CN118126115A

CN118126115A - Compound combined with E3 ubiquitination ligase substrate recognition protein and application thereof

Info

Publication number: CN118126115A
Application number: CN202410255844.4A
Authority: CN
Inventors: 董城; 苏向东; 齐非; 辛超; 刘士林; 曾江蒙; 王金戌
Original assignee: Taibidi Pharmaceutical Technology Shijiazhuang Co ltd
Current assignee: Taibidi Pharmaceutical Technology Shijiazhuang Co ltd
Priority date: 2024-03-06
Filing date: 2024-03-06
Publication date: 2024-06-04

Abstract

The invention relates to the technical field of biological medicines, in particular to a compound combining E3 ubiquitination ligase substrate recognition protein or pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof. The invention designs and develops the inhibitor with novel and unique structure aiming at the E3 ubiquitin ligase substrate recognition protein ZYG B or ZER1, can effectively combine or inhibit the E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B, regulate and control the related key protein and further play a role in diagnosing, preventing and treating related diseases. Meanwhile, the compound developed by the invention can form a compound with CLR2-ZYG B or CLR2-ZER1, becomes a part of a PROTAC molecule or a molecular gel molecule with a target protein degradation function for recruiting E3 enzyme, and has great significance for developing novel medicines.

Description

Compound combined with E3 ubiquitination ligase substrate recognition protein and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a compound combined with E3 ubiquitin ligase substrate recognition protein or pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof, which can regulate and control related key proteins and further play a role in diagnosing, preventing and treating related diseases by combining with E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B.

Background

Ubiquitin-protease system (UPS) is a major approach for cells to achieve selective protein degradation. Organisms maintain their normal state by selective degradation of damaged, misfolded, dysfunctional or unwanted proteins through UPS systems. Aberrations in UPS-related proteins can lead to a variety of human diseases including neurodegeneration, abnormal aging, and cancer. In addition, research proves that the ubiquitin-proteinase system has important pathophysiological significance in cardiovascular diseases, and can regulate the occurrence and development of important diseases such as atherosclerosis, reperfusion injury after ischemia, familial cardiomyopathy, cardiac hypertrophy, heart failure and the like. UPS is reported to be composed of ubiquitin (Ub), ubiquitin activating enzyme (ubiquitin-ACTIVATING ENZYME, E1), ubiquitin binding enzyme (ubiquitin-conjugating enzyme, E2), ubiquitin ligase (ubiquitin-protein ligase, E3), proteasome and its substrate (protein). The substrates of the UPS are polyubiquitinated by the successive actions of these three enzymes (E1, E2 and E3) and subsequently degraded by the 26S proteasome (Balchin,D.,Science 2016;353,aac4354;Sontag,E.M.Annu.Rev.Biochem.2017;86,97–122;Pickart,C.M.Annu.Rev.Biochem.2001;70,503–533).

In a UPS system, E3 ubiquitin ligase is a key component for regulating the selectivity of the UPS system, and the specific recognition capability of the E3 ubiquitin ligase on a substrate protein determines that ubiquitin-mediated protein degradation has specificity. Ubiquitin is linked to E1 through its carboxyl group on the C-terminal glycine and the necessary cysteine sulfhydryl group on ubiquitin activating enzyme E1 to form a high energy thioester bond, becoming ubiquitin in an activated state; second, the activated ubiquitin is transferred from ubiquitin activating enzyme E1 to ubiquitin binding enzyme E2; finally, under the action of E3 ubiquitin ligase, ubiquitin molecules linked to ubiquitin binding enzyme E2 are linked to the underlying protein by covalent linkage means of isopeptide bonds. The substrate protein may be delivered to the 26S proteasome after ubiquitination or may enter the lysosome (lysosome) for digestion and degradation. To date, more than 600E 3 ubiquitin ligases have been identified in the human proteome (Bachmair, A., cell 1989;56, 1019-1032; lucas, X., curr. Opin. Struct. Biol.2017;44, 101-110). E3 ubiquitin ligase is capable of specifically recognizing degradation signals of the substrate (degron), determining the specificity of the UPS system. Thus, E3 ligase is also a key target for current cancer targeted therapies. The E3 ligase recognizes, through its recognition protein portion, specifically the degradation signal of the substrate, which is typically a specific sequence located in its cognate substrate. When the signal is recognized at the N-terminus of the protein, it is referred to as an N-terminal degradation (N-degron). The N-terminal mediated proteasome degradation pathway is a more specific protein degradation pathway in cells; when the protein is hydrolyzed in peptide, a new N-terminal initial sequence is generated, so that the new protein sequence is unstable, and the relevant functional protein can rapidly mediate the hydrolyzed peptide segment to enter a proteasome for degradation. Since all 20 natural amino acids are unstable N-terminal residues and can be separated into different branches of the N-degron pathway, for example, arg/N-degron, pro/N-degron, gly/N-degron, ac/N-degron and fMet/N-degron pathway. The Pro/N-degron pathway recognizes degradation of proteins containing N-terminal prolines. The Ac/N-degron pathway recognizes N- α -terminal acetylation (acetylmethionine, acetylalanine, acetylserine, acetylthreonine, acetylvaline, acetylcysteine). The fMet/N-degron pathway recognizes the N-terminal formylated methionine. The Gly/N-degron pathway degrades proteins that are not myristoylated and thus monitors their acylation process. In the N-degron pathway, ZYG B and ZER1 act as substrate receptors for the Cullin 2-RING E3 ubiquitin ligase (CRL 2), responsible for recognizing the N-terminal glycine and some small side chain amino acids, such as alanine, serine, cysteine, threonine (Varshavsky,A.Protein Sci.2011;20,1298–1345;Chen,S.J.,Science 2017;355,eaal3655;Timms,R.T.Science 2019;365aaw4912;Hwang,C.S.,Science 2010;327,973–977;Kim,J.M.Science 2018;aat0174;Varshavsky A.Proc Natl Acad Sci USA.2019;116(2),358-366;Y,Li,Nat Commun.2022Dec 10;13(1):7636.).

As shown by the crystal structures of ZER1 and ZYG B obtained in the experiments, ARM2-ARM8 of ZYG B were stacked together and Gly/N-degron formed an arched conformation. Each ARM repeat consists of three alpha helices (H1, H2 and H3) arranged in a right triangle. However, in ARM6 and ARM7, the regions involved in ARM6-H3 helix and ARM7-H1 helix formation (ZYG B residues W638-F651) form an elongated loop (e-loop) rather than the typical helix. This e-loop bends toward the center of the body, resulting in a 90 deg. twisted arrangement of ARM7-ARM 8. Thus, the internal H3 helix of ARM3-ARM4 forms a deep cavity with the e-loop for recognition of Gly/N-degron. The crystal structure of ZER1 is very similar to that of ZYG B, the upstream portion of e-loop is structurally irregular due to the difference in sequence, but the downstream region of e-loop can be well overlapped to facilitate interaction with Gly/N-degron. Furthermore, the amino acid sequences of the N-degron binding pocket, consisting of ZYG B and ZER1, are highly overlapping, demonstrating that they have an approximate substrate recognition mechanism in the Gly/N-degron pathway (Xiaojie Y.; molecular Cell 2021;81, 3262-3274).

In general, post-translational modification (PTM) is often required for most proteins to function properly. Studies have shown that PTM plays a key role in regulating various physiological and pathological processes, such as protein stability maintenance and tumorigenesis. Among them, the ZER1 and ZYG B mediated Gly/N-degron pathway plays an important role in regulating protein homeostasis in cells. For example, small side chain amino acids at the N-terminus of certain proteins (e.g., alanine, serine, cysteine, threonine) may be acetylated to avoid recognition by the E3 ligase, and if N-terminal acetylation is incorrect, the non-acetylated proteins may be recognized by ZER1 and ZYG B and degraded by the proteasome, thereby maintaining quality control of N-terminal acetylation of the protein. In addition, the glycine at the N-terminus of the protein is typically myristoylated to achieve precise membrane localization. The Gly/N-degron pathway degrades proteins that are not myristoylated and allows quality monitoring of myristoylation modification processes. N-myristoylation (N-myristoylation) is a lipid modification that has attracted attention in the field of cancer. Inhibition of ZER1 or ZYG B may affect the quality monitoring process of N-myristoylation to exert a corresponding bioregulation effect. Cleavage by other endopeptidases such as caspases also exposes the N-terminal glycine, and the Gly/N-degron pathway can degrade caspase enzymatic products during apoptosis. More importantly, this pathway activates human NLRP1 inflammatory corpuscles by degrading the NLRP1 self-inhibiting fragment after cleavage by enterovirus 3C enzyme, playing an important role in the immune response to viral infection (Kim S., science 2020;370, eaay2002).

Inflammatory body sensors detect pathogen and risk related molecular patterns and promote inflammation and cell apoptosis. NLRP1 is the first described inflammatory body sensor, whose over-activation is associated with auto-inflammatory diseases and cancers. Inflammatory bodies act as intracellular sensors for pathogen infection or cellular perturbation and thus play a central role in a variety of diseases. Patients with acquired mutations in NLRP1 function exhibit an inflammatory phenotype of the skin or respiratory tract, primarily affecting keratinocytes and respiratory epithelial cells. This suggests that human NLRP1 plays an important role in barrier tissue epithelial cells rather than in non-self recognition within the intramedullary canal. The inflammatory corpuscles induce maturation of the inflammatory cytokines IL-1 beta and IL-18, the activity of which is associated with the pathophysiology of a variety of infections and inflammatory diseases. As a validated therapeutic target for the treatment of acute and chronic inflammatory diseases, there is a great interest in developing small molecule inhibitors to target inflammatory body activity and reduce the inflammatory burden associated with the disease. ZER1 and ZYG B promote NLRP1 activation through UPS systems, so development of ZER1 and ZYG B inhibitors can find novel compounds preventing NLRP1 activation for prevention and treatment of various inflammatory or immune diseases.

In addition, human Papillomavirus (HPV) E7 protein binds to host cell proteins to promote viral replication, and interactions between HPV E7 and host cell proteins can also drive the progression of cancer. The cellular protein ZER1 interacts with the E7 protein from HPV16, a genotype most commonly associated with human cancers. Inhibition of ZER1 would impair the growth of primary keratinocytes expressing HPV16E7 or HPV 18E 7, and HPV16 and HPV18 positive cervical cancer cell lines. ZER1 contributes to HPV-mediated canceration and is critical for the growth of HPV-positive cells (White EA, proc NATL ACAD SCI USA 2012;109: E260-E267; joangela N, mbio 2022; E0203322). ZER1 inhibitors can potentially be developed as an effective means of preventing HPV viral carcinogenesis. The ORF10 protein encoded by the genome of the novel coronavirus SARS-CoV-2 is closely combined with ZYG B, and the ORF10 is related to inhibiting the internal viral immune response process of the organism, thereby promoting the pathogenicity and transmission capacity of the virus. Studies have shown that inhibition of ZYG B complex formation with ORF10 may be a method of modulating immune responses (Bing Z., biochemical and Biophysical Research Communications,2022;616, 14-18).

In recent years, methods for regulating intracellular protein levels using the ubiquitin-protease system (UPS) in eukaryotic cells have received great attention. The protein degradation targeting chimeric PROTAC (proteolic TARGETING CHIMERA) technology utilizes UPS to specifically degrade target proteins, can effectively induce degradation of target proteins for treating various diseases, comprises enzymes, transcription factors, epigenetic factors, scaffold proteins and the like, and initially shows excellent curative effects and safety in clinic. PROTAC bind to the small molecule ligand of a specific targeting protein, and form a bifunctional compound by bridging the chain fragment to the ligand of E3 ubiquitin ligase. Through the optimized linking position and the length of the bridging chain, the ligands at two ends of PROTAC molecules are simultaneously combined with the target protein and the E3 ubiquitin ligase to form a target protein-PROTACs-E3 ligase ternary complex, so that the ubiquitination marking of the target protein is promoted, and the target protein is further degraded by a protease system. Because of the characteristic of ternary complex formation, the target protein ligand adopted by PROTAC technology does not need to have strong target binding activity, so that target proteins, such as skeleton proteins, transcription factors and the like, which cannot be prepared by the traditional inhibitors can be targeted; in addition, the integral degradation of the target protein is helpful to overcome the drug resistance problem of the small molecule inhibitor; PROTAC molecules are cycled repeatedly by a catalytic mechanism, PROTAC-induced degradation is event-driven, not site-occupying driven, and after complex formation and ubiquitin transfer is completed, the drug dissociates and enzymatically transfers to the next target. To some extent, low doses can be achieved to maintain effective pharmaceutical activity. PROTAC are technically difficult to control because of the effect of the degradation and production rate of the target protein, the ability of the target protein to bind ubiquitin, the conformation and site of the attachment of the target protein ligand and the E3 ubiquitin ligase ligand, the modification of the length and composition of the bridging chain and the concentration, etc. on the formation of ternary complexes and their stability. Of the over 600E 3 ubiquitin ligases known, there are only a limited number of practical applications in PROTAC compound designs, including CRBN class, VHL class, MDM2 class, cIAP1 class. These E3 ubiquitin ligases confer substrate specificity to achieve ubiquitination of the target protein. The Von Hippel-Lindau (VHL) tumor suppressor in the common E3 ubiquitin ligase consists of the elongin B and C, cul and Rbx1, the major substrates of which are hypoxia inducible factor 1 (HIF-1). The ligand study of E3 ubiquitin ligase VHL in Chinese patent CN108601764A, for example, has resulted in the crystal structure of the complex, thereby confirming that small molecule compounds can mimic the major substrate of transcription factor HIF-1. In the application process, the ligand of VHL and E3 ubiquitin ligase have relatively weak binding property, which is easy to cause incomplete degradation of target protein and cause off-target effect. Another important E3 ligase Cereblon (CRBN) is the protein encoded by the human CRBN gene, which is highly conserved, showing its physiological importance. Cereblon together with the damaged DNA binding protein 1 (DDB 1), cullin-4A (CUL 4A) and the Cullin-1 regulator (ROC 1) constitute the E3 ubiquitin ligase complex. The complex is capable of ubiquitinating a range of proteins. Given the binding capacity of a phthalimide building block to CRBN, this building block is often used as an E3 ligase recruitment ligand to hijack CRBN to degrade the protein of interest. Currently, protein degrading agents entering the clinical stage mainly utilize two E3 ligases, CRBN and VHL. These PROTAC compounds are likely to exhibit off-target effects and drug resistance during treatment, and therefore development of new E3 ligase tools is highly desirable. ZYG11B or ZER1 is a substrate protein recognition factor in the E3 ligase CRL2 complex, so the art expects to develop more novel small molecule compounds capable of binding ZYG B or ZER1, which provides a basis for developing unique PROTAC molecules or molecular gel molecules.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to provide a compound that binds to E3 ubiquitination ligase substrate recognition protein or a pharmaceutically acceptable salt, stereoisomer, solvate or polymorph thereof; the compounds regulate and control related key proteins by combining E3 ubiquitination ligase substrate recognition protein ZER1 or ZYG B and further play roles in diagnosing, preventing and treating related diseases; meanwhile, the compounds can be combined with CLR2-ZYG B or a CLR2-ZER1 complex to become a PROTAC molecule or a molecular part for recruiting E3 enzyme in a molecular gel molecule;

The second technical problem solved by the invention is to provide a preparation method and application of the compound combined with the E3 ubiquitination ligase substrate recognition protein, which have great significance in developing novel medicines.

In order to solve the above technical problems, the present invention provides a compound that binds to an E3 ubiquitinated ligase substrate recognition protein, or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, oxynitride, prodrug or polymorph thereof, the compound having a structure represented by the following formula (I):

Wherein,

The R ¹ is selected from H, CH ₂ X; wherein X is selected from H, OH, SH, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon radicals; preferably, the X is H, OH or CH ₃;

the R ² is selected from H, OH, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon groups; preferably, R ² is H;

The R ³ is selected from H, C-C8 saturated or unsaturated linear or branched hydrocarbon group ,-(CH₂)_m-Cyc¹、-(CH₂)_p-N(CO)_qR⁵(R⁶)-、-COR⁵;, wherein m is optionally from natural number 0-4; p is optionally from a natural number of 1 to 5; q is optionally from a natural number 0-1; cyc ¹ is selected from benzene ring, substituted or unsubstituted 5-6 membered aromatic heterocycle, substituted or unsubstituted condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, substituted or unsubstituted 4-7 membered monocycloalkyl, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered bridged cycloalkyl; the substitution includes substitution of 0 to 3C 1-4 linear or branched alkyl groups independently of each other with 0 to 3C 1-4 linear or branched alkyl groups selected from halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、–(CH₂)_pOR⁷,C1-4 linear or branched alkyl groups, halogen substituted C1-4 linear or branched alkyl groups, hydroxy substituted C1-4 linear or branched alkyl groups, or alkoxy substituted C1-4 linear or branched alkyl groups; preferably, the aromatic heterocycle, aromatic fused heterocycle, carboheteromonocyclic contains 1 to 3 heteroatoms selected from O, S, N; r ⁵、R⁶、R⁷、R⁸ is independently selected from H, C C4 linear or branched alkane, C1C 4 linear or branched alkene, C1C 4 linear or branched alkyne;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; the substitutions include each independently being substituted with 0 to 3 linear or branched hydrocarbyl groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂,-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; the above-mentioned C1-4 saturated or unsaturated straight-chain or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 1 to 4 heteroatoms selected from O, S, N; r ⁷、R⁸ is independently selected from H, C-C4 saturated or unsaturated straight-chain or branched hydrocarbon groups.

The invention also provides a compound which binds to the E3 ubiquitination ligase substrate recognition protein, as shown in the formula (I):

When A-G are connected by a double bond and G-J are connected by a single bond, wherein G is a carbon atom, J is a nitrogen atom, and A is selected from O, S, CH ₂、CF₂; or alternatively

When A-G are connected by a single bond and G-J are connected by a double bond, wherein G, J is a carbon atom, A is selected from OH, SH, CH ₃ and halogen; or alternatively

When no covalent bond is formed between Q ² and M, M is selected from CR ¹³ or N, wherein R ¹³ is selected from H, -CH ₂ Ar, ar is selected from benzene ring, pyridine ring; q ² is selected from O, NH, NHOH, CF ₂ and is linked to the adjacent carbon atom by a double bond; q ¹ is NR ¹⁰ and is linked to an adjacent carbon atom by a single bond, wherein R ¹⁰ is selected from H, C-3 saturated or unsaturated straight or branched hydrocarbon groups; or alternatively

When a covalent single bond is formed between Q ² and M, M is a carbon atom, Q ² is a nitrogen or oxygen atom, Q ¹ is N or NR ¹⁰, wherein R ¹⁰ is selected from H, C1-3 saturated or unsaturated straight or branched hydrocarbon groups;

Preferably, L is selected from N or CR ⁹ or absent, wherein R ⁹ is selected from H, C1-4 saturated or unsaturated straight or branched hydrocarbon, - (CH ₂)_r-Cyc³), wherein R is selected from a natural number 0-1, cyc ³ is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocycloalkane, a substituted or unsubstituted 5-10 membered acene, or R ⁴ forms a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring with R ⁹ closed ring, the substitutions including but not limited to, being substituted independently of each other with 0-3 straight or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated straight or branched hydrocarbon; preferably, the above mentioned C1-4 saturated or unsaturated straight or branched hydrocarbon groups may be optionally substituted by halogen, hydroxy, alkoxy, said carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic containing 1 to 3 heteroatoms selected from O, S, N; preferably, R ⁷、R⁸ is each independently selected from H, C to C4 saturated or unsaturated straight or branched hydrocarbon groups; preferably, when L is absent, the carbon atom to which R ⁴ is attached forms a covalent single bond with M.

The invention also provides a compound which binds to the E3 ubiquitination ligase substrate recognition protein, and the compound has a structure shown as the following formula (II):

Wherein,

The R ¹ is selected from H, CH ₂ X; x is selected from H, OH, SH, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; preferably, the X is H, OH or CH ₃;

The R ³ is selected from H, C-C8 saturated or unsaturated linear or branched hydrocarbon, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵) wherein m is optionally selected from a natural number of 0-4;p, a natural number of 0-1, cyc1 is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted condensed ring formed by a benzene ring and a 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carbon heteromonocyclic ring, a substituted or unsubstituted 5-10 membered carbon heteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocyclic alkyl, a substituted or unsubstituted 5-10 membered cycloalkyl, a substituted or unsubstituted 7-10 membered bridged cycloalkyl, wherein the substitution includes a C1-4 linear or branched alkyl substituted by 0-3C 1-4 linear or branched alkyl, hydroxy-substituted C1-4 linear or branched alkyl or alkoxy independently of each other, a substituted C1-4 linear or branched alkyl, preferably the heterocyclic ring, the substituted C1-4 linear or branched alkyl, C1-4, C4-membered aromatic heterocycle, C1-4, C3, C1-4-branched C1-4 linear or branched alkyl, C1-4-C4-membered heteroaromatic ring independently selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、-(CH2)pOR⁷;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; the substitution includes substitution independently of each other with 0 to 3 linear or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; the above-mentioned C1-4 saturated or unsaturated straight-chain or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 1 to 4 heteroatoms selected from O, S, N; r ⁷、R⁸ is independently selected from H, C-C4 saturated or unsaturated straight-chain or branched hydrocarbon groups.

The invention also provides a compound which binds to an E3 ubiquitinated ligase substrate recognition protein, the compound having a structure as shown in the following formula (III):

Wherein,

The R ¹¹ is selected from H, OH, NH ₂, halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂,-(CH₂)_rNHCOCH＝CR⁷(R⁸)、C1-C7 saturated or unsaturated linear or branched alkyl ,-(CH₂)_m-Cyc¹、-(CH₂)_p-N(CO)_qR⁵(R⁶)-;, wherein m is optionally from a natural number of 0-4; p is optionally from a natural number of 1 to 5; q is optionally from a natural number 0-1; r is optionally from a natural number of 0 to 6; cyc ¹ is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted fused ring formed from a benzene ring and a 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, a substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocycloalkyl group, a substituted or unsubstituted 5-10 membered cycloalkyl group, a substituted or unsubstituted 7-10 membered bridged cycloalkyl group; the substitution includes C1-4 linear or branched alkyl groups which must be independently substituted with 0-3 groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸, C1-4 linear or branched alkyl groups substituted with halogen, C1-4 linear or branched alkyl groups substituted with hydroxy, or C1-4 linear or branched alkyl groups substituted with alkoxy; preferably, the aromatic heterocycle, aromatic fused heterocycle, carboheteromonocyclic contains 1 to 3 heteroatoms selected from O, S, N; r ⁵、R⁶、R⁷、R⁸ is independently selected from H, C-C4 linear or branched alkane, C1-C4 linear or branched alkene, C1-C4 linear or branched alkyne;

The E ring is selected from a substituted or unsubstituted 5-8 membered aromatic ring, a substituted or unsubstituted 5-8 membered aromatic heterocyclic ring, a substituted or unsubstituted 4-7 membered carbon heteromonocyclic ring; wherein the aromatic heterocycle or the carboheteromonocyclic ring contains 1-3 nitrogen atoms, and the E ring is substituted by halogen, C1-C4 straight-chain or branched alkyne, -OH, -COOH, -CN and-NH ₂、-CONH₂ besides the substituent R ¹¹; preferably, the E ring is selected from 6 membered aromatic heterocycles, wherein the aromatic heterocycle contains 1-2 nitrogen atoms.

The invention also provides a compound which binds to an E3 ubiquitinated ligase substrate recognition protein, the compound having a structure represented by the following formula (IV):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated straight chain or branched hydrocarbon radicals, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵; wherein m is optionally from a natural number of 0 to 4;p, optionally from a natural number of 0 to 5;q, optionally from a natural number of 0 to 1, cyc1 is selected from the group consisting of substituted or unsubstituted benzene rings, substituted or unsubstituted 5-6 membered aromatic heterocycles, substituted or unsubstituted fused rings formed by benzene rings and 5-6 membered aromatic heterocycles, substituted or unsubstituted 4-7 membered carboheteromonocyclic rings, substituted or unsubstituted 5-10 membered carboheteromonocyclic rings, substituted or unsubstituted 4-7 membered monocyclic alkyl groups, substituted or unsubstituted 5-10 membered cycloalkyl groups, substituted or unsubstituted 7-10 membered bridged cycloalkyl groups, said substitution including C1-4 straight or branched alkyl groups which must be independently substituted with 0 to 3C 1-4 straight or branched alkyl groups selected from the group consisting of halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、–(CH₂)pOR⁷,C1-4, halogen substituted C1-4 straight or branched alkyl groups, hydroxy substituted C1-4 straight or branched alkyl groups or alkoxy groups, preferably said aromatic heterocycles, aromatic fused heterocycles, carboheteromonocyclic rings contain 1 to 3 heteroatoms selected from the group consisting of O, S, N;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; the substitution includes substitution independently of each other with 0 to 3 linear or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂,-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; preferably, the above C1-4 saturated or unsaturated straight or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 0 to 4 heteroatoms selected from O, S, N; preferably, R ⁷、R⁸ is each independently selected from H, C1-C4 saturated or unsaturated straight or branched hydrocarbon groups;

The Q ² is selected from nitrogen or oxygen atoms;

The Q ¹ is selected from N or NR ¹⁰, wherein the R ¹⁰ is selected from H, C1-3 saturated or unsaturated straight or branched hydrocarbon groups.

The invention also provides a compound which binds to an E3 ubiquitinated ligase substrate recognition protein, the compound having a structure represented by the following formula (V):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated straight chain or branched hydrocarbon radicals, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵; wherein m is optionally from a natural number of 0 to 4;p, optionally from a natural number of 1 to 5;q, optionally from a natural number of 0 to 1, cyc1 is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted fused ring formed from a benzene ring and a 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, a substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocycloalkyl, a substituted or unsubstituted 5-10 membered cycloalkyl, a substituted or unsubstituted 7-10 membered bridged cycloalkyl; the substitution includes substitution of 0 to 3C 1-4 linear or branched alkyl groups independently of each other with 0 to 3C 1-4 linear or branched alkyl groups selected from halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、–(CH₂)pOR⁷,C1-4 linear or branched alkyl groups, halogen substituted C1-4 linear or branched alkyl groups, hydroxy substituted C1-4 linear or branched alkyl groups, alkoxy substituted C1-4 linear or branched alkyl groups, preferably the aromatic heterocycle, carboheteromonocyclic, carboheterobicyclic ring contains 1 to 3 heteroatoms selected from O, S, N, preferably the R ⁵、R⁶、R⁷、R⁸ is independently selected from H, C1-C4 linear or branched alkane, C1-C4 linear or branched alkene, C1-C4 linear or branched alkyne;

The R ¹² is selected from H, OH, NH ₂, halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂, C1-C4 saturated or unsaturated straight or branched hydrocarbon group.

The present invention also provides a compound that binds to an E3 ubiquitination ligase substrate recognition protein, or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof, said compound being selected from at least one of the following compounds:

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The invention also provides a pharmaceutical composition comprising said compound that binds to the E3 ubiquitination ligase substrate recognition protein or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof.

In particular, the pharmaceutical composition comprises an excipient, diluent, co-solvent, adjuvant, vehicle, or combination thereof.

Specifically, the compounds containing chiral centers form different stereo configurations, and therefore can exist in more than one stereoisomer form. The stereoisomers to which the invention relates are present either in optically pure form, such as greater than 95% ee, or mixtures thereof, including racemic mixtures. These optically pure isomers can be prepared starting with optically pure starting materials by asymmetric synthesis or by chiral resolution.

The invention also provides the use of said compound or salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof which binds to the E3 ubiquitin ligase substrate recognition protein for the preparation of a medicament which can bind to the E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B.

The invention also provides the use of the compound or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitrogen oxide, prodrug or polymorph thereof which binds to E3 ubiquitination ligase substrate recognition protein for preparing a medicament for inhibiting the protein complex of cullin2-ZER1 and cullin 2-ZYG B.

The invention also provides the use of the compound or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitrogen oxide thereof which binds to the E3 ubiquitination ligase substrate recognition protein, a prodrug or a polymorph thereof for preparing a medicament for inhibiting the combination of a protein N-terminal degradation molecule and the E3 ubiquitination ligase substrate recognition protein ZER1 or ZYG B and the ubiquitination and proteasome degradation of the protein.

The invention also provides the use of the compound that binds to the E3 ubiquitination ligase substrate recognition protein or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitrogen oxide, prodrug or polymorph thereof for the preparation of an inhibitor of inflammatory small NLRP1 activation.

The invention also provides application of the compound combined with the E3 ubiquitination ligase substrate recognition protein or salts, enantiomers, geometric isomers, solvates, isotopically enriched analogues, nitrogen oxides, prodrugs or polymorphs thereof in preparing medicaments for preventing or treating inflammation or complications triggered by factors such as microbial infection, gene mutation and the like.

Specifically, the inflammation or complication triggered by a microorganism infection, a gene mutation and the like includes gout, pneumonia, parkinsonism, alzheimer's disease, diabetes, cancer or a virus infectious disease.

