophene-2-amine as a white solid with a yield of 52%. 1 H NMR (600 MHz, DMSO-d 6 )δ11.85(s,1H),10.73(s,1H),8.12(d,J=8.4Hz,2H),7.95(d,J=8.4Hz,2H),7.68(d,J=7.1Hz,2H), 7.63(d,J=7.6Hz,2H),7.45–7.37(m,5H),7.29(t,J=7.3Hz,1H),7.25(d,J=1.4Hz,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.75,162.68,142.37,140.85,138.35,138.15,137.55,135.72,130.31,129.41,129.2 1,127.57,127.45,126.16,125.10,124.92,119.31,113.65,111.50,27.19.HRMS(ESI):[MH] - Calculate for C 25 H 19 N 2 O 4 S 2 - 475.0792,found 475.0797.HPLCpurity,95%.

[0137] Embodiment 28

[0138] The synthesis steps of compound C2 are the same as those of compound B16, and it is prepared from compound 11 and 5-methyl-4-phenylthiazol-2-amine as a white solid with a yield of 40%. 1 H NMR (600 MHz, DMSO-d 6)δ12.85(s,1H),10.75(s,1H),8.21(d,J=8.5Hz,2H),7.92(d,J=8.5Hz,2H),7.70–7.66(m,4H ),7.47(t,J=7.7Hz,2H),7.43(t,J=7.8Hz,1H),7.41–7.34(m,2H),2.51(s,3H),2.50(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ196.66,141.72,137.23,137.08,129.25,128.66,127.79,127.47,126.78,126.22,124.03,123.84,118.25,26.11,11.23.HRMS(ESI):[MH] - Calculate for C 25 H 20 N 3 O 4 S - 490.0901, found 490.0910. HPLC purity, 96%.

[0139] Embodiment 29

[0140] The synthesis steps of compound C3 are the same as those of compound B16, and it is prepared from compound 11 and 4-phenyl-1H-imidazole-2-amine as a white solid with a yield of 36%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.06(s,1H),11.95(s,1H),10.72(s,1H),8.18(d,J=8.3Hz,2H),7.90(d,J=8.4Hz,2H),7.76(d,J=7.6Hz,2H),7.67(dd, J=6.5,5.1Hz,2H),7.43(t,J=7.8Hz,1H),7.39(d,J=8.3Hz,2H),7.36(t,J=7.7Hz,2H),7.20(t,J=7.3Hz,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.75,138.38,138.15,130.30,129.48,129.02,127.21,125.05,124.89,124.55,119.29,27.19.HRMS(ESI):[MH] - Calculate for C 24 H19 N 4 O 4 S - 459.1132,found 459.1135.HPLC purity,97%.

[0141] Embodiment 30

[0142] The synthesis steps of compound C4 are the same as those of compound B16, and it is prepared from compound 11 and 1-methyl-3-phenyl-1H-pyrazole-5-amine as a white solid with a yield of 64%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.76(s,1H),10.64(s,1H),8.12(d,J=8.3Hz,2H),7.95(d,J=8.4Hz,2H),7.78(d,J=7.3Hz,2H), 7.71–7.65(m,2H),7.46–7.38(m,4H),7.30(t,J=7.4Hz,1H),6.72(s,1H),3.75(s,3H),2.52(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.79,164.97,148.69,142.64,138.42,138.16,137.84,137.59,133.75,130.33,129.51,1 29.12,127.96,127.37,125.24,125.05,124.81,119.19,98.07,36.41,27.21.HRMS(ESI):[MH] - Calculate for C 25 H 21 N 4 O 4 S - 473.1289,found 473.1291.HPLCpurity,96%.

[0143] Embodiment 31

[0144] The synthesis steps of compound C5 are the same as those of compound B16, and it is prepared from compound 11 and 5-phenylthiazol-2-amine as a white solid with a yield of 47%. 1 H NMR (600 MHz, DMSO-d 6)δ12.93(s,1H),10.75(s,1H),8.21(d,J=8.4Hz,2H),7.99(s,1H),7.93(d,J=8.5Hz,2H),7.6 9–7.65(m,4H),7.44(t,J=7.8Hz,3H),7.41–7.38(m,1H),7.33(t,J=7.4Hz,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.75,142.84,138.31,138.16,131.85,130.33,129.79,129.71,129.4 0,128.20,127.32,126.24,125.11,124.91,119.31,27.20.HRMS(ESI):[MH] - Calculate for C 24 H 18 N 3 O 4 S 2 - 476.0744, found 476.0764. HPLC purity, 95%.

[0145] Embodiment 32

[0146] The synthesis steps of compound C6 are the same as those of compound B16, and it is prepared from compound 11 and 5-phenyl-4H-1,2,4-triazole-3-amine as a white solid with a yield of 45%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.79(s,1H),8.25–8.22(m,2H),7.97–7.94(m,2H),7.92–7.89(m,3H),7.70(dd,J=7.1,1.4Hz,2H),7.49–7.40(m,5H),2.52(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.72,166.85,160.40,159.29,142.85,138.32,138.17,136.76,132.02,130.70,13 0.37,130.33,129.16,126.97,126.74,125.13,125.01,119.40,27.19.HRMS(ESI):[MH] - Calculate for C 23 H 18 N5 O 4 S - 460.1085,found 460.1088.HPLCpurity,96%.

[0147] Embodiment 33

[0148] The synthesis steps of compound D1 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2-fluorophenyl)thiazol-2-amine as a white solid with a yield of 57%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.03(s,1H),10.76(s,1H),8.23(d,J=8.4Hz,2H),8.09(dd,J=8.1,6.8Hz,1H),7.94(d,J=8.4Hz,2H),7.70 –7.66(m,2H),7.63(d,J=2.1Hz,1H),7.44(t,J=7.8Hz,1H),7.42–7.36(m,2H),7.36–7.30(m,2H),2.52(s,3H). 13 CNMR (150MHz, DMSO-d 6 )δ197.74,164.66,160.85,159.20,158.14,143.36,142.92,138.31,138.16,136.51,130.33,130.05,129.8 3,129.78,127.31,125.26,125.11,124.92,122.30,119.32,116.76,116.61,113.76,27.20.HRMS(ESI):[MH] - Calculate for C 24 H 17 FN 3 O 4 S 2 - 494.0650, found 494.0656. HPLC purity, 97%.

