E3 ubiquitin ligase ligand compound and application thereof

Abstract

The invention provides an E3 ubiquitin ligase ligand compound and application thereof. Specifically, a series of small molecules which have unique structures and can be specifically combined with the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31 are designed and developed, the small molecules can serve as inhibitors aiming at the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31, and the purposes of preventing and treating diseases can be achieved by interfering combination of the SCF-FBXO31 and specific protein; meanwhile, due to the fact that the small molecule compound can be combined with an SCF-FBXO31 compound, the small molecule compound can become a molecular part for recruiting E3 enzyme in PROTAC molecules or molecular glue molecules, the corresponding PROTAC molecules or molecular glue molecules can be prepared through the small molecule compound, and great significance is achieved for developing novel drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry and relates to an E3 ubiquitin ligase ligand, and in particular, to an E3 ubiquitin ligase ligand compound and application thereof. Background Art

[0002] The ubiquitin-proteasome system (UPS) is the main pathway for intracellular protein degradation, and is involved in the degradation of more than 80% of intracellular proteins. Organisms maintain their normal state by selectively degrading damaged, misfolded, dysfunctional or unnecessary proteins through the UPS system. Distortion of UPS-related proteins can lead to various human diseases, including neurodegeneration, abnormal aging and cancer. In addition, studies have confirmed that the ubiquitin-proteasome system has important pathophysiological significance in cardiovascular diseases, and can regulate the occurrence and development of important diseases such as atherosclerosis, post-ischemia reperfusion injury, familial cardiomyopathy, myocardial hypertrophy and heart failure. UPS is composed of ubiquitin (Ub), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-protein ligase (E3), proteasome and its substrate (protein). The substrates of the UPS are ubiquitinated through the sequential action of these three enzymes (E1 enzyme, E2 enzyme, and E3 enzyme) and subsequently degraded by the 26S proteasome.

[0003] The method of using the ubiquitin-protease system in eukaryotic cells to regulate intracellular protein levels has received great attention in the past decade. Among them, the protein degradation targeting chimera (PROTAC) technology uses UPS to specifically degrade target proteins, which can effectively induce the degradation of target proteins for the treatment of various diseases, including enzymes, transcription factors, epigenetic factors, and scaffold proteins, etc., and has initially shown superior efficacy and safety in clinical practice. PROTAC is a heterobifunctional small molecule composed of two ligands connected by a linker: one ligand recruits and binds to a specific target protein, while the other ligand recruits and binds to a specific E3 ubiquitin ligase. By optimizing the connection position and the length of the connection chain, the ligands at both ends of the PROTAC molecule bind to the target protein and the E3 ubiquitin ligase at the same time, forming a target protein-PROTAC-E3 ligase ternary complex, thereby promoting the ubiquitination tagging of the target protein, and then being degraded by the proteasome system. Due to the characteristics of ternary complex formation, the target protein ligand used in PROTAC technology does not need to have strong target binding activity, so it can target target proteins that traditional inhibitors cannot drug, such as skeleton proteins and transcription factors; in addition, the overall degradation of the target protein helps to overcome the drug resistance problem of small molecule inhibitors; PROTAC molecules act repeatedly in a catalytic mechanism, and PROTAC-induced degradation is event-driven rather than occupancy-driven. After the complex is formed and ubiquitin transfer is completed, the drug dissociates and transfers to the next target in an enzymatic manner. To a certain extent, it is possible to maintain effective drug activity at low doses. The technical difficulty of PROTAC lies in the relationship between target protein degradation and generation rate, the ability of target protein to bind ubiquitin, the conformation and site of the connection between target protein ligand and E3 ubiquitin ligase ligand, the modification of the length and composition of the connection chain, and the concentration affect the formation and stability of the ternary complex, so it is more challenging to regulate. Among the more than 600 known E3 ubiquitin ligases, only a limited number of them are actually used in the design of PROTAC compounds, including CRBN, VHL, MDM2, and cIAP1. The protein degraders currently in clinical stages mainly utilize two E3 ubiquitin ligases, CRBN and VHL. These PROTAC compounds may have off-target effects and drug resistance during treatment, so new E3 ubiquitin ligase tools are urgently needed to be developed.