The invention also provides the use of the compound or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitrogen oxide, prodrug or polymorph thereof which binds to the E3 ubiquitination ligase substrate recognition protein for preparing a medicament which interferes with the binding of ZYG B to ORF10 protein of the novel coronavirus SARS-CoV-2 and affects the immune response to the novel coronavirus.

The invention also provides application of the compound combined with the E3 ubiquitination ligase substrate recognition protein or salts, enantiomers, geometric isomers, solvates, isotopically enriched analogues, nitrogen oxides, prodrugs or polymorphs thereof in preparing medicines capable of forming a complex with the cullin2-ZER1 or the cullin2-ZYG B and playing a role in regulating and controlling the quality of the N-myristoylation protein.

The invention also provides the use of said compound or salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide thereof which binds to E3 ubiquitination ligase substrate recognition protein, prodrug or polymorph for the preparation of a medicament for the degradation of a specific target protein by non-covalent or covalent bonding to cullin2 (ZER 1/ZYG B) and constituting part of a proteolytically targeted chimeric (PROTAC) molecule or molecular glue (molecular glue) molecule, recruiting ubiquitin-proteinase system.

The invention also provides the use of the compound that binds to the E3 ubiquitination ligase substrate recognition protein or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitrogen oxide, prodrug or polymorph thereof for the preparation of a cancer, inflammatory disease, autoimmune disease, fibrotic disease or neurodegenerative disease.

Specifically, the use is as follows:

Such cancers include gastric cancer, intestinal cancer, esophageal cancer, head and neck cancer, lung cancer, liver cancer, brain cancer, breast cancer, colorectal cancer, skin cancer, thyroid cancer, prostate cancer, soft tissue cancer, endometrial cancer, uterine cancer, testicular cancer, cervical cancer, ovarian cancer, fallopian tube tumor, leukemia, squamous cell cancer, basal cell cancer, adenocarcinoma, renal cell cancer, bladder cancer, renal cancer, pancreatic cancer, lymphoma, non-hodgkin's lymphoma, melanoma, myeloproliferative disorder, sarcoma, angiosarcoma, peripheral nerve epithelial tumor, glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, gangliocytoma, neuroblastoma, pineal tumor, meningioma, neurofibroma or schwannoma; and/or the number of the groups of groups,

The inflammatory or autoimmune disease includes rheumatoid arthritis, autoimmune encephalomyelitis, ankylosing spondylitis, central axis spondyloarthritis, psoriasis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, recurrent oral ulceration, kawasaki disease, spondyloarthritis, neuromyelitis optica, behcet's disease, lupus nephritis, familial mediterranean fever, ulcerative colitis, autoimmune hepatitis, asthma, arteriosclerosis, or crohn's disease; and/or the number of the groups of groups,

The fibrotic disease includes cystic fibrosis or cystic fibrosis, endocardial fibrosis, liver fibrosis (cirrhosis), idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis), interstitial lung disease (Diffuse parenchymal lung disease), mediastinal fibrosis (MEDIASTINAL FIBROSIS), peritoneal fibrosis (Retroperitoneal fibrosis), pneumoconiosis (Pneumoconiosis), tumor fibrosis (Neoplastic fibrosis), or spleen fibrosis; and/or the number of the groups of groups,

The neurodegenerative disease includes Alzheimer's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, bovine spongiform encephalopathy, creutzfeldt-Jakob disease, huntington's chorea, cerebellar atrophy, multiple sclerosis, parkinson's disease, primary lateral sclerosis, spinal muscular atrophy, cerebral ischemia, spastic paraplegia or myasthenia gravis.

Specifically, the medicament further comprises one or more than one bioactive agent;

Preferably, the bioactive agent comprises at least one of an anti-cancer agent, an immunomodulator, an immune checkpoint inhibitor, a kinase inhibitor, an anti-infective agent, a neuroprotective agent or an anti-inflammatory agent.

Among the above-mentioned uses of the present invention, the pharmaceutical preparation according to the present invention may be selected from the types of preparations suitable for oral administration, injection administration, and inhalation administration, and it will be apparent to those skilled in the art that the following dosage forms may contain as an active ingredient to prepare a desired pharmaceutical preparation.

For the preparation of a suitable pharmaceutical formulation according to the invention, the pharmaceutically acceptable carrier may be solid or liquid. Solid form preparations include powders, tablets, nine agents, capsules, cachets, and dispersible granules. The solid carrier may be one or more substances that also function as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is a finely divided solid which is admixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

In particular, liquid formulations include solutions, suspensions and emulsions, for example, aqueous solutions or water-propylene glycol solutions. For example, parenteral injection liquid preparations may be formulated as a solution of water-polyethylene glycol.

Thus, the medicaments for use in the present invention may be formulated together into a formulation for parenteral administration (e.g. injection, such as bolus injection or continuous infusion) and may be presented in unit dose form with added preservative in ampoules, pre-filled syringes, small volume infusion bags or in multi-dose containers. The compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in the form of a powder, obtainable from sterile solid sterile isolation or from solution lyophilization for reconstitution with a suitable carrier such as sterile, pyrogen-free water just prior to use.

Aqueous solutions suitable for oral administration may be prepared by dissolving the active ingredient in water and adding the desired colorants, flavors, stabilizers, and thickeners.

Aqueous suspensions suitable for oral administration can be prepared by dispersing the finely divided active ingredient in water with viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.

The pharmaceutical formulations of the present invention also include solid formulations designed to be converted to liquid formulations for oral administration shortly before use. Such liquid formulations include solutions, suspensions and emulsions. Such formulations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersing agents, thickeners, solubilizing agents, and the like.

Respiratory administration of the pharmaceutical formulations of the present invention may also be achieved by aerosol formulations wherein the active ingredient is contained in a pressurized package with a suitable propellant, including chlorofluorocarbons (CFCs) such as dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gases. The aerosol formulation may also suitably contain a surfactant, such as lecithin. The dosage of the drug may be controlled by a throughput valve.

Alternatively the active ingredient may be in the form of a dry powder, for example a powder mixture of the compound with a suitable powder base such as lactose, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). The powder carrier may conveniently form a gel within the nasal cavity. The powder composition may be present in unit dosage form, for example in a capsule or cartridge (such as a gelatin gum or cartridge) or in a blister pack where the powder may be administered via an inhaler.

Alternatively, compositions suitable for sustained release of the active ingredient may be employed, if desired.

In the therapeutic use of the pharmaceutical formulation, the daily dosage of the compound is in accordance with conventional dosages. These dosages may vary depending on the patient's needs, the severity of the condition being treated and the compound being used, and generally treatment will begin with a smaller dosage than the optimum dosage of the compound, after which the dosage is increased by a small amount to achieve the optimum effect, and the total daily dosage may be subdivided for administration in portions of the day if desired, for convenience.

The invention designs and develops the inhibitor with novel and unique structure aiming at the E3 ubiquitin ligase substrate recognition protein ZYG B or ZER1, can effectively combine or inhibit the E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B, regulate and control the related key protein and further play a role in diagnosing, preventing and treating related diseases. Meanwhile, the compound developed by the invention can form a compound with CLR2-ZYG B or CLR2-ZER1, becomes a part of a PROTAC molecule or a molecular gel molecule with a target protein degradation function for recruiting E3 enzyme, and has great significance for developing novel medicines.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,

FIG. 1 is a graph showing the results of the measurement of the binding capacity of a compound of the invention to ZER1 protein-isothermal calorimetric titration (ITC);

FIG. 2 is a graph showing the results of the binding capacity of a compound of the invention to ZYG B protein-isothermal calorimetric titration (ITC);

FIG. 3 shows the results of a competition immunoblotting experiment of the compound of the present invention against a substrate in HEK293T-GAGN cells;

FIG. 4 is a graph showing the effect of the compounds of the present invention on the stability of a substrate protein in HEK293T-GFWC cells;

FIG. 5 is a mass spectrometry result of covalent binding of a compound of the invention to ZYG B protein;

FIG. 6 shows the results of immunoblotting experiments of the degradation activity of representative compounds of the present invention on target proteins in tumor cells MKN 45;

FIG. 7 is a graph showing the results of inhibition of MKN45 tumor cells by representative compounds of the present invention; wherein (a) - (e) represent the growth inhibitory effect of compounds 79, 80, 81, 82, 83, respectively, on tumor cell MKN 45;

FIG. 8 shows the results of a study of the mechanism of degradation of a target protein by a representative compound of the present invention.

Detailed Description

The invention is further described below with reference to examples and figures of examples. These examples are merely for more detailed description and should not be construed as limiting the invention in any way. The invention may be embodied in a number of different forms, which are defined and covered by the claims.

Although many materials and methods of operation are known in the art, the invention is still described in as much detail herein. Hereinafter, the materials used and the methods of operation are well known in the art, unless otherwise indicated. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The synthesis of the target compound adopts a method shown in a reaction flow chart. The product undergoes nuclear magnetic resonance, mass spectrometry and liquid chromatography to confirm the structure and purity. The starting materials used in the preparation of the compounds of the present invention are commercially available or may be prepared according to known methods described in the art or herein unless otherwise specified.

In the following examples of the present invention, the general synthetic procedures for the compounds involved may be used to prepare the compounds of the present disclosure in view of the present disclosure or by the illustrative methods shown in the following general schemes, using methods known to those skilled in the art. If desired, in any of the general schemes, suitable protecting groups may be used in the synthesis. It should be understood that the embodiments and examples are not intended to limit the scope of the present disclosure, and that the claims presented herein are intended to cover all embodiments and examples, whether or not explicitly presented herein. The embodiments of the present invention should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.

EXAMPLE 1 Synthesis of Compound 1

Synthesis of Compounds 1-3

In a 250mL single vial, compound SM1 (5.0 g,23.2 mmol), DMSO (50 mL), KOH (1.69 g,30.2 mmol) and iodoisopropyl (5.13 g,30.2 mmol) were added sequentially. The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (200 mL), extracted five times with EtOAc (500 mL), concentrated to dryness, and the residue was purified by chromatography on a column with an eluent ratio PE/EtOAc/dcm=5/1/1 to 1/1//1 to give compound 1-3 (1.5 g, yellow oil, purity 95%). The yield was calculated: 23.65%.

LCMS(ESI)m/z calcd.for C₁₃H₂₄N₂O₃[M+H]⁺257.2;found 257.1;¹H NMR(400MHz,DMSO_d₆):δ＝6.80(d,J＝7.5Hz,1H),4.67-4.48(m,1H),3.87(s,1H),3.18-3.07(m,2H),1.97-1.84(m,1H),1.84-1.50(m,3H),1.38(s,9H),1.03(d,J＝6.8Hz,6H).

Synthesis of Compounds 1-4

In a 100mL single vial was added compound 1-3 (1.5 g,5.85 mmol) in sequence 4N HCl/1, 4-dioxane (30 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give crude compound 1-4 (1.1 g, yellow solid, purity 80.625%). Yield: 98.28%.

LCMS(ESI)m/z calcd.for C₈H₁₆N₂O[M+H]⁺157.1；found 157.1。

Synthesis of Compounds 1-7

In a 100mL single vial were added compound 1-4 (1.5 g,9.60 mmol), DCM (30 mL), boc-D-serine (1.98 g,9.60 mmol), EDCI (2.76 g 14.4 mmol), HOBt (1.95 g,14.4 mmol) and TEA (3.89 g,38.4 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, concentrated to dryness, and the residue was purified by chromatography with eluent ratio DCM/meoh=100/1 to 20/1 to give compound 1-7 (600 mg, white solid, purity 79.255%). Yield: 14.58%.

LCMS(ESI)m/z calcd.for C₁₆H₂₉N₃O₅[M+H]⁺344.2;found 344.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.94(d,J＝6.3Hz,1H),6.69-6.50(m,1H),4.89-4.71(m,1H),4.66-4.55(m,1H),4.25-4.09(m,1H),3.98(s,1H),3.31(s,3H),3.17(t,J＝5.2Hz,2H),2.10-1.95(m,1H),1.76(d,J＝5.1Hz,2H),1.38(s,9H),1.04(t,J＝5.8Hz,6H).

Synthesis of Compound 1

Compounds 1-7 (450 mg,1.75 mmol) in 4N HCl/methanol (15 mL) were added sequentially in a 25mL single-port bottle. After the reaction was completed at 25℃for 2 hours, the reaction mixture was concentrated to dryness to give compound 1 (167 mg, yellow solid, purity 95%). The yield was calculated: 48.91%.

LCMS(ESI)m/z calcd.for C₁₁H₂₂ClN₃O₃[M+H]⁺244.2;found 244.2;¹H NMR(400MHz,DMSO_d₆):δ＝8.67(d,J＝6.9Hz,2H),8.21(s,2H),4.69-4.52(m,1H),4.30-4.12(m,1H),3.90-3.61(m,3H),3.13(d,J＝44.0Hz,2H),2.11-1.92(m,1H),1.79(d,J＝5.5Hz,2H),1.59(s,1H),1.07(s,6H).

EXAMPLE 2 Synthesis of Compound 2

Synthesis of Compound 2-3

To a 100mL three-necked flask, compound SM1 (5 g,35.7 mmol), potassium carbonate (14.8 g,107.1 mmol), bromoisopropyl (6.59 g,53.6 mmol) and DMF (50 mL) were sequentially added. The reaction was carried out at 60℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (200 mL), and extracted three times with EA (200 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatography with an eluent ratio of PE/ea=5/1 to 1/1 to give compound 2-3 (1.8 g, yellow solid, purity 99.9%). The yield was calculated: 27.73%.

LCMS(ESI)m/z calcd.for C₃₃H₃₃FN₄O₅[M+H]⁺183.2;found 183.0;¹H NMR(400MHz,CDCl₃):δ＝8.34(d,J＝7.6Hz,1H),8.25(d,J＝6.7Hz,1H),6.47(t,J＝7.2Hz,1H),5.12(dt,J＝13.6,6.8Hz,1H),1.34(d,J＝6.8Hz,6H).

Synthesis of Compounds 2-4

To a 100mL three-necked flask were added 2-3 (1.8 g,9.88 mmol), pd/C (200 mg, 10%) and MeOH (30 mL). The reaction was carried out at 20℃for 2 hours under a hydrogen pressure of 1 atm. The reaction solution was filtered and concentrated to dryness to give crude compound 2-4 (1.5 g, yellow solid, 98.691% purity). The yield was calculated: 97.98%.

LCMS(ESI)m/z calcd.for C₃₃H₃₁FN₄O₅[M+H]⁺153.2;found 153.1;¹H NMR(400MHz,CDCl₃):δ＝6.91(dd,J＝7.0,1.5Hz,1H),6.40(dd,J＝7.0,1.6Hz,1H),6.08(t,J＝7.0Hz,1H),5.16-5.01(m,3H),1.26(d,J＝6.8Hz,6H).

Synthesis of Compounds 2-5

In a 50mL three-necked flask, 2-4 (500 mg,2.42 mmol), DMF (10 mL), HATU (1.38 g,3.64 mmol) and DIEA (627 mg,4.85 mmol) were added sequentially. The reaction was carried out at 20℃for 16 hours. After the completion of the reaction, the reaction mixture was poured into water (50 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and the residue was purified by silica gel chromatography with an eluent ratio of PE/ea=5/1 to 1/1 to give compound 2-5 (240 mg, yellow solid, purity 97.648%). Yield: 28.40%.

LCMS(ESI)m/z calcd.for C₁₁H₂₀N₂O₃[M+H]⁺340.4;found 340.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.35(s,1H),8.30(d,J＝7.3Hz,1H),7.07(d,J＝7.0Hz,1H),6.27(t,J＝7.2Hz,1H),5.61(br.s,1H),5.34-5.20(m,1H),4.41(br.s,1H),4.17(d,J＝11.6Hz,1H),3.79(dd,J＝11.0,5.0Hz,1H),1.48(s,9H),1.37(d,J＝6.8Hz,6H).

Synthesis of Compound 2

To a 50mL three-necked flask, compound 2-5 (100 mg,0.29 mmol) and HCl (g)/1, 4-dioxane (5 mL, 4N) were sequentially added. The reaction was carried out at 20℃for 4 hours. The reaction solution is concentrated to dryness to obtain crude products. The crude product was slurried with methyl tertiary ether to give compound 2 (50 mg, yellow solid, 96.293% purity). Yield: 59.43%.

LCMS(ESI)m/z calcd.for C₂₄H₃₃N₅O₅[M+H]⁺240.3;found 239.9;¹H NMR(400MHz,CDCl₃):δ＝9.94(s,1H),8.29(br.s,3H),8.20(d,J＝7.3Hz,1H),7.52(d,J＝7.0Hz,1H),6.35(t,J＝7.1Hz,1H),5.17-5.05(m,1H),4.29(s,1H),3.85-3.74(m,2H),3.49(br.s,1H),1.32(d,J＝6.7Hz,6H).

In various embodiments of the present invention, compounds 3-5 may be synthesized in a similar manner as described above.

EXAMPLE 3 Synthesis of Compound 6

Synthesis of Compound 6-1

A100 mL single vial was charged with compound SM1 (4.9 g,0.0224 mol), pinacol vinylborate (10.35g,0.0672mol)、K₂CO₃(9.29g,0.0672mol)、Pd(PPh₃)₄(2.59g,0.0022mol), and 1,4-dioxane/H ₂ O=10/1 (80 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (250 mL), and extracted three times with EA (100 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by chromatography on a silica gel column with eluent ratio DCM/meoh=80/1 to 10/1 to give compound 6-1 (1.7 g, brown oil, 53.145% purity). Yield: 24.11%.

LCMS(ESI)m/z calcd.for C₇H₆N₂O₃[M-H]⁺165.04；found 165.1。

Synthesis of Compound 6-2

To a 50mL single vial was added, in order, compound 6-1 (800 mg,4.82 mmol), 2-iodopropane (1637 mg,9.63 mmol), K ₂CO₃ (1331 mg,9.63 mmol), and DMF (10 mL). The reaction was carried out at 60℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (50 mL), and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with eluent ratio PE/etoac=20/1 to 5/1 to give compound 6-2 (200 mg, brown oil, 92.852% purity). Yield: 18.52%.

LCMS(ESI)m/z calcd.for C₁₀H₁₂N₂O₃[M+H]⁺209.08;found 209.1;¹H NMR(400MHz,DMSO_d6):δ＝8.61(s,1H),8.28(s,1H),6.63(dd,J＝17.7,11.0Hz,1H),5.23(d,J＝11.1Hz,1H),5.16-5.06(m,1H),1.36(d,J＝6.8Hz,6H),1.19-1.10(m,1H).

Synthesis of Compound 6-3

To a 25mL single vial was added compound 6-2 (200 mg,0.96 mmol), pd/C (30 mg) and MeOH (6 mL) in sequence. The reaction was carried out at 25℃for 16 hours under the protection of hydrogen. After the reaction was completed, it was concentrated to dryness to give crude compound 6-3 (180 mg, brown solid, purity 85.898%). Yield: 89.30%.

LCMS(ESI)m/z calcd.for C₁₀H₁₆N₂O[M+H]⁺181.13;found 181.2;¹H NMR(400MHz,DMSO_d6):δ＝6.71(s,1H),6.33(s,1H),5.10-5.02(m,2H),2.33-2.25(m,2H),1.25(d,J＝6.8Hz,6H),1.23(s,1H),1.10-1.05(m,3H).

Synthesis of Compound 6-4

A25 mL single vial was charged with compound 6-3 (180 mg,1.00 mmol), boc-L-serine (247 mg,1.20 mmol), HATU (570 mg,1.50 mmol), DIEA (387 mg,3.00 mmol) and DMF (4 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developing reagent DCM/meoh=14/1 to give compound 6-4 (30 mg, yellow solid, 96.908% purity). Yield: 7.90%.

LCMS(ESI)m/z calcd.for C₁₈H₂₉N₃O₅[M+H]⁺368.21；found 368.3。

Synthesis of Compound 6

A25 mL single vial was charged with compound 6-4 (30 mg,0.0814 mmol) and 4N HCl/1, 4-dioxane (6 mL) in sequence. The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, it was concentrated to dryness to give Compound 6 (19.6 mg, yellow solid, purity 95.421%). Yield: 62.78%.

LCMS(ESI)m/z calcd.for C₁₃H₂₁N₃O₃[M+H]⁺268.16;found268.2;1H NMR(400MHz,DMSO_d6):δ＝9.94(s,1H),8.31(s,3H),8.17(d,J＝2.0Hz,1H),7.32(s,1H),5.61(br.s,1H),5.15-5.04(m,1H),4.29(s,1H),3.86-3.72(m,2H),2.42(q,J＝7.5Hz,2H),1.32(d,J＝6.4Hz,6H),1.12(t,J＝7.5Hz,3H).

EXAMPLE 4 Synthesis of Compound 7

Synthesis of Compound 7-1

To a 250mL single vial was added compound SM1 (10 g,80.6 mmol), pivCl (10.69 g,88.7 mmol), TEA (9.79 g,96.7 mmol) and DCM (150 mL) in this order. The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was poured into an aqueous sodium hydrogencarbonate solution (200 mL) and washed, and the organic phase was separated and concentrated to dryness to give compound 7-1 (17 g, brown oil, purity 97.828%). Yield: 99.13%.

LCMS(ESI)m/z calcd.for C₁₁H₁₆N₂O₂[M+H]⁺209.12;found 209.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.48(s,1H),8.11(d,J＝7.7Hz,1H),7.91(dd,J＝5.0,1.7Hz,1H),7.01-6.90(m,1H),3.92(s,3H),1.22(s,9H).

Synthesis of Compound 7-2

To a 250mL three-necked flask, compound 7-1 (10 g,0.048 mol), TMEDA (5.58 g,0.048 mol) and THF (250 mL) were sequentially added, and after the reaction was cooled to-78℃n-Buli (1045 g,0.1632 mol) was added dropwise, the reaction was warmed to 0℃for 1 hour, and then benzaldehyde (15.28 g,0.144 mol) was added dropwise while continuing to cool to-78 ℃. Naturally heating to 25 ℃ under the protection of nitrogen, and reacting for 16 hours. After the reaction was completed, the reaction mixture was poured into water (200 mL), extracted five times with EtOAc (500 mL), the reaction mixture was concentrated to dryness, and the residue was purified by chromatography on a column with an eluent ratio of PE/etoac=30/1 to 2//1 to give compound 7-2 (1.6 g, yellow oil, purity 62.099%). Yield: 33.33%.

LCMS(ESI)m/z calcd.for C₁₈H₂₂N₂O₃[M+H]⁺315.16;found 315.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.69(s,1H),7.31-7.28(m,3H),7.22-7.19(m,2H),7.12(d,J＝5.3Hz,1H),5.93(d,J＝4.6Hz,1H),5.76(d,J＝5.9Hz,2H),3.79(s,3H),1.18(s,9H).

Synthesis of Compound 7-3

To a 100mL single vial was added compound 7-2 (1.6 g,0.0051 mol), pd/C (0.5 g, 10%) and MeOH (20 mL) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, filtered, concentrated to dryness, and the residue was purified by chromatography with an eluent ratio of PE/etoac=15/1 to 1//1 to give compound 7-3 (700 mg, brown oil, purity 84.537%). Yield: 39.22%.

LCMS(ESI)m/z calcd.for C₁₈H₂₂N₂O₂[M+H]⁺299.17;found 299.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.84(s,1H),7.92(d,J＝5.2Hz,1H),7.28(t,J＝7.3Hz,2H),7.19(t,J＝8.3Hz,3H),6.69(d,J＝5.2Hz,1H),3.82(s,3H),3.80(s,2H),1.21(s,9H).

Synthesis of Compound 7-4

A100 mL single-port flask was charged with compound 7-3 (700 mg,2.35 mmol), sodium ethanethiol (1010 mg,11.73 mmol), and DMF (10 mL). The reaction was carried out at 80℃for 16 hours under nitrogen protection. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (50 mL), extracted eight times with DCM (160 mL) and concentrated to dryness to give compound 7-4 (500 mg, brown oil, 88.451% purity). Yield: 66.30%.

LCMS(ESI)m/z calcd.for C₁₇H₂₀N₂O₂[M+H]⁺285.15；found 285.1。

Synthesis of Compound 7-5

To a 100mL single-port flask were added, in order, compound 7-4 (500 mg,1.76 mmol), isopropyl iodide (747 mg,4.40 mmol), K ₂CO₃ (4816 mg,3.52 mmol), and DMF (10 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (50 mL), extracted five times with EtOAc (150 mL), concentrated to dryness, and the residue was purified by chromatography on a column with an eluent ratio PE/etoac=5/1 to 1//1 to give compound 7-5 (500 mg, yellow oil, purity 82.792%). Yield: 28.85%.

LCMS(ESI)m/z calcd.for C₂₀H₂₆N₂O₂[M+H]⁺327.20;found 327.1;¹H NMR(400MHz,CDCl₃):δ＝7.76(s,1H),7.29(d,J＝6.9Hz,2H),7.25-7.20(m,1H),7.15(d,J＝7.0Hz,2H),7.05(d,J＝7.3Hz,1H),5.96(d,J＝7.3Hz,1H),5.23-5.16(m,1H),3.85(s,2H),1.35-1.33(m,6H),1.33(s,9H).

Synthesis of Compound 7-6

To a 25mL single vial was added compound 7-5 (200 mg,0.613 mmol) followed by EtOH/HCl=2/1 (3 mL). The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and concentrated to dryness to give compound 7-6 (180 mg, brown oil, purity 81.119%). Yield: 98.35%.

LCMS(ESI)m/z calcd.for C₁₅H₁₈N₂O[M+H]⁺243.14;found 243.1;¹H NMR(400MHz,CDCl₃):δ＝7.29(d,J＝7.5Hz,2H),7.26-7.18(m,4H),6.81(d,J＝7.2Hz,1H),6.00(d,J＝7.2Hz,1H),5.32-5.26(m,1H),3.83(s,2H),3.79-3.65(m,1H),1.35(d,J＝6.7Hz,6H).

Synthesis of Compound 7-7

A100 mL single vial was charged with compound 7-6 (180 mg,0.84 mol), boc-L-serine (184 mg,0.89 mol), HATU (424 mg,1.11 mol), DIEA (288 mg,2.23 mmol), and DMF (2 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (10 mL), extracted five times with EtOAc (25 mL), filtered, concentrated to dryness, and the residue was prepared by hplc with the following parameters: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% fa); gradient: 10-55/8 min, compound 7-7 (20 mg, yellow solid, 95.981% purity) was obtained. Yield: 6.00%.

LCMS(ESI)m/z calcd.for C₂₃H₃₁N3O₅[M+H]⁺430.23;found 430.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.20(s,1H),7.53(d,J＝7.3Hz,1H),7.31-7.19(m,5H),6.73(d,J＝8.0Hz,1H),5.03-4.91(m,2H),4.23(d,J＝7.0Hz,1H),3.73-3.61(m,4H),1.38(s,9H),1.25(d,J＝6.8Hz,6H).

Synthesis of Compound 7

To a 100mL single vial was added compound 7-7 (20 mg,0.0465 mmol) in sequence 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, the reaction was concentrated to dryness to give compound 7 (11.8 mg, yellow solid, purity 90.799%). Yield: 57.20%.

LCMS(ESI)m/z calcd.for C₁₈H₂₃N₃O₃[M+H]⁺330.17;found 330.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.83(s,1H),8.24(s,3H),7.60(d,J＝7.3Hz,1H),7.34-7.20(m,6H),6.00(d,J＝7.3Hz,1H),5.04-4.93(m,1H),4.10(s,1H),3.93-3.87(m,2H),3.75-3.71(m,2H),1.27(dd,J＝6.6,2.5Hz,6H).

In various embodiments of the present invention, compounds 8-13 may be synthesized in a similar manner as described above.

EXAMPLE 5 Synthesis of Compound 14

Synthesis of Compound 14-2

A500 mL single vial was charged with Compound H ₂SO₄ (250 mL), 14-1 (25 g,115.7 mmol), and NIS (46.85 g,208.2 mmol) in sequence. The reaction was carried out at 0℃for 2 hours under nitrogen protection. After the reaction was completed, the mixture was poured into ice, and extracted three times with EA (500 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. Compound 14-2 (30 g, brown oil, 85.039% purity) was obtained. Yield: 64.48%.

¹H NMR(400MHz,DMSO_d₆):δ＝8.38(d,J＝1.7Hz,1H),8.14(d,J＝1.7Hz,1H),2.42(s,3H)。

Synthesis of Compound 14-3

A1L single vial was charged with compound 14-2 (22 g,64.3 mmol), i-PrOH: H ₂O＝3:1(300mL)、NH₄ Cl (13.76 g,257.2 mmol), and Fe (14.36 g,257.2 mmol) in sequence. The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (900 mL), and extracted three times with EA (500 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio PE/etoac=80/1 to 30/1 to give compound 14-3 (11 g, brown oil, 94.300% purity). Yield: 51.79%.

LCMS(ESI)m/z calcd.for C₇H₇BrIN[M+H]⁺311.88;found 312.0;¹H NMR(400MHz,DMSO_d₆):δ＝7.12(d,J＝1.5Hz,1H),6.82(d,J＝1.4Hz,1H),5.44(s,2H),2.16(s,3H).