[0149] Embodiment 34

[0150] The synthesis steps of compound D2 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2-chlorophenyl)thiazol-2-amine as a white solid with a yield of 38%. 1 H NMR (600 MHz, Acetone-d 6)δ8.29(d,J=8.2Hz,2H),8.01(d,J=8.4Hz,2H),7.92(d,J=7.7Hz,1H),7.84(s,1H),7.74(d,J=7. 7Hz,1H),7.71(s,1H),7.54–7.49(m,2H),7.47–7.40(m,2H),7.36(t,J=7.6Hz,1H),2.53(s,3H). 13 C NMR (150 MHz, Acetone-d 6 )δ198.20,165.64,158.87,148.25,144.88,140.12,139.73,138.25,135.13,133.31,133.15,132.2 2,131.48,130.90,130.70,129.21,128.84,126.76,126.55,121.76,115.12,27.62.HRMS(ESI):[MH] - Calculate for C 24 H 19 C1N 3 O 4 S 2 - 512.0511,found512.0506.HPLC purity,99%.

[0151] Embodiment 35

[0152] The synthesis steps of compound D3 are the same as those of compound B16, and it is prepared from compound 11 and 4-(o-tolyl)thiazol-2-amine as a white solid with a yield of 59%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.93(s,1H),10.75(s,1H),8.21(d,J=8.5Hz,2H),7.92(d,J=8.5Hz,2H),7.70–7.65(m,2H),7.64 –7.58(m,1H),7.43(t,J=7.8Hz,1H),7.41-7.35(m,2H),7.31–7.24(m,3H),2.51(s,3H),2.44(s,3H). 13 C NMR (150 MHz, DMSO-d 6)δ197.74,142.85,138.33,138.16,135.92,131.27,130.33,129.88,129.78,128.3 0,127.31,126.30,125.11,124.91,119.32,112.11,27.19,21.44.HRMS(ESI):[MH] - Calculate for C 25 H 20 N 3 O 4 S 2 - 490.0901,found 490.0916.HPLC purity,100%.

[0153] Embodiment 36

[0154] The synthesis steps of compound D4 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2-(trifluoromethyl)phenyl)thiazol-2-amine as a white solid with a yield of 49%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.99(s,1H),10.75(s,1H),8.21(d,J=8.5Hz,2H),7.92(d,J=8.5Hz,2H),7.85(d,J=7.9Hz,1H),7.75(t,J =7.5Hz,1H),7.70–7.62(m,4H),7.43(t,J=7.8Hz,1H),7.39(dd,J=8.1,1.0Hz,1H),7.35(s,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.73,164.67,157.98,147.17,142.90,138.31,138.16,136.53,134.86,132.77,132.61,130.33,129.80,12 9.22,127.58,127.39,127.29,126.75,125.45,125.10,124.90,123.63,119.31,113.28,27.19.HRMS(ESI):[MH] - calcdfor C 25 H 17 F3N 3 O 4 S 2 -544.0618,found 544.0628.HPLC purity,99%.

[0155] Embodiment 37

[0156] The synthesis steps of compound D5 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2-methoxyphenyl)thiazol-2-amine as a white solid with a yield of 65%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.91(s,1H),10.76(s,1H),8.23(d,J=8.3Hz,2H),8.13(d,J=7.3Hz,1H),7.94(d,J=8.3Hz,2H),7.75(s,1H),7.69(d,J=6.9Hz,2H),7. 44(t,J=8.0Hz,1H),7.40(d,J=8.0Hz,1H),7.33(t,J=7.6Hz,1H),7.14(d,J=8.3Hz,1H),7.05(t,J=7.5Hz,1H),3.93(s,3H),2.52(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.73,157.11,142.84,138.32,138.17,130.33,129.78,129.45,129.39,127.2 9,125.10,124.92,120.91,119.33,112.95,112.16,55.94,27.19.HRMS(ESI):[MH] - calcdfor C 25 H 20 N 3 O 5 S 2 - 506.0850,found 506.0864.HPLC purity,99%.

[0157] Embodiment 38

[0158] The synthesis steps of compound D6 are the same as those of compound B16, and it is prepared from compound 11 and 2-(2-aminothiazol-4-yl)phenol as a white solid with a yield of 18%. 1 H NMR (600 MHz, Acetone-d 6)δ8.27(d,J=8.4Hz,2H),8.02(d,J=8.3Hz,2H),7.86–7.80(m,2H),7.75(d,J=7.6Hz,1H),7.68(s,1H),7 .50(dd,J=8.1,1.1Hz,1H),7.45(t,J=7.9Hz,1H),7.21(t,J=7.2Hz,1H),6.92–6.86(m,2H),2.53(s,3H). 13 C NMR (150 MHz, Acetone-d 6 )δ198.20,157.68,149.85,145.06,140.12,139.69,131.49,130.64,129.34,12 8.27,126.81,126.60,121.78,121.05,119.20,109.09,27.62.HRMS(ESI):[M+H] + Calculate for C 24 H 21 N 4 O 4 S 2 + 493.0999,found 493.1008.HRMS(ESI):[M+H] + calcdfor C 24 H 20 N 3 O 5 S 2 + 494.0839,found 494.0848.HPLC purity,99%.

[0159] Embodiment 39

[0160] The synthesis steps of compound D7 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2-nitrophenyl)thiazol-2-amine as a white solid with a yield of 31%. 1 H NMR (600 MHz, Acetone-d 6 )δ8.26(d,J=8.4Hz,2H),7.99(d,J=8.5Hz,2H),7.87–7.81(m,3H),7.76–7.71(m,2H), 7.65–7.60(m,1H),7.52(s,1H),7.51–7.48(m,1H),7.44(t,J=7.9Hz,1H),2.52(s,3H). 13 C NMR (150 MHz, Acetone-d 6)δ198.19,165.76,159.82,151.11,147.79,144.88,140.11,139.71,138.14,133.94,132.64,131.46 ,130.90,130.70,130.51,129.19,126.81,126.55,125.68,121.83,113.93,27.61.HRMS(ESI):[M+Na] + Calculate for C 24 H 18 N 4 NaO 6 S 2 + 545.0560,found 545.0573.HPLC purity,97%.