[0004] FBXO31 is a member of the FBPs family and is a senescence-related gene located on chromosome 16q24.3. It consists of 539 amino acids and has a molecular weight of 60,533Da. FBXO31 contains a 40-amino acid F-box domain and is an important component of the SCF ubiquitin E3 ligase, involved in ligase-mediated ubiquitination of target proteins and subsequent proteasomal degradation. In the SCF-FBXO31 E3 ligase, cullin 1 plays a molecular scaffolding role, interacting with the adaptor subunit Skp1 at the amino terminus and the RING finger protein ring box 1 (Rbx1) at the carboxyl terminus. Rbx1 recruits specific E2 ubiquitin conjugating enzymes (UBCs), including Ubc3, Ubc4, and Ubc5. In contrast, Skp1 binds to FBXO31, which interacts with it through the F-box domain and participates in the recognition of a variety of substrates and ubiquitination modification of them through the SCF ubiquitin ligase complex.

[0005] FBXO31 is a cell cycle regulatory protein that is highly expressed from the G2 phase to the early G1 phase. It is expressed in most tissue types, especially in the brain, and plays an important role in regulating the neuronal cell cycle, dendritic growth, and neuronal migration. Diseases associated with it include mental retardation, autosomal recessive non-syndromic intellectual disability, and cerebral palsy. FBXO31 plays an important role in a variety of diseases such as mental retardation, autosomal recessive non-syndromic intellectual disability, cerebral palsy, DNA damage response, and tumorigenesis by degrading related substrates.

[0006] Initial detection found that FBXO31 was lowly expressed in breast cancer and was identified as a candidate tumor suppressor gene for the first time. As the research continues to deepen and expand, FBXO31 plays two completely different roles in different types of cancer: tumor suppressor and tumor promoter. It plays a tumor suppressor role in breast cancer, melanoma, hepatocellular carcinoma, gastric cancer, ovarian cancer, prostate cancer and colon cancer. It plays a tumor promoter role in esophageal squamous cell carcinoma, pancreatic cancer and lung cancer. In breast cancer cell lines and primary breast tumors, the expression of FBXO31 is reduced, and ectopic expression of FBXO31 induces breast cancer cell senescence. In pancreatic cancer patients, FBXO31 is highly expressed and negatively correlated with patient survival. Therefore, finding new small molecules that can bind to SCF-FBXO31 and developing unique PROTAC molecules or molecular glue molecules based on this can provide new experimental means for the study of diseases such as cancer, and provide technical support for cancer treatment and target verification. Summary of the invention

[0007] In view of the deficiencies of the prior art, the object of the present invention is to provide E3 ubiquitin ligase ligand compounds and applications thereof.

[0008] In the first aspect of the present invention, a compound capable of binding to SCF-FBXO31 protein is provided, wherein the compound is as shown in (I):

[0009]

[0010] In formula (I):

[0011] m is any natural number from 0 to 3;

[0012] R 1 selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; the above C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, aromatic heterocycle, aromatic condensed ring, aromatic condensed heterocycle, monocyclic alkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocycle, carbon heterocyclic ring can be further optionally and independently replaced by 0 to 3 halogens, -OH, -COOH, -CN, -NO 2 、-CH3、-CHO、-NH 2 、-SH、-SCH 3 、-CONH 2 、-NHCOR 10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH 2 ) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is selected from natural numbers 1-5; the aromatic heterocycle, aromatic condensed heterocycle, carbon heteromonocycle, carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N;

[0013] R 2 Selected from -XR 4 (R 5 ), X is selected from CH, N, preferably X is N; Y is selected from CH 2 , C=O, preferably Y is C=O; Z is selected from CH 2 , NH or O; R4 Selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon group, -COR 7 、-CONR 8 (R 9 );R 5 , R 6 , R 7 , R 8 and R 9 selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; the above C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, aromatic heterocycle, aromatic condensed ring, aromatic condensed heterocycle, monocyclic alkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocycle, carbon heterocyclic ring can be further optionally and independently replaced by 0 to 3 halogens, -OH, -COOH, -CN, -NO 2 、-CH 3 , -CHO, -NH 2 、-SH、-SCH 3 、-CONH 2 、-NHCOR 10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH 2 ) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is selected from natural numbers 1-5; the aromatic heterocycle, aromatic condensed heterocycle, carbon heteromonocycle, carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N;

[0014] The A ring is selected from a 5-6 membered aromatic ring, a 5-6 membered aromatic heterocycle, a benzene ring and a fused ring formed by a 5-6 membered aromatic heterocycle, a 4-7 membered carbon heteromonocycle, a 5-10 membered carbon heterocyclic ring, a 4-7 membered monocycloalkyl, a 5-10 membered cycloalkyl, and a 7-10 membered bridged cycloalkyl; the aromatic heterocycle, aromatic fused heterocycle, carbon heteromonocycle, and carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N; the above aromatic ring, aromatic heterocycle, aromatic fused ring, aromatic fused heterocycle, monocycloalkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocycle, and carbon heterocyclic ring may be further optionally and independently substituted by 0 to 3 halogens, -OH, -COOH, -CN, -NO 2 、-CH 3 , -CHO, -NH 2 、-SH、-SCH 3 、-CONH 2 、-NHCOR 10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH 2 ) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is selected from natural numbers 1-5; the aromatic heterocycle, aromatic condensed heterocycle, carbon heteromonocycle, carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N;