Synthesis of Compound 14-4

A250 mL single port flask was charged with compound 14-3 (11 g,0.0353 mol), ACN (120 mL), tributyl (1-ethoxyethylene) tin (19.12 g,52.9 mmol), cuI (0.67 g,3.5 mmol) and Pd (PPh ₃)₂Cl₂ (2.48 g,3.5 mmol) were reacted at 80℃for 16 hours under nitrogen protection, after the reaction was completed, concentrated to dryness, the residue was purified by silica gel chromatography column with eluent ratio PE/EtOAc=80/1 to 20/1 to give compound 14-4 (4.3 g, brown oil, purity 96.64%). Yield 45.89%.

LCMS(ESI)m/z calcd.for C₁₁H₁₄BrNO[M+H]⁺256.03；found 256.0。

Synthesis of Compound 14-5

A100 mL single vial was charged with compound 14-4 (3 g,11.7 mmol) followed by 1N HCl (30 mL). The reaction was carried out at 25℃for 1 hour under nitrogen. After the reaction was completed, the mixture was poured into water (100 mL) and extracted three times with EA (80 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with eluent ratio PE/etoac=40/1 to 10/1 to give compound 14-5 (1.2 g, yellow solid, purity 98.595%). Yield: 44.44%.

LCMS(ESI)m/z calcd.for C₉H₁₀BrNO[M+H]⁺227.99;found 228.0;¹H NMR(400MHz,DMSO_d₆):δ＝6.93(d,J＝2.0Hz,2H),5.39(s,2H),2.46(s,3H),1.98(s,3H).

Synthesis of Compound 14-6

To a 25mL single port flask were added, in order, compound 14-5 (400 mg,1.75 mmol), 1, 4-dioxane:H2O=10:1 (10 mL), pyridine-3-boronic acid (431 mg,3.51 mmol), K ₂CO₃ (727 mg,5.26 mmol) and Pd (PPh ₃)₄ (203 mg,0.175 mmol) reacted at 100℃for 16 hours under nitrogen, after completion of the reaction, poured into water (30 mL), extracted three times with EA (20 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, the residue was purified by prep. plates with a ratio of developing solvent DCM/MeOH=14/1 to give compound 14-6 (300 mg, yellow solid, purity 93.659%). Yield: 70.83%.

LCMS(ESI)m/z calcd.for C₁₄H₁₄N₂O[M+H]⁺227.11;found 227.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.84(d,J＝1.6Hz,1H),8.56(d,J＝3.9Hz,1H),8.00(d,J＝7.9Hz,1H),7.47(dd,J＝7.8,4.8Hz,1H),7.18(s,1H),7.10(s,1H),5.25(s,2H),2.56(s,3H),2.11(s,3H).

Synthesis of Compound 14-7

To a 25mL single vial was added compound 14-6 (300 mg,1.33 mmol), DMF (6 mL), boc-L-serine (328 mg,1.59 mmol), HATU (7516 mg,1.99 mmol) and DIEA (514 mg,3.98 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (30 mL), and extracted three times with EA (25 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=14/1 to give compound 14-7 (60 mg, yellow solid, 99.635% purity). Yield: 10.88%.

LCMS(ESI)m/z calcd.forC₂₂H₂₇N₃O₅[M+H]⁺414.2;found 414.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.59(s,1H),8.91(s,1H),8.60(d,J＝3.8Hz,1H),8.09(d,J＝7.9Hz,1H),7.86(d,J＝29.3Hz,2H),7.52(dd,J＝7.7,4.8Hz,1H),6.82(d,J＝7.5Hz,1H),5.02(t,J＝5.6Hz,1H),4.21(d,J＝6.9Hz,1H),3.70(t,J＝5.5Hz,2H),2.64(s,3H),2.23(s,3H),1.41(s,9H).

Synthesis of Compound 14

A25 mL single vial was charged with compound 14-7 (60 mg,0.145 mmol) followed by 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, the reaction was concentrated to dryness to give compound 14 (41.45 mg, yellow solid, purity 95.750%). Yield: 78.38%.

LCMS(ESI)m/z calcd.for C₁₇H₁₉N₃O₃[M+H]⁺314.14;found314.2;1H NMR(400MHz,DMSO_d₆):δ＝10.42(s,1H),9.15(s,1H),8.80(d,J＝5.0Hz,1H),8.55(d,J＝7.5Hz,1H),8.35(s,3H),8.06(s,1H),7.98-7.87(m,2H),4.18(d,J＝4.5Hz,1H),3.99-3.90(m,2H),2.67(s,3H),2.29(s,3H).

EXAMPLE 6 Synthesis of Compound 15

Synthesis of Compound 15-1

To a 25mL single vial was added compound 14-5 (300 mg,1.32 mmol), pyridine-4-boronic acid (323 mg,2.63 mmol), K ₂CO₃(545mg,3.95mmol)、Pd(PPh₃)₄ (152 mg,0.13 mmol), and 1, 4-dioxane:H2O=10:1 (6 mL) in sequence. The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the mixture was poured into water (20 mL) and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developing reagent DCM/meoh=14/1 to give compound 15-1 (180 mg, yellow solid, 67.789% purity). Yield: 41.00%.

LCMS(ESI)m/z calcd.for C₁₄H₁₄N₂O[M+H]⁺227.11;found 227.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.62(d,J＝4.9Hz,2H),7.63(d,J＝5.1Hz,2H),7.24(s,1H),7.17(s,1H),5.29(s,2H),2.56(s,3H),2.10(s,3H).

Synthesis of Compound 15-2

To a 25mL single vial was added compound 15-1 (180 mg,0.80 mmol), boc-L-serine (197mg, 0.95 mmol), HATU (454 mg,1.19 mmol), DIEA (308 mg,2.39 mmol) and DMF (4 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developing reagent DCM/meoh=14/1 to give compound 15-2 (30 mg, yellow solid, 97.222% purity). Yield: 8.85%.

LCMS(ESI)m/z calcd.forC₂₂H₂₇N₃O₅[M+H]⁺414.2;found 414.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.61(s,1H),8.66(d,J＝4.5Hz,2H),7.95(s,1H),7.90(s,1H),7.73(d,J＝4.4Hz,2H),6.83(d,J＝7.5Hz,1H),5.03(t,J＝5.6Hz,1H),4.21(d,J＝6.7Hz,1H),3.70(t,J＝5.4Hz,2H),2.64(s,3H),2.24(s,3H),1.41(s,9H).

Synthesis of Compound 15

A25 mL single vial was charged with compound 15-2 (30 mg,0.0724 mmol) followed by 4N HCl/1, 4-dioxane (4 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give crude compound 15 (9.91 mg, yellow solid, purity 95.603%). Yield: 41.71%.

LCMS(ESI)m/z calcd.for C₁₇H₁₉N₃O₃[M+H]⁺314.14;found314.2;1H NMR(400MHz,DMSO_d₆):δ＝10.60(s,1H),8.97(d,J＝6.3Hz,2H),8.41(s,3H),8.32(d,J＝5.9Hz,2H),8.19(s,1H),8.10(s,1H),4.22(d,J＝4.2Hz,1H),4.05-3.89(m,2H),2.69(s,3H),2.33(s,3H).

EXAMPLE 7 Synthesis of Compound 16

Synthesis of Compound 16-1

A250 mL single vial was charged with compound SM1 (10 g,0.068 mol), 2-bromopropane (16.7 g,0.136 mol), K ₂CO₃ (18.8 g,0.136 mol) and DMF (100 mL) in this order. The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (500 mL), and extracted three times with EA (400 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/etoac=50/1 to 10/1 to give compound 16-1 (9 g, red solid, 99.607% purity). Yield: 69.71%.

LCMS(ESI)m/z calcd.for C₁₁H₁₁NO₂[M-H]⁺190.08;found 190.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.64(t,J＝7.8Hz,1H),7.55(d,J＝7.4Hz,1H),7.30(d,J＝8.0Hz,1H),7.11(t,J＝7.5Hz,1H),4.49-4.39(m,1H),1.43(d,J＝6.9Hz,6H).

Synthesis of Compound 16-2

A100 mL single vial was charged with compound 16-1 (3 g,0.0159 mol), NH ₂ OH HCl (2.54 g,0.0365 mol), naOAc (3.26 g,0.0397 mol) and EtOH: H2O=2:1 (60 mL). The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (180 mL), and extracted three times with EA (150 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/etoac=15/1 to 3/1 to give compound 16-2 (3 g, white solid, purity 97.533%). Yield: 89.94%.

LCMS(ESI)m/z calcd.for C₁₁H₁₂N₂O₂[M+H]⁺205.09;found 205.2;¹H NMR(400MHz,DMSO_d₆):δ＝13.39(s,1H),8.03(d,J＝7.1Hz,1H),7.42(t,J＝7.9,1.0Hz,1H),7.24(d,J＝8.0Hz,1H),7.08(t,J＝7.5Hz,1H),4.61-4.49(m,1H),1.43(d,J＝7.0Hz,6H).

Synthesis of Compound 16-3

A50 mL single vial was charged with compound 16-2 (1 g,4.90 mmol), pd/C (0.16 g, 10%) and MeOH (10 mL). The reaction was carried out at 25℃for 16 hours under 1atm hydrogen. After the reaction was completed, the mixture was concentrated to dryness to give crude compound 16-3 (700 mg, purple oil, purity 78.369%). Yield: 59.18%.

LCMS(ESI)m/z calcd.for C₁₁H₁₄N₂O[M+H]⁺191.11;found 191.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.36(d,J＝7.3Hz,1H),7.24(t,J＝7.6Hz,1H),7.11(d,J＝7.9Hz,1H),7.03-6.97(m,1H),4.53-4.43(m,1H),4.14(s,1H),2.33(br.s,1H),1.38(dd,J＝7.0,2.3Hz,6H).

Synthesis of Compound 16-4

To a 25mL single vial was added compound 16-3 (300 mg,1.58 mmol), boc-L-serine (390 mg,1.89 mmol), HATU (899 mg,2.37 mmol), DIEA (611 mg,4.73 mmol) and DMF (6 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (30 mL), and extracted three times with EA (25 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=14/1 to give compound 16-4 (200 mg, yellow solid, 97.056% purity). Yield: 32.53%.

LCMS(ESI)m/z calcd.for C₁₉H₂₇N₃O₅[M+H]⁺378.20;found 322.2;1H NMR(400MHz,DMSO_d₆):δ＝8.74-8.56(m,1H),7.25(t,J＝7.6Hz,1H),7.13(d,J＝7.8Hz,2H),6.97(t,J＝7.4Hz,1H),6.65(d,J＝5.4Hz,1H),5.14(dd,J＝26.1,8.1Hz,1H),4.83(t,J＝5.1Hz,1H),4.60-4.40(m,1H),4.09-3.86(m,1H),3.67-3.42(m,2H),1.40-1.38(m,15H).

Synthesis of Compound 16

A25 mL single vial was charged with compound 16-4 (60 mg,0.16 mmol) followed by 4N HCl/1, 4-dioxane (3 mL). After the reaction was completed at 25℃for 2 hours, the mixture was concentrated to dryness to give compound 16 (41.27 mg, yellow solid, purity 97.998%). Yield: 91.99%.

LCMS(ESI)m/z calcd.for C₁₄H₁₉N₃O₃[M+H]⁺278.14;found278.1;1H NMR(400MHz,DMSO_d₆):δ＝9.36(dd,J＝37.1,7.8Hz,1H),8.26(d,J＝13.8Hz,3H),7.33-7.20(m,2H),7.16(t,J＝8.0Hz,1H),7.07-6.93(m,1H),5.15(dd,J＝73.6,7.7Hz,1H),4.59-4.46(m,1H),3.93-3.79(m,2H),3.76-3.68(m,1H),3.39(br.s,1H),1.41(d,J＝6.9Hz,6H).

In various embodiments of the present invention, compounds 17-18 may be synthesized in a similar manner as described above.

EXAMPLE 8 Synthesis of Compound 19

Synthesis of Compound 19-1

To a 250mL single vial was added compound SM1 (6.3 g,0.0232 mol), 2-aminobenzaldehyde (17.19 g,0.0302 mol) and AcOH/H2O=1/1 (50 mL) in order. The reaction was carried out at 70℃for 16 hours under nitrogen. After the completion of the reaction, the reaction solution was cooled to room temperature, directly filtered and dried by spin to give compound 19-1 (4.5 g, yellow solid, purity 53.651%). Yield: 26.85%.

LCMS(ESI)m/z calcd.for C₉H₆N₂O₃[M+H]⁺191.04；found 191.0。

Synthesis of Compound 19-2

A100 mL single-port flask was charged with compound 19-1 (4.5 g,0.0237 mol), isopropyl iodide (10.07 g,0.0593 mol), K ₂CO₃ (4.91 g,0.0356 mmol), and DMF (50 mL) in this order. The reaction was carried out at 60℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (200 mL), extracted five times with EtOAc (500 mL), concentrated to dryness, and the residue was purified by chromatography on a column with an eluent ratio of PE/etoac=15/1 to 1//1 to give compound 19-2 (0.3 g, yellow oil, purity 73.485%). Yield: 3.80%.

LCMS(ESI)m/z calcd.for C₁₂H₁₂N₂O₃[M+H]⁺233.08;found 233.0;¹H NMR(400MHz,DMSO_d₆):δ＝8.84(s,1H),7.92(dd,J＝21.1,8.4Hz,2H),7.83-7.76(m,1H),7.40(t,J＝7.4Hz,1H),5.48-5.32(m,1H),1.58(d,J＝6.9Hz,6H).

Synthesis of Compound 19-3

To a 100mL single vial was added compound 19-2 (300 mg,1.29 mmol), pd/C (41 mg), and MeOH (10 mL) in order. The reaction was carried out at 25℃for 16 hours under a hydrogen pressure of 1 atm. After the completion of the reaction, the reaction mixture was filtered and concentrated to dryness to give compound 19-3 (170 mg, brown oil, purity 78.828%). Yield: 51.29%.

LCMS(ESI)m/z calcd.for C₁₂H₁₄N₂O[M+H]⁺203.11;found 203.0;¹H NMR(400MHz,DMSO_d₆):δ＝7.61(d,J＝8.5Hz,1H),7.39(d,J＝7.7Hz,1H),7.26-7.18(m,1H),7.14-7.07(m,1H),6.72(s,1H),5.44(s,2H),4.09-3.97(m,1H),1.56(d,J＝6.9Hz,6H).

Synthesis of Compound 19-4

To a 100mL single vial was added compound 19-3 (170 mg,0.84 mmol), boc-L-serine (173 mg,0.84 mmol), HATU (639 mg,1.68 mmol), DIEA (326 mg,2.52 mmol), and DMF (2 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, concentrated to dryness, and the residue was purified by silica gel plate with a ratio of DCM/meoh=10/1 to give compound 19-4 (30 mg, yellow oil, purity 91.906%). Yield: 9.14%.

LCMS(ESI)m/z calcd.for C₂₀H₂₇N₃O₅[M+H]⁺390.20；found 390.1。

Synthesis of Compound 19

To a 100mL single vial was added sequentially compound 19-4 (30 mg,0.077 mmol) 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give crude compound 19 (2.9 mg, yellow solid, purity 94.001%). Yield: 18.36%.

LCMS(ESI)m/z calcd.for C₁₅H₁₉N₃O₃[M+H]⁺290.1;found 290.2;¹H NMR(400MHz,CD₃OD_d₆):δ＝8.67(s,1H),7.76(d,J＝8.7Hz,1H),7.63(d,J＝7.5Hz,1H),7.54(t,J＝7.9Hz,1H),7.28(t,J＝7.5Hz,1H),4.31-4.26(m,1H),4.15-4.07(m,1H),4.02-3.98(m,2H),3.89(s,1H),3.85(s,1H),1.66(d,J＝6.9Hz,6H).

EXAMPLE 9 Synthesis of Compound 20

Synthesis of Compound 20-1

A100 mL single-port flask was charged with compound SM1 (1.5 g,10.7 mmol), 3-bromomethylpyridine hydrobromide (3.25 g,12.8 mmol), K ₂CO₃ (3.7 g,26.7 mol) and DMF (30 mL) in this order. The reaction was carried out at 60℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (150 mL), and extracted three times with EA (100 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by chromatography on a silica gel column with eluent ratio DCM/meoh=80/1 to 30/1 to give compound 20-1 (1.3 g, yellow solid, purity 99.31%). Yield: 52.34%.

LCMS(ESI)m/z calcd.for C₁₁H₉N₃O₃[M-H]⁺232.06;found 232.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.65(s,1H),8.53(d,J＝4.5Hz,1H),8.51-8.40(m,2H),7.78(d,J＝7.8Hz,1H),7.41(dd,J＝7.7,4.8Hz,1H),6.53(t,J＝7.1Hz,1H),5.30(s,2H).

Synthesis of Compound 20-2

A50 mL single-necked flask was charged with compound 20-1 (500 mg,2.16 mmol), reduced iron powder (604 mg,10.81 mmol), NH ₄ Cl (578 mg,10.81 mmol) and EtOH/H ₂ O=3/1 (10 mL) in this order. The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated to dryness. The residue was purified by chromatography on a silica gel column with eluent ratio DCM/meoh=80/1 to 30/1 to give crude compound 20-2 (300 mg, brown solid, 99.147% purity). Yield: 68.35%.

LCMS(ESI)m/z calcd.for C₁₁H₁₁N₃O[M+H]⁺202.09;found202.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.67-8.46(m,2H),7.73(d,J＝7.8Hz,1H),7.50(s,1H),7.44-7.33(m,1H),7.24(s,1H),7.11(dd,J＝6.8,1.6Hz,1H),6.47(dd,J＝7.1,1.6Hz,1H),6.09(t,J＝7.0Hz,1H),5.14(s,2H).

Synthesis of Compound 20-3

To a 25mL single vial was added compound 20-2 (120 mg,0.60 mmol), boc-L-serine (148 mg,0.72 mmol), HATU (457 mg,1.19 mmol), DIEA (308 mg,2.39 mmol) and DMF (4 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developing reagent DCM/meoh=14/1 to give compound 20-3 (10 mg, yellow solid, 87.102% purity). Yield: 3.76%.

LCMS(ESI)m/z calcd.for C₁₉H₂₄N₄O₅[M+H]⁺389.18;found 389.3;1H NMR(400MHz,DMSO_d₆):δ＝9.32(s,1H),8.59(d,J＝1.4Hz,1H),8.49(dd,J＝4.7,1.4Hz,1H),8.24(dd,J＝7.4,1.5Hz,1H),7.70(d,J＝7.9Hz,1H),7.63(dd,J＝6.9,1.7Hz,1H),7.39-7.35(m,1H),6.34(t,J＝7.1Hz,1H),5.20(s,2H),5.02(t,J＝5.8Hz,1H),4.12(br.s,1H),3.65-3.62(m,2H),3.21-3.08(m,1H),1.39(s,9H).

Synthesis of Compound 20

A25 mL single vial was charged with compound 20-3 (10 mg,0.026 mmol) followed by 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, it was concentrated to dryness to give compound 20 (6.37 mg, yellow solid, purity 98.679%). Yield: 74.32%.

LCMS(ESI)m/z calcd.for C₁₄H₁₆N₄O₃[M+H]⁺289.12;found289.1;1H NMR(400MHz,DMSO_d₆):δ＝10.00(s,1H),8.87(s,1H),8.76(d,J＝5.0Hz,1H),8.33(s,3H),8.28-8.12(m,1H),7.92-7.70(m,2H),6.41(t,J＝7.1Hz,1H),5.33(s,2H),4.25(d,J＝4.5Hz,1H),3.82-3.74(m,2H).

EXAMPLE 10 Synthesis of Compound 21

Synthesis of Compound 21-1

A25 mL single vial was charged with compound 20-2 (120 mg,0.60 mmol), boc-L-alanine (136 mg,0.72 mmol), HATU (457 mg,1.19 mmol), DIEA (308 mg,2.39 mmol) and DMF (4 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=14/1 to give compound 21-1 (30 mg, yellow solid, 98.392% purity). Yield: 13.25%.

LCMS(ESI)m/z calcd.for C₁₉H₂₄N₄O₄[M+H]⁺373.18;found 373.2;1H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.60(d,J＝1.4Hz,1H),8.49(dd,J＝4.7,1.4Hz,1H),8.23(dd,J＝7.4,1.5Hz,1H),7.70(d,J＝7.9Hz,1H),7.63(dd,J＝6.9,1.7Hz,1H),7.47(d,J＝6.7Hz,1H),7.36(dd,J＝7.8,4.8Hz,1H),6.33(t,J＝7.1Hz,1H),5.25-5.16(m,2H),4.18-4.06(m,1H),1.38(s,9H),1.23(d,J＝7.1Hz,3H).

Synthesis of Compound 21

A25 mL single vial was charged with compound 21-1 (30 mg,0.080 mmol) followed by 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, it was concentrated to dryness to give compound 21 (12.85 mg, yellow solid, purity 98.938%). Yield: 51.31%.

LCMS(ESI)m/z calcd.for C₁₄H₁₆N₄O₂[M+H]⁺273.13;found273.2;1H NMR(400MHz,DMSO_d₆):δ＝10.01(s,1H),8.93(s,1H),8.82(d,J＝5.0Hz,1H),8.41(d,J＝6.1Hz,4H),8.24(dd,J＝7.4,1.4Hz,1H),7.95(dd,J＝7.9,5.6Hz,1H),7.82(dd,J＝6.8,1.5Hz,1H),6.41(t,J＝7.2Hz,1H),5.37(s,2H),4.32-4.21(m,1H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 12 Synthesis of Compound 22

Synthesis of Compound 22-1

To a 100mL single port flask were added, in order, compound SM1 (1.5 g,10.7 mmol), 2- (bromomethyl) pyridine hydrobromide (3.25 g,12.8 mmol), K ₂CO₃ (3.7 g,26.8 mmol), and DMF (10 mL). The reaction was carried out at 60℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (50 mL), extracted five times with EtOAc (150 mL), concentrated to dryness, and the residue was purified by chromatography on a column with eluent ratio DCM/meoh=80/1 to 30/1 to give compound 22-1 (1.8 g, yellow solid, purity 96.731%). Yield: 70.09%.

LCMS(ESI)m/z calcd.for C₁₁H₉N₃O₃[M+H]⁺232.06;found 232.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.53-8.42(m,2H),8.39-8.32(m,1H),7.86-7.76(m,1H),7.40(d,J＝7.8Hz,1H),7.35-7.28(m,1H),6.58-6.47(m,1H),5.35(s,2H).

Synthesis of Compound 22-2

To a 100mL single vial was added compound 22-1 (500 mg,2.16 mmol), fe (604 mg,10.81 mmol), NH ₄ Cl (578 mg,10.81 mmol), and IPA/H ₂ O=3/1 (15 mL) in order. The reaction was carried out at 80℃for 4 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and concentrated to dryness to give compound 22-2 (400 mg, black solid, purity 98.278%). Yield: 90.34%.

LCMS(ESI)m/z calcd.for C₁₁H₁₁N₃O[M+H]⁺202.09；found 202.1。

Synthesis of Compound 22-3

To a 100mL single vial was added compound 22-2 (200 mg,0.99 mmol), boc-L-serine (246 mg,1.19 mmol), HATU (756 mg,1.99 mmol), DIEA (514 mg,3.98 mmol) and DMF (5 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted five times with EA (50 mL), concentrated to dryness, and the residue was purified by chromatography column with eluent ratio DCM/meoh=80/1 to 30/1, crude product was isolated by high pressure preparation (column type: XBridge-1,5 μm, 19 to 150 mm; detector: 254nm; mobile phase: acetonitrile/water (0.1% aqueous ammonia solution), elution gradient: 20 to 53/9 min, 95 to 95/2 min, acetonitrile ratio in water; retention time: 9.0 min) to give compound 22-3 (29 mg, yellow solid, purity 92.44%). The yield was 4.86%.

LCMS(ESI)m/z calcd.for C₁₉H₂₄N₄O₅[M+H]⁺389.17;found 389.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.29(s,1H),8.55-8.41(m,1H),8.27(dd,J＝7.4,1.7Hz,1H),7.85-7.71(m,1H),7.52(dd,J＝6.9,1.7Hz,1H),7.34-7.24(m,1H),7.22(d,J＝7.9Hz,1H),7.14(d,J＝7.4Hz,1H),6.33(t,J＝7.1Hz,1H),5.33-5.19(m,2H),5.01(t,J＝5.8Hz,1H),4.17-4.06(m,1H),3.71-3.56(m,2H),1.34(s,9H).

Synthesis of Compound 22

To a 100mL single vial was added compound 22-3 (29 mg,0.0745 mmol) in sequence 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give crude compound 22 (24.57 mg, yellow solid, purity 95.316%). Yield: 77.18%.

LCMS(ESI)m/z calcd.for C₁₄H₁₆N₄O₃[M+H]⁺289.12;found 289.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.96(s,1H),8.75-8.64(m,1H),8.49-8.31(m,3H),8.31-8.25(m,1H),8.19-8.07(m,1H),7.74-7.67(m,1H),7.65-7.57(m,1H),7.53-7.44(m,1H),6.43-6.36(m,1H),5.50-5.37(m,2H),4.29-4.18(m,1H),3.87-3.71(m,2H).

EXAMPLE 13 Synthesis of Compound 23

Synthesis of Compound 23-1

To a 100mL single vial was added compound 22-2 (200 mg,0.99 mmol), N-Boc-L-alanine (227 mg,1.19 mmol), HATU (7516 mg,1.99 mmol), DIEA (514 mg,3.98 mmol) and DMF (5 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted five times with EA (50 mL), the reaction solution was concentrated to dryness, and the residue was separated by a preparative plate (dichloromethane/methanol=15/1) to give compound 23-1 (100 mg, yellow solid, purity 99.473%). Yield: 26.70%.

LCMS(ESI)m/z calcd.for C₁₉H₂₄N₄O₄[M+H]⁺373.18;found 373.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.18(s,1H),8.49(d,J＝4.6Hz,1H),8.28-8.21(m,1H),7.80-7.70(m,1H),7.56-7.48(m,1H),7.45(d,J＝6.7Hz,1H),7.33-7.25(m,1H),7.22(d,J＝7.8Hz,1H),6.32(t,J＝7.1Hz,1H),5.32-5.19(m,2H),4.19-4.07(m,1H),1.37(s,9H),1.23(d,J＝7.1Hz,3H).

Synthesis of Compound 23

To a 100mL single vial was added compound 23-1 (50 mg,0.13 mmol) 4N HCl/1, 4-dioxane (5 mL) in sequence. After the reaction was completed at 25℃for 2 hours, the mixture was concentrated to dryness to give compound 23 (33.51 mg, yellow solid, purity: 98.796%). Yield: 64.75%.

LCMS(ESI)m/z calcd.for C₁₄H₁₆N₄O₂[M+H]⁺273.13;found 272.9;¹H NMR(400MHz,DMSO_d₆):δ＝9.97(s,1H),8.67(d,J＝4.8Hz,1H),8.40(s,3H),8.26(dd,J＝7.4,1.4Hz,1H),8.09(t,J＝7.5Hz,1H),7.70(dd,J＝6.8,1.5Hz,1H),7.62-7.55(m,1H),7.46(d,J＝7.9Hz,1H),6.40(t,J＝7.1Hz,1H),5.43(s,2H),4.33-4.19(m,1H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 14 Synthesis of Compound 24

Synthesis of Compound 24-1

To a 100mL single vial was added compound 22-2 (250 mg,1.24 mmol), DMF (5 mL), boc-D-serine (284 mg,1.49 mmol), HATU (945 mg,2.49 mmol) and DIEA (640 mg,4.97 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), extracted five times with EA (50 mL), concentrated to dryness, and the residue was purified by prep. plate with a ratio of DCM/meoh=15/1 to give compound 24-1 (100 mg, yellow solid, purity 99.87%). The yield was 21.53%.

LCMS(ESI)m/z calcd.for C1₉H₂₄N₄O₄[M+H]⁺373.18;found 373.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.19(s,1H),8.57-8.39(m,1H),8.26(dd,J＝7.4,1.7Hz,1H),7.84-7.67(m,1H),7.52(dd,J＝6.9,1.8Hz,1H),7.46(d,J＝6.9Hz,1H),7.35-7.25(m,1H),7.23(d,J＝7.8Hz,1H),6.33(t,J＝7.1Hz,1H),5.36-5.17(m,2H),4.21-4.00(m,1H),1.31(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 24

A100 mL single vial was charged with compound 24-1 (90 mg,0.214 mmol) and 4N HCl/1, 4-dioxane (35 mL) sequentially. The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give compound 24 (38.8 mg, yellow solid, purity 98.823%). Yield: 66.01%.

LCMS(ESI)m/z calcd.for C₁₄H₁₆N₄O2[M+H]⁺273.13;found 273.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.99(s,1H),8.81(d,J＝5.2Hz,1H),8.53(S,3H),8.35(t,J＝7.7Hz,1H),8.28(dd,J＝7.4,1.3Hz,1H),7.82(t,J＝6.3Hz,2H),7.62(d,J＝8.0Hz,1H),6.44(t,J＝7.2Hz,1H),5.57(s,2H),4.32-4.18(m,1H),1.40(d,J＝6.9Hz,3H).