[0161] Embodiment 40

[0162] The synthesis steps of compound D8 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-fluorophenyl)thiazol-2-amine as a white solid with a yield of 63%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(s,1H),10.73(s,1H),8.23(d,J=8.5Hz,2H),7.93(d,J=8.5Hz,2H),7.86(s,1H),7.80(d,J=7.8Hz,1H),7.76–7.73(m, 1H),7.70–7.67(m,2H),7.52–7.47(m,1H),7.44(t,J=7.8Hz,1H),7.41–7.38(m,1H),7.18(td,J=8.5,2.4Hz,1H),3.34(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.74,164.65,163.87,162.26,158.75,148.37,142.93,138.30,138.16,136.48,131.34,130.33 ,129.82,127.31,125.11,124.93,122.27,119.32,114.97,112.70,110.73,27.20.HRMS(ESI):[M+H] + calcdfor C 24 H 19 FN 3 O 4 S 2+ 496.0796,found 496.0798.HPLC purity,100%.

[0163] Embodiment 41

[0164] The synthesis steps of compound D9 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-chlorophenyl)thiazol-2-amine as a white solid with a yield of 29%. 1 H NMR (600 MHz, Acetone-d 6 )δ8.30(d,J=8.4Hz,2H),8.02(d,J=8.4Hz,2H),7.97(s,1H),7.88(d,J=7.5Hz,1H),7.85(s,1H),7.74(d,J= 7.6Hz,1H),7.69(d,J=2.0Hz,1H),7.53–7.49(m,1H),7.47–7.41(m,2H),7.35(d,J=7.8Hz,1H),2.53(s,3H). 13 CNMR(150MHz,Acetone-d 6 )δ198.22,165.64,159.92,150.09,144.90,140.10,139.72,138.38,138.17,135.96,132.14,131.48 ,130.71,129.35,129.20,127.59,126.77,126.55,125.96,121.76,111.35,27.62.HRMS(ESI):[M+Na] + Calculate for C 24 H 18 C1N 3 NaO 4 S 2 + 534.0319,found 534.0323.HPLC purity,98%.

[0165] Embodiment 42

[0166] The synthesis steps of compound D10 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-bromophenyl)thiazol-2-amine as a white solid with a yield of 61%. 1 H NMR (600 MHz, Acetone-d 6)δ8.31(d,J=8.3Hz,2H),8.14(s,1H),8.02(d,J=8.3Hz,2H),7.94(d,J=7.8Hz,1H),7.84(s,1H),7.74(d,J =7.6Hz,1H),7.71(s,1H),7.51(d,J=7.9Hz,2H),7.45(t,J=7.9Hz,1H),7.39(t,J=7.9Hz,1H),2.53(s,3H). 13 CNMR(150MHz,Acetone-d 6 )δ198.19,165.64,159.94,149.97,144.92,140.12,139.73,138.64,138.18,132.43,132.33,131.48 ,130.72,130.58,129.22,126.77,126.56,126.36,124.17,121.77,111.36,27.62.HRMS(ESI):[M+H] + Calculate for C 24 H 19 Bn 3 O 4 S 2 + 555.9995,found 555.9999.HPLC purity,99%.

[0167] Embodiment 43

[0168] The synthesis steps of compound D11 are the same as those of compound B16, and compound 11 and 4-(m-tolyl)thiazol-2-amine are used to prepare the compound D11 as a white solid with a yield of 69%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.96(s,1H),10.75(s,1H),8.23(d,J=8.5Hz,2H),7.92(d,J=8.5Hz,2H),7.78(s,1H),7.73(d,J=7.8Hz,1H),7.71–7.6 6(m,3H),7.44(t,J=7.8Hz,1H),7.41–7.38(m,1H),7.33(t,J=7.6Hz,1H),7.16(d,J=7.5Hz,1H),2.51(s,3H),2.36(s,3H). 13 C NMR (150 MHz, DMSO-d 6)δ197.73,164.52,158.50,149.81,142.89,138.31,138.16,136.56,134.62,130.33,129.79,129.1 4,129.00,127.30,126.90,125.11,124.93,123.41,119.33,109.14,27.19,21.59.HRMS(ESI):[MH] - Calculate for C 25 H 20 N 3 O 4 S 2 - 490.0901,found 490.0917.HPLC purity,99%.

[0169] Embodiment 44

[0170] The synthesis steps of compound D12 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-(trifluoromethyl)phenyl)thiazol-2-amine as a white solid with a yield of 53%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.01(s,1H),10.75(s,1H),8.30(s,1H),8.27-8.21(m,3H),7.99(s,1H),7.93(d,J =8.5Hz,2H),7.73–7.65(m,4H),7.44(t,J=7.8Hz,1H),7.41–7.38(m,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.73,164.64,158.97,147.93,142.97,138.30,138.16,136.42,135.60,130.46,130.33,130.22,130.01,12 9.96,129.82,127.32,125.62,125.12,124.94,124.80,123.81,122.65,119.33,111.16,27.19.HRMS(ESI):[MH] - Calculate for C 25 H 17 F 3 N 3 O 4 S 2 -544.0618, found544.0632. HPLC purity, 97%.

[0171] Embodiment 45

[0172] The synthesis steps of compound D13 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-methoxyphenyl)thiazol-2-amine as a white solid with a yield of 63%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(s,1H),10.76(s,1H),8.24(d,J=8.5Hz,2H),7.94(d,J=8.5Hz,2H),7.77(s,1H),7.69(t,J=4.2Hz,2H),7.52(dd,J=8.3,5.0Hz ,2H),7.44(t,J=7.8Hz,1H),7.40(dd,J=5.8,4.1Hz,1H),7.36(t,J=7.9Hz,1H),6.92(dd,J=8.1,2.2Hz,1H),3.82(s,3H),2.52(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ196.66,163.50,159.03,157.41,148.49,141.82,137.24,137.09,135.48,135.01,129.26,128.7 4,126.23,124.04,123.86,118.26,117.55,113.12,110.38,108.64,54.53,26.12.HRMS(ESI):[MH] - Calculate for C 25 H 20 N 3 O 5 S 2 - 506.0850,found 506.0865.HPLCpurity,99%.