[0015] R 3 selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; the above C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, aromatic heterocycle, aromatic condensed ring, aromatic condensed heterocycle, monocyclic alkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocycle, carbon heterocyclic ring can be further optionally and independently replaced by 0 to 3 halogens, -OH, -COOH, -CN, -NO 2 、-CH3、-CHO、-NH 2 、-SH、-SCH 3 、-CONH 2 、-NHCOR10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH 2 ) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is selected from natural numbers 1-5; the aromatic heterocycle, aromatic condensed heterocycle, carbon heteromonocycle, carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N;

[0016] R 10 , R 11 They are independently selected from H, C1-C4 straight or branched alkane, C1-C4 straight or branched olefin, C1-C4 straight or branched alkyne, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6 membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle; the aromatic heterocycle and aromatic condensed heterocycle contain 1 to 3 heteroatoms selected from O, S and N.

[0017] Furthermore, the compound of formula (I) has the following structure:

[0018]

[0019]

[0020] In the second aspect of the present invention, a pharmaceutically acceptable salt of the compound of formula (I) according to the first aspect of the present invention is provided.

[0021] Furthermore, the pharmaceutically acceptable salt refers to a salt prepared from the parent compound by addition of a non-toxic acid or base.

[0022] In the third aspect of the present invention, a protein degrading agent is provided, wherein the protein degrading agent is: target protein ligand + linker + SCF-FBXO31 protein conjugate (ligand);

[0023] The structural formula of the protein degradation agent is shown in formula (II):

[0024]

[0025] L of the Linker substructure in Formula II 1 Can be connected to R 2 The connected C, N, O or S;

[0026] Wherein, the SCF-FBXO31 conjugate (ligand) is the compound described in the first aspect of the present invention;

[0027] Wherein, the target protein ligand can be selected from the following:

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] One or more of .

[0036] Among them, the substructure L of the Linker 1 , L 3 and L 5 Can be selected from one or more of the following:

[0037]

[0038] Among them, R a Selected from H, alkyl, SO 2 (alyl) or SO 2 (aryl) One or more.

[0039] The Linker substructure L 2 , L 4 You can choose one or more of the following:

[0040]

[0041] Furthermore, the protein degradation agent of formula (II) has the following structure:

[0042]

[0043]

[0044]

[0045] In the fourth aspect of the present invention, a pharmaceutical composition is provided, comprising a combination of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention and a pharmaceutically acceptable diluent or carrier.

[0046] Furthermore, the pharmaceutical composition may also comprise a combination of an enantiomer, a diastereomer, a stereoisomer of the compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0047] In the fifth aspect of the present invention, a pharmaceutical composition for targeted protein degradation is provided, which comprises a combination of the protein degrading agent of formula (II) or a pharmaceutically acceptable salt thereof according to the third aspect of the present invention and a pharmaceutically acceptable diluent or carrier.

[0048] Furthermore, the pharmaceutical composition targeted for protein degradation may also comprise a combination of an enantiomer, a diastereomer, a stereoisomer of the protein degradation agent of formula (II) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0049] In the sixth aspect of the present invention, provided is the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention and the pharmaceutical composition according to the fourth aspect of the present invention in the preparation of a drug.

[0050] Furthermore, the drug is a drug for treating SCF-FBXO31 related diseases.

[0051] Furthermore, the SCF-FBXO31-related diseases include mental retardation, autosomal recessive non-syndromic intellectual disability, cerebral palsy, spastic-dystonia cerebral palsy syndrome, glioma, melanoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, prostate cancer, colon cancer, esophageal cancer, esophageal squamous cell carcinoma, lung cancer, cervical cancer, pancreatic cancer, diabetes, cardiac hypertrophy or heart failure, etc.

[0052] In the seventh aspect of the present invention, provided is the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, the pharmaceutical composition according to the fourth aspect of the present invention, the protein degrader of formula (II) or a pharmaceutically acceptable salt thereof according to the third aspect of the present invention, and the pharmaceutical composition for targeted protein degradation according to the fifth aspect of the present invention in the preparation of drugs.

[0053] Furthermore, the medicine includes medicine for treating tumors and / or neurodegenerative diseases.