In various embodiments of the invention, compound 25 may be synthesized in a similar manner as described above.

EXAMPLE 15 Synthesis of Compound 26

Synthesis of Compound 26-1

A100 mL single-necked flask was charged with compound SM1 (2 g,0.0215 mol), t-butoxybis (dimethylamino) methane (11.24 g,0.0645 mol) and DMF (40 mL) in this order. The reaction was carried out at 140℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was concentrated to dryness to give crude compound 26-1 (0.5 g, brown oil, purity 64.514%). Yield: 10.23%.

LCMS(ESI)m/z calcd.for C9H12N2[M+H]⁺149.10；found 149.1。

Synthesis of Compound 26-2

To a 100mL single-necked flask were added sequentially 26-1 (550 mg,3.69 mmol), 2-phenyl-5-oxazolone (1188 mg,10.81 mmol) and AcOH (3 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (50 mL), extracted three times with EA (60 mL), the reaction solution was concentrated to dryness, and the residue was purified by chromatography, eluting with PE/ea=3/1 to 1/1, filtered, and concentrated to dryness to give compound 26-2 (200 mg, yellow solid, purity 97.832%). Yield: 20.01%.

LCMS(ESI)m/z calcd.for C₁₆H₁₂N₂O₂[M+H]⁺265.09;found 265.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.53(s,1H),8.93(d,J＝7.3Hz,1H),8.65(d,J＝8.3Hz,1H),8.04-7.93(m,2H),7.79(d,J＝8.9Hz,1H),7.66-7.59(m,1H),7.59-7.51(m,2H),7.48-7.37(m,1H),7.26-7.16(m,1H),7.00(d,J＝8.3Hz,1H).

Synthesis of Compound 26-3

To a 100mL single vial was added compound 26-2 (200 mg,0.76 mmol) followed by 2N HCl (10 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, adjusted to pH to alkaline with aqueous sodium bicarbonate, extracted five times with EA (100 mL), and concentrated to dryness to give Compound 26-3 (140 mg, brown solid, purity 84.216%). Yield: 97.26%.

LCMS(ESI)m/z calcd.for C₉H₈N₂O[M+H]⁺161.06;found 161.2;¹H NMR(400MHz,DMSO_d₆):δ＝8.69-8.56(m,1H),7.49-7.39(m,1H),7.11(d,J＝7.9Hz,1H),6.93-6.84(m,2H),6.74(d,J＝7.9Hz,1H),5.32(s,2H).

Synthesis of Compound 26-4

To a 100mL single vial was added, in order, compound 26-3 (140 mg,0.87 mmol), N-t-butoxycarbonyl-L-alanine (216 mg,1.049 mmol), HATU (6615 mg,1.75 mmol), DIEA (399 mg,2.62 mmol), and DMF (5 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted three times with EA (50 mL), and the organic phase was washed with water, dried, concentrated to dryness to give a residue, which was separated by a preparative plate (PE/ea=1/1) to give a crude product, which was separated by high pressure preparative separation (column type: XBridge-1,5 μm, 19-150 mm; detector: 254nm; mobile phase: acetonitrile/water (0.1% aqueous ammonia solution), elution gradient: 20-53/9 min, ratio of acetonitrile in water; retention time: 9.0 min) to give compound 26-4 (90 mg, yellow solid, purity 98.934%). The yield was 30.65%.

LCMS(ESI)m/z calcd.for C₁₇H₂₁N₃O₄[M+H]⁺332.15;found 332.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.33(s,1H),8.87(d,J＝7.4Hz,1H),8.71(d,J＝8.3Hz,1H),7.73(d,J＝8.9Hz,1H),7.49(d,J＝6.7Hz,1H),7.41-7.31(m,1H),7.20-7.11(m,1H),6.93(d,J＝8.4Hz,1H),4.30-4.13(m,1H),1.42(s,9H),1.30(d,J＝7.1Hz,3H).

Synthesis of Compound 26

To a 100mL single vial was added compound 26-4 (90 mg,0.27 mmol) in sequence 4N HCl/1, 4-dioxane (5 mL). The reaction is carried out for 2 hours at 25 ℃, and after the reaction is finished, the mixture is concentrated to dryness to obtain a crude product. The crude product was added to water, adjusted to pH 10 with sodium carbonate, extracted three times with EA (50 mL), dried on organic phase and concentrated to dryness to give compound 26 (25.9 mg, yellow solid, 98.701% purity). Yield: 22.68%.

LCMS(ESI)m/z calcd.for C₁₂H₁₃N₃O₂[M+H]⁺232.10;found 232.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.26(s,1H),8.90(d,J＝7.3Hz,1H),8.69(d,J＝8.3Hz,1H),8.20(s,0H),7.75(d,J＝8.8Hz,1H),7.44-7.35(m,1H),7.18(t,J＝6.9Hz,1H),6.95(d,J＝8.4Hz,1H),4.07-3.97(m,2H),3.45(s,2H),1.39(d,J＝6.9Hz,3H).

In various embodiments of the invention, compound 27 may be synthesized in a similar manner as described above.

EXAMPLE 16 Synthesis of Compound 28

Synthesis of Compound 28-1

A100 mL single-port flask was charged with compound SM1 (3 g,31.9 mmol), t-butoxybis (dimethylamino) methane (16.68 g,95.7 mmol), and DMF (20 mL). The reaction was carried out at 140℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was concentrated to dryness to give compound 28-1 (0.5 g, brown oil, purity 77.878%). Yield: 32.60%.

LCMS(ESI)m/z calcd.for C₈H₁₁N₃[M+H]⁺150.10；found 150.1。

Synthesis of Compound 28-2

A100 mL single vial was charged with compound 28-1 (3 g,20.1 mmol), 2-phenyl-5-oxazolone (6.48 g,40.2 mmol), and AcOH (3 mL) in sequence. The reaction was carried out at 100℃for 16 hours under nitrogen. After completion of the reaction, the reaction solution was cooled to room temperature, adjusted to ph=7, poured into water (50 mL), extracted five times with EA (100 mL), the organic phase was concentrated to dryness, and the residue was purified by chromatography column with eluent ratio DCM/meoh=120/1 to 70/1 to give compound 28-2 (0.23 g, yellow solid, purity 86.916%). Yield: 3.98%.

LCMS(ESI)m/z calcd.for C₁₅H₁₁N₃O₂[M+H]⁺266.09；found 266.1。

Synthesis of Compound 28-3

To a 100mL single vial was added compound 28-2 (200 mg,0.754 mmol) followed by 2N HCl (10 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, adjusted to pH to be alkaline with aqueous sodium bicarbonate, extracted five times with EA (50 mL), and the organic phase was concentrated to dryness to give compound 28-3 (120 mg, brown solid, purity 87.818%). Yield: 86.72%.

LCMS(ESI)m/z calcd.for C₈H₇N₃O[M+H]⁺162.06;found 162.0;¹H NMR(400MHz,DMSO_d₆):δ＝8.32(d,J＝2.3Hz,1H),7.78(d,J＝8.8Hz,1H),7.02(d,J＝7.8Hz,1H),6.71(d,J＝7.8Hz,1H),6.61(dd,J＝8.9,4.1Hz,1H),3.38(s,2H).

Synthesis of Compound 28-4

To a 100mL single vial was added, in order, compound 28-3 (200 mg,1.24 mmol), N-t-butoxycarbonyl-L-alanine (307 mg,1.49 mmol), HATU (944 mg,2.48 mmol), DIEA (481mg, 3.72 mmol) and DMF (10 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted five times with EA (100 mL), the reaction solution was concentrated to dryness, and the residue was separated by a preparative plate (PE/ea=1/1) to give a yellow solid product, and the crude product was separated by high-pressure preparation (column type: XBridge-1,5 μm, 19 to 150 mm; detector: 254nm; mobile phase: acetonitrile/water (0.1% aqueous ammonia solution), elution gradient: 20 to 58/9 min, 95 to 95/2 min, ratio of acetonitrile in water; retention time: 8.5 min), to give compound 28-4 (80 mg, yellow solid, purity 94.775%). The yield was 18.32%.

LCMS(ESI)m/z calcd.for C₁₆H₂₀N₄O₄[M+H]⁺333.15;found 333.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.48(s,1H),8.70(d,J＝8.1Hz,1H),8.58(dd,J＝4.1,1.8Hz,1H),8.05(dd,J＝9.2,1.7Hz,1H),7.55(d,J＝6.9Hz,1H),7.07(dd,J＝9.0,4.1Hz,1H),6.87(d,J＝8.1Hz,1H),4.30-4.17(m,1H),3.36(s,1H),1.41(s,9H),1.29(d,J＝7.2Hz,3H).

Synthesis of Compound 28

To a 100mL single vial was added compound 28-4 (80 mg,0.24 mmol) in sequence 4N HCl/1, 4-dioxane (4 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, the reaction was concentrated to dryness to give compound 28 (60.04 mg, yellow solid, purity 99.561%). Yield: 92.71%.

LCMS(ESI)m/z calcd.for C₁₁H₁₂N₄O₂[M+H]⁺233.10;found 233.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.32(s,1H),8.72-8.63(m,1H),8.67-8.58(m,1H),8.43(s,3H),8.09(dd,J＝9.1,1.7Hz,1H),7.14(dd,J＝9.0,4.1Hz,1H),6.90(d,J＝8.2Hz,1H),4.47-4.33(m,1H),1.46(d,J＝6.9Hz,3H).

In various embodiments of the present invention, compounds 29-33 may be synthesized in a similar manner as described above.

EXAMPLE 17 Synthesis of Compound 34

Synthesis of Compound 34-1

A100 mL single vial was charged with compound SM1 (3 g,31.9 mmol), SM2 (16.68 g,95.7 mmol), and DMF (30 mL) in order. The reaction was carried out at 140℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was concentrated to dryness to give compound 34-1 (3 g, brown oil, purity 90.00%). Yield: 56.74%.

LCMS(ESI)m/z calcd.for C₈H₁₁N₃[M+H]⁺150.10;found 150.1;1H NMR(400MHz,DMSO_d₆):δ＝8.38(d,J＝4.8Hz,2H),7.74(d,J＝13.1Hz,1H),6.79(t,J＝4.8Hz,1H),5.11(d,J＝13.1Hz,1H),2.90(s,6H).

Synthesis of Compound 34-2

A100 mL single vial was charged with compound 34-1 (3 g,20.1 mmol), SM3 (6.48 g,40.2 mmol), and AcOH (30 mL) in sequence. The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, adjusted to ph=8 using saturated aqueous sodium bicarbonate, extracted 3 times with EA (100 mL), concentrated to dryness, and the residue was purified by chromatography on a column with an eluent ratio PE/etoac=5/1 to 1/1, filtered, concentrated to dryness to give compound 34-2 (0.6 g, yellow solid, purity 74.718%). Yield: 8.46%.

LCMS(ESI)m/z calcd.for C₁₅H₁₁N₃O₂[M+H]⁺266.09;found 266.1;1H NMR(400MHz,DMSO_d₆):δ＝9.56(s,1H),9.15(d,J＝7.3Hz,1H),8.78(d,J＝8.5Hz,1H),8.69(dd,J＝3.5,1.7Hz,1H),7.99(d,J＝7.1Hz,2H),7.63(d,J＝7.1Hz,1H),7.57(t,J＝7.4Hz,2H),7.21(dd,J＝7.4,3.7Hz,1H),7.02(d,J＝8.5Hz,1H).

Synthesis of Compound 34-3

To a 100mL single vial was added compound 34-2 (300 mg,1.13 mmol) followed by EtOH/HCl=2/1 (4 mL). The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the reaction was completed, the reaction solution was cooled to room temperature, adjusted to ph=8 using saturated aqueous sodium bicarbonate, extracted 3 times with EA (15 mL), and the organic phase was concentrated to dryness to give compound 34-3 (180 mg, brown solid, purity 62.066%). Yield: 61.30%.

LCMS(ESI)m/z calcd.for C₈H₇N₃O[M+H]⁺162.06;found 162.0;1H NMR(400MHz,DMSO_d₆):δ＝12.17(s,1H),8.16(d,J＝3.5Hz,1H),7.79(d,J＝7.6Hz,1H),7.12-7.06(m,1H),6.69(s,1H),5.71(s,2H).

Synthesis of Compound 34-4

To a 25mL single port flask were added compound 34-3 (100 mg,0.62 mmol), N-t-butoxycarbonyl-L-alanine (141.6 mg,0.74 mmol), HATU (471.9 mg,1.24 mmol), DIEA (320.8 mg,2.48 mmol) and DMF (4 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), extracted 3 times with EA (20 mL), concentrated to dryness, and the residue was isolated by preparative plate, developing solvent ratio DCM/meoh=15/1 to give compound 34-4 (35 mg, yellow solid, purity 95.740%). The yield was 16.20%.

LCMS(ESI)m/z calcd.for C₁₆H₂₀N₄O₄[M+H]⁺333.15;found 333.1;¹H NMR(400MHz,DMSO_d₆):δ＝12.70(s,1H),9.43(s,1H),8.63(s,1H),8.44(d,J＝3.3Hz,1H),8.15(d,J＝7.6Hz,1H),7.55(d,J＝6.3Hz,1H),7.27(dd,J＝7.7,4.7Hz,1H),4.32-4.13(m,1H),1.41(s,9H),1.28(d,J＝7.1Hz,3H).

Synthesis of Compound 34

To a 25mL single vial was added compound 34-4 (30 mg,0.09 mmol) in sequence 4N HCl/1, 4-dioxane (3 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, concentrated to dryness to give compound 34 (22.74 mg, brown solid, purity 96.169%). Yield: 90.44%.

LCMS(ESI)m/z calcd.for C₁₁H₁₂N₄O₂[M+H]⁺233.10;found 233.1;¹H NMR(400MHz,DMSO_d₆):δ＝12.73(s,1H),10.26(s,1H),8.67(s,1H),8.46(d,J＝4.8Hz,4H),8.18(dd,J＝7.7,1.3Hz,1H),7.28(dd,J＝7.7,4.7Hz,1H),4.46-4.33(m,1H),1.45(d,J＝6.9Hz,3H).

In various embodiments of the invention, compound 35 may be synthesized in a similar manner as described above.

EXAMPLE 18 Synthesis of Compound 36

Synthesis of Compound 36-1a

To 25mL was added, in order, compound 36-1 (800 mg,3.69 mmol), bnOH (552 mg,5.14 mmol), naH (123 mg,5.14 mmol), and THF (10 mL). The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the mixture was poured into water (30 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/etoac=6/1 to 2/1 to give compound 36-1a (500 mg, yellow solid, 86.364% purity). Yield: 60.61%.

LCMS(ESI)m/z calcd.for C₁₅H₁₆N₂O₄[M+H]⁺289.1;found289.0;¹H NMR(400MHz,DMSO_d₆):δ＝8.06(d,J＝8.0Hz,1H),7.46-7.31(m,5H),6.60(d,J＝8.0Hz,1H),5.40(s,2H),5.07-4.91(m,1H),1.31(d,J＝6.8Hz,6H).

Synthesis of Compound 36-2a

To a 25mL single vial was added compound 36-1a (300 mg,1.04 mmol), iron powder (290 mg,5.20 mmol), NH ₄ Cl (278 mg,5.20 mmol), and EtOH/H ₂ O=3/1 (10 mL) in this order. The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the reaction was completed, the organic phase in the reaction solution was filtered, dried by spin-drying, extracted three times with EA (20 mL), and concentrated to dryness. The residue was purified by prep. plate with a ratio of PE/etoac=1/1 to give compound 36-2a (200 mg, white solid, 78.177% purity). Yield: 58.17%.

LCMS(ESI)m/z calcd.for C₁₅H₁₈N₂O₂[M+H]⁺259.14;found259.2;¹H NMR(400MHz,DMSO_d₆):δ＝7.47(d,J＝7.1Hz,2H),7.39(t,J＝7.3Hz,2H),7.33(d,J＝7.2Hz,1H),7.06(d,J＝7.8Hz,1H),6.35(d,J＝7.8Hz,1H),5.16(s,2H),5.11-5.03(m,1H),4.25(s,2H),1.25(d,J＝6.8Hz,6H).

Synthesis of Compound 36-3a

To a 25mL single vial was added compound 36-2a (200 mg,0.77 mmol), SM1 (177 mg,0.93 mmol), HATU (589 mg,1.55 mmol), DIEA (400 mg,3.10 mmol) and DMF (4 mL) in order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of DCM/meoh=15/1 to give compound 36-3a (220 mg, white solid, 99.895% purity). Yield: 65.94%.

LCMS(ESI)m/z calcd.for C₂₃H₃₁N₃O₅[M+H]⁺430.23;found 430.4;¹H NMR(400MHz,DMSO_d₆):δ＝8.76(s,1H),7.68(d,J＝7.8Hz,1H),7.44(d,J＝7.1Hz,2H),7.36(t,J＝7.3Hz,2H),7.31(d,J＝7.1Hz,1H),6.80(d,J＝7.7Hz,1H),6.35(d,J＝7.9Hz,1H),5.17(s,2H),5.10-4.94(m,1H),4.29-4.13(m,1H),1.38(s,9H),1.27(d,J＝6.8Hz,6H),1.20(d,J＝7.0Hz,3H).

Synthesis of Compound 36-5

A25 mL single vial was charged with compound 36-3a (220 mg,0.511 mmol), pd/C (16 mg, 10%) and MeOH (5 mL). The reaction was carried out at 25℃for 3 hours under the protection of hydrogen. After the completion of the reaction, the mixture was concentrated to dryness to give compound 36-5 (160 mg, white solid, purity 99.893%). Yield: 91.88%.

LCMS(ESI)m/z calcd.for C₁₆H₂₅N₃O₅[M+H]⁺340.18;found 340.2;¹H NMR(400MHz,DMSO_d₆):δ＝11.52(s,1H),9.35(s,1H),7.53(d,J＝7.7Hz,1H),7.43(d,J＝6.3Hz,1H),6.01(d,J＝7.7Hz,1H),5.11-4.86(m,1H),4.31-4.17(m,1H),1.40(s,9H),1.27(dd,J＝9.4,3.5Hz,9H).

Synthesis of Compound 36

To a 25mL single vial was added compound 36-5 (160 mg,0.47 mmol) followed by Eton's reagent (4 mL). After the reaction was completed at 80℃for 16 hours, the reaction mixture was cooled to room temperature, poured into saturated aqueous NaHCO ₃ (20 mL) and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 36 (11.91 mg, gray solid, 96.849% purity). Yield: 11.09%.

LCMS(ESI)m/z calcd.for C₁₁H₁₅N₃O₂[M+H]⁺222.1;found222.2;1H NMR(400MHz,DMSO_d₆):δ＝7.78(d,J＝7.5Hz,1H),6.83(d,J＝7.5Hz,1H),5.26-5.14(m,1H),4.13(q,J＝6.8Hz,1H),2.29(s,2H),1.41(d,J＝6.8Hz,3H),1.32(d,J＝6.8Hz,6H).

In various embodiments of the invention, compound 37 may be synthesized in a similar manner as described above.

EXAMPLE 19 Synthesis of Compound 38

Synthesis of Compound 36-1

A100 mL single-port flask was charged with compound SM1 (5 g,0.0286 mol), bromoisopropyl (7.91 g,0.0572 mol), potassium carbonate (12.15 g,0.0715 mol), and ACN (20 mL) in this order. The reaction was carried out at 80℃for 16 hours under nitrogen protection. After the reaction, the reaction mixture was concentrated to dryness, the residue was purified by chromatography with an eluent ratio of PE/ea=5/1 to 1/1, filtered and concentrated to dryness to give compound 36-1 (0.45 g, yellow solid, purity 92.812%). Yield: 6.64%.

LCMS(ESI)m/z calcd.for C₈H₉ClN₂O₃[M+H]⁺217.03;found 217.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.14(d,J＝7.5Hz,1H),6.73(d,J＝7.5Hz,1H),5.05-4.92(m,1H),1.35(d,J＝6.8Hz,6H).

Synthesis of Compound 38-1

To a 100mL single vial was added compound 36-1 (500 mg,2.31 mmol), NH ₃H₂ O (8 mL), and MeOH (8 mL) in order. The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was concentrated to dryness to give crude compound 38-1 (450 mg, yellow solid, purity 98.596%). Yield: 97.48%.

LCMS(ESI)m/z calcd.for C₈H₁₁N₃O₃[M+H]⁺198.08；found 198.2。

Synthesis of Compound 38-2

To a 100mL single vial was added compound 38-1 (500 mg,2.54 mmol) followed by Pd/C (50 mg). The reaction was carried out at 25℃for 16 hours under 1atm hydrogen. After the completion of the reaction, the reaction solution was concentrated to dryness by filtration to give crude compound 38-2 (400 mg, yellow solid, purity 93.576%). Yield: 88.28%.

LCMS(ESI)m/z calcd.for C₈H₁₃N₃O[M+H]⁺167.2;found 168.2;¹H NMR(400MHz,DMSO_d₆):δ＝7.25(br.s,2H),6.95(d,J＝7.5Hz,1H),5.83(d,J＝7.5Hz,1H),5.25(br.s,2H),5.09-4.94(m,1H),1.21(d,J＝7.5Hz,6H).

Synthesis of Compound 38-3

To a 100mL single vial was added compound 38-2 (500 mg,1.24 mmol), N-Boc-L-alanine (569 mg,2.99 mmol), HATU (2.27 g,5.98 mmol), DIEA (1.16 g,8.97 mmol) and DMF (10 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, poured into water (20 mL), extracted five times with EA (100 mL), the reaction concentrated to dryness and the residue isolated by prep. plate (DCM/meoh=15/1) to give compound 38-3 (400 mg, brown solid, 94.600% purity). The yield was 37.28%.

LCMS(ESI)m/z calcd.forC₁₆H₂₆N₄O₄[M+H]⁺339.2;found 339.2;¹H NMR(400MHz,DMSO_d₆):δ＝8.71(s,1H),7.32(d,J＝7.6Hz,1H),7.09(d,J＝6.1Hz,1H),5.84(d,J＝7.5Hz,1H),5.66(s,2H),5.03-4.85(m,1H),4.17-3.96(m,1H),3.61(br.s,1H),3.14(br.s,1H),1.37(s,3H),1.28-1.19(m,15H).

Synthesis of Compound 38-4

To a 100mL single vial was added compound 38-3 (400 mg,0.24 mmol) followed by AcOH (6 mL). After reaction at 120 ℃ for 16 hours, the reaction was adjusted to ph=7, concentrated to dryness, and the residue was isolated by prep. plate (DCM/meoh=10/1) to give compound 38-4 (95 mg, yellow solid, 98.955% purity). Yield: 30.41%.

LCMS(ESI)m/z calcd.for C₁₃H₁₈N₄O₂[M+H]⁺263.14;found 263.1;¹H NMR(400MHz,DMSO_d₆):δ13.04(s,0.54H),12.37(s,0.40H),8.44(d,J＝7.5Hz,0.42H),8.33(d,J＝7.4Hz,0.52H),7.43(d,J＝7.4Hz,1H),6.60(d,J＝7.3Hz,0.57H),6.49(d,J＝7.2Hz,0.41H),5.33-5.14(m,1H),5.11-4.94(m,1H),1.86(s,3H),1.55-1.31(m,3H),1.36-1.21(m,6H).

Synthesis of Compound 38

To a 100mL single vial was added compound 38-4 (80 mg,0.305 mmol) followed by 2N HCl (5 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, adjusted to pH to alkaline with aqueous sodium bicarbonate, extracted five times with DCM (50 mL), and the organic phase was concentrated to dryness to give compound 38 (30.7 mg, yellow solid, purity 98.519%). Yield: 45.02%.

LCMS(ESI)m/z calcd.for C₁₁H₁₆N₄O[M+H]⁺221.13;found 221.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.42(d,J＝7.4Hz,1H),6.56(d,J＝7.3Hz,1H),5.31-5.15(m,1H),4.15-4.04(m,1H),3.43(br.s,3H),1.39(d,J＝6.8Hz,3H),1.30(d,J＝6.8Hz,6H).

In various embodiments of the present invention, compounds 39-41 may be synthesized in a similar manner as described above.

EXAMPLE 20 Synthesis of Compound 42

Synthesis of Compound 42-1

To a 100mL single-port flask, compound SM1 (20 g,0.143 mol), methanol (200 mL), and Pd/C (200 mg) were sequentially added. The reaction was carried out at 25℃for 16 hours under a hydrogen pressure of 1 atm. After the completion of the reaction, the reaction mixture was concentrated to dryness by filtration to give compound T42-1 (12 g, white solid, purity 90.00%). Yield: 68.70%.

LCMS(ESI)m/z calcd.for C₅H₆N₂O[M+H]⁺111.05;found 111.2;¹H NMR(400MHz,DMSO_d₆):δ＝11.31(s,1H),6.60(dd,J＝6.5,1.8Hz,1H),6.44(dd,J＝7.0,1.8Hz,1H),5.99(t,J＝6.8Hz,1H),4.99(s,2H).

Synthesis of Compound 42-2

A250 mL single vial was charged with compound 42-1 (12 g,0.109 mol), DMF (50 mL), N-t-butoxycarbonyl-L-alanine (24.88 g,0.131 mol), HATU (82.89 g,0.218 mol) and DIEA (56.35 g,0.436 mol). The reaction was carried out at 50℃for 3 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (250 mL), extracted eight times with EA (800 mL), concentrated to dryness, and the residue was separated by chromatography (DCM/meoh=100/1 to 30/1) to give compound 42-2 (10 g, yellow solid, purity 79.156%). The yield was 25.69%.

LCMS(ESI)m/z calcd.for C₁₃H₁₉N₃O₄[M+H]⁺282.14;found 282.1;¹H NMR(400MHz,DMSO_d₆):δ＝11.99(s,1H),9.15(s,1H),8.22(dd,J＝7.3,1.6Hz,1H),7.56-7.41(m,1H),7.19-7.01(m,1H),6.22(t,J＝6.9Hz,1H),4.18-4.07(m,1H),1.35(s,9H),1.24(d,J＝9.5Hz,3H).

Synthesis of Compound 42-3

A100 mL single vial was charged with compound 42-2 (1 g,3.55 mmol), DMF (20 mL), bromoethanol (0.66 g,5.33 mmol), and potassium carbonate (0.97 g,7.10 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (100 mL), extracted three times with EA (150 mL), the reaction solution was concentrated to dryness, and the residue was purified by chromatography on a column with eluent ratio DCM/meoh=100/1 to 20/1, concentrated to dryness to give compound 42-3 (0.7 g, white solid, purity 96.432%). Yield: 58.85%.

LCMS(ESI)m/z calcd.for C₁₅H₂₃N₃O₅[M+H]⁺326.16;found 326.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.20(s,1H),8.21(dd,J＝7.4,1.8Hz,1H),7.49(d,J＝6.7Hz,1H),7.33(dd,J＝6.8,1.8Hz,1H),6.24(t,J＝7.1Hz,1H),4.89(t,J＝5.4Hz,1H),4.24-4.04(m,1H),4.04-3.89(m,2H),3.65(q,J＝5.5Hz,2H),1.35(s,9H),1.25(d,J＝7.2Hz,3H).

Synthesis of Compound 42-4

To a100 mL single vial was added compound 42-3 (400 mg,1.23 mmol), DCM (5 mL), and dessmartin oxidant (1039 mg,2.45 mmol) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (10 mL), extracted five times with DCM (50 mL), concentrated to dryness, and the residue was isolated by preparative plate (DCM/meoh=15/1) to give compound 42-4 (180 mg, yellow solid, purity 82.833%). The yield was 37.51%.

LCMS(ESI)m/z calcd.forC₁₅H₂₁N₃O₅[M+H]⁺324.15;found 356.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.19(s,1H),8.35-8.18(m,1H),8.13-7.93(m,1H),7.48(s,1H),7.39-7.22(m,1H),6.36-6.20(m,1H),4.79-4.60(m,1H),4.22-4.09(m,2H),1.36(s,9H),1.24(d,J＝7.4Hz,3H).

Synthesis of Compound 42-5

To a 100mL single vial was added compound 42-4 (120 mg,0.37 mmol), DCE (5 mL), dimethylamine hydrochloride (22.2 mg,0.74 mmol), STAB (157 mg,0.74 mmol) and AcOH (67 mg,1.11 mmol) in order. After reaction at 25 ℃ for 16 hours, the reaction was cooled to room temperature, poured into water (10 mL), extracted five times with DCM (50 mL), the organic phase concentrated to dryness and the residue isolated by prep. plate (DCM/meoh=15/1) to give compound 42-5 (50 mg, yellow solid, 94.166% purity). Yield: 36.00%.

LCMS(ESI)m/z calcd.for C₁₇H₂₈N₄O₄[M+H]⁺353.21;found 353.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.20(s,1H),8.19(dd,J＝7.4,1.7Hz,1H),7.50(d,J＝6.9Hz,1H),7.37(dd,J＝6.9,1.8Hz,1H),6.25(t,J＝7.1Hz,1H),4.28-4.07(m,1H),4.08-3.92(m,2H),2.51(s,2H),2.16(s,6H),1.34(s,9H),1.25(d,J＝7.3Hz,3H).