[0173] Embodiment 46

[0174] The synthesis steps of compound D14 are the same as those of compound B16, and it is prepared from compound 11 and 3-(2-aminothiazol-4-yl)phenol as a white solid with a yield of 27%. 1 H NMR (600 MHz, Acetone-d 6)δ8.32(d,J=8.1Hz,2H),8.04(d,J=8.4Hz,2H),7.86(s,1H),7.82–7.73(m,3H),7.54– 7.50(m,1H),7.48–7.42(m,2H),7.19(dd,J=8.0,2.2Hz,1H),7.04(s,1H),2.54(s,3H). 13 C NMR (150 MHz, Acetone-d 6 )δ198.22,169.93,165.23,153.03,151.47,145.85,140.12,139.66,138.74,135.43,132.46,131.50 ,131.25,129.32,126.78,126.62,125.14,122.17,121.78,120.89,104.48,27.63.HRMS(ESI):[M+H] + Calculate for C 24 H 20 N 3 O 5 S 2 + 494.0839,found 494.0845.HPLC purity,95%.

[0175] Embodiment 47

[0176] The synthesis steps of compound D15 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3-nitrophenyl)thiazol-2-amine as a white solid with a yield of 22%. 1 H NMR (600 MHz, Acetone-d 6 )δ8.78(s,1H),8.36(d,J=7.3Hz,1H),8.19(d,J=8.1Hz,1H),8.03(d,J=8.4Hz,2H),7.88–7.7 2(m,2H),7.76–7.71(m,2H),7.52(dd,J=8.1,1.2Hz,1H),7.45(t,J=7.9Hz,1H),2.53(s,3H). 13 C NMR (150 MHz, Acetone-d 6)δ196.40,163.95,158.50,148.88,147.46,143.19,138.32,137.94,136.29,131.58,130.09,129 .69,128.96,127.44,125.00,124.77,122.25,120.46,119.99,110.66,25.82.HRMS(ESI):[M+Na] + Calculate for C 24 H 18 N 4 NaO 6 S 2 + 545.0560,found 545.0568.HPLCpurity,99%.

[0177] Embodiment 48

[0178] The synthesis steps of compound D16 are the same as those of compound B16, and it is prepared from compound 11 and 4-(4-fluorophenyl)thiazol-2-amine as a white solid with a yield of 53%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.00(s,1H),10.76(s,1H),8.25–8.21(m,2H),8.01–7.96(m,2H),7.96–7.93(m,2H),7.72(s, 1H),7.70–7.67(m,2H),7.44(t,J=7.8Hz,1H),7.42–7.39(m,1H),7.32–7.26(m,2H),2.52(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ196.66,163.49,162.03,160.41,157.65,147.62,141.83,137.24,137.09,135.47,129.26,128.7 2,127.21,127.16,126.24,124.04,123.85,118.25,115.11,114.97,108.04,26.12.HRMS(ESI):[MH] - Calculate for C 24 H 17 FN 3 O 4 S 2 - 494.0650,found 494.0667.HPLCpurity,97%.

[0179] Embodiment 49

[0180] The synthesis steps of compound D17 are the same as those of compound B16, and it is prepared from compound 11 and 4-(4-chlorophenyl)thiazol-2-amine as a white solid with a yield of 49%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(s,1H),10.74(s,1H),8.22(d,J=8.4Hz,2H),7.96(d,J=8.5Hz,2H),7.93(d,J=8.4Hz,2H),7.79(s ,1H),7.70–7.66(m,2H),7.51(d,J=8.5Hz,2H),7.43(t,J=7.8Hz,1H),7.39(d,J=8.3Hz,1H),2.51(s,3H). 13 CNMR (150MHz, DMSO-d 6 )δ197.75,164.61,158.82,148.47,142.97,138.34,138.18,136.51,133.58,132.82,130.32 ,129.78,129.27,127.96,127.31,125.08,124.95,119.36,110.06,27.18.HRMS(ESI):[M+H] + Calculate for C 24 H 19 C1N 3 O 4 S 2 + 512.0500,found 512.0507.HPLCpurity,99%.

[0181] Embodiment 50

[0182] The synthesis steps of compound D18 are the same as those of compound B16, and it is prepared from compound 11 and 4-(4-bromophenyl)thiazol-2-amine as a white solid with a yield of 49%. 1 H NMR (600 MHz, DMSO-d 6)δ12.99(s,1H),10.74(s,1H),8.23(d,J=8.5Hz,2H),7.93(d,J=8.5Hz,2H),7.90(d,J=8.5Hz,2H),7.80(s, 1H),7.68(d,J=8.4Hz,2H),7.65(d,J=8.5Hz,2H),7.44(t,J=7.8Hz,1H),7.39(d,J=8.3Hz,1H),2.51(s,3H). 13 CNMR (150MHz, DMSO-d 6 )δ197.72,164.59,158.82,148.52,142.97,138.32,138.19,136.50,133.93,132.18,130.32 ,129.78,128.26,127.31,125.08,124.94,121.43,119.37,110.15,27.18.HRMS(ESI):[M+H] + Calculate for C 24 H 19 Bn 3 O 4 S 2 + 555.9995,found556.0005.HPLC purity,96%.

[0183] Embodiment 51

[0184] The synthesis steps of compound D19 are the same as those of compound B16, and it is prepared from compound 11 and 4-(p-tolyl)thiazol-2-amine as a white solid with a yield of 52%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.94(s,1H),10.73(s,1H),8.22(d,J=8.4Hz,2H),7.92(d,J=8.4Hz,2H),7.83(d,J=8.0Hz,2H),7.70–7.66(m ,2H),7.64(s,1H),7.43(t,J=7.8Hz,1H),7.39(d,J=8.4Hz,1H),7.25(d,J=8.0Hz,2H),2.51(s,3H),2.33(s,3H). 13 C NMR (150 MHz, DMSO-d 6)δ197.72,164.50,158.48,149.81,142.90,138.32,138.18,137.67,136.58,132.07,130.32,12 9.79,129.77,127.29,126.19,125.08,124.94,119.37,108.46,27.18,21.28.HRMS(ESI):[M+H] + Calculate for C 25 H 22 N 3 O 4 S 2 + 492.1046,found492.1057.HPLC purity,99%.