[0054] Furthermore, the tumor includes melanoma, renal cancer, adrenocortical carcinoma, bladder cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, uterine cancer, testicular cancer, fallopian tube tumors, gastrointestinal cancer, colorectal cancer, lymphoma, Hodgkin's lymphoma, liver cancer, intrahepatic bile duct carcinoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, esophageal squamous cell carcinoma, neuroendocrine tumor, glioma, astrocytoma, oligodendroglioma, glioblastoma, neuroblastoma, gangliocytoma, ganglioglioma, medulloblastoma, neurofibroma, neurotheliomas, peripheral neuroepithelioma, brain cancer, meningioma, meningiosarcoma or ependymoma, etc.

[0055] Furthermore, the neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), bovine spongiform encephalopathy, multiple sclerosis, cerebral ischemia, amyotrophic lateral sclerosis, ataxia telangiectasia, Creutzfeldt-Jakob disease, cerebellar atrophy, primary lateral sclerosis, spastic paraplegia or myasthenia gravis, etc.

[0056] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0057] Compared with the prior art, the present invention has the following advantages and progress:

[0058] The present invention designs and develops a series of small molecules with unique structures that can specifically bind to the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31, which can be used as inhibitors for the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31, and can prevent and treat diseases by interfering with the binding of SCF-FBXO31 to specific proteins; at the same time, since the above-mentioned small molecule compounds can bind to the SCF-FBXO31 complex, they can become the molecular part of the PROTAC molecule or the molecular glue molecule that recruits the E3 enzyme, which is of great significance for the development of new drugs. Specifically, the present invention first innovatively designs the above-mentioned small molecules with unique structures that can specifically bind to the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31 and completes its preparation by chemical synthesis; at the same time, some small molecules are selected for PROTACs chemical synthesis. Furthermore, the present invention verified the binding ability of the above-mentioned small molecules to the E3 ubiquitin ligase substrate recognition protein SCF-FBXO31 by isothermal titration calorimetry; and verified that the PROTACs prepared by the above-mentioned small molecules can effectively degrade the target protein through Western blot experiments of target protein degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0060] Figure 1 : Degradation effect of BRD4 or SMARCA2 after 4 h of treatment with 1 μM different PROTACs in A375 cell line (Western blot).

[0061] Figure 2 : Degradation effect of BRD4 or SMARCA2 in A375 cell line after 8 h of treatment with 1 μM different PROTACs (Western blot).

[0062] Figure 3 : The degradation effect of different PROTACs at 5 μM on BRD4 in A375 cell line for 6 h (Western blot).

[0063] Figure 4 : The degradation effect of 1 μM NEP215-1 on BRD4 in A375 cell line after treatment for different time periods (Western blot).

[0064] Figure 5 : The degradation effect of different concentrations of NEP215-2 or NEP215-6 on BRD4 in A375 cell line after 8 h of treatment (Western blot).

[0065] Figure 6 : The degradation effect of different concentrations of NEP215-1 or NEP215-2 on BRD4 in A375 cell line after 12 h of treatment (Western blot).

[0066] Figure 7 : The degradation effects of different PROTACs at different concentration levels on BRD4 or SMARCA2 in H1299 cell lines for different periods of time (Western blot).

[0067] Figure 8 : The degradation effect of 10 μM NEP215-16 or NEP215-17 treatment on ERα in MCF7 cell line for 12 h (Western blot). DETAILED DESCRIPTION

[0068] The present invention provides E3 ubiquitin ligase ligand compounds and their applications. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0069] Example 1: Ligand Synthesis

[0070] 1. Synthesis of Compound NEP203

[0071]

[0072] Compound NEP203 (60 mg, 0.37 mmol) was added to a 50 mL single-necked bottle and dissolved in 2 mL DMF. 2 Purge, then add Et 3 N (156 μL, 1.11 mmol) and isopropyl isocyanate (43 μL, 0.44 mmol). The reaction was carried out at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was poured into a saturated saline solution (10 mL) and extracted three times with EA (15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP203 (71 mg, white solid, yield: 78%). 1 H NMR (400 MHz, DMSO-d 6 )δ7.40(s,1H),7.31–7.23(m,2H),7.23–7.15(m,3H),6.96(s,1H),5.92(d,J=7.6Hz,1H),5.83(d,J=8.5Hz,1H) ,4.38–4.28(m,1H),3.67–3.54(m,1H),2.98–2.89(m,1H),2.79–2.69(m,1H),1.03–0.95(m,6H).HRMS(ESI)m / z calcd.for C 13 H 19 N 3 O 2 Na[M+Na] + 272.1477; found 272.1367.