Synthesis of Compound 42

A100 mL single vial was charged with compound 42-5 (40 mg,0.113 mmol) followed by HCl (g)/1, 4-dioxane (5 mL). After the reaction was completed at 25℃for 2 hours, the reaction mixture was concentrated to dryness to give compound 42 (24.11 mg, white solid, purity 98.28%). Yield: 82.95%.

LCMS(ESI)m/z calcd.for C₁₂H₂₀N₄O₂[M+H]⁺252.16;found 252.8;¹H NMR(400MHz,DMSO_d₆):δ＝10.93(s,1H),10.03(s,1H),8.47(s,3H),8.24(dd,J＝7.4,1.5Hz,1H),7.58(dd,J＝6.9,1.6Hz,1H),6.38(t,J＝7.2Hz,1H),4.48-4.37(m,2H),4.34-4.27(m,1H),3.50-3.33(m,2H),2.82(d,J＝4.6Hz,6H),1.41(d,J＝6.9Hz,3H).

In various embodiments of the present invention, compounds 43-44 may be synthesized in a similar manner as described above.

EXAMPLE 21 Synthesis of Compound 45

Synthesis of Compound 45-1

A50 mL single vial was charged with compound SM1 (100 mg,0.562 mmol), DMF (5 mL), 42-2 (237.82 mg,0.8424 mmol) and potassium carbonate (155.24 mg,1.123 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (5 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developing solvent DCM/meoh=10/1 to give compound 42-1 (120 mg, white solid, purity 98.607%). Yield: 54.38%.

LCMS(ESI)m/z calcd.for C₂₀H₂₆N₄O₄[M+H]⁺387.2;found 387.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.18(s,1H),8.33(s,1H),8.24(dd,J＝7.4,1.7Hz,1H),7.57(dd,J＝7.9,1.7Hz,1H),7.53-7.38(m,2H),7.13(d,J＝7.9Hz,1H),6.31(t,J＝7.1Hz,1H),5.32-5.10(m,J＝4.6Hz,2H),4.23-4.03(m,J＝5.3Hz,1H),3.17(d,J＝5.3Hz,1H),2.26(s,3H),1.34(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 45

A25 mL single vial was charged with compound 45-1 (100 mg,0.258 mmol) and 4N HCl/1,4-dioxane (5 mL) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 45 (38.05 mg, white solid, purity 98.936%). Yield: 50.95%.

LCMS(ESI)m/z calcd.for C₁₅H₁₈N₄O₂[M+H]⁺287.1;found 287.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.98(s,1H),8.70(s,1H),8.51(d,J＝4.1Hz,3H),8.26(dd,J＝7.4,1.4Hz,1H),8.20(d,J＝8.0Hz,1H),7.80(dd,J＝6.8,1.4Hz,1H),7.55(d,J＝8.2Hz,1H),6.43(t,J＝7.2Hz,1H),5.52(s,2H),4.32-4.14(m,1H),2.42(s,3H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 22 Synthesis of Compound 46

Synthesis of Compound 46-1

A50 mL single vial was charged with compound SM1 (100 mg,0.56 mmol), 42-2 (190 mg,0.67 mmol), potassium carbonate (155 mg,1.12 mmol), and DMF (3 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (5 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 46-1 (100 mg, off-white solid, 99.695% purity). Yield: 45.82%.

LCMS(ESI)m/z calcd.for C₂₀H₂₆N₄O₄[M+H]⁺387.2;found 387.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.47(d,J＝1.9Hz,1H),8.21(dd,J＝7.4,1.6Hz,1H),7.62-7.59(m,2H),7.48(d,J＝6.9Hz,1H),7.21(d,J＝8.0Hz,1H),6.32(t,J＝7.1Hz,1H),5.15(d,J＝2.3Hz,2H),4.17-4.11(m,1H),2.42(s,3H),1.39(s,9H),1.23(d,J＝7.2Hz,3H).

Synthesis of Compound 46

A25 mL single vial was charged with compound 46-1 (50 mg,0.129 mmol) followed by 4N HCl/1,4-dioxane (1 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction was completed, it was concentrated to dryness to give compound 46 (21.45 mg, light brown solid, purity 95.084%). Yield: 48.99%.

LCMS(ESI)m/z calcd.for C₁₅H₁₈N₄O₂[M+H]⁺287.1;found 287.2;¹H NMR(400MHz,DMSO_d₆):δ＝10.02(s,1H),8.80(d,J＝1.6Hz,1H),8.40-8.38(m,4H),8.24(dd,J＝7.4,1.6Hz,1H),7.86(d,J＝8.3Hz,1H),7.82(dd,J＝6.9,1.7Hz,1H),6.40(t,J＝7.2Hz,1H),5.33(s,2H),4.29-4.24(m,1H),2.72(s,3H),1.38(d,J＝6.9Hz,3H).

In various embodiments of the invention, compound 47 may be synthesized in a similar manner as described above.

EXAMPLE 23 Synthesis of Compound 48

Synthesis of Compound 48-1

To a100 mL single-port flask, compound 42-2 (500 mg,1.78 mmol), DMF (10 mL), benzyl 4- (bromomethyl) piperidinecarboxylate (555 mg,1.78 mmol) and potassium carbonate (489 mg,3.54 mmol) were added sequentially. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (50 mL), extracted three times with EA (120 mL), the reaction solution was concentrated to dryness, and the residue was purified by chromatography, the eluent ratio was PE/ea=20/1 to 10/1, and concentrated to dryness to give compound 48-1 (550 mg, white solid, purity 89.792%). Yield: 60.47%.

LCMS(ESI)m/z calcd.for C₂₇H₃₆N₄O₆[M+H]⁺513.26;found 513.3;¹H NMR(400MHz,DMSO_d₆):δ＝8.21(d,J＝7.3Hz,1H),7.48(s,1H),7.43-7.28(m,6H),6.26(dd,J＝9.1,5.1Hz,1H),5.05(s,2H),4.13(s,1H),3.98(d,J＝11.6Hz,2H),3.87(d,J＝7.4Hz,2H),2.81-2.66(m,2H),2.01-1.95(m,1H),1.49(s,3H),1.38(s,9H),1.24(d,J＝7.1Hz,4H).

Synthesis of Compound 48-3

To a 100mL single vial was added compound 48-1 (200 mg,0.3894 mmol), meOH (10 mL) paraformaldehyde (5.8 mg,0.195 mmol), and Pd/C (20 mg,10%, wet) in sequence. The reaction was carried out at 25℃for 16 hours under a hydrogen pressure of 1 atm. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and the residue was isolated by preparative plate (DCM/meoh=15/1) to give compound 48-3 (50 mg, yellow solid, purity 98.713%). The yield was 31.97%.

LCMS(ESI)m/z calcd.for C₂₀H₃₂N₄O₄[M+H]⁺393.24;found 393.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.20(s,1H),8.21(dd,J＝7.4,1.7Hz,1H),7.50(d,J＝6.9Hz,1H),7.38(dd,J＝6.9,1.8Hz,1H),6.27(t,J＝7.1Hz,1H),4.21-4.03(m,1H),3.99-3.73(m,2H),2.93(d,J＝10.3Hz,2H),2.31(s,3H),2.10(s,2H),1.81(s,1H),1.52(d,J＝12.8Hz,3H),1.34(s,9H),1.26-1.08(m,4H).

Synthesis of Compound 48

A50 mL single vial was charged with compound 48-3 (40 mg,0.127 mmol) followed by HCl (g)/1, 4-dioxane (5 mL). After the reaction was completed at 25℃for 2 hours, the reaction mixture was concentrated to dryness to give compound 48 (34.68 mg, white solid, purity 98.687%). Yield: 89.06%.

LCMS(ESI)m/z calcd.for C₁₅H₂₄N₄O₂[M+H]⁺293.19;found 293.2;¹H NMR(400MHz,DMSO_d₆):δ＝10.65(s,1H),9.97(s,1H),8.36(s,3H),8.21(dd,J＝7.4,1.6Hz,1H),7.58-7.42(m,1H),6.32(dd,J＝12.2,5.1Hz,1H),4.35-4.22(m,1H),4.00-3.75(m,2H),3.48-3.23(m,2H),2.95-2.74(m,2H),2.74-2.57(m,3H),2.06-1.87(m,1H),1.83-1.47(m,4H),1.40(d,J＝6.9Hz,3H).

EXAMPLE 24 Synthesis of Compound 49

Synthesis of Compound 49-1

A50 mL single vial was charged with compound SM1 (300 mg,3.06 mmol), CBr ₄(1.01g,3.06mmol)、PPh₃ (802 mg,3.06 mmol), and DCM (10 mL). The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was concentrated to dryness to give crude 49-1 (300 mg, brown oil, purity 18.763%). Yield: 11.43%.

LCMS(ESI)m/z calcd.for C₅H₅BrO[M+H]⁺160.9；found 355.0and 357.0。

Synthesis of Compound 49-2

A50 mL single vial was charged with compound 49-1 (100 mg,0.621 mmol), 42-2 (210 mg,0.745 mmol), potassium carbonate (172 mg,1.242 mmol) and DMF (5 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (5 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of PE/etoac=1/1 to give compound 49-2 (120 mg, off-white solid, 97.164% purity). Yield: 51.80%.

LCMS(ESI)m/z calcd.for C₁₈H₂₃N₃O₅[M+H]⁺362.2;found 362.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.23(s,1H),8.20(dd,J＝7.4,1.7Hz,1H),7.68(s,1H),7.61(t,J＝1.6Hz,1H),7.51-7.47(m,2H),6.46(br.s,1H),6.29(t,J＝7.1Hz,1H),4.99(d,J＝2.7Hz,2H),4.18-4.11(m,1H),1.40(s,9H),1.25(d,J＝7.3Hz,3H).

Synthesis of Compound 49

A25 mL single vial was charged with compound 49-2 (50 mg,0.138 mmol) followed by 4N HCl/1,4-dioxane (1 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction was completed, it was concentrated to dryness to give compound 49 (10.14 mg, light brown solid, purity 99.338%). Yield: 25.65%.

LCMS(ESI)m/z calcd.for C₁₃H₁₅N₃O₃[M+H]⁺262.1;found 262.1;¹H NMR(400MHz,CD₃OD):δ＝8.56(s,0.5H),8.31(d,J＝7.0Hz,1H),7.59(s,1H),7.46-7.42(m,2H),6.45(s,1H),6.35(t,J＝7.0Hz,1H),5.07(s,2H),3.98(d,J＝5.0Hz,1H),1.49(d,J＝6.7Hz,3H).

EXAMPLE 25 Synthesis of Compound 50

Synthesis of Compound 50-1

To a 100mL three-necked flask was added compound SM1 (2 g,13.1 mmol) followed by MeOH/THF=2/1 (30 mL). After cooling to 0deg.C, sodium borohydride (0.5 g,13.1 mmol) was added in portions. The reaction was carried out at 25℃for 4 hours under nitrogen. After the reaction was completed, ph=8 was adjusted with 1N hydrochloric acid, and water (100 mL) was added, and extracted three times with EA (100 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/etoac=3/1 to 1/1 to give compound 50-1 (1.2 g, white solid, purity 96.514%). Yield: 70.99%.

LCMS(ESI)m/z calcd.for C₆H₈N₂O[M+H]⁺125.1;found 125.3;¹H NMR(400MHz,DMSO_d₆):δ＝7.62-7.55(m,2H),5.59(t,J＝5.9Hz,1H),4.72(d,J＝5.9Hz,2H),2.60(s,3H).

Synthesis of Compound 50-2

A50 mL single vial was charged with compound 50-1 (800 mg,6.44 mmol), THF (16 mL), CBr ₄ (2.35 g,7.09 mmol), and PPh ₃ (1.77 g,6.77 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 50-2 (800 mg, brown solid, purity 27.925%). Yield: 18.53%.

LCMS(ESI)m/z calcd.for C₆H₇BrN₂[M+H]⁺186.9；found 187.1。

Synthesis of Compound 50-3

A50 mL single vial was charged with compound 50-2 (800 mg,4.28 mmol), 42-2 (1.45 g,5.13 mmol), potassium carbonate (1.18 g,8.55 mmol) and DMF (20 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (60 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with eluent ratio DCM/meoh=100/1 to 30/1 to give compound 50-3 (150 mg, white solid, purity 98.107%). Yield: 8.86%.

LCMS(ESI)m/z calcd.for C₁₉H₂₅N₅O₄[M+H]⁺388.2;found 388.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.17(s,1H),8.26(dd,J＝7.4,1.7Hz,1H),7.58(dd,J＝6.9,1.8Hz,1H),7.54(d,J＝8.6Hz,1H),7.46(d,J＝8.6Hz,2H),6.35(t,J＝7.1Hz,1H),5.41(d,J＝2.3Hz,2H),4.15-4.12(m,1H),2.58(s,3H),1.36(s,9H),1.23(d,J＝7.2Hz,3H).

Synthesis of Compound 50

A25 mL single vial was charged with compound 50-3 (50 mg,0.129 mmol) and 4N HCl in 1,4-dioxane (1 mL) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 50 (26.22 mg, yellow solid, purity 98.224%). Yield: 61.77%.

LCMS(ESI)m/z calcd.for C₁₄H₁₇N₅O₂[M+H]⁺288.1;found 288.1;1H NMR(400MHz,DMSO_d₆):δ＝9.96(s,1H),8.41(d,J＝4.2Hz,3H),8.27(dd,J＝7.4,1.7Hz,1H),7.98-7.93(m,2H),7.70(dd,J＝6.9,1.7Hz,1H),6.40(t,J＝7.2Hz,1H),5.50(s,2H),4.28-4.23(m,1H),2.72(s,3H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 26 Synthesis of Compound 51

Synthesis of Compound 51-1

A50 mL single vial was charged with compound SM1 (100 mg, 0.560 mmol), DMF (5 mL), 42-2 (239 mg,0.847 mmol) and potassium carbonate (155 mg,1.123 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 51-1 (100 mg, yellow solid, 99.331% purity). Yield: 46.48%.

LCMS(ESI)m/z calcd.for C₁₈H₂₃N₃O₄S[M+H]⁺378.14;found 378.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.24(s,1H),8.21(dd,J＝7.4,1.6Hz,1H),7.64-7.45(m,3H),7.43(d,J＝1.8Hz,1H),7.10(dd,J＝4.9,1.0Hz,1H),6.30(t,J＝7.1Hz,1H),5.23-5.05(m,2H),4.25-4.02(m,1H),1.52-1.28(m,9H),1.25(d,J＝7.2Hz,3H).

Synthesis of Compound 51

A25 mL single vial was charged with compound 51-1 (90 mg,0.2378 mmol) followed by 4N HCl/1,4-dioxane (5 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 51 (36.8 mg, yellow solid, purity 96.3%). Yield: 53.74%.

LCMS(ESI)m/z calcd.forC₁₃H₁₅N₃O₂S[M+H]⁺278.09;found 278.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.01(s,1H),8.45(s,3H),8.20(dd,J＝7.4,1.4Hz,1H),7.64(dd,J＝6.8,1.6Hz,1H),7.52(dd,J＝4.9,3.0Hz,1H),7.46(d,J＝1.9Hz,1H),7.12(d,J＝4.2Hz,1H),6.33(t,J＝7.1Hz,1H),5.17(s,2H),4.41-4.17(m,1H),1.41(d,J＝6.8Hz,3H).

EXAMPLE 27 Compound 52

Synthesis of Compound 52-1

A50 mL single vial was charged with compound SM1 (300 mg, 2.6278 mmol), CBr ₄(871mg,2.628mmol)、PPh₃ (689 mg, 2.6278 mmol), and DCM (10 mL). The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 52-1 (300 mg, brown oil, purity 14.103%). Yield: 9.10%.

LCMS(ESI)m/z calcd.for C₅H₅BrS[M+H]⁺176.9；found 355.0and 357.0。

Synthesis of Compound 52-2

A50 mL single vial was charged with compound 52-1 (100 mg, 0.560 mmol), 42-2 (191 mg,0.678 mmol), potassium carbonate (156 mg,1.130 mmol), and DMF (5 mL) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (5 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of PE/etoac=1/1 to give compound 52-2 (130 mg, off-white solid, 98.485% purity). Yield: 59.90%.

LCMS(ESI)m/z calcd.for C₁₈H₂₃N₃O₄S[M+H]⁺378.1;found 378.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.25(s,1H),8.20(dd,J＝7.4,1.7Hz,1H),7.56(dd,J＝6.9,1.8Hz,1H),7.50(d,J＝6.8Hz,1H),7.46(dd,J＝5.1,1.2Hz,1H),7.19(d,J＝2.6Hz,1H),6.98(dd,J＝5.1,3.5Hz,1H),6.30(t,J＝7.1Hz,1H),5.33(d,J＝3.0Hz,2H),4.19-4.12(m,1H),1.41(s,9H),1.25(d,J＝7.2Hz,3H).

Synthesis of Compound 52

A25 mL single vial was charged with compound 52-2 (50 mg,0.132 mmol) followed by 4N HCl/1,4-dioxane (1 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction was completed, it was concentrated to dryness to give compound 52 (23.27 mg, light brown solid, purity 98.744%). Yield: 55.41%.

LCMS(ESI)m/z calcd.for C₁₃H₁₅N₃O₂S[M+H]⁺278.1;found 278.3;¹H NMR(400MHz,DMSO_d₆):δ＝10.07(s,1H),8.30(br.s,3H),8.21(dd,J＝7.4,1.7Hz,1H),7.66(dd,J＝6.9,1.7Hz,1H),7.47(dd,J＝5.1,1.1Hz,1H),7.21(d,J＝2.8Hz,1H),6.99(dd,J＝5.1,3.5Hz,1H),6.34(t,J＝7.1Hz,1H),5.34(s,2H),4.33-4.29(m,1H),1.40(d,J＝6.9Hz,3H).

EXAMPLE 28 Synthesis of Compound 53

Synthesis of Compound 53-1

To 25mL was added, in order, compound SM1 (1 g,8.92 mmol), CBr ₄(3.25g,9.80mmol)、PPh₃ (2.45 g,9.30 mmol), and DCM (20 mL). The reaction was carried out at 25℃for 16 hours under nitrogen. After completion of the reaction, the mixture was poured into water (60 mL) and extracted three times with DCM (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/etoac=5/1 to 2/1 to give compound 53-1 (600 mg, yellow solid, purity 34.83%). Yield: 60.61%.

LCMS(ESI)m/z calcd.for C₅H₇BrN₂[M+H]⁺174.98;found175.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.64(d,J＝2.0Hz,1H),6.29(d,J＝2.1Hz,1H),4.59(s,2H),3.79(d,J＝15.0Hz,3H).

Synthesis of Compound 53-2

A25 mL single vial was charged with compound 53-1 (100 mg,0.57 mmol), 42-2 (194 mg,0.69 mmol), K ₂CO₃ (158 mg,1.14 mmol), and DMF (4 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (20 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 53-2 (150 mg, yellow solid, 98.398% purity). Yield: 68.63%.

LCMS(ESI)m/z calcd.For C₁₈H₂₅N₅O₄[M+H]⁺376.19;found 376.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.19(dd,J＝7.4,1.7Hz,1H),7.60(d,J＝2.1Hz,1H),7.49(d,J＝6.9Hz,1H),7.41(dd,J＝6.9,1.8Hz,1H),6.27(t,J＝7.1Hz,1H),6.12(d,J＝2.1Hz,1H),5.09(d,J＝3.6Hz,2H),4.23-4.07(m,1H),3.78(s,3H),1.40(s,9H),1.25(d,J＝7.2Hz,3H).

Synthesis of Compound 53

To a 25mL single vial was added sequentially compound 53-2 (100 mg,0.266 mmol) 4N HCl/1, 4-dioxane (5 mL). The reaction was carried out at 25℃for 2 hours, and after completion of the reaction, the reaction was concentrated to dryness to give compound 53 (43.66 mg, yellow solid, purity 98.843%). Yield: 59.01%.

LCMS(ESI)m/z calcd.for C₁₃H₁₇N₅O₂[M+H]⁺276.1;found276.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.29(s,1H),8.24(dd,J＝7.3,1.7Hz,1H),7.60(d,J＝2.0Hz,1H),7.39(dd,J＝6.9,1.7Hz,1H),6.25(t,J＝7.1Hz,1H),6.12(d,J＝2.1Hz,1H),5.09(s,2H),3.78(s,3H),3.47-3.38(m,1H),2.37(d,J＝35.6Hz,2H),1.22(d,J＝7.0Hz,3H).

EXAMPLE 29 Synthesis of Compound 54

Synthesis of Compound 54-1

A50 mL single vial was charged with compound SM1 (500 mg,5.05 mmol), DCM (15 mL), CBr ₄ (1.673 g,5.046 mmol) and PPh ₃ (1.323 g,5.046 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 54-1 (500 mg, brown oil, purity 33.342%). Yield: 20.40%.

LCMS(ESI)m/z calcd.for C₄H₄BrNO[M+H]⁺161.9；found 355.1。

Synthesis of Compound 54-2

A50 mL single-port flask was charged with compound 54-1 (500 mg,3.09 mmol), 2-hydroxy-3-nitropyridine (719 mg,3.70 mmol), potassium carbonate (853 mg,6.17 mmol), and DMF (10 mL). The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (30 mL), and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=15/1 to give 54-2 (80 mg, yellow solid, 84.362% purity). Yield: 9.88%.

LCMS(ESI)m/z calcd.for C₉H₇N₃O₄[M+H]⁺222.0;found 222.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.50(dd,J＝7.7,2.0Hz,1H),8.34(dd,J＝6.6,2.0Hz,1H),8.12(s,1H),7.19(s,1H),6.56(t,J＝7.2Hz,1H),5.41(s,2H).

Synthesis of Compound 54-3

To a 50mL single vial was added compound 54-2 (80 mg,0.36 mmol), etOH/H2 ₂ O=2/1 (6 mL), iron powder (101 mg,1.81 mmol), and ammonium chloride (97 mg,1.81 mmol) in sequence. The reaction was carried out at 60℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=10/1 to give compound 54-3 (60 mg, brown solid, 95.990% purity). Yield: 83.27%.

LCMS(ESI)m/z calcd.for C₉H₉N₃O₂[M+H]⁺192.1;found 192.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.05(d,J＝0.6Hz,1H),7.16(d,J＝0.6Hz,1H),6.98(dd,J＝6.8,1.7Hz,1H),6.46(dd,J＝7.1,1.7Hz,1H),6.09(t,J＝7.0Hz,1H),5.23(s,2H),5.12(s,2H).

Synthesis of Compound 54-4

To a 50mL single vial was added compound 54-3 (40 mg,0.21 mmol), DMF (3 mL), N-fluorenylmethoxycarbonyl-L-alanine (98 mg,0.31 mmol), HATU (1599 mg,0.42 mmol) and DIEA (54 mg,0.42 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (10 mL), and extracted three times with EA (3 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 54-4 (70 mg, brown solid, 92.132% purity). Yield: 63.48%.

LCMS(ESI)m/z calcd.for C₂₇H₂₄N₄O₅[M+H]⁺485.2;found 485.3;1H NMR(400MHz,DMSO_d₆):δ＝9.27(s,1H),8.26(d,J＝6.8Hz,1H),8.07(s,1H),7.90(t,J＝7.3Hz,3H),7.73-7.70(m,2H),7.54(d,J＝5.5Hz,1H),7.41(t,J＝6.6Hz,2H),7.31(dd,J＝14.9,7.4Hz,2H),7.17(d,J＝0.6Hz,1H),6.36(t,J＝7.1Hz,1H),5.33(s,2H),4.30-4.21(m,4H),1.28(d,J＝7.2Hz,3H).

Synthesis of Compound 54

To a 25mL single vial was added compound 54-4 (90 mg,0.19 mmol) followed by piperidine/DCM=1/5 (3 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction, and preparing residues by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% NH ₃H₂ O); gradient: 20-40/9 min, compound 54 (5.25 mg, white solid, 97.480% purity) was obtained. Yield: 10.52%.

LCMS(ESI)m/z calcd.for C₁₂H₁₄N₄O₃[M+H]⁺263.1;found 262.7;1H NMR(400MHz,CD₃OD):δ＝8.40(dd,J＝7.5,1.7Hz,1H),7.89(br.s,1H),7.43(dd,J＝6.9,1.7Hz,1H),7.13(s,1H),6.41(t,J＝7.2Hz,1H),5.36(s,2H),3.59(q,J＝7.0Hz,1H),1.34(d,J＝7.0Hz,3H).

EXAMPLE 30 Synthesis of Compound 55

Synthesis of Compound 55-1

To a 50mL three-necked flask, compound SM1 (400 mg,4.04 mmol), THF (10 mL), 2-hydroxy-3-nitropyridine (679 mg,4.8 mmol) and triphenylphosphine (1.59 g,6.06 mmol) were added in this order, and after the system had cooled to 0deg.C, DIAD (1.22 g,6.06 mmol) was added. The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was poured into water (30 mL) and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=15/1 to give 55-1 (500 mg, yellow solid, 94.468% purity). Yield: 52.90%.

LCMS(ESI)m/z calcd.for C₉H₇N₃O₄[M+H]⁺222.0;found 222.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.43(dd,J＝7.7,2.0Hz,1H),8.36(s,1H),8.31(dd,J＝6.7,2.0Hz,1H),7.24(s,1H),6.51(dd,J＝7.6,6.7Hz,1H),5.36(s,2H).

Synthesis of Compound 55-2

To a 50mL single vial was added compound 55-1 (200 mg,0.90 mmol), etOH/H2 ₂ O=2/1 (6 mL), iron powder (255 mg,4.52 mmol), and ammonium chloride (242 mg,4.52 mmol) in sequence. The reaction was carried out at 60℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give 55-2 (130 mg, brown solid, 99.533% purity). Yield: 74.84%.

LCMS(ESI)m/z calcd.for C₉H₉N₃O₂[M+H]⁺192.1;found 192.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.31(s,1H),7.13(s,1H),6.95(dd,J＝6.8,1.7Hz,1H),6.43(dd,J＝7.1,1.7Hz,1H),6.07(t,J＝7.0Hz,1H),5.20(s,2H),5.15(s,2H).

Synthesis of Compound 55-3

A50 mL single vial was charged with compound 55-2 (130 mg,0.68 mmol), DMF (3 mL), N-fluorenylmethoxycarbonyl-L-alanine (318 mg,1.02 mmol), HATU (517mg, 1.36 mmol) and DIEA (176 mg,1.36 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was poured into water (10 mL) and extracted three times with EA (3 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with a ratio of DCM/meoh=100/1 to 50/1 to give compound 55-3 (240 mg, brown solid, 97.632% purity). Yield: 70.97%.

LCMS(ESI)m/z calcd.for C₂₇H₂₄N₄O₅[M+H]⁺485.2;found 485.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.30(s,1H),8.32(s,1H),8.22(d,J＝6.6Hz,1H),7.93-7.88(m,3H),7.74-7.72(m,2H),7.53-7.50(m,1H),7.41(t,J＝7.1Hz,2H),7.35-7.30(m,2H),7.17(s,1H),6.33(t,J＝7.1Hz,1H),5.29(s,2H),4.32-4.24(m,4H),1.29(d,J＝7.2Hz,3H).

Synthesis of Compound 55

To a 25mL single vial was added compound 55-3 (240 mg,0.49 mmol) followed by piperidine/DCM=1/5 (5 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction is finished, and preparing the residue by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% NH ₃H₂ O); gradient: 20-40/9 min, compound 55 (5.35 mg, brown solid, 97.494% purity) was obtained. Yield: 4.03%.

LCMS(ESI)m/z calcd.for C₁₂H₁₄N₄O₃[M+H]⁺263.1;found 262.7;1H NMR(400MHz,CD₃OD):δ＝8.35(dd,J＝7.5,1.7Hz,1H),8.17(s,1H),7.43(dd,J＝6.9,1.8Hz,1H),7.19(s,1H),6.38(t,J＝7.2Hz,1H),5.32(s,2H),3.61(d,J＝6.6Hz,1H),1.35(d,J＝7.0Hz,3H).

EXAMPLE 31 Synthesis of Compound 56

Synthesis of Compound 56-1

To a 50mL single vial was added compound SM1 (500 mg,5.05 mmol), DCM (10 mL), CBr ₄ (1.67 g,5.05 mmol) and PPh ₃ (1.32 g,5.05 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 56-1 (900 mg, brown oil). Yield: 55.05%.

LCMS(ESI)m/z calcd.for C₄H₄BrNO[M+H]⁺161.9；found 357.1。

Synthesis of Compound 56-2

A50 mL single-port flask was charged with compound 56-1 (500 mg,3.09 mmol), DMF (10 mL), 2-hydroxy-3-nitropyridine (649 mg,4.63 mmol), and potassium carbonate (853 mg,6.17 mmol) in this order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (30 mL), and extracted three times with EA (10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/ea=5/1 to 1/1 to give 56-2 (420 mg, yellow solid, purity 97.028%). Yield: 59.69%.

LCMS(ESI)m/z calcd.for C₉H₇N₃O₄[M+H]⁺222.0;found 222.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.43(dt,J＝7.7,2.3Hz,1H),8.37(s,1H),8.31(dd,J＝6.6,2.0Hz,1H),8.15(d,J＝0.5Hz,1H),6.52-6.45(m,1H),5.17(s,2H).