[0185] Embodiment 52

[0186] The synthesis steps of compound D20 are the same as those of compound B16, and it is prepared from compound 11 and 4-(4-(trifluoromethyl)phenyl)thiazol-2-amine as a white solid with a yield of 26%. 1 H NMR (600 MHz, Acetone-d 6 )δ8.32–8.28(m,2H),8.16(d,J=8.1Hz,2H),8.02(d,J=8.5Hz,2H),7.84(t,J=1.9Hz,1H),7.81(s,1 H),7.78(d,J=8.3Hz,2H),7.76–7.74(m,1H),7.53–7.49(m,1H),7.45(t,J=7.9Hz,1H),2.53(s,3H). 13 CNMR(150MHz,Acetone-d 6 )δ198.21,165.77,155.01,150.13,144.90,140.09,138.18,131.50,130.75,12 9.22,128.16,127.40,126.76,126.57,121.73,112.46,27.63.HRMS(ESI):[M+H] + Calculate for C 25 H 19 F 3 N 3 O 4 S 2 + 546.0764,found 546.0773.HPLC purity,99%.

[0187] Embodiment 53

[0188] The synthesis steps of compound D21 are the same as those of compound B16, and it is prepared from compound 11 and 4-(4-isopropylphenyl)thiazol-2-amine as a white solid with a yield of 63%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.96(s,1H),10.73(s,1H),8.22(d,J=8.4Hz,2H),7.92(d,J=8.4Hz,2H),7.86(d,J=8.1Hz,2H),7.70–7.63(t,J=12. 7Hz,3H),7.43(t,J=7.8Hz,1H),7.39(d,J=8.5Hz,1H),7.31(d,J=8.1Hz,2H),2.96–2.87(m,1H),1.23(d,J=6.9Hz,6H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.73,164.51,158.50,149.83,148.60,142.91,138.34,138.18,136.59,132.44,130.31,129.7 8,127.28,127.13,126.26,125.07,124.94,119.37,108.53,33.66,27.18,24.27.HRMS(ESI):[M+H] + calcdfor C 27 H 26 N 3 O 4 S 2 + 520.1359,found 520.1364.HPLC purity,99%.

[0189] Embodiment 54

[0190] The synthesis steps of compound D22 are the same as those of compound B16, and it is prepared from compound 11 and 4-(2,4-difluorophenyl)thiazol-2-amine as a white solid with a yield of 28%. 1 H NMR (600 MHz, DMSO-d 6)δ13.00(s,1H),10.74(s,1H),8.23(d,J=8.4Hz,2H),8.13–8.07(m,1H),7.93(d,J=8.4Hz,2H),7.68 (d,J=8.5Hz,2H),7.60(d,J=2.1Hz,1H),7.47–7.37(m,3H),7.23(td,J=8.5,2.3Hz,1H),2.51(s,3H). 13 C NMR (150 MHz, DMSO-d 6 )δ197.73,164.67,161.21,159.18,158.27,142.98,142.59,138.32,138.19,136.49,131.03,130.32,12 9.81,127.30,125.08,124.95,119.37,113.26,113.17,112.56,112.42,105.17,27.18.HRMS(ESI):[M+H] + Calculate for C 24 H 18 F 2 N 3 O 4 S 2 + 514.0701,found 514.0712.HPLC purity,99%.

[0191] Embodiment 55

[0192] The synthesis steps of compound D23 are the same as those of compound B16, and it is prepared from compound 11 and 4-(3,4-difluorophenyl)thiazol-2-amine as a white solid with a yield of 44%. 1 H NMR (600 MHz, Acetone-d 6 )δ8.29(d,J=8.4Hz,2H),8.01(d,J=8.4Hz,2H),7.89–7.82(m,2H),7.81–7.76(m,1H),7.74(d,J=7.7Hz ,1H),7.65(s,1H),7.53–7.49(m,1H),7.45(t,J=7.9Hz,1H),7.38(dd,J=18.9,8.5Hz,1H),2.53(s,3H). 13 C NMR (150 MHz, Acetone-d 6)δ198.22,165.73,160.05,152.92,152.38,151.29,150.75,149.52,144.93,140.12,139.75,138.21,13 4.10,131.49,130.72,129.21,126.78,126.56,124.20,121.77,119.45,116.60,27.62.HRMS(ESI):[M+H] + Calculate for C 24 H 18 F 2 N 3 O 4 S 2 + 514.0701,514.0711.HPLC purity,96%.

[0193] Test Example 1

[0194] PHGDH inhibitory activity test

[0195] CBR-5884 and BI-03-002 were used as positive controls.

[0196] Test method: Centrifuge the powder of the compound to be tested at high speed, 12000rpm, 2min. Add DMSO to make a 10mM stock solution and store it temporarily in a -20℃ refrigerator. Prepare PHGDH enzyme reaction buffer (200mM Tris, pH 8.1, 400mM KCl and 10mM EDTA). NAD + Prepare 24mM stock solution, 3-PG prepare 100mM stock solution, and diaphorase prepare 100unit / mL stock solution and store in -20℃ refrigerator for use. Prepare resazurin prepare 10mM stock solution and store in 4℃ refrigerator for use (dilute 200 times before use). All dilutions of compounds in this detection method are then made with PHGDH enzyme reaction buffer.

[0197] The test compound can be diluted to the required concentration in a 96-well plate in a gradient as needed. Prepare PHGDH enzyme reaction solution (16ng / μL) and substrate mixture (1mM 3-PG, 50μM NAD+) according to the required volume. Add 5μL of PHGDH enzyme buffer, 2.5μL of compound, and 2.5μL of substrate mixture to a 384-well white microwell reaction plate, and incubate in the dark at 37°C for 60 minutes. The experiment sets up wells without enzyme and inhibitor supplements and the corresponding volume of enzyme reaction solution as Blank and Control. Then add 5μL of a mixture containing Diaphroase (0.25μM) and Resazurin (0.001unit / μL) to each well and incubate in the dark at room temperature for 10 minutes. Use a multifunctional microplate reader MD The fluorescence signal was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0198] The test results are shown in Table 1 below:

[0199] Table 1

[0200]

[0201]