[0073] 2. Synthesis of Compound NEP204

[0074]

[0075] Compound SM-1 (50 mg, 0.31 mmol), DMF (3 mL), 1-naphthoic acid (64 mg, 0.37 mmol), HATU (180 mg, 0.47 mmol) and DIPEA (280 μL, 1.6 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP204 (39 mg, white solid, yield: 40%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.68–8.61(m,1H),8.02–7.91(m,2H),7.83–7.76(m,1H),7.60(s,1H),7.56–7.48(m,3H),7.47–7.42(m,1H),7.42–7.36(m,2 H),7.36–7.30(m,2H),7.30–7.23(m,1H),7.20(s,1H),4.88–4.78(m,1H),3.22–3.17(m,1H),2.98–2.88(m,1H).HRMS(ESI)m / z calcd.for C 20 H 18 N 2 O 2 Na[M+Na] + 341.1368; found 341.1256. III. Synthesis of compound NEP205

[0076]

[0077] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1H-pyrrole-2-carboxylic acid (41 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP205 (29 mg, transparent solid, yield: 37%). 1 H NMR (400 MHz, Methanol-d 4)δ7.20–7.10(m,4H),7.10–7.01(m,1H),6.81–6.76(m,1H),6.71–6.65(m,1H),6.07– 6.01(m,1H),4.71–4.63(m,1H),3.16–3.08(m,1H),2.96–2.87(m,1H).HRMS(ESI)m / z calcd.for C 14 H 15 N 3 O 2 Na[M+Na] + 280.1164; found 280.1254.

[0078] 4. Synthesis of Compound NEP206

[0079]

[0080] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1H-indazole-3-carboxylic acid (59 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP206 (47 mg, white solid, yield: 50%). 1 H NMR (400 MHz, DMSO-d 6 )δ13.61(s,1H),8.12(d,J=8.2Hz,1H),8.06(d,J=8.4Hz,1H),7.67–7.59(m,2H),7.48–7.38(m,1H),7.30–7.26(m, 3H),7.26–7.22(m,3H),7.21–7.13(m,1H),4.81–4.71(m,1H),3.20–3.15(m,3H),3.14–3.05(m,1H).HRMS(ESI)m / z calcd.for C 17 H 16 N 4 O 2 Na[M+Na] + 331.1273; found 331.1164.

[0081] 5. Synthesis of Compound NEP207

[0082]

[0083] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1H-imidazole-5-carboxylic acid (41 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP207 (41 mg, white solid, yield: 52%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.70(s,1H),7.65(s,1H),7.33–7.27(m,2H),7.27–7.13(m,3H),4.83–4.79(m,1H),3.29–3.20(m,1H),3.15–3.02(m,1H).HRMS(ESI)m / zcalcd.for C 13 H 14 N 4 O 2 Na[M+Na] + 281.1117; found 281.1007.

[0084] VI. Synthesis of Compound NEP208

[0085]

[0086] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by benzoic acid (45 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP208 (52 mg, white solid, yield: 63%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.53–8.46(m,1H),7.84–7.77(m,2H),7.56(s,1H),7.55–7.48(m,1H),7.48–7.40(m,2H),7.39–7.32(m,2H),7.31– 7.22(m,2H),7.21–7.14(m,1H),7.12(s,1H),4.71–4.61(m,1H),3.18–3.09(m,1H),3.06–2.95(m,1H).HRMS(ESI)m / z calcd.for C 16 H 16 N 2 O 2 Na[M+Na] + 291.1212; found 291.1100.

[0087] VII. Synthesis of Compound NEP209

[0088]

[0089] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 6-hydroxypyridine-2-carboxylic acid (51 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP209 (32 mg, white solid, yield: 37%). 1 H NMR (400 MHz, DMSO-d 6 )δ11.18(s,1H),8.41(s,1H),7.73(s,2H),7.52–7.10(m,7H),6.79(m,1H ),4.75–4.70(m,1H),3.19–3.10(m,1H),3.03–2.93(m,1H).HRMS(ESI)m / z calcd.for C 15 H 15 N 3 O 3 Na[M+Na] + 308.1113; found 308.1004.

[0090] 8. Synthesis of Compound NEP210

[0091]

[0092] Prepared according to the preparation steps of NEP203, except that isopropyl isocyanate was replaced with tert-butyl isocyanate (51 μL, 0.44 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH=40 / 1 to obtain a crude compound NEP210. The crude NEP210 was dissolved in EA, stirred at room temperature for 10 min, filtered, and the filter residue was washed with EA three times to obtain pure NEP210 (47 mg, white solid, yield: 49%). 1 H NMR (400 MHz, DMSO-d 6 )δ7.37(s,1H),7.31–7.23(m,2H),7.23–7.15(m,3H),6.95(s,1H),5.91(s,1H),5.85–5.78( m,1H),4.35–4.25(m,1H),2.98–2.88(m,1H),2.78–2.68(m,1H),1.18(s,9H).HRMS(ESI)m / z calcd.forC 14 H 21 N 3O 2 Na[M+Na] + 286.1634; found 286.1523.