Synthesis of Compound 56-3

A50 mL single-port flask was charged with compound 56-2 (400 mg,1.81 mmol), etOH/H2 ₂ O=2/1 (12 mL), iron powder (505 mg,9.04 mmol), and ammonium chloride (254 mg,9.04 mmol) in this order. The reaction was carried out at 60℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to dryness. Compound 56-3 (350 mg, brown solid, 88.792% purity) is obtained. Yield: 89.88%.

LCMS(ESI)m/z calcd.for C₉H₉N₃O₂[M+H]⁺192.1;found 192.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.33(s,1H),8.00(s,1H),6.94(dd,J＝6.8,1.5Hz,1H),6.42(dd,J＝7.1,1.5Hz,1H),6.04(t,J＝7.0Hz,1H),5.10(s,2H),5.00(s,2H).

Synthesis of Compound 56-4

A50 mL single vial was charged with compound 56-3 (400 mg,2.09 mmol), DMF (10 mL), N-fluorenylmethoxycarbonyl-L-alanine (1307 mg,4.18 mmol), HATU (1591 mg,4.18 mmol) and DIEA (1082 mg,8.37 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (50 mL), and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 56-4 (300 mg, yellow solid, 67.963% purity). Yield: 20.07%.

LCMS(ESI)m/z calcd.for C₂₇H₂₄N₄O₅[M+H]⁺485.2;found 485.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.27(s,1H),8.35(s,1H),8.22(d,J＝7.0Hz,1H),8.04(s,1H),7.99-7.79(m,3H),7.81-7.67(m,2H),7.60-7.23(m,5H),6.31(t,J＝7.1Hz,1H),5.10(s,2H),4.36-4.07(m,4H),1.30(d,J＝7.2Hz,3H).

Synthesis of Compound 56

To a 25mL single vial was added compound 56-4 (200 mg,0.185 mmol) followed by piperidine/DCM=1/5 (6 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction, and preparing residues by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% NH ₃H₂ O); gradient: 25-45/10 min, compound 56 (5.5 mg, white solid, 99.562% purity) was obtained. Yield: 5.07%.

LCMS(ESI)m/z calcd.for C₁₂H₁₄N₄O₃[M+H]⁺263.1;found 262.7;¹H NMR(400MHz,CD₃OD):δ＝8.35(dd,J＝7.5,1.7Hz,1H),8.15(s,1H),7.94(s,1H),7.44(dd,J＝6.9,1.7Hz,1H),6.37(t,J＝7.2Hz,1H),5.18-5.12(m,2H),3.66-3.52(m,1H),1.35(d,J＝7.0Hz,3H).

EXAMPLE 32 Synthesis of Compound 57

Synthesis of Compound 57-1

A50 mL single vial was charged with compound SM1 (1 g,8.68 mmol), triphenylphosphine (2.28 g,8.68 mmol), carbon tetrabromide (2.89 g,8.68 mmol) and DCM (10 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction, the reaction mixture was concentrated to dryness, the residue was purified by chromatography with an eluent ratio of PE/ea=20/1 to 10/1, filtered and concentrated to dryness to give compound 57-1 (0.2 g, white solid, purity 94.037%). Yield: 12.64%.

LCMS(ESI)m/z calcd.for C₄H₄BrNS[M+H]⁺177.92and 179.92；found 177.9and 179.9。

Synthesis of Compound 57-2

A100 mL single vial was charged with compound 57-1 (150 mg,0.84 mmol), 42-2 (356 mg,1.26 mmol), K ₂CO₃ (233 mg,1.68 mmol), and ACN (5 mL) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After completion of the reaction, poured into water (5 mL), extracted three times with EA (15 mL), the reaction concentrated to dryness and the residue isolated by prep. plate (DCM/meoh=15/1) to give compound 57-2 (90 mg, yellow solid, 90.495% purity). The yield was 25.47%.

LCMS(ESI)m/z calcd.forC₁₇H₂₂N₄O₄S[M+H]⁺379.14;found 379.0;¹H NMR(400MHz,DMSO_d₆):δ＝9.23(s,1H),8.25(dd,J＝7.4,1.7Hz,1H),7.76(d,J＝3.3Hz,1H),7.70(d,J＝3.3Hz,1H),7.57(dd,J＝6.9,1.8Hz,1H),7.47(d,J＝7.0Hz,1H),6.35(t,J＝7.1Hz,1H),5.54-5.40(m,2H),4.22-4.07(m,1H),1.39(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 57

A100 mL single vial was charged with compound 57-2 (90 mg,0.237 mmol) followed by HCl (g)/1, 4-dioxane (5 mL). After the reaction was completed at 25℃for 2 hours, the reaction mixture was concentrated to dryness to give compound 57 (49.1 mg, white solid, purity 99.530%). Yield: 74.03%.

LCMS(ESI)m/z calcd.for C₁₂H₁₄N₄O₂S[M+H]⁺279.08;found 279.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.06(s,1H),8.31-8.25(m,3H),8.24(d,J＝1.7Hz,1H),7.77(d,J＝3.3Hz,1H),7.71(d,J＝3.3Hz,1H),7.66(dd,J＝6.9,1.8Hz,1H),6.38(t,J＝7.2Hz,1H),5.50(s,2H),4.32-4.24(m,1H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 33 Synthesis of Compound 58

Synthesis of Compound 58-1

A50 mL single vial was charged with compound SM1 (100 mg,0.588 mmol), DMF (5 mL), 42-2 (249 mg,0.882 mmol) and potassium carbonate (162.53 mg,1.176 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 58-1 (100 mg, yellow solid, 99.010% purity). Yield: 44.37%.

LCMS(ESI)m/z calcd.for C₁₇H₂₂N₄O₄S[M+H]⁺379.14;found 379.0;¹H NMR(400MHz,DMSO_d₆):δ＝9.21(s,1H),9.06(s,1H),8.23(dd,J＝7.4,1.7Hz,1H),7.51(dd,J＝4.9,1.8Hz,2H),7.47(d,J＝6.9Hz,1H),6.31(t,J＝7.1Hz,1H),5.38-5.21(m,2H),4.21-4.06(m,1H),1.33(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 58

A25 mL single vial was charged with compound 58-1 (60 mg,0.1581 mmol) followed by 4N HCl/1,4-dioxane (5 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 58 (37.03 mg, yellow solid, purity 97.12%). Yield: 72.23%.

LCMS(ESI)m/z calcd.forC₁₂H1₄N₄O₂S[M+H]⁺279.08;found 279.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.98(s,1H),9.07(d,J＝1.9Hz,1H),8.36(s,3H),8.21(dd,J＝7.4,1.5Hz,1H),7.59(dd,J＝6.8,1.7Hz,1H),7.55(d,J＝1.7Hz,1H),6.34(t,J＝7.1Hz,1H),5.37-5.25(m,2H),4.33-4.20(m,1H),1.40(d,J＝6.9Hz,3H).

EXAMPLE 34 Synthesis of Compound 59

Synthesis of Compound 59-1a

To a 50mL single vial was added compound SM1 (200 mg,2.04 mmol) followed by DCM (5 mL), msCl (279 mg,2.45 mmol), TEA (309 mg,3.06 mmol) at 0deg.C. The reaction was carried out at 0℃for 2 hours under nitrogen protection. After the completion of the reaction, the reaction mixture was concentrated to dryness to give crude 59-1a (500 mg, yellow oil, purity 28.763%). Yield: 21.45%.

LCMS(ESI)m/z calcd.for C6H8O4S[M+H]⁺177.01found 173.0。

Synthesis of Compound 59-2

A50 mL single vial was charged with compound 59-1a (200 mg,1.25 mmol), DMF (5 mL), 42-2 (423 mg,1.50 mmol), and potassium carbonate (345 mg,2.50 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (10 mL), and extracted three times with EA (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 59-2 (100 mg, yellow solid, 98.368% purity). Yield: 21.75%.

LCMS(ESI)m/z calcd.for C₁₈H₂₃N3O₅[M+H]⁺362.16;found 362.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.20(dd,J＝7.4,1.7Hz,1H),7.61(dd,J＝1.5,0.9Hz,1H),7.56-7.34(m,2H),6.46-6.36(m,2H),6.30(t,J＝7.1Hz,1H),5.24-5.11(m,2H),4.23-4.03(m,1H),1.35(s,9H),1.24(d,J＝7.1Hz,3H).

Synthesis of Compound 59

A25 mL single vial was charged with compound 59-2 (50 mg,0.138 mmol) and 4N HCl (g)/1, 4-dioxane (1 mL) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 59 (20.73 mg, light brown solid, purity 99.055%). Yield: 39.64%.

LCMS(ESI)m/z calcd.for C₁₃H₁₅N₃O₃[M+H]⁺262.1;found 262.1;¹H NMR(400MHz,DMSO_d₆):δ＝10.02(s,1H),8.26(s,3H),8.20(dd,J＝7.4,1.4Hz,1H),7.62(s,1H),7.53(dd,J＝6.8,1.5Hz,1H),6.43(s,2H),6.34(t,J＝7.1Hz,1H),5.21(s,2H),4.32(s,1H),1.40(d,J＝6.9Hz,3H).

EXAMPLE 35 Synthesis of Compound 60

Synthesis of Compound 60-1

To a 25mL single vial was added compound 42-2 (200 mg,0.708 mmol), SM1 (131 mg,1.06 mmol), K ₂CO₃ (196 mg,1.42 mmol), and DMF (5 mL) in order. The reaction was carried out at 50℃for 16 hours under nitrogen. After the completion of the reaction, the reaction mixture was cooled to room temperature, poured into water (40 mL), and extracted three times with EA (40 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of PE/etoac=1/1 to give compound 60-1 (100 mg, yellow solid, 99.019% purity). Yield: 43.08%.

LCMS(ESI)m/z calcd.For C₁₆H₂₅N₃O₄[M+H]⁺324.19;found 324.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.95(s,1H),8.40(d,J＝7.4Hz,1H),7.85(dd,J＝4.9,1.7Hz,1H),7.49(d,J＝6.7Hz,1H),6.94(dd,J＝7.8,5.0Hz,1H),5.30(dt,J＝12.3,6.2Hz,1H),4.31-4.11(m,1H),1.41(s,9H),1.34(t,J＝5.9Hz,6H),1.27(d,J＝7.2Hz,3H).

Synthesis of Compound 60

To a 25mL single vial was added compound 60-1 (80 mg,0.247 mmol) followed by DCM/TFA=2/1 (3 mL). After the reaction was completed at 25℃for 2 hours, the reaction mixture was concentrated to dryness to give 60.56 mg of a yellow solid having a purity of 99.808%). Yield: 91.65%.

LCMS(ESI)m/z calcd.for C₁₁H₁₇N₃O₂[M+H]⁺224.1;found224.1;1H NMR(400MHz,DMSO_d₆):δ＝10.10(s,1H),8.54(dd,J＝7.8,1.8Hz,1H),7.82(dd,J＝5.0,1.8Hz,1H),6.93(dd,J＝7.8,5.0Hz,1H),5.38-5.23(m,1H),3.44(q,J＝7.0Hz,1H),1.34(dd,J＝6.2,2.3Hz,6H),1.25(d,J＝7.0Hz,3H).

EXAMPLE 36 Synthesis of Compound 61

Synthesis of Compound 61

A50 mL single vial was charged with compound 42-2 (40 mg,0.142 mmol) followed by HCl (g)/1, 4-dioxane (5 mL). After the completion of the reaction, the reaction mixture was concentrated to dryness to give compound 61 (21.1 mg, yellow solid, purity 98.32%). Yield: 67.25%.

LCMS(ESI)m/z calcd.forC₈H₁₁N₃O₂[M+H]⁺182.09;found 182.1;¹H NMR(400MHz,DMSO_d₆):δ＝12.10(s,1H),9.94(s,1H),8.36(s,3H),8.23(dd,J＝7.3,1.6Hz,1H),7.17(d,J＝6.1Hz,1H),6.25(t,J＝6.9Hz,1H),4.42-4.19(m,1H),1.38(dd,J＝15.6,7.0Hz,3H).

EXAMPLE 37 Synthesis of Compound 62

Synthesis of Compound 62

A25 mL single vial was charged with compound 62-1 (50 mg,0.116 mmol) and 4N HCl/1,4-dioxane (1 mL) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the reaction was completed, it was concentrated to dryness to give compound 62 (22.59 mg, light brown solid, purity 96.812%). Yield: 57.12%.

LCMS(ESI)m/z calcd.for C₁₆H₁₈N₄O₄[M+H]⁺331.1;found 331.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.97(s,1H),9.00(d,J＝1.6Hz,1H),8.39(d,J＝4.1Hz,3H),8.31-8.25(m,2H),7.64(dd,J＝6.8,1.7Hz,1H),7.42(d,J＝8.2Hz,1H),6.39(t,J＝7.1Hz,1H),5.38(s,2H),4.28-4.23(m,1H),3.88(s,3H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 38 Synthesis of Compound 63

Synthesis of Compound 63

A25 mL single vial was charged with compound 63-1 (50 mg,0.124 mmol) followed by 4N HCl/1,4-dioxane (1 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, it was concentrated to dryness to give compound 63 (23.6 mg, light brown solid, purity 97.475%). Yield: 61.42%.

LCMS(ESI)m/z calcd.for C₁₅H₁₈N₄O₃[M+H]⁺303.1;found 303.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.97(s,1H),8.62(s,1H),8.40(s,3H),8.26(d,J＝7.3Hz,1H),8.10(d,J＝7.8Hz,1H),7.72(d,J＝6.8Hz,1H),7.49(d,J＝8.1Hz,1H),6.41(t,J＝7.1Hz,1H),5.43(s,2H),4.59(s,2H),4.30-4.23(m,1H),1.38(d,J＝6.8Hz,3H).

EXAMPLE 39 Synthesis of Compound 64

Synthesis of Compound 64-1

To a 25mL single-port flask, compound 42-2 (340 mg,1.21 mmol), DMF (5 mL), 3- (bromomethyl) benzaldehyde (200 mg,1.00 mmol) and potassium carbonate (278 mg,2.01 mmol) were added sequentially. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (25 mL), and extracted three times with EA (60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate to give compound 64-1 (300 mg, yellow solid, 98.607% pure) with a ratio of PE/ea=2/1. Yield: 73.52%.

LCMS(ESI)m/z calcd.for C₂₁H₂₅N₃O₅[M+H]⁺400.18;found 400.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.99(s,1H),9.24(s,1H),8.25(dd,J＝7.4,1.7Hz,1H),7.84(dd,J＝6.6,5.3Hz,2H),7.73-7.56(m,3H),7.48(d,J＝6.8Hz,1H),6.35(t,J＝7.1Hz,1H),5.30-5.25(m,1H),4.21-4.09(m,2H),1.38(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 64-2

A50 mL single port flask was charged with compound 64-1 (200 mg,0.50 mmol), DMF (5 mL), dimethylamine hydrochloride (81 mg,1.00 mmol), STAB (212 mg,1.00 mmol) and acetic acid (90 mg,1.50 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into saturated sodium bicarbonate solution (20 mL), and extracted three times with DCM (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=10/1 to give compound 64-2 (150 mg, white solid, purity 99.23%). Yield: 69.38%.

LCMS(ESI)m/z calcd.for C₂₃H₃₂N₄O₄[M+H]⁺429.24;found 429.2;¹H NMR(400MHz,CDCl₃):δ＝8.84(s,1H),8.28(dd,J＝7.4,1.8Hz,1H),7.30-7.16(m,4H),7.10(d,J＝7.1Hz,1H),6.97(dd,J＝6.9,1.7Hz,1H),6.16(t,J＝7.2Hz,1H),5.24-5.01(m,2H),4.27(s,1H),3.41(s,2H),2.21(s,6H),1.38(s,9H),1.31(s,3H).

Synthesis of Compound 64

A25 mL single vial was charged with compound 64-2 (70 mg,0.163 mmol) followed by 4N HCl/dioxane (3 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 64 (46.73 mg, yellow solid, purity 97.569%). Yield: 69.69%.

LCMS(ESI)m/z calcd.for C₁₈H₂₄N₄O₂[M+H]⁺329.19;found 328.9;¹H NMR(400MHz,DMSO_d₆):δ＝11.31(s,1H),9.98(s,1H),8.50(s,J＝4.1Hz,3H),8.23(dd,J＝7.4,1.3Hz,1H),7.66(dd,J＝6.8,1.4Hz,1H),7.62-7.48(m,2H),7.43(t,J＝7.6Hz,1H),7.34(d,J＝7.7Hz,1H),6.35(t,J＝7.1Hz,1H),5.22(s,2H),4.38-4.20(m,3H),2.65(d,J＝4.8Hz,6H),1.41(d,J＝6.9Hz,3H).

In various embodiments of the present invention, compound 65 may be synthesized in a similar manner as described above.

EXAMPLE 40 Synthesis of Compound 66

Synthesis of Compound 66-1

To a 25mL single vial was added compound 44-2 (170 mg,0.60 mmol), DMF (5 mL), 3- (bromomethyl) benzaldehyde (100 mg,0.50 mmol) and potassium carbonate (139 mg,1.00 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, poured into a saturated NH ₄ Cl solution (25 mL), and extracted three times with EA (60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate to give compound 66-1 (150 mg, yellow solid, 98.789% pure) in the ratio PE/ea=2/1. Yield: 73.65%.

LCMS(ESI)m/z calcd.for C₂₁H₂₅N₃O₅[M+H]⁺400.18;found 400.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.98(s,1H),9.23(s,1H),8.26(dd,J＝7.4,1.7Hz,1H),7.89(d,J＝8.2Hz,2H),7.58(dd,J＝6.9,1.7Hz,1H),7.47(d,J＝8.0Hz,3H),6.35(t,J＝7.1Hz,1H),5.32-5.22(m,2H),4.20-4.04(m,1H),1.38(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 66-2

A50 mL single port flask was charged with compound 66-1 (200 mg,0.50 mmol), DMF (5 mL), dimethylamine hydrochloride (81 mg,1.00 mmol), STAB (212 mg,1.00 mmol) and acetic acid (90 mg,1.50 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into saturated sodium bicarbonate solution (20 mL), and extracted three times with DCM (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 66-2 (90 mg, white solid, 95.889% purity). Yield: 40.23%.

LCMS(ESI)m/z calcd.for C₂₃H₃₂N₄O₄[M+H]⁺429.24;found 429.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.23(s,1H),8.22(dd,J＝7.4,1.7Hz,1H),7.55(dd,J＝6.9,1.7Hz,1H),7.51(dd,J＝19.9,4.2Hz,1H),7.26(s,4H),6.31(t,J＝7.1Hz,1H),5.27-5.04(m,2H),4.24-4.01(m,1H),3.43(s,2H),2.14(s,6H),1.37(s,9H),1.24(d,J＝7.2Hz,3H).

Synthesis of Compound 66

To a 25mL single vial was added compound 66-2 (40 mg,0.093 mmol) followed by 4N HCl/dioxane (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 66 (12.16 mg, yellow solid, purity 98.619%). Yield: 32.129%.

LCMS(ESI)m/z calcd.for C₁₈H₂₄N₄O₂[M+H]⁺329.19;found 328.9;¹H NMR(400MHz,DMSO_d₆):δ＝8.23(dd,J＝7.4,1.5Hz,1H),7.65(dd,J＝6.8,1.6Hz,1H),7.54(d,J＝8.1Hz,2H),7.39(d,J＝8.1Hz,2H),6.39(t,J＝7.1Hz,1H),5.22(s,2H),4.26(d,J＝4.0Hz,3H),2.69(s,6H),1.41(d,J＝6.9Hz,3H).

In various embodiments of the present invention, compounds 67-69 may be synthesized in a similar manner as described above.

EXAMPLE 41 Synthesis of Compound 70

Synthesis of Compound 70-1

To a 50mL single vial was added compound 64-1 (300 mg,0.75 mmol), meOH (5 mL), methylamine hydrochloride (233 mg,7.49 mmol), and sodium cyanoborohydride (94 mg,1.50 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=10/1 to give compound 70-1 (150 mg, white solid, 92.769% purity). Yield: 44.70%.

LCMS(ESI)m/z calcd.for C₂₂H₃₀N₄O₄[M+H]⁺415.23;found 415.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.13(s,1H),8.14(dd,J＝7.4,1.7Hz,1H),7.48-7.35(m,2H),7.35-7.13(m,4H),6.23(t,J＝7.1Hz,1H),5.66(s,1H),5.17-4.95(m,2H),4.10-3.97(m,1H),3.84(s,2H),2.35(s,3H),1.22(s,9H),1.14(d,J＝7.1Hz,3H).

Synthesis of Compound 70

A25 mL single vial was charged with compound 70-1 (30 mg,0.077 mmol) followed by 4N HCl/dioxane (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 70 (7.35 mg, yellow solid, purity 95.525%). Yield: 25.07%.

LCMS(ESI)m/z calcd.for C₁₇H₂₂N₄O₂[M+H]⁺315.17;found 314.8;¹H NMR(400MHz,DMSO_d₆):δ＝9.99(s,1H),9.36(s,2H),8.36(d,J＝3.8Hz,3H),8.23(dd,J＝7.4,1.7Hz,1H),7.67-7.62(m,1H),7.54-7.40(m,2H),7.41(d,J＝7.7Hz,1H),7.34-7.22(m,1H),6.35(t,J＝7.1Hz,1H),5.20(s,2H),4.34-4.20(m,1H),4.06(dd,J＝10.7,4.9Hz,2H),3.37(s,3H),1.40(d,J＝6.9Hz,3H).

EXAMPLE 42 Synthesis of Compound 71

Synthesis of Compound 71-1

To a 25mL single vial was added compound 66-1 (280 mg,0.70 mmol), meOH (2 mL), methylamine hydrochloride (217 mg,7.00 mmol), and sodium cyanoborohydride (88 mg,1.40 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated to dryness. The residue was purified by prep. plate with the ratio of DCM/meoh=10/1 to give compound 71-1 (90 mg, white solid, 97.266% purity). Yield: 30.13%.

LCMS(ESI)m/z calcd.for C₂₂H₃₀N₄O₄[M+H]⁺415.23;found 415.4;¹H NMR(400MHz,DMSO_d₆):δ＝9.13(s,1H),8.12(dd,J＝7.3,1.5Hz,1H),7.45(dd,J＝6.9,1.7Hz,1H),7.42-7.20(m,5H),6.22(t,J＝7.1Hz,1H),5.09(s,2H),4.07-3.98(m,1H),3.86(s,2H),3.34(s,1H),2.33(s,3H),1.30(s,9H),1.14(d,J＝7.1Hz,3H).

Synthesis of Compound 71

A25 mL single vial was charged with compound 71-1 (20 mg,0.048 mmol) and 4N HCl/dioxane (2 mL) in sequence. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 71 (6.05 mg, white solid, purity 97.363%). Yield: 31.60%.

LCMS(ESI)m/z calcd.for C₁₇H₂₂N₄O₂[M+H]⁺315.17;found 314.8;¹H NMR(400MHz,DMSO_d₆):δ＝9.99(s,1H),9.40(s,2H),8.35(s,3H),8.22(dd,J＝7.4,1.7Hz,1H),7.66(dd,J＝6.9,1.7Hz,1H),7.52(d,J＝8.1Hz,2H),7.38(t,J＝14.4Hz,2H),6.35(t,J＝7.1Hz,1H),5.20(s,2H),4.38-4.19(m,1H),4.10-3.95(m,2H),3.39(s,3H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 43 Synthesis of Compound 72

Synthesis of Compound 72-1

To a 100mL three-necked flask was added compound SM1 (500 mg,3.14 mmol) followed by MeOH (10 mL). After cooling to 0deg.C, naBH ₄ (178 mg,4.71 mmol) was added. The reaction was carried out at 25℃for 16 hours under nitrogen. After completion of the reaction, the reaction was quenched by addition of water (20 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/ea=10/1 to 1/1 to give compound 72-1 (4500 mg, yellow oil, 97.043% pure). Yield: 86.25%.

LCMS(ESI)m/z calcd.for C₁₀H₁₁NO[M+H]⁺162.08;found 162.1;¹H NMR(400MHz,DMSO_d₆):δ＝7.47(d,J＝0.7Hz,1H),7.35(d,J＝8.4Hz,1H),7.26(d,J＝3.0Hz,1H),7.12(dd,J＝8.4,1.4Hz,1H),6.37(dd,J＝3.0,0.7Hz,1H),5.03(s,1H),4.56(s,2H),3.73(s,3H).

Synthesis of Compound 72-2

A100 mL single vial was charged with compound 72-1(450mg,2.7916mmol)、42-2(785.3mg,2.7916mmol)、DBAD(1928.38mg,8.3748mmol)、PPh3(2196.63mg,8.3748mmol) followed by THF (5 mL). The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was poured into a saturated sodium bicarbonate solution (60 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/ea=10/1 to 1/1 to give compound 72-2 (120 mg, white solid, purity 66.829%). Yield: 5.63%.

LCMS(ESI)m/z calcd.for C₂₃H₂₈N₄O₄[M+H]⁺425.21；found 425.3。

Synthesis of Compound 72

To a 50mL single vial was added compound 72-2 (100 mg,0.235 mmol), DCM (4 mL), and trifluoroacetic acid (1 mL) in this order. The reaction was carried out at room temperature for 2 hours under nitrogen protection. After the reaction, the reaction solution was concentrated to dryness, and the residue was prepared by high performance liquid chromatography with the following parameters: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% TFA); gradient: 25-50/8 min, 72 (14.84 mg, yellow solid, 99.319% purity) was obtained. Yield: 15.28%.

LCMS(ESI)m/z calcd.for C₁₈H₂₀N₄O₂[M+H]⁺325.16;found 324.8;¹H NMR(400MHz,DMSO_d₆):δ＝10.02(s,1H),8.31-8.05(m,4H),7.64(dd,J＝6.9,1.8Hz,1H),7.57(s,1H),7.40(d,J＝8.4Hz,1H),7.33(d,J＝3.1Hz,1H),7.21(dd,J＝8.5,1.6Hz,1H),6.41-6.30(m,1H),6.32-6.25(m,1H),5.26-5.15(m,2H),4.37-4.25(m,1H),3.75(s,3H),1.39(d,J＝6.9Hz,3H).

EXAMPLE 44 Synthesis of Compound 73

Synthesis of Compound 73-1

To a 250mL single vial was added, in order, compound SM1 (5 g,28.54 mmol), DCM (100 mL), tetrabutylammonium bisulfate (1.21 g,3.42 mmol), tsCl (10.87 g,57.08 mmol), DMAP (0.28 g,2.28 mmol), and TEA (5.77 g,57.08 mmol). The reaction was carried out at 25℃for 16 hours under nitrogen. After completion of the reaction, the reaction was quenched by addition of water (200 mL) and extracted three times with DCM (450 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. Concentrating to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/ea=20/1 to 5/1 to give compound 73-1 (2 g, yellow solid, purity 94.238%). Yield: 20.00%.

LCMS(ESI)m/z calcd.for C₁₇H₁₅NO₄S[M+H]⁺330.07;found 352.0;¹H NMR(400MHz,DMSO_d₆):δ＝8.27(d,J＝1.2Hz,1H),8.06(d,J＝8.7Hz,1H),7.97-7.85(m,4H),7.40(d,J＝8.1Hz,2H),6.98(d,J＝3.3Hz,1H),3.86(s,3H),2.32(s,3H).

Synthesis of Compound 73-2

To a 100mL three-necked flask, compound 73-1 (500 mg,1.51 mmol) and THF (10 mL) were sequentially added. After cooling to 0deg.C, LAH (57 mg,1.51 mmol) was added. The reaction was carried out at 0℃for 2 hours under nitrogen protection. After completion of the reaction, the reaction was quenched by addition of water (20 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by preparative plate to give compound 73-2 (380 mg, off-white solid, 90.949% pure) in the ratio PE/ea=2/1. Yield: 75.53%.

LCMS(ESI)m/z calcd.for C₁₇H₁₅NO₄S[M+H]⁺302.08；found 284.1。

Synthesis of Compound 73-3

A100 mL single vial was charged with compound 73-2 (400 mg,1.32 mmol), THF (5 mL), 42-2 (447 mg,1.59 mmol), DIAD (401 mg,7.08 mmol), and PPh ₃ (520 mg,1.98 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was poured into a saturated sodium bicarbonate solution (60 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with eluent ratio DCM/meoh=100/1 to 50/1 to give compound 73-3 (120 mg, white solid, purity 98.673%). Yield: 15.82%.

LCMS(ESI)m/z calcd.for C₂₉H₃₂N₄O₆S[M+H]⁺565.20;found 565.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.21(s,1H),8.20(dd,J＝7.4,1.7Hz,1H),8.02-7.70(m,4H),7.70-7.16(m,6H),6.81(d,J＝3.6Hz,1H),6.29(t,J＝7.1Hz,1H),5.34-5.06(m,2H),4.23-4.02(m,1H),2.30(s,3H),1.38(s,9H),1.23(d,J＝7.1Hz,3H).