[0202] It can be seen from the data in Table 1 that the compounds provided in the present application have an inhibitory effect on PHGDH. By comparing A2 with B1-B25, it can be seen that substitution with secondary amine groups (such as morpholine (B1), thiomorpholine (B2), 1,1-dioxo-4-thiomorpholine (B3)) leads to a decrease in inhibitory activity, IC 50 However, the introduction of a carboxyl group (B4) or an ethoxy group (B5) at the para-position of piperidine resulted in a slight increase in the inhibitory activity, with an IC 50 The values ​​were 12.96 μM and 12.21 μM, respectively. For secondary amine substituents with non-planar rings, cyclohexylamine (B6), 4-aminotetrahydropyran (B7), and 1-methylpiperidin-4-amine (B8) resulted in decreased PHGDH inhibitory activity, whereas 1-acetylpiperidin-tetraamine (B9) resulted in increased activity (IC 50 =6.81±0.46 μM). However, increasing the length or flexibility of the substituents generated compounds B10-B14, which only maintained inhibitory activity, IC 50 The values ​​ranged from 10.85 μM to 17.84 μM. For the aromatic amine substituents, aniline (B15) and 4,6-dimethylpyrimidin-2-amine (B16) increased the inhibitory activity, IC 50The values ​​were 6.50±0.80μM and 3.19±1.00μM, respectively. The 2-aminopyrazole substituent (B17) resulted in decreased inhibitory activity. Different substituents were introduced at the meta position of the phenyl group, such as hydroxymethyl (B18), hydroxyethyl (B19), morpholine (B20), N-methylpiperazine (B21), carboxyl (B22), carboxymethyl (B23), acetyl (B24), and N-methylacetamide (B25). The inhibitory activities of compounds B18, B19, B20, B21, B23, and B24 were similar to those of compound B15. The inhibitory activities of compounds B22 and B25 were slightly increased, and the IC 50 The values ​​were 3.59 ± 0.79 μM and 4.96 ± 0.63 μM, respectively.

[0203] By comparing compound B24 with C1-C6, it can be seen that 4-phenylthiazole has a great influence on the PHGDH inhibitory activity. By comparing compound B24 with D1-D23, it can be seen that the introduction of different substituents on the ortho, meta or para position of the phenyl group of compound B24 can maintain the PHGDH inhibitory activity, IC 50 The values ​​ranged from 2.2 μM to 6.7 μM. However, the other compounds resulted in a significant decrease in inhibitory activity. When two fluorine atoms were introduced simultaneously, there was no further increase in activity compared to compounds D1, D8, or D16 (D22, IC 50 =6.7±1.6μM; D23, IC 50 =17.2±3.9 μM).

[0204] Test Example 2

[0205] Anti-MDA-MB-468 and MDA-MB-231 cell proliferation activity test

[0206] MDA-MB-468 is a PHGDH gene amplified cell line, and MDA-MB-231 is a PHGDH low-expressing cell line.

[0207] BI-03-002, BI-01-013, and CBR5884 were used as positive controls.

[0208] Test method: discard the supernatant culture medium from the cells in the culture dish, add 1mL PBS to rinse, add trypsin and digest in the incubator. When most of the cells are observed to be digested under the microscope, add the same volume of cell culture medium to stop and gently blow into single cells with a pipette, transfer to a centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant and add 1-2mL culture medium to resuspend, take 100μL to count. According to the required density of seeding, add 100μL culture medium to each well of the 96-well plate, set up a separate well with only 100μL culture medium without seeding cells as the Blank well. The next day, prepare the required concentration of compound in the 96-well plate with serum-free culture medium, take 10μL and add it to the cells. For the control group, prepare the culture medium containing DMSO according to the highest DMSO concentration in the drug-dosing group, take 10μL and add it to the cells, and add 10μL culture medium to the Blank well. The outermost wells are not used for experiments due to the large edge effect. The 96-well plate treated with drugs was returned to the cell culture incubator for continued culture. After three days, the plate was collected and 10 μL of CCK8 dye was added to each well. The plate was read at a wavelength of 450 nm using an ELISA reader. The plate can be read multiple times within 4 hours. The reading value of the control group is more accurate if it falls within the range of 0.8 to 1.2. The experiment was set up with 2 replicates. After deducting Blank from all values, the cell survival rate / inhibition rate was calculated:

[0209] Survival rate (%) = [OD (drug well) / OD (control well)] × 100%

[0210] Inhibition rate (%) = 100 - survival rate (%)

[0211] Where, SI = IC for MDA-MB-231 50 Value / IC for MDA-MB-468 50 value.

[0212] The test results are shown in Table 2 below:

[0213] Table 2

[0214]

[0215] As shown in Table 2 , several compounds (B19, B21, B24, D5, D8, D11, and D13) showed significant inhibitory activity against the growth of PHGDH-dependent cancer cells (MDA-MB-468) and had good selectivity against human normal cells MCF10A.

[0216] Test Example 3

[0217] In vitro metabolic stability test

[0218] Test method: The test was carried out according to the method disclosed in the literature Gao, DD; Dou, HX; Su, HX; Zhang, MM; Wang, T.; Liu, QF; Cai, HY; Ding, HP; Yang, Z.; Zhu, WL; Xu, YC; Wang, HY; Li, YX From Hit to Lead: Structure-based Discovery of Naphthalene-1-sulfonamide Derivatives as Potent and Selective Inhibitors of Fatty Acid Binding Protein 4. Eur. J. Med. Chem. 2018, 154, 44–59.

[0219] The test results are shown in Table 3 below:

[0220] Table 3

[0221]

[0222]

[0223] As shown in Table 3, compound D8 showed better liver microsomal stability than the other three compounds in all three species.

[0224] Test Example 4

[0225] Inhibition kinetics test of D8 on PHGDH

[0226] Test method:

[0227] Inhibition kinetics test method: setting up different concentration gradients of NAD + Or 3-PG, add 5μL to a 384-well white microplate, mix the enzyme with another substrate in the same tube according to the above concentration, add 5μL to a 384-well white microplate, and finally the mixture of Diaphroase and Resazurin, and immediately test on the machine. This reaction is set to measure the fluorescence value every 2 minutes. Finally, the reaction speed needs to be calculated based on the reaction time and the fluorescence value, and a double reciprocal graph of the enzyme reaction rate and the substrate concentration is drawn.

[0228] To characterize the inhibitory mechanism of PHGDH inhibitor D8, different concentrations of D8, 3-PG and NAD + Competitive testing was performed. Lineweaver-Burk Figure 1 It shows that D8 is combined with NAD + Competitive mode of action inhibits PHGDH, NAD+ It is a cofactor of PHGDH. Lineweaver-Burk Figure 2 This indicates that D8 is in a non-competitive mode for the substrate 3-PG.