[0093] IX. Synthesis of Compound NEP211

[0094]

[0095] Compound SM-1 (60 mg, 0.37 mmol) was added to a 50 mL single-necked bottle and dissolved in 2 mL DMF. 2 Purge, then add Et 3 N (156 μL, 1.11 mmol) and benzyl isocyanate (55 μL, 0.44 mmol). The reaction was carried out at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was poured into a saturated saline solution (100 mL) and extracted three times with EA (5 mL). The organic phases were combined and filtered, and the residue was washed three times with EA to obtain pure NEP211 (37 mg, white solid, yield: 34%). 1 H NMR (400 MHz, DMSO-d 6 )δ7.44(s,1H),7.36–7.26(m,4H),7.23–7.14(m,6H),7.00(s,1H),6.58–6.51(m,1H),6.17–6.10( m,1H),4.45–4.35(m,1H),4.28–4.11(m,2H),3.04–2.94(m,1H),2.83–2.73(m,1H).HRMS(ESI)m / z calcd.for C 17 H 19 N 3 O 2 Na[M+Na] + 320.1477; found 320.1369. X. Synthesis of compound NEP212

[0096]

[0097] Compound SM-1 (60 mg, 0.37 mmol) was added to a 50 mL single-necked bottle and dissolved in 2 mL DMF. 2 Purge, then add Et 3 N (156 μL, 1.11 mmol) and cyclohexyl isocyanate (51 μL, 0.44 mmol) were added and reacted at room temperature for 1.5 hours. After the reaction was completed, the reaction solution was filtered and washed with ddH 2 The mixture was washed with O and EA three times in sequence, and the residue was collected to obtain the pure product of compound NEP212 (83 mg, white solid, yield: 78%).1 H NMR (400 MHz, DMSO-d 6 )δ7.41(s,1H),7.30–7.22(m,2H),7.22–7.15(m,3H),6.97(s,1H),6.01(d,J=7.9Hz,1H),5.89(d,J=8.5Hz,1H),4.38–4. 28(m,1H),2.99–2.88(m,1H),2.79–2.70(m,1H),1.74–1.58(m,5H),1.54–1.47(m,1H),1.32–0.97(m,5H).HRMS(ESI)m / z calcd.for C 16 H 23 N 3 O 2 Na[M+Na] + 312.1790; found 312.1681. XI. Synthesis of compound NEP213

[0098]

[0099] Prepared according to the preparation steps of NEP203, except that isopropyl isocyanate was replaced with ethyl isocyanate (35 μL, 0.44 mmol), and purified by column chromatography, the developing solvent ratio was DCM / MeOH=30 / 1, to obtain a crude compound NEP213. The crude NEP213 was dissolved in EA, stirred at room temperature for 10 min, filtered, and the filter residue was washed with EA three times to obtain pure NEP213 (39 mg, white solid, yield: 50%). 1 H NMR (400 MHz, DMSO-d 6 )δ7.42(s,1H),7.31–7.23(m,2H),7.23–7.15(m,3H),6.99(s,1H),6.05–5.98(m,1H),5.97–5.90(m,1H),4. 38–4.28(m,1H),3.02–2.97(m,1H),2.97–2.89(m,2H),2.79–2.69(m,1H),0.99–0.91(t,3H).HRMS(ESI)m / z calcd.for C 12 H 17 N 3 O 2 Na[M+Na] + 258.1790; found 258.1211.

[0100] 12. Synthesis of Compound NEP214

[0101]

[0102] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 6-amino-2-pyridinecarboxylic acid (46 mg, 0.33 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP214 (26 mg, white solid, yield: 31%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.54–7.42(m,1H),7.35–7.29(m,1H),7.28–7.10(m,5H),6.72–6.66(m,1 H),4.82–4.79(m,1H),3.29–3.16(m,1H),3.14–3.04(m,1H).HRMS(ESI)m / z calcd.for C 15 H 17 N 4 O 2 [M+H] + 285.1352; found 285.1356.

[0103] 13. Synthesis of Compound NEP215

[0104]

[0105] 1) Compound SM-2 (200 mg, 1.3 mmol), LiOH·H 2 O (546 mg, 13 mmol), MeOH (6 mL), H 2 O (3 mL). The reaction was stirred at room temperature for 5 h. After the reaction was completed, the pH was adjusted to neutral, and the product was concentrated under reduced pressure to remove methanol. The residue was washed with dilute hydrochloric acid, extracted three times with EA (5 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product of compound 1 (148 mg, yellow solid) was obtained and directly carried out to the next step.