Synthesis of Compound 73-4

To a 50mL single vial was added compound 73-3 (110 mg,0.19 mmol), DCM (4 mL), and trifluoroacetic acid (1 mL) in this order. The reaction was carried out at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 73-4 (90 mg, yellow solid, purity 98.043%). Yield: 97.43%.

LCMS(ESI)m/z calcd.for C₂₄H₂₄N₄O₄S[M+H]⁺465.15;found 465.0;¹H NMR(400MHz,DMSO_d₆):δ＝9.98(s,1H),8.38-7.94(m,4H),7.94-7.76(m,4H),7.66(dd,J＝6.9,1.7Hz,1H),7.54(s,1H),7.36(dd,J＝11.8,4.8Hz,3H),6.82(d,J＝3.7Hz,1H),6.33(t,J＝7.1Hz,1H),5.22(s,2H),2.31(s,3H),1.37(d,J＝6.9Hz,3H).

Synthesis of Compound 73

To a 50mL three-necked flask were added, in order, compound 73-4 (110 mg,0.89 mmol), meOH (3 mL), and K ₂CO₃ (163 mg,1.18 mmol). The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, it was filtered and concentrated to dryness. The residue was prepared by high performance liquid chromatography with the following parameters: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% NH ₃H₂ O); gradient: 28-48/8 min, compound 73 (9.6 mg, yellow solid, 98.681% pure) was obtained. Yield: 12.91%.

LCMS(ESI)m/z calcd.for C₁₇H₁₈N₄O₂[M+H]⁺311.14;found 311.1;¹H NMR(400MHz,DMSO_d₆):δ＝11.13(s,1H),10.32(s,1H),8.24(dd,J＝7.3,1.8Hz,1H),7.54-7.44(m,2H),7.35(d,J＝8.4Hz,2H),7.11(dd,J＝8.3,1.5Hz,1H),6.40(s,1H),6.29-6.19(m,1H),5.20(d,J＝6.3Hz,2H),3.53-3.37(m,1H),3.34(s,2H),1.19(dd,J＝23.9,7.1Hz,3H).

EXAMPLE 45 Synthesis of Compound 74

Synthesis of Compound 74-1

A50 mL single vial was charged with compound SM1 (150 mg,0.545 mmol), DCE (5 mL), 42-2 (184 mg, 0.650 mmol), copper acetate (294 mg, 1.345 mmol), and pyridine (216 mg,2.725 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into saturated aqueous sodium bicarbonate (10 mL) and extracted three times with DCM (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. And concentrated to dryness. The residue was purified by preparative plate to give compound 74-1 (90 mg, yellow solid, 97.850% pure) in the ratio PE/ea=2/1. Yield: 30.63%.

LCMS(ESI)m/z calcd.for C₂₇H₃₈N₄O₄Si[M+H]⁺511.27;found 511.4;¹H NMR(400MHz,DMSO_d₆):δ＝9.27(s,1H),8.36(dd,J＝7.4,1.8Hz,1H),7.75-7.38(m,5H),7.23-7.00(m,1H),6.73(d,J＝2.7Hz,1H),6.40(t,J＝7.2Hz,1H),4.27-4.10(m,1H),1.40(s,9H),1.31(d,J＝7.0Hz,3H),0.92(s,9H),0.68(s,6H).

Synthesis of Compound 74

To a 25mL single vial was added compound 74-1 (50 mg,0.0979 mmol) followed by TFA/DCM=1/5 (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction, and preparing residues by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% TFA); gradient: 25-50/8 min, compound 74 (3.01 mg, brown solid, 97.978% pure, TFA salt) was obtained. Yield: 8.78%.

LCMS(ESI)m/z calcd.for C₁₆H₁₆N₄O₂[M+H]⁺297.13;found 296.8;¹H NMR(400MHz,DMSO_d₆):δ＝11.38(s,1H),10.03(s,1H),8.32(dd,J＝7.4,1.5Hz,1H),8.19(s,3H),7.56(d,J＝1.5Hz,1H),7.53-7.34(m,3H),7.08(dd,J＝8.6,1.9Hz,1H),6.52(s,1H),6.38(t,J＝7.1Hz,1H),4.34(s,1H),1.41(d,J＝6.9Hz,3H).

EXAMPLE 46 Synthesis of Compound 75

Synthesis of Compound 75-1

A50 mL single vial was charged with compound SM1 (200 mg,0.727 mmol), DCE (5 mL), 42-2 (248 mg,0.872 mmol), copper acetate (390 mg,2.180 mmol), and pyridine (287 mg,3.633 mmol) in sequence. The reaction was carried out at 50℃for 16 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into saturated aqueous sodium bicarbonate (10 mL) and extracted three times with DCM (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. And concentrated to dryness. The residue was purified by preparative plate to give compound 75-1 (90 mg, yellow solid, 98.735% pure) in the ratio PE/ea=2/1. Yield: 23.94%.

LCMS(ESI)m/z calcd.for C₂₇H₃₈N₄O₄Si[M+H]⁺511.27;found 511.4;¹H NMR(400MHz,DMSO_d₆):δ＝9.29(s,1H),8.49-8.34(m,1H),7.71(d,J＝8.4Hz,1H),7.61-7.52(m,1H),7.48-7.38(m,2H),7.35-7.23(m,1H),7.09(dd,J＝28.5,7.4Hz,1H),6.43(t,J＝7.1Hz,1H),6.29(d,J＝2.6Hz,1H),4.23-4.14(m,1H),1.40(s,9H),1.35-1.27(m,3H),0.91(s,9H),0.68(s,6H).

Synthesis of Compound 75

To a 25mL single vial was added compound 75-1 (50 mg,0.0979 mmol) followed by TFA/DCM=1/5 (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction, and preparing residues by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% TFA); gradient: 25-50/8 min, compound 75 (3.01 mg, brown solid, 97.978% purity) was obtained. Yield: 8.78%.

LCMS(ESI)m/z calcd.for C₁₆H₁₆N₄O₂[M+H]⁺297.13;found 297.1;¹H NMR(400MHz,DMSO_d₆):δ＝11.49(s,1H),10.06(s,1H),8.36(dd,J＝7.4,1.7Hz,1H),8.22(s,3H),7.54(d,J＝8.2Hz,1H),7.47-7.36(m,2H),7.22(t,J＝7.8Hz,1H),7.02(d,J＝7.3Hz,1H),6.42(t,J＝7.1Hz,1H),6.10(s,1H),4.40-4.25(m,1H),1.42(d,J＝6.9Hz,3H).

EXAMPLE 47 Synthesis of Compound 76

Synthesis of Compound 76-1

A50 mL single vial was charged with compound 70-1 (90 mg,0.217 mmol), DCM (5 mL), acryloyl chloride (39 mg,0.433 mmol) and TEA (66 mg,0.650 mmol) in sequence. The reaction was carried out at 25℃for 4 hours under nitrogen protection. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate (10 mL) and extracted three times with DCM (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. And concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 76-1 (60 mg, white solid, 93.727% purity). Yield: 55.31%.

LCMS(ESI)m/z calcd.for C₂₅H₃₂N₄O₅[M+H]⁺469.24;found 469.2;¹H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.22(dd,J＝7.4,1.7Hz,1H),7.70-7.40(m,2H),7.42-7.22(m,1H),7.25-7.03(m,3H),6.77(m,1H),6.31(t,J＝7.1Hz,1H),6.24-6.04(m,1H),5.74-5.69(m,1H),5.17(s,2H),4.55(t,J＝34.4Hz,2H),4.21-4.03(m,1H),2.87(dd,J＝38.4,35.1Hz,3H),1.34(s,9H),1.24(d,J＝7.1Hz,3H).

Synthesis of Compound 76

To a 25mL single vial was added compound 76-1 (70 mg,0.149 mmol) followed by TFA/DCM=1/5 (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. Concentrating to dryness after the reaction, and preparing residues by high performance liquid chromatography, wherein the related parameters are as follows: chromatographic column: XBIdge-1 um 19-150mm; mobile phase: acetonitrile-water (0.1% TFA); gradient: 20-40/8 min, compound 76 (12.19 mg, yellow solid, 99.938% purity) was obtained. Yield: 16.97%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₃[M+H]⁺368.18;found 368.9;¹H NMR(400MHz,DMSO_d₆):δ＝9.73(s,1H),8.45-7.80(m,J＝7.3Hz,4H),7.53(d,J＝6.8Hz,1H),7.33(t,J＝7.5Hz,1H),7.27-7.05(m,J＝21.0,10.1Hz,3H),6.84-6.60(m,1H),6.31(t,J＝7.1Hz,1H),6.12(d,J＝16.7Hz,1H),5.66(d,J＝10.2Hz,1H),5.20(s,2H),4.59(s,2H),4.39-4.25(m,J＝6.8Hz,1H),2.94(s,3H),1.45(d,J＝6.9Hz,3H).

Example 48: synthesis of Compound 77

Synthesis of Compound 77-1

A50 mL single vial was charged with compound 71-1 (60 mg,0.144 mmol), DCM (2 mL), acryloyl chloride (26 mg,0.289 mmol) and TEA (44 mg,0.432 mmol) in sequence. The reaction was carried out at 25℃for 3 hours under nitrogen protection. After the reaction was completed, the reaction solution was poured into saturated aqueous sodium bicarbonate (10 mL) and extracted three times with DCM (15 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. And concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 77-1 (40 mg, white solid, 98.815% purity). Yield: 58.31%.

LCMS(ESI)m/z calcd.for C₂₅H₃₂N₄O₅[M+H]⁺469.24;found 369.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.22(s,1H),8.21(dd,J＝7.4,1.7Hz,1H),7.51(dd,J＝25.2,6.8Hz,2H),7.38-7.02(m,4H),6.92-6.65(m,1H),6.41-6.17(m,1H),6.22-6.08(m,1H),5.84-5.58(m,1H),5.15(s,2H),4.54(t,J＝36.9Hz,2H),4.25-4.00(m,1H),2.87(dd,J＝40.2,34.8Hz,3H),1.34(s,9H),1.24(d,J＝8.4Hz,3H).

Synthesis of Compound 77

To a 25mL single vial was added compound 77-1 (40 mg,0.085 mmol) followed by TFA/DCM=1/5 (2 mL). The reaction was carried out at 25℃for 2 hours under nitrogen protection. After the completion of the reaction, the mixture was concentrated to dryness to give compound 77 (12.16 mg, yellow solid, purity 98.191%). Yield: 28.99%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₃[M+H]⁺368.18;found 369.1;¹H NMR(400MHz,DMSO_d₆):δ＝8.15(d,J＝7.1Hz,1H),7.52(d,J＝6.6Hz,1H),7.24(dd,J＝40.8,6.7Hz,3H),6.81-6.61(m,1H),6.32(t,J＝6.9Hz,1H),6.12(d,J＝16.8Hz,1H),5.66(d,J＝9.2Hz,1H),5.16(s,2H),4.62-4.42(m,2H),4.37-4.17(m,1H),2.93(s,3H),1.44(d,J＝6.8Hz,3H).

EXAMPLE 49 Synthesis of Compound 78

Synthesis of Compound 78-1

A250 mL single vial was charged with compound 42-1 (4 g,0.0363 mol), DMF (80 mL), boc-glycine (12.79 g,0.0726 mol), HATU (27.6 g,0.0726 mol) and DIEA (18.77 g,0.1452 mol). The reaction was carried out at 50℃for 3 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (400 mL), extracted eight times with EA (800 mL), concentrated to dryness, and the residue was separated by chromatography (DCM: meoh=100:1 to 30/1) to give compound 78-1 (3.4 g, white solid, purity 93.449%). The yield was 32.51%.

LCMS(ESI)m/z calcd.for C₁₂H₁₇N₃O₄[M+H]⁺268.12;found 268.2;¹H NMR(400MHz,DMSO_d₆):δ＝12.00(s,1H),9.17(s,1H),8.22(dd,J＝7.2,1.6Hz,1H),7.46-7.25(m,1H),7.10(dd,J＝6.6,1.7Hz,1H),6.22(t,J＝6.9Hz,1H),3.74(d,J＝6.1Hz,2H),1.39(s,9H).

Synthesis of Compound 78-2

A50 mL single vial was charged with compound 78-1 (300 mg,1.12 mmol), THF (10 mL), 2-pyridinemethanol (183 mg,1.68 mmol), DBAD (772 mg,3.35 mmol), and PPh ₃ (480 mg,3.35 mmol) in sequence. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was poured into a saturated sodium bicarbonate solution (60 mL) and extracted three times with EA (30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with an eluent ratio of PE/ea=10/1 to 1/1 to give compound 78-2 (120 mg, white solid, purity 98.659%). Yield: 29.46%.

LCMS(ESI)m/z calcd.for C₁₈H₂₂N₄O₄[M+H]⁺359.16;found 359.1;¹H NMR(400MHz,DMSO_d₆):δ＝9.19(s,1H),8.49(d,J＝4.7Hz,1H),8.25(d,J＝6.4Hz,1H),7.83-7.70(m,1H),7.58-7.45(m,1H),7.45-7.14(m,3H),6.32(t,J＝7.1Hz,1H),5.27(s,2H),3.73(d,J＝5.9Hz,2H),1.38(s,9H).

Synthesis of Compound 78

To a 50mL single vial was added compound 78-2 (60 mg,0.167 mmol), DCM (1.5 mL), and trifluoroacetic acid (0.3 mL) in this order. The reaction was carried out at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated to dryness and lyophilized to give 78 (43.5 mg, yellow solid, purity 99.211%). Yield: 93.23%.

LCMS(ESI)m/z calcd.for C₁₃H₁₄N4O₂[M+H]⁺259.11;found 258.7;¹H NMR(400MHz,DMSO_d₆):δ＝9.94(s,1H),8.51(d,J＝4.4Hz,1H),8.27(d,J＝7.1Hz,1H),8.10(s,3H),7.81(t,J＝7.6Hz,1H),7.59(d,J＝6.7Hz,1H),7.43-7.21(m,2H),6.36(t,J＝7.1Hz,1H),5.29(s,2H),3.98-3.76(m,2H).

EXAMPLE 50 Synthesis of Compound 79

Synthesis of Compound 79-1

To a 250mL single vial was added, in order, compound Int A (2 g,3.228 mmol), 1-Boc-piperazine (1.2 g, 6.458 mmol), pd ₂(dba)₃ (591 mg, 0.640 mmol), BINAP (254 mg, 1.29mmol), cesium carbonate (2.1 g, 6.458 mmol), and 1, 4-dioxane (80 mL). The reaction was carried out at 100℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated to dryness. The residue was purified by chromatography on a silica gel column with eluent ratio DCM/meoh=60/1 to 30/1 to give compound 79-1 (1.8 g, yellow solid, purity 86.364%). Yield: 73.33%.

LCMS(ESI)m/z calcd.for C₃₆H₃₈FN₅O₆[M+H]⁺656.3;found 656.3;¹H NMR(400MHz,DMSO_d₆):δ＝10.20(s,1H),10.07(s,1H),8.45(d,J＝5.2Hz,1H),7.76(d,J＝8.9Hz,2H),7.66-7.63(m,2H),7.50(s,1H),7.35(s,1H),7.22(d,J＝9.0Hz,2H),7.18-7.13(m,2H),6.41(d,J＝5.2Hz,1H),3.96(s,3H),3.53(br.s,4H),3.11-3.09(m,4H),1.48(s,4H),1.44(s,9H).

Synthesis of Compound 79-2

To a 50mL single vial was added compound 79-1 (300 mg,0.457 mmol), DCM (4 mL), and trifluoroacetic acid (2 mL) in sequence. The reaction was carried out at room temperature for 1 hour under nitrogen protection. After the reaction was completed, the reaction mixture was concentrated to dryness to give crude 79-2 (300 mg, yellow solid, purity 78.798%). Yield: 93.15%.

LCMS(ESI)m/z calcd.for C₃₁H₃₀FN₅O₄[M+H]⁺556.2；found 556.2。

Synthesis of Compound 62-1

To a 100mL single-port flask were added compound 42-2 (1 g, 3.552 mmol), methyl 6-bromomethylnicotinate (978 mg,4.251 mmol), potassium carbonate (979 mg,7.084 mmol), and acetonitrile (20 mL) in this order. The reaction was carried out at 25℃for 16 hours under nitrogen. After the reaction was completed, the reaction mixture was poured into a saturated NH ₄ Cl solution (60 mL) and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by chromatography on a silica gel column with eluent in the ratio DCM/meoh=100/1 to 50/1 to give compound 62-1 (1.2 g, white solid, purity 98.385%). Yield: 77.25%.

LCMS(ESI)m/z calcd.for C₂₁H₂₆N₄O₆[M+H]⁺431.2;found 431.3;¹H NMR(400MHz,DMSO_d₆):δ＝9.18(s,1H),9.00(d,J＝1.7Hz,1H),8.29-8.26(m,2H),7.55(dd,J＝6.9,1.8Hz,1H),7.45(d,J＝7.0Hz,1H),7.37(d,J＝8.2Hz,1H),6.35(t,J＝7.1Hz,1H),5.36(d,J＝4.8Hz,2H),4.18-4.11(m,1H),3.87(s,3H),1.36(s,9H),1.23(d,J＝7.2Hz,3H).

Synthesis of Compound 63-1

To a 100mL three-necked flask, compound 62-1 (1 g,2.317 mmol) and THF (20 mL) were sequentially added. After cooling to 0deg.C, LAH (132 mg,3.476 mmol) was added in portions. The reaction was carried out at 0℃for 4 hours under nitrogen protection. After completion of the reaction, the reaction was quenched by addition of water (60 mL) and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by silica gel chromatography with eluent ratio DCM/meoh=100/1 to 30/1 to give compound 63-1 (490 mg, off-white solid, 91.319% purity). Yield: 47.85%.

LCMS(ESI)m/z calcd.for C₂₀H₂₆N₄O₅[M+H]⁺403.2；found 403.2。

Synthesis of Compound 79-3

To a 50mL three-necked flask, compound 63-1 (360 mg,0.892 mmol) and DCM (10 mL) were sequentially added. After cooling to 0deg.C, DMP (757 mg,1.785 mmol) was added. The reaction was carried out at 25℃for 2 hours under nitrogen protection. After completion of the reaction, the reaction was quenched by addition of water (30 mL) and extracted three times with DCM (10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 79-3 (320 mg, yellow solid, 96.293% purity). Yield: 86.02%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₅[M+H]⁺401.2;found 401.3;¹H NMR(400MHz,DMSO_d₆):δ＝10.07(s,1H),9.17(s,1H),8.99(d,J＝1.6Hz,1H),8.27(dd,J＝7.4,1.8Hz,1H),8.22(dd,J＝8.1,2.1Hz,1H),7.56(dd,J＝6.9,1.8Hz,1H),7.44(d,J＝8.1Hz,2H),6.36(t,J＝7.1Hz,1H),5.37(d,J＝4.4Hz,2H),4.17-4.10(m,1H),1.35(s,9H),1.23(d,J＝7.1Hz,3H).

Synthesis of Compound 79-4

To a 50mL single-port flask, compound 62-1 (300 mg,0.54 mmol), 63-1 (217 mg,0.54 mmol), sodium triacetoxyborohydride (229 mg,1.08 mmol), acetic acid (65 mg,1.08 mmol) and DCE (10 mL) were added sequentially. The reaction was carried out at 25℃for 4 hours under nitrogen protection. After the reaction was completed, the reaction solution was poured into water (15 mL), and a proper amount of saturated NaHCO ₃ solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted three times with DCM (10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=10/1 to give compound 79-4 (160 mg, yellow solid, 98.889% purity). Yield: 31.13%.

LCMS(ESI)m/z calcd.for C₅₁H₅₄FN₉O₈[M+H]⁺940.4;found 940.3;¹H NMR(400MHz,DMSO_d₆):δ＝10.18(s,1H),10.06(s,1H),9.20(s,1H),8.45-8.43(m,2H),8.27-8.25(m,1H),7.77-7.74(m,3H),7.66-7.62(m,2H),7.54(dd,J＝6.9,1.6Hz,1H),7.77-7.46(m,2H),7.30(s,1H),7.23-7.20(m,3H),7.15(t,J＝8.9Hz,2H),6.40(d,J＝5.2Hz,1H),6.34(t,J＝7.1Hz,1H),5.27(d,J＝4.4Hz,2H),4.16-4.12(m,1H),3.93(s,3H),3.57(s,2H),3.14(s,4H),2.57(s,4H),1.47(s,4H),1.37(s,9H),1.24(d,J＝6.9Hz,3H).

Synthesis of Compound 79

To a 50mL single vial was added compound 79-4 (50 mg,0.0531 mmol), DCM (1 mL), and trifluoroacetic acid (0.5 mL) in this order. The reaction was carried out at room temperature for 2 hours under nitrogen protection. After the reaction was completed, the reaction solution was concentrated to dryness, added to water (15 mL), and a proper amount of saturated NaHCO ₃ solution was added to make the reaction solution alkaline. The organic phase was separated and the aqueous phase was extracted three times with DCM (10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with a ratio of DCM/meoh=6/1 to give compound 79 (18.16 mg, off-white solid, 96.022% purity). Yield: 39.17%.

LCMS(ESI)m/z calcd.for C₄₆H₄₆FN₉O₆[M+H]⁺840.4;found 840.3;¹H NMR(400MHz,CD₃OD):δ＝8.51(br.s,1H),8.41-8.37(m,2H),7.84(dd,J＝8.0,2.0Hz,1H),7.70(d,J＝8.9Hz,2H),7.59-7.54(m,3H),7.49-7.47(m,1H),7.35(s,1H),7.31(d,J＝8.0Hz,1H),7.21-7.18(m,2H),7.08-7.04(m,2H),6.49(d,J＝5.3Hz,1H),6.41(t,J＝7.2Hz,1H),5.33(s,2H),3.99(s,3H),3.69-3.64(m,3H),3.25(s,4H),2.69(br.s,4H),1.63(s,4H),1.37(d,J＝7.0Hz,3H).

EXAMPLE 51 Synthesis of Compound 80

Synthesis of Compound 80-1

To a 50mL three-necked flask, compound Int A-4 (500 mg,1.026 mmol), toluene (5 mL) and CMBP (2.475 g,10.257 mmol) were successively added, and the mixture was reacted at a temperature of 100℃under nitrogen protection for 16 hours. After cooling the reaction, the reaction was quenched by addition of water (20 mL) and extracted three times with EA (10 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by a silica gel column with an eluent ratio of PE/ea=5/1 to 1/1 to give compound 80-1 (1 g, brown oil, purity 20%, containing CMBP derivatives). Yield: 28.35%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₅[M+H]⁺687.3；found 687.3。

Synthesis of Compound 80-2

To a25 mL single vial was added compound 80-1 (300 mg, 0.433 mmol), DCM (3 mL), and trifluoroacetic acid (3 mL) in sequence. The reaction was carried out at 25℃for 30 minutes. After the reaction was completed, the system was concentrated to dryness, ph=8 was adjusted with saturated aqueous sodium bicarbonate, and extracted three times with EA (20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give compound 80-2 (250 mg, brown oil, 83.459% purity). Yield: 81.55%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₅[M+H]⁺587.3；found 587.3。

Synthesis of Compound 80-3

A25 mL single vial was charged with compound 80-2 (250 mg,0.426 mmol), DCE (5 mL), 79-3 (171 mg,0.426 mmol), STAB (271mg, 1.276 mmol), and acetic acid (77 mg, 1.276 mmol) in sequence. The reaction was carried out at 25℃for 16 hours. After the completion of the reaction, the reaction was quenched by addition of saturated aqueous sodium bicarbonate (10 mL) and extracted three times with EA (50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The residue was purified by prep. plate with the ratio of the developer DCM/meoh=15/1 to give compound 80-3 (14 mg, brown solid, 98.151% purity). Yield: 3.31%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₅[M+H]⁺971.4；found 971.4。

Synthesis of Compound 80

To a 25mL single vial was added compound 80-3 (14 mg,0.0144 mmol), DCM (1 mL) and trifluoroacetic acid (0.5 mL) in order. The reaction was carried out at 25℃for 1 hour. After the reaction was completed, the system was concentrated to dryness to give compound 80 (16.86 mg, brown solid, purity 99.044%). Yield: 80.56%.

LCMS(ESI)m/z calcd.for C₂₀H₂₄N₄O₅[M+H]⁺871.4;found 871.4;¹H NMR(400MHz,DMSO_d₆):δ＝10.32(s,1H),9.98(d,J＝17.9Hz,3H),8.78(d,J＝6.4Hz,1H),8.62(s,1H),8.26(d,J＝6.0Hz,1H),8.17(d,J＝3.9Hz,3H),7.95(dd,J＝8.1,2.0Hz,1H),7.85(d,J＝8.9Hz,2H),7.73(s,1H),7.67-7.58(m,4H),7.37(dd,J＝16.9,8.5Hz,3H),7.16(t,J＝8.9Hz,2H),6.80(d,J＝6.4Hz,1H),6.38(t,J＝7.1Hz,1H),5.33(q,J＝15.4Hz,2H),4.45(d,J＝13.5Hz,1H),4.25(dd,J＝12.4,6.3Hz,4H),4.03(s,3H),3.12(d,J＝38.4Hz,2H),2.68(s,3H),1.83(dd,J＝45.1,13.2Hz,4H),1.51(dd,J＝10.9,7.5Hz,6H),1.38(d,J＝6.9Hz,3H).

In various embodiments of the present invention, compounds 81-83 may be synthesized in a similar manner as described above.

Example 52

In this example, the nuclear magnetic resonance spectra of each of the compounds 1 to 83 were measured by liquid chromatography-mass spectrometry and high resolution mass spectrometry. Wherein, the microwave reactor adopted in the synthesis reaction is Biotage Initiator +; the medium-pressure preparation instrument used for separation is Isolera Prime manufactured by Biotage company; the preparation type high performance liquid chromatography adopts a Gilson GX-281 type instrument, and a reverse phase silica gel column is from Agela Technologies company; the purification conditions of the high performance liquid chromatography are as follows: separation column type XBLID-1, filler particle diameter 5um, diameter 13mm, length 150mm; the ultraviolet detector is arranged at 254 or 220nm; eluent: 0.1% TFA in water and solvent methanol or acetonitrile; the flow rate is 20mL/min; the LC-MS adopts Agilent Technologies 1260Infinity II; wherein the chromatographic column is SYMMETRY C m column, the filler has a particle diameter of 5um, a diameter of 4.6mm and a length of 50mm; the mobile phase adopts 0.05% formic acid aqueous solution and 0.05% formic acid acetonitrile solution; the mass spectrum ionization source is electrospray; the mass spectrum acquisition is Agilent Technologies 6120; high resolution mass spectrometry using a Waters Q-Tof instrument, the molecular weight of the compound was determined to be within ±5ppm of the calculated value (table one); the nuclear magnetic resonance spectrum is measured by a Bruker Assnd 400; the internal standard is tetramethylsilane and deuterated DMSO; the units parts per million (ppm) are indicated by the symbol delta. Example 53

In this example, the binding capacity of compounds 1-83 to the ZER1 protein and ZYG B protein was measured using isothermal titration calorimetry.

This example measures the binding capacity of the compounds of the invention to the E3 ubiquitinated ligase substrate recognition proteins ZER1 and ZYG B, respectively. Isothermal titration calorimetry (Isothermal Titration Calorimetry, ITC) was used for the measurement, and the activity was expressed as the dissociation constant Kd (nM). This example demonstrates that compounds have significant binding capacity for both recognition proteins ZER1 and ZYG B.

Isothermal titration thermal (ITC) measurements were performed at 16℃using a MicroCal PEAQ-ITC instrument (MALVEN PANALYTICAL).

The ZER1 (amino acid residues 469-766) fragment and ZYG B (amino acid residues 490-728) were cloned into the pNIC-CH (Addgene plasmid # 26117) vector and the MEEL sequence at its N-terminus was used for ITC detection. 70ul of the test compound at a concentration of 1mM and 330ul of ZYG b and ZER1 protein at a concentration of 50uM were prepared prior to the assay, both protein and compound were prepared and stored in 20mM Tris-HCl pH 7.5,150mM NaCl solution, and centrifuged at 12000rpm, 4℃for 30min prior to titration to remove air bubbles and sediment. In each experiment, the test compound was titrated into the protein 15 times in a single titration, with a 90s interval between each titration, the first drop of compound was titrated with 0.5uL, the remaining 14 times 1.5uL per drop, and the entire titration process was performed at 16 ℃. After the titration was completed, the interaction of the compound with the protein was analyzed using MicroCal PEAQ-ITC analysis software, and the fitted model was one set of sites. The partial compound dose curves obtained by isothermal titration calorimetry are shown in figure 1 (ZER 1) and figure 2 (ZYG B).

In this example, isothermal titration calorimeter measurements are shown in table 1 below; when the dissociation constant Kd is 1000nM or less, compound Activity targets is "+) ++"; when the dissociation constant Kd is more than 1000nM and less than 5000nM, compound Activity marked as' ++ "; when the dissociation constant Kd is greater than 5000nM and less than 10000nM, the compound activity is marked as "++"; when the dissociation constant Kd is greater than 10000nM, the compound activity is marked as "+".

Example 54

This example describes the measurement of the binding capacity of compounds 1-83 to the ZER1 protein, i.e., using Surface Plasmon Resonance (SPR).