[0229] Test Example 5

[0230] Molecular dynamics simulation

[0231] Test method: Molecular docking was first performed using the Glide module in Schrödinger Maestro 11.4 software. (1) Preparation of protein and grid files: The crystal complex structure of PHGDH protein was downloaded from the Protein Data Bank, with the PDB number 2G76. The Protein Preparation Wizard module was used to hydrogenate the protein, add missing side chains, adjust the protein protonation state, delete the B chain, D-malic acid and water molecules, and then perform energy optimization (OPLS2005 force field, RMSD is ). Use the Receptor Grid Generation module to create a grid file with NAD+ as the center, and set the box size to (2) Compound preparation: The compound was processed by the LigPrep module and the corresponding 3D structure was output. (3) Molecular docking: Molecular docking was performed in the Ligand docking module in extraprecision mode, and the conformation with the best score was selected. (4) Molecular dynamics simulation: Homology modeling of human full-length PHGDH was performed using SWISS-MODEL, and then the above-mentioned optimal docking conformation was subjected to 100ns molecular dynamics simulation in the full-length PHGDH protein structure.

[0232] like Figure 3 and Figure 4 The final model after 100 ns MD simulation is shown in Figure 2. D8 is consistent in both the cis-PHGDH and trans-PHGDH models. After 20 ns, the NH of the amide bond of D8 is in hydrogen bond interaction with D175 most of the time, with a distance of Figure 3 In d1 and Figure 4 The d2 in the graphs represents the distance of the hydrogen bond between compound D8 and D175. Meanwhile, the sulfonyl group in compound D8 has a weak interaction with G157.

[0233] Test Example 6

[0234] D8 inhibits serine synthesis in MDA-MB-468 cells

[0235] Since PHGDH is an important enzyme in SSP, inhibition or deficiency of this protein would limit the de novo synthesis of serine from glucose.

[0236] Test method: Take an appropriate amount of MDA-MB-468 cells in the logarithmic growth phase and plant them in a six-well plate, with 2mL DMEM / F-12 medium in each well. After 24 hours, replace the fresh medium and add compound GDD-04-35 or DMSO for pretreatment for 1 hour, then wash 3 times with sterile PBS solution, suck out the residual liquid and add 1mL of three-free DMEM medium containing 15mM C13 glucose label (supplemented with 1mM sodium pyruvate, 4mM glutamine) and the corresponding concentration of compounds, return to the incubator and culture for 2 hours, then collect cell metabolites. Prepare and precool the lysis solution in advance (methanol: acetonitrile: water = 4:4:2, add internal standard 1:150), and precool it in a -80℃ refrigerator for at least 30 minutes. Add 500μL lysis buffer to each well, shake gently to make the lysis buffer spread all over the bottom of the well, place at -80℃ for 30min, scrape the cells on ice with a cell scraper (Biyuntian), collect them in a 1.5mL EP tube, rinse with pre-cooled 250μL lysis buffer, and combine the wash buffer. Vortex 3 times, 30s each time, place the sample on ice in the middle to ensure the quality of the sample. Then centrifuge at 4℃, 14000g for 15min, take 500μL of the supernatant, and place it in a freeze dryer to evaporate the sample. Take out the evaporated sample, add 40μL of methoxyamine solution to each sample to re-dissolve, vortex 3 times, react on a constant temperature shaker at 37℃ for 1h, then add 30μL of the derivatization reagent MtBSTFA, vortex twice, gently shake off the liquid on the wall with a centrifuge, and react on a constant temperature sample cooker at 55℃ for 1.5h. Finally, centrifuge at 2000 rcf for 3 min, take 55 μL of supernatant and add it to the injection bottle, and use GC-MS to detect the proportion of labeled serine.

[0237] The test results are as follows Figure 5 As shown, t-Test was used, ***p≤0.001; D8 at 10μM significantly reduced 13 The results of serine synthesis from C-glucose were better than those of CBR-5884, indicating that the SSP of MDA-MB-468 cells can be inhibited by compound D8.

[0238] Test Example 7

[0239] Antiproliferative activity test of compound D8 on cancer cell lines

[0240] PHGDH inhibitors can inhibit the proliferation of cell lines with PHGDH gene amplification (MDA-MB-468 and BT-20) or PHGDH overexpression (PC9, H1975, HCT116, and HCC827). Therefore, these cell lines were selected for cell proliferation evaluation to prove that the antitumor activity of D8 is related to PHGDH. At the same time, PHGDH low-expressing cell lines (H23, Panc-1, MDA-MB-231, ZR-75-1, and MCF-7) were used for comparison.

[0241] Test method: Cancer cells were placed in 96-well plates in appropriate culture medium containing 10% FBS overnight. Afterwards, cells were treated with a series of diluted compounds for 72 hours. Antiproliferative activity was assessed using CCK-8 cell viability assay (Dojindo, Japan). The absorbance at 450 nm was measured using a microplate reader.

[0242] Figure 6 The results showed that D8 was significantly sensitive to cell lines with PHGDH gene amplification or overexpression, and IC 50 The values ​​ranged from 3.5 μM to 21.6 μM. However, the antiproliferative activity of D8 against PHGDH-independent cell lines was reduced, with an IC 50 The values ​​ranged from 28.6 μM to 39.2 μM. These antiproliferative data preliminarily demonstrated that D8 is a potential selective inhibitor of PHGDH.

[0243] Test Example 8

[0244] Pharmacokinetic testing

[0245] Since compound D8 exhibited excellent enzymatic and cellular activities, its pharmacokinetic properties were evaluated in ICR mice using oral (po) and intravenous (iv) administration.

[0246] Test method: Six-week-old ICR male mice were housed in a 12-hour light / 12-hour dark cycle at 18 to 29°C and 30-70% relative humidity. The mice were randomly divided into three groups of 6 mice each, weighing 21-25 grams. Compound D8 was dissolved in water containing DMSO / 0.5% HPMC (5 / 95, v / v / ) and administered orally at a dose of 3 mg / kg; blood samples were collected at seven time points (0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 24 hours). In addition, compound D8 was dissolved in DMSO / EtOH / PEG300 / 0.9% NaCl (5 / 5 / 40 / 50, v / v / v / v) and administered intravenously at a dose of 1 mg / kg; blood samples were collected at seven time points (0.05, 0.25, 0.75, 2.0, 4.0, 8.0 and 24 hours). Plasma concentrations of D8 were analyzed using AQUITY UPLC.