[0106] 2) Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1 (148 mg, 1.1 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP215 (164 mg, light yellow solid, yield: 53%). 1 H NMR (400 MHz, Methanol-d 4)δ9.59(s,1H),7.34–7.30(m,1H),7.30–7.22(m,3H),7.22–7.15(m,1H),7.05–6.97(m,1H) ,6.94–6.86(m,1H),4.85–4.82(m,1H),3.32–3.19(m,1H),3.09–2.98(m,1H).HRMS(ESI)m / z calcd.for C 15 H 16 N 3 O 3 [M+H] + 286.1192; found 286.1203.

[0107] 14. Synthesis of Compound NEP216

[0108]

[0109] 1) Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 5-nitro-2-pyridinecarboxylic acid (100 mg, 0.59 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH=80 / 1 to obtain compound 2 (75 mg, white solid, yield: 40%).

[0110] 2) Compound 2 (50 mg, 0.16 mmol), MeOH (4 mL), and H 2 / Pd (10 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP216 (27 mg, orange solid, yield: 61%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.21–8.14(m,1H),7.92–7.87(s,1H),7.69–7.62(m,1H),7.60(s,1H),7.27–7.20(m,2H),7.20–7.12(m,4 H),6.99–6.91(m,1H),5.96(s,2H),4.72–4.62(m,1H),3.14–3.05(m,1H),3.05–2.95(m,1H).HRMS(ESI)m / z calcd.forC 15 H 17 N 4 O 2 [M+H] +285.1352; found 285.1384.

[0111] 15. Synthesis of Compound NEP217

[0112]

[0113] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 2-pyridinecarboxylic acid (45 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP217 (34 mg, white solid, yield: 41%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.68–8.58(m,2H),8.03–7.95(m,2H),7.69(s,1H),7.66–7.58(m,1H),7.28–7.24(m,2H) ,7.24–7.14(m,4H),4.79–4.69(m,1H),3.22–3.12(m,1H),3.12–3.02(m,1H).HRMS(ESI)m / z calcd.for C 15 H 15 N 3 O 2 Na[M+Na] + 292.1164; found 292.1051.

[0114] 16. Synthesis of Compound NEP218

[0115]

[0116] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by pyrazole-3-carboxylic acid (41 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain a crude compound NEP218. The crude NEP218 was slurried with EA and then filtered to obtain pure NEP218 (23 mg, white solid, yield: 29%). 1 H NMR (400 MHz, DMSO-d 6 )δ13.27(s,1H),7.87–7.79(m,2H),7.67–7.60(m,1H),7.35–7.15(m,6H),6.65–6.59(m ,1H),4.72–4.62(m,1H),3.15–3.07(m,1H),3.07–2.91(m,1H).HRMS(ESI)m / zcalcd.for C13 H 15 N 4 O 2 [M+H] + 259.1117; found 259.1185.

[0117] 17. Synthesis of Compound NEP219

[0118]

[0119] Compound SM-1 (50 mg, 0.31 mmol), DMF (3 mL), imidazole-2-carboxylic acid (41 mg, 0.37 mmol), HATU (180 mg, 0.47 mmol) and DIPEA (280 μL, 1.6 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), and the organic phases were combined. Solids precipitated from the organic phases, which were filtered and the residue was washed three times with EA to obtain pure NEP219 (24 mg, transparent solid, yield: 30%). 1 HNMR (400 MHz, DMSO-d 6 )δ13.00(s,1H),8.09–8.03(m,1H),7.63(s,1H),7.29–7.23(m,5H),7.22–7.15(m,2H) ,7.03(s,1H),4.71–4.61(m,1H),3.18–3.09(m,1H),3.09–2.99(m,1H).HRMS(ESI)m / z calcd.for C 13 H 15 N 4 O 2 [M+H] + 259.1117; found 259.1184.

[0120] 18. Synthesis of Compound NEP220

[0121]

[0122] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1H-pyrazole-5-carboxylic acid (41 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP220 (27 mg, white solid, yield: 34%). 1 H NMR (400 MHz, Methanol-d 4)δ7.69(m,1H),7.33–7.15(m,5H),6.74(m,1H),4.85–4.81(m,1H),3.29–3.20(m,1H),3.12–3.02(m,1H).HRMS(ESI)m / z calcd.for C 13 H 15 N 4 O 2 [M+H] + 259.1117; found 259.1185.