This example demonstrates that compounds have significant binding capacity for the recognition protein ZER 1. Chip Surface Plasmon Resonance (SPR) experiments used a Biacore X100 instrument (Cytiva) and CM5 sensor chip (Cytiva). The binding of the compound to the ZER1 protein was detected, the ZER1 protein was immobilized on a sensor chip (CM 5), and 30ug/ml of the ZER1 protein was passed through the chip at a rate of 10ul/min for saturation coupling according to the manual amino coupling method. The compound was dissolved in 1.05XHBS-EP with 5% DMSO and solvent corrected (1.05 XHBS-EP solution with 4.5%,5%,5.5%,6.0% DMSO). Experiments were performed using 1.05XHBS-EP with 5% DMSO, and the analyte was injected into the flow system at a flow rate of 30. Mu.l/min as a serial dilution of the compound (up to 500. Mu.M down five-fold dilution). The binding time was 120s and the dissociation time was 180s. Kd values were calculated using the Affinity/kinetics analysis option of Biacore evaluation software. The specific operation steps are as follows: 1) Dock chip: taking out a new CM5 chip to be docked into a Biacore X100 instrument (Cytiva); 2) Preparing an amino coupling reagent NHS and EDC, wherein 100ul of each amino coupling reagent NHS and EDC are respectively placed in a 1.5ml uncovered EP tube, diluting ZER1 protein to 30ug/ml, placing the diluted ZER1 protein in the 1.5ml uncovered EP tube, and enabling the solution to flow through a chip at a speed of 10ul/min for saturated coupling; 3) Compound powder was dissolved to 10mM using DMSO and diluted 20-fold with 1.05X HBS-EP to 500uM compound solution containing 5% DMSO; 4) The 500uM compound solution was diluted five times down to 100, 20, 4, 0.8, 0.16, 0.032, 0.0064uM using 1.05 XHBS-EP; 5) Solvent correction solutions (1.05X HBS-EP solutions containing 4.5%,5%,5.5%,6.0% dmso were prepared; . 6) Preparation of extra wash solution: DMSO was diluted to 50% with deionized water; 7) Different 1.5ml uncovered EP tubes were placed to the indicated positions according to the system prompt; 8) Analytes were injected into the flow system at a flow rate of 30 μl/min, binding time of 120s and dissociation time of 180s; 9) Kd values were calculated using the Affinity/kinetics analysis option of Biacore evaluation software.

In this example, the measurement results of the surface plasmon resonance method are shown in table 1: when the dissociation constant Kd is 1000nM or less, compound Activity targets is "+) ++"; when the dissociation constant Kd is more than 1000nM and less than 5000nM, compound Activity marked as' ++ "; when the dissociation constant Kd is greater than 5000nM and less than 10000nM, the compound activity is marked as "++"; when the dissociation constant Kd is greater than 10000nM, the compound activity is marked as "+".

Molecular formula of the compound in Table 1, mass Spectrometry data and results of binding ability to ZER1 protein and ZYG B protein

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Example 55

This example measures the competing effects of compounds 1-83 described above on GAGN substrate proteins.

Human HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (BioInd) supplemented with 10% fetal bovine serum (BioInd) and penicillin/streptomycin (HyClone). To produce lentiviruses, HEK293T cells were co-transfected with pCDH-Puro or pCDH-Ub-GPS (containing Ub-peptide-GFP-P2A-RFP cascade) transfer vector with third generation packaging vectors pMDLg/pRRE, pMD2.VSVG and pRSV-Rev. After 48h, the lentiviruses were filtered through a 0.45 μm filter, followed by the addition of 8mg/mL polybrene to HEK293T cells. The cells were washed 24 hours prior to further manipulation to give the 293-GAGN cell line. Western Blot detects GFP expression. Western blot experiment steps: steady state cells (HEK 293-GAGN) were seeded into 6-well plates at a concentration of 2 x 105 cells/mL, 2mL was added per well and incubated for 24 hours.

Cells were examined microscopically every other day to confirm cell status. Preparing a compound, namely weighing a certain amount of serial compounds of the patent or a control drug cabotinib respectively, preparing a mother solution with higher concentration by using pure DMSO, and preparing a solution containing 0.5% DMSO by adopting a gradient dilution method, wherein the concentration of the compound is as follows: 20mM, 6mM, 2mM, 600 mM, 200 mM, 60 mM, 20mM, 0nM solution, and shaking to mix them thoroughly. Mu.l of the liquid medicine was added to each well. After 24 hours incubation, the cells were removed and the suspension cells were pipetted into a 1.5ml centrifuge tube and centrifuged at 5000rpm for 10min to pellet the cells. The medium was carefully aspirated to leave a cell pellet, 1ml of PBS was added and the wash was repeated 3 times, and the residual solution was blotted dry. 150 μl of RIPA lysate premixed with 1% PMSF was added, and the lysate was allowed to come into full contact with the cells by pipetting and incubated on ice for 10min. Centrifugation at 12000rpm for 10min, aspiration of supernatant, assay with BCA protein quantification kit and sample loading homogenization.

According to 50: mixing BCA-A and B solutions in proportion to prepare working solution, diluting a sample 5 times, sucking 20ul to 96-hole ELISA plates, adding 200 ul of premixed BCA working solution, incubating at 37 ℃ for 20min, measuring an A562nm light absorption value by using an ELISA meter, and substituting into a protein standard curve formula to obtain the concentration of the sample. After the required volume for loading 20. Mu.g was calculated, the samples were dispensed into 1.5ml centrifuge tubes. 10 μl of loading buffer was added to each tube and mixed well, and the mixture was heated in a metal bath at 95deg.C for 5min to denature the proteins.

And (3) fixing 4-20% gradient prefabricated glue on a glue frame, adding electrophoresis liquid in a groove, pulling out a comb, loading a Marker and a protein sample, performing 80V electrophoresis for 30min, adjusting the voltage to 120V electrophoresis, and stopping electrophoresis from the Marker strip to the forefront of the glue. And taking out the glue block, cutting to a proper size, placing the PVDF film in methanol for activation, and placing the activated PVDF film in a film transferring balance liquid for balancing for 1min. The foam cushion, PVDF film, glue and foam cushion are placed in the clamping plate in sequence and put into eBlot equipment for quick wet rotation. The membrane is taken out and put into 5% skimmed milk powder dissolved in 1xTBST for sealing for 1h, the membrane is cut according to the molecular weight of target protein and reference protein, and put into 1xTBST diluted primary antibody and shaken overnight at 4 ℃.

Transferring the strip to 1xTBST for 3 times in the next day, adding a secondary antibody diluted in a specific proportion, shaking and incubating for 1.5 hours, transferring the strip to 1xTBST again for 3 times, preparing ECL color development liquid, placing the film into an imaging system, spreading the ECL color development liquid on the surface, and taking a picture of the film by using the imaging system.

And measuring the gray values of each band of the target protein and the internal reference protein by utilizing SHST ANALYSIS system analysis results, normalizing the results by the gray value of the target protein/the gray value of the corresponding lane GAPDH, and carrying out quantitative analysis according to the gray value of the protein quantity = experimental group/the gray value of a blank control group. The examples demonstrate that the concentration of natural substrate GAGN in cells shows a positive correlation with the concentration of drug, i.e. the compounds of the invention compete with GAGN substrate for binding to the ZER1 protein, inhibiting the degradation of GAGN by competitive binding, stabilizing the natural substrate protein. The experimental results are shown in figure 3.

Example 56

This example measures the effect of compounds 1-83 described above on the stability of HEK293T cell substrate protein GFWC.

The stability analysis of the substrate protein fused with the green fluorescent protein adopts a flow cytometry method for determining the proportion of the green fluorescent protein GFP to the red fluorescent protein RFP, wherein the red fluorescent protein RFP is used as an internal reference. The test compound (10 mM) was dissolved in molecular sieve buffer 20mM Tris7.5+150mM NaCl. HEK293T-SNX11 (GFWC-) stable cell line was plated with cells of the same density in seven wells of a six-well plate, and after 12-24h, inhibitor was added at a concentration of 50uM, respectively, and an equal volume of buffer was added to the control well. Culturing in an incubator until the cells are digested for 24 hours, and performing first flow detection. Passaging was continued with the addition of inhibitor, placed in an incubator for culturing to 72h, cells were digested and subjected to a second flow assay using ACEA NovoCyte instrument (ACEA Biosciences, inc.). The 72h detection result is shown in FIG. 4. Experiments prove that the compound has obvious influence on the stability of the substrate protein.

Example 57

This example describes the mass spectrum of covalent binding of the above compounds 1-83 to ZYG B protein.

A concentration of 5. Mu.M of the X1C2 ZYG B protein was incubated with 20. Mu.M of compound 76 for 16 hours at 25 ℃. Sample injection LC/MS (Waters Xevo G2-XS Qtof coupled with ACUITY UPLC class I) was performed using electrospray ionization in positive ion mode. Sample is passed through C4 chromatographic column1.7Mm, 21X 50 mm) was separated and the column temperature was maintained at 60 ℃. The temperature of the autosampler was 10 ℃. The desolvation temperature was 450℃and the flow rate was 800L/hour. The capillary voltage was 3.0kV and the cone voltage was 40V.

The results are shown in FIG. 5, wherein a comparison of the mass spectrum 5, a, and FIG. 5, B, shows that the compound forms a stable covalently bound complex with protein ZYG B.

Example 58

This example measures the degradation of a portion of the compounds on the target protein.

Western blot experiment: gastric cancer cells (MKN 45) were inoculated into 6-well plates at a concentration of 2×105 cells/mL, 2mL was added to each well, and incubated for 24 hours. Cells were examined microscopically every other day to confirm cell status. Preparing a compound, respectively weighing a certain amount of the compound or a control drug of cabotinib, preparing a mother solution with higher concentration by using pure DMSO, and adopting a gradient dilution method to prepare the compound with the concentration of: 2mM, 200. Mu.M, 100. Mu.M, 20. Mu.M, 2. Mu.M, 200nM, 0nM solution, where the final concentration of DMSO is 0.5%. Shaking to fully mix the materials. 10. Mu.L of the liquid medicine was added to each well. After 24 hours incubation, the cells were removed and the suspension cells were pipetted into a 1.5ml centrifuge tube and centrifuged at 5000rpm for 10min to pellet the cells. The medium was carefully aspirated to leave a cell pellet, 1ml of PBS was added and the wash was repeated 3 times, and the residual solution was blotted dry. 150. Mu.L of RIPA lysate premixed with 1% PMSF was added, and the lysate was allowed to come into full contact with the cells by pipetting and incubated on ice for 10min. Centrifugation at 12000rpm for 10min, aspiration of supernatant, assay with BCA protein quantification kit and sample loading homogenization.

According to 50: mixing BCA-A and B solutions in proportion to prepare working solution, diluting a sample 5 times, sucking 20 mu L to 96-well ELISA plates, adding 200 mu L of premixed BCA working solution, incubating at 37 ℃ for 20min, measuring an A562 nm light absorption value by using an ELISA meter, and substituting into a protein standard curve formula to obtain the concentration of the sample. After the required volume for loading 20 μg was calculated, the samples were dispensed into 1.5mL centrifuge tubes. 10 mu L of loading buffer solution is added into each tube, and the mixture is placed in a metal bath at 95 ℃ and heated for 5min to denature protein.

And (3) fixing 4-20% gradient prefabricated glue on a glue frame, adding electrophoresis liquid in a groove, pulling out a comb, loading a Marker and a protein sample, performing 80V electrophoresis for 30min, adjusting the voltage to 120V electrophoresis, and stopping electrophoresis from the Marker strip to the forefront of the glue. And taking out the glue block, cutting to a proper size, placing the PVDF film in methanol for activation, and placing the activated PVDF film in a film transferring balance liquid for balancing for 1min. The foam cushion, PVDF film, glue and foam cushion are placed in the clamping plate in sequence and put into eBlot equipment for quick wet rotation. The membrane was removed and blocked in 1 XTBE-dissolved 5% nonfat milk powder for 1h, the membrane was cut according to the molecular weight of the target protein and the reference protein, placed in 1 XTBE-diluted primary antibody, and shaken overnight at 4 ℃.

Transferring the strip to 1 XTBE for 3 times in the next day, adding a secondary antibody diluted in a specific proportion, shaking and incubating for 1.5 hours, transferring the strip to 1 XTBE again for 3 times, preparing ECL color development liquid, placing the film into an imaging system, spreading the ECL color development liquid on the surface, and taking a picture of the film by using the imaging system.

And measuring the gray values of each band of the target protein and the internal reference protein by utilizing SHST ANALYSIS system analysis results, normalizing the results by the gray value of the target protein/the gray value of the corresponding lane GAPDH, and carrying out quantitative analysis according to the gray value of the protein quantity = experimental group/the gray value of a blank control group. As a result, as shown in FIG. 6, this example demonstrates that the compounds of the present invention can degrade target proteins in tumor cells after PROTAC is formed.

Example 59

This example measures the growth inhibitory effect of some compounds on cancer cells.

Gastric cancer cells (MKN 45) were seeded into 96-well plates at a concentration of 4X 104 cells/mL, with 100. Mu.L added per well. Incubate for 30 minutes. The cell status was examined by a microscope. Preparing a compound, respectively weighing a certain amount of series of compounds of the patent or a control drug of cabatinib, preparing mother liquor with higher concentration by pure DMSO, preparing medicinal liquor with the concentration of 200 mu M, 60 mu M, 20 mu M, 6 mu M, 2 mu M, 600nM, 200nM and 0nM by adopting a gradient dilution method, and vibrating to fully and uniformly mix the medicinal liquor. The control group was prepared with cabatinib in the same solvent and manner.

After preparation, 1. Mu.L of the drug solution was added to each well of a 96-well plate, and 100. Mu.L of the medium was added thereto, and each dose was repeated three times. The cells were allowed to come into sufficient contact with the drug solution by gentle shaking, and incubated in an incubator for 72 hours. After 72 hours, the cells were removed, developed with CCK-8 kit, 20. Mu.L of CCK-8 reagent was added to each well, gently shaken and incubated in an incubator for one hour. After one hour, the sample was taken out, and absorbance was measured at 450nm using an enzyme-labeled instrument, according to the formula: the inhibition rate of cell growth is = [1 (-absorbance of experimental group-absorbance of culture medium control group)/(absorbance of blank control group-absorbance of culture medium control group) ]multipliedby 100%, and the inhibition capability of the compound on the growth of gastric cancer cells under the current dose and time can be obtained. The half maximal inhibitory concentration (IC 50) was calculated using GRAPHPAD PRISM software (graphpad software, la Jolla, CA) as a function of the gradient of the concentration. The results are shown in Table 2 below and in FIG. 7, which demonstrate that the compounds of the present invention inhibit the proliferation of tumor cells MKN 45.

Inhibitory Activity of the Compounds of Table 2 on proliferation of tumor cells

Compounds of formula (I)	Cell proliferation inhibitory Activity (IC 50, nM)
		79	161.5
80	207.9
		81	209.3
82	115.4
		83	266

Example 60

The mechanism of degrading proteins by partial compounds is studied in this example, namely ubiquitin-protease system inhibitor experiments.

The degradation mechanism of the compound on the target protein is verified by adding a proteasome inhibitor MG132 and a protein ubiquitination inhibitor MLN4924 into the system. Tumor cells were seeded in 6-well plates with 2 x 10 ⁵ cells per well and the following day the cell growth status was observed. When the cells grew to about 80% confluence, the protease inhibitor MG132 (working concentration 10 uM) or the protein ubiquitination inhibitor MLN4924 (working concentration 1 uM) was added to the cell culture solution of the experimental group, and the culture was continued for 6 hours by adding an equal volume of DMSO to the control group. The medium in the well plate was discarded, washed twice with PBS, fresh medium was added, and the compound (working concentration 100 nM) or an equal volume of DMSO was added to each group of cells and the incubation was continued for 18 hours. Western Blot detects protein expression such as c-Kit, AXL, met (same as above). Experiments were repeated three times and more, and statistical analysis was performed on the experimental data using GRAPHPAD PRISM software.

The results are shown in FIG. 8, which shows that the proteasome inhibitor MG132 or the protein ubiquitination inhibitor MLN4924 can counteract the degradation of the target protein by the compound 79, and the protein degradation is proved to be through the ubiquitination proteasome system.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. A compound that binds to an E3 ubiquitinated ligase substrate recognition protein or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug, or polymorph thereof, wherein said compound has a structure represented by formula (I):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated linear or branched hydrocarbon group 、-(CH₂)_m-Cyc¹、-(CH₂)_p-N(CO)_qR⁵(R⁶)-、-COR⁵;, wherein m is optionally from natural number 0-4; p is optionally from a natural number of 1 to 5; q is optionally from a natural number 0-1; wherein Cyc ¹ is selected from benzene ring, substituted or unsubstituted 5-6 membered aromatic heterocycle, substituted or unsubstituted condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, substituted or unsubstituted 4-7 membered monocycloalkyl, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered bridged cycloalkyl; wherein the substitution includes substitution of 0 to 3C 1-4 linear or branched alkyl groups independently of each other with 0 to 3C 1-4 linear or branched alkyl groups selected from halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、-(CH₂)_pOR⁷,C1-4 linear or branched alkyl groups, halogen substituted C1-4 linear or branched alkyl groups, hydroxy substituted C1-4 linear or branched alkyl groups, or alkoxy substituted C1-4 linear or branched alkyl groups; preferably, the aromatic heterocycle, aromatic fused heterocycle, carboheteromonocyclic contains 1-3 heteroatoms selected from O, S, N; r ⁵、R⁶、R⁷、R⁸ is independently selected from H, C to C4 linear or branched alkane, C1 to C4 linear or branched alkene, C1 to C4 linear or branched alkyne;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; wherein said substitution includes substitution independently of each other with 0-3 straight or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂,-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; wherein the above-mentioned C1-4 saturated or unsaturated straight or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 1 to 4 heteroatoms selected from O, S, N; the R ⁷、R⁸ groups are each independently selected from H, C-C4 saturated or unsaturated straight or branched hydrocarbon groups.

2. The compound that binds to E3 ubiquitinated ligase substrate recognition protein of claim 1, wherein, in the compound of formula (i):

When A-G are connected by a double bond and G-J are connected by a single bond, wherein G is a carbon atom, J is a nitrogen atom, A is selected from O, S, CH ₂、CF₂; or alternatively

When no covalent bond is formed between Q ² and M, M is selected from CR ¹³ or N, wherein R ¹³ is selected from H, -CH ₂ Ar, wherein Ar is selected from benzene ring, pyridine ring; q ² is selected from O, NH, NHOH, CF ₂ and is linked to the adjacent carbon atom by a double bond; q ¹ is NR ¹⁰ and is linked to an adjacent carbon atom by a single bond, wherein R ¹⁰ is selected from H, C-3 saturated or unsaturated straight or branched hydrocarbon groups; or alternatively

Preferably, L is selected from N or CR ⁹ or absent, wherein R ⁹ is selected from H, C1-4 saturated or unsaturated straight or branched hydrocarbon, - (CH ₂)_r-Cyc³), wherein R is selected from a natural number 0-1, cyc ³ is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocycloalkane, a substituted or unsubstituted 5-10 membered acene, or R ⁴ forms a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring with R ⁹ closed ring, the substitutions including but not limited to, being substituted independently of each other with 0-3 straight or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated straight or branched hydrocarbon; preferably, the above mentioned C1-4 saturated or unsaturated straight or branched hydrocarbon groups may be optionally substituted by halogen, hydroxy, alkoxy, said carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic containing 1 to 3 heteroatoms selected from O, S, N; preferably, each R ⁷、R⁸ is independently selected from H, C C4 saturated or unsaturated straight or branched hydrocarbon groups; preferably, when L is absent, the carbon atom to which R ⁴ is attached forms a covalent single bond with M.

3. The compound that binds to E3 ubiquitinated ligase substrate recognition protein of claim 1, wherein the compound has a structure represented by the following formula (ii):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated straight-chain or branched hydrocarbon, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵) wherein m is optionally from natural number 0-4;p is optionally from natural number 1-5;q is optionally from natural number 0-1, wherein Cyc1 is selected from substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered aromatic heterocycle, substituted or unsubstituted condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, substituted or unsubstituted 4-7 membered monocycloalkyl, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered bridged cycloalkyl, wherein the substitution comprises C1-4 straight-chain or branched alkyl substituted by 0-3C 1-4 straight-chain or branched alkyl, hydroxy-substituted C1-4 straight-chain or branched alkyl independently from each other, wherein the substitution comprises C1-4 straight-chain or branched alkyl substituted by 0-3C 1-4 straight-chain or branched alkyl, halogen, C1-4 straight-chain or branched alkyl, preferably the straight-chain or branched heterocycle comprises R1-C4, and each independently selected from the group consisting of straight-chain, branched C1-chain, C1-4, and branched C3, and branched C1-C3, straight-membered heterocycle, and C3, and each independently selected from the R is selected from the group consisting of R and C1-4;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; wherein said substitution comprises substitution independently of each other with 0-3 straight or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; the above-mentioned C1-4 saturated or unsaturated straight-chain or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 1 to 4 heteroatoms selected from O, S, N; the R ⁷、R⁸ groups are each independently selected from H, C-C4 saturated or unsaturated straight or branched hydrocarbon groups.

4. The compound that binds to E3 ubiquitinated ligase substrate recognition protein of claim 1, wherein the compound has a structure represented by the following formula (iii):

Wherein,

The R ¹¹ is selected from H, OH, NH ₂, halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂,-(CH₂)_rNHCOCH＝CR⁷(R⁸)、C1-C7 saturated or unsaturated linear or branched alkyl ,-(CH₂)_m-Cyc¹、-(CH₂)_p-N(CO)_qR⁵(R⁶)-;, wherein m is optionally from a natural number of 0-4; p is optionally from a natural number of 1 to 5; q is optionally from a natural number 0-1; r is optionally from a natural number of 0 to 6; wherein Cyc ¹ is selected from a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, a substituted or unsubstituted condensed ring formed from a benzene ring and a 5-6 membered aromatic heterocycle, a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, a substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, a substituted or unsubstituted 4-7 membered monocycloalkyl group, a substituted or unsubstituted 5-10 membered cycloalkyl group, a substituted or unsubstituted 7-10 membered bridged cycloalkyl group; wherein the substitution includes substitution with 0 to 3C 1-4 linear or branched alkyl groups independently of each other selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂、-NHCOCH＝CR⁷(R⁸), C1-4 linear or branched alkyl groups substituted with halogen, C1-4 linear or branched alkyl groups substituted with hydroxy, or C1-4 linear or branched alkyl groups substituted with alkoxy; preferably, the aromatic heterocycle, aromatic fused heterocycle, carboheteromonocyclic contains 1-3 heteroatoms selected from O, S, N; r ⁵、R⁶、R⁷、R⁸ is independently selected from H, C-C4 linear or branched alkane, C1-C4 linear or branched alkene, C1-C4 linear or branched alkyne;

5. A compound that binds to an E3 ubiquitinated ligase substrate recognition protein according to claim 1, wherein the compound has a structure represented by the following formula (IV):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated linear or branched hydrocarbon, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵) wherein m is optionally from natural number 0-4;p is optionally from natural number 1-5;q is optionally from natural number 0-1, wherein Cyc1 is selected from substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered aromatic heterocycle, substituted or unsubstituted condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, substituted or unsubstituted 4-7 membered monocycloalkyl, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered bridged cycloalkyl, wherein said substitution comprises, independently of each other, C1-4 linear or branched alkyl substituted by 0-3C 1-4 linear or branched alkyl selected from halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、-(CH₂)pOR⁷、C1-4, hydroxy-substituted C1-4 linear or branched alkyl or alkoxy, preferably said aromatic heterocycle, C1-4 heteromonocyclic ring, substituted or C1-4 linear alkyl substituted by alkoxy, preferably said aromatic heterocycle, C1-4 heteromonocyclic ring, C3-4-branched C1-4 linear or branched C1-4 alkyl independently selected from 523C 1-4 straight chain or branched C1-C4 alkyl independently of each other;

The R ⁴ is selected from H, halogen, C1-C3 saturated or unsaturated straight-chain or branched hydrocarbon; or the R ⁴ and R ³ are closed to form a substituted or unsubstituted benzene ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, and a substituted or unsubstituted 4-7 membered carboheteromonocyclic ring; the substitution includes substitution independently of each other with 0 to 3 linear or branched hydrocarbon groups selected from halogen, -OH, -COOH, -CN, -NH ₂、-CONH₂,-NHCOCH＝CR⁷(R⁸), C1-4 saturated or unsaturated; preferably, the above C1-4 saturated or unsaturated straight or branched hydrocarbon groups may be optionally substituted with halogen, hydroxy, alkoxy; preferably, the carbon heteromonocyclic, carbon heterobicyclic, aromatic heterocyclic ring contains 0 to 4 heteroatoms selected from O, S, N; preferably, R ⁷、R⁸ is each independently selected from H, C to C4 saturated or unsaturated straight or branched hydrocarbon groups;

The Q ² is selected from nitrogen or oxygen atoms;

6. A compound that binds to E3 ubiquitinated ligase substrate recognition protein according to claim 1, wherein the compound has a structure represented by the following formula (V):

Wherein,

The R ³ is selected from H, C-C8 saturated or unsaturated linear or branched hydrocarbon, - (CH ₂)m-Cyc1、-(CH₂)p-N(CO)qR⁵(R⁶)-、-COR⁵) wherein m is optionally from natural number 0-4;p is optionally from natural number 1-5;q is optionally from natural number 0-1, wherein Cyc1 is selected from substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered aromatic heterocycle, substituted or unsubstituted condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, substituted or unsubstituted 4-7 membered carboheteromonocyclic ring, substituted or unsubstituted 5-10 membered carboheteromonocyclic ring, substituted or unsubstituted 4-7 membered monocycloalkyl, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered bridged cycloalkyl, wherein said substitution comprises, independently of each other, C1-4 linear or branched alkyl substituted by 0-3C 1-4 linear or branched alkyl selected from halogen 、-OH、-COOH、-CN、-NH₂、-CONH₂、-NHCOCH＝CR⁷(R⁸)、-(CH₂)pOR⁷,C1-4, hydroxy-substituted C1-4 linear or branched alkyl or alkoxy-substituted C1-4 linear or branched alkyl substituted, preferably said aromatic heterocycle, C1-4 heteromonocyclic ring, substituted or unsubstituted 5-10 membered cycloalkyl, substituted or unsubstituted 7-10 membered cycloalkyl independently of each other is selected from C1-4 linear or branched C1-4 alkyl independently of one another;

7. The compound that binds to E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-6, wherein the compound is selected from at least one of the following:

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8. A pharmaceutical composition comprising a compound that binds to an E3 ubiquitination ligase substrate recognition protein of any one of claims 1-7, or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug, or polymorph thereof.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises an excipient, diluent, co-solvent, adjuvant, vehicle, or combination thereof.

10. Use of a compound that binds to an E3 ubiquitin ligase substrate recognition protein according to any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament that can bind to E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B.

11. Use of a compound that binds to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament for inhibiting a cullin2-ZER1 and/or cullin 2-ZYG B protein complex.

12. Use of a compound that binds to an E3 ubiquitin ligase substrate recognition protein according to any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament for inhibiting the binding of a protein N-terminal degradation terminator to E3 ubiquitin ligase substrate recognition protein ZER1 or ZYG B, and the ubiquitination and proteasome degradation of a protein.

13. Use of a compound binding to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug or polymorph thereof for the preparation of an inhibitor of inflammatory small NLRP1 activation.

14. Use of a compound that binds to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament for the prevention or treatment of inflammation or complications triggered by factors such as microbial infection, genetic mutation or the like.

15. The use according to claim 14, wherein the inflammation or complications triggered by microbial infection, genetic mutation, etc. comprise gout, pneumonia, parkinson, alzheimer, diabetes, cancer or viral infectious diseases.

16. Use of a compound that binds to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament for interfering with the binding of ZYG B to ORF10 protein of the novel coronavirus SARS-CoV-2 affecting the immune response to the novel coronavirus.

17. Use of a compound that binds to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament that can form a complex with cullin2-ZER1 and/or cullin2-ZYG B for modulating the quality control of an N-myristoylated protein.

18. Use of a compound that binds to an E3 ubiquitinated ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analog, nitroxide, prodrug or polymorph thereof for the manufacture of a medicament for binding cullin2 (ZER 1/ZYG B) via non-covalent or covalent bonds and forming part of a proteolytically targeted chimeric (PROTAC) molecule or molecular glue (molecular glue) molecule for degrading a specific target protein using the ubiquitin-proteinase system.

19. Use of a compound binding to an E3 ubiquitination ligase substrate recognition protein of any one of claims 1-7 or a salt, enantiomer, geometric isomer, solvate, isotopically enriched analogue, nitroxide, prodrug or polymorph thereof for the preparation of a cancer, inflammatory disease, autoimmune disease, fibrotic disease or neurodegenerative disease.

20. Use according to claim 19, characterized in that:

21. The use according to claim 19 or 20, wherein the medicament further comprises one or more additional bioactive agents;