[0247] The pharmacokinetic test results of compound D8 are shown in Table 4 below, where AUC represents the average area under the curve, T 1 / 2 represents the mean half-life, T max Indicates the peak time, CL_ obs represents clearance rate, Cmax represents maximum plasma drug concentration, and F represents bioavailability.

[0248] Table 4

[0249]

[0250] The test results are shown in Table 4 and Figure 7 As shown, for intravenous administration of 1 mg / kg, the average half-life of D8 was 4.94 hours, and the average area under the curve (AUC) value was 38358 h*ng / mL. After oral administration of D8 at a dose of 3 mg / kg, it showed the best average half-life (T 1 / 2 =4.74h), high drug exposure (AUC last = 94386h*ng / mL) and good bioavailability (F% = 82.0%).

[0251] Test Example 9

[0252] In vivo anti-tumor test

[0253] Test method: PC-9 cells were suspended in serum-free medium and 0.2 mL containing 5×10 6 The cells were injected into the right flank of BALB / c nude mice. The tumor growth of each mouse was monitored using a digital caliper and calculated using the equation: tumor volume = 0.5 × length × width 2 When the tumor volume of each mouse reaches 50-100mm 3 At 4 pm, tumor-bearing nude mice were randomly divided into three different treatment groups (blank control group 0 mg / kg, 12.5 mg / kg and 25 mg / kg; n = 6 in each group). The experimental groups were intraperitoneally injected with compound D8 once a day for 31 consecutive days. Tumor size was measured twice a week. All experiments were performed in accordance with animal ethics guidelines and approved by the Animal Ethics and Use Committee of the Shanghai Institute of Materia Medica.

[0254] like Figure 8 As shown, one way ANOVA test, *p<0.05, **p<0.01, D8 exhibited antitumor effect in vivo and significantly delayed tumor growth, with TGI of 41.4% at 12.5 mg / kg and 68.9% at 25 mg / kg.

[0255] like Fig. 9 As shown in Figure 2, D8 at a dose of 25 mg / kg can significantly reduce the tumor weight of mice. Fig.10 As shown, D8 did not cause weight loss in mice at both doses of 12.5 mg / kg and 25 mg / kg, indicating that compound D8 is safe.

[0256] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An application of a para-amidobenzenesulfonyl compound in the preparation of a phosphoglycerate dehydrogenase inhibitor, characterized in that: The para-amidobenzenesulfonyl compound is selected from any one of the following structures: Among them, R 11 Selected from unsubstituted or R d Any one of substituted C4-C12 nitrogen-containing heterocycloalkyl; The R d Any one selected from carboxyl, C1-C10 straight or branched alkyl, C1-C10 alkoxy; R 12 Selected from unsubstituted or R e Substituted C2-C12 heterocycloalkyl, unsubstituted or R e Substituted C3-C12 cycloalkyl, unsubstituted or R e Substituted C6-C12 aryl, unsubstituted or R e Substituted C2-C12 heteroaryl, unsubstituted or R e Substituted C1-C10 straight or branched alkyl, unsubstituted or R e Any one of substituted C1-C10 alkylcarboxyl groups; The R e Selected from hydroxyl, carboxyl, C1-C10 straight or branched alkyl, C1-C10 alkoxy, C1-C10 alkylhydroxy, C2-C12 heterocycloalkyl, C1-C10 alkylcarbonyl, C1-C10 alkylamide, C1-C10 alkylcarboxyl, NR N3 R N4 Any of the following; The R N3 , R N4 Each is independently selected from any one of hydrogen, C1-C10 straight chain or branched alkyl; R2 is selected from any one of substituted thiazolyl, substituted thienyl, and substituted triazole; The substituted substituents are each independently selected from unsubstituted or R c Substituted phenyl, unsubstituted C1-C10 straight chain or branched alkyl; The R c Any one selected from halogen, hydroxyl, nitro, C1-C10 straight or branched alkyl, C1-C10 alkoxy or C1-C10 haloalkyl.

2. Use of a para-amidobenzenesulfonyl compound in the preparation of a phosphoglycerate dehydrogenase inhibitor, characterized in that: The p-amidobenzenesulfonyl compound has any one of the structures shown in A2, B1-B25, C1-C6, and D1-D23 below:

3. Use of a para-amidobenzenesulfonyl compound in the preparation of an anti-tumor drug, characterized in that: The para-amidobenzenesulfonyl compound is selected from any one of the following structures: Among them, R 11 Selected from unsubstituted or R d Any one of substituted C4-C12 nitrogen-containing heterocycloalkyl; The R d Any one selected from carboxyl, C1-C10 straight or branched alkyl, C1-C10 alkoxy; R 12 Selected from unsubstituted or R e Substituted C2-C12 heterocycloalkyl, unsubstituted or R e Substituted C3-C12 cycloalkyl, unsubstituted or R e Substituted C6-C12 aryl, unsubstituted or R e Substituted C2-C12 heteroaryl, unsubstituted or R e Substituted C1-C10 straight or branched alkyl, unsubstituted or R e Any one of substituted C1-C10 alkylcarboxyl groups; The R e Selected from hydroxyl, carboxyl, C1-C10 straight or branched alkyl, C1-C10 alkoxy, C1-C10 alkylhydroxy, C2-C12 heterocycloalkyl, C1-C10 alkylcarbonyl, C1-C10 alkylamide, C1-C10 alkylcarboxyl, NR N3 R N4 Any of the following; The R N3 , R N4 Each is independently selected from any one of hydrogen, C1-C10 straight chain or branched alkyl; R2 is selected from any one of substituted thiazolyl, substituted thienyl, and substituted triazole; The substituted substituents are each independently selected from unsubstituted or R c Substituted phenyl, unsubstituted C1-C10 straight chain or branched alkyl; The R c Any one selected from halogen, hydroxyl, nitro, C1-C10 straight or branched alkyl, C1-C10 alkoxy or C1-C10 haloalkyl.

4. Use of a para-amidobenzenesulfonyl compound in the preparation of an anti-tumor drug, characterized in that: The p-amidobenzenesulfonyl compound has any one of the structures shown in A2, B1-B25, C1-C6, and D1-D23 below:

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