[0123] 19. Synthesis of Compound NEP221

[0124]

[0125] 1) Compound SM-3 (400 mg, 1.40 mmol), DMF (8 mL), NH 4 Cl (100 mg, 1.78 mmol), HBTU (693 mg, 1.82 mmol) and DIPEA (1217 μL, 7 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 3 (244 mg, white solid, yield: 60%).

[0126] 2) Compound 3 (244 mg, 0.86 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 4 (104 mg, transparent solid, yield: 66%).

[0127] 3) Compound 4 (40 mg, 0.23 mmol), acetonitrile (3 mL), 1H-pyrrole-2-carboxylic acid (31 mg, 0.276 mmol), HBTU (114 mg, 0.299 mmol) and DIPEA (200 μL, 1.15 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP221 (25 mg, white solid, yield: 40%).1 H NMR (400 MHz, DMSO-d 6 )δ11.36(s,1H),8.06–7.99(m,1H),7.49(s,1H),7.33–7.24(m,1H),7.19–7.12(m,2H),7.10(s,1H),7.03–6.94(m,1H) ,6.84(s,1H),6.11–6.05(m,1H),5.77(s,1H),4.66–4.62(m,1H),3.14–3.06(m,1H),3.01–2.90(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 FN 3 O 2 Na[M+Na] + 298.1117; found 298.0956.

[0128] 20. Synthesis of Compound NEP222

[0129]

[0130] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-4 (200 mg, 0.7 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 100 / 1 to obtain compound 5 (112 mg, white solid, yield: 60%).

[0131] 2) Compound 5 (112 mg, 0.39 mmol), DCM (2 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 6 (50 mg, transparent solid, yield: 71%).

[0132] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by 6 (50 mg, 0.25 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP222 (20 mg, yellow solid, yield: 29%). 1 H NMR (400 MHz, Methanol-d 4)δ7.33–7.25(m,2H),7.02–6.94(m,2H),6.94–6.88(m,1H),6.84–6.79(m,1H),6.20– 6.14(m,1H),4.82–4.74(m,1H),3.27–3.19(m,1H),3.07–2.97(m,1H).HRMS(ESI)m / z calcd.for C 14 H 15 FN 3 O 2 [M+H] + 298.1477; found 298.0958.

[0133] 21. Synthesis of Compound NEP223

[0134]

[0135] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-5 (200 mg, 0.67 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 7 (127 mg, white solid, yield: 63%).

[0136] 2) Compound 7 (127 mg, 0.42 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 8 (97 mg, transparent solid, yield: 84%).

[0137] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by 8 (50 mg, 0.25 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP223 (23 mg, white solid, yield: 32%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.22(s,1H),7.16–7.03(m,3H),6.84–6.76(m,1H),6.76–6.67(m,1H),6.08–6. 01(m,1H),4.70–4.67(m,1H),3.16–3.10(m,1H),2.95–2.87(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 C1N3 O 2 Na[M+Na] + 314.0775; found 314.0661.

[0138] 22. Synthesis of Compound NEP224

[0139]

[0140] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-6 (200 mg, 0.51 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 100 / 1 to obtain compound 9 (130 mg, white solid, yield: 67%).

[0141] 2) Compound 9 (130 mg, 0.33 mmol), DCM (2 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 10 (61 mg, transparent solid, yield: 64%).

[0142] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by 10, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP224 (21 mg, brown solid, yield: 26%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.64–7.58(d,2H),7.11–7.05(d,2H),6.95–6.89(m,1H),6.85–6.79(m,1H),6.21– 6.14(m,1H),4.81–4.73(m,1H),3.25–3.15(m,1H),3.04–2.94(m,1H).HRMS(ESI)m / z calcd.for C 14 H 15 IN 3 O 2 [M+H] + 384.0131; found 384.0233.

[0143] 23. Synthesis of Compound NEP225

[0144]

[0145] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-7 (200 mg, 0.72 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound 11 (109 mg, white foamy solid, yield: 54%).

[0146] 2) Compound 11 (109 mg, 0.39 mmol), DCM (2 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP225-2 (30 mg, transparent solid, yield: 43%).

[0147] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by 12, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP225 (15 mg, transparent solid, yield: 35%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.12–7.03(d,2H),6.89(s,1H),6.82–6.77(m,1H),6.68(d,J=8.0Hz,2H),6.18–6 .12(m,1H),4.74–4.66(m,1H),3.19–3.07(m,1H),2.98–2.84(m,1H).HRMS(ESI)m / z calcd.for C 14 H 16 N 3 O 3 [M+H] + 274.1113; found 274.1193.

[0148] 24. Synthesis of Compound NEP226

[0149]

[0150] 1) Compound SM-6 (200 mg, 0.51 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 13 (111 mg, transparent solid, yield: 75%).

[0151] 2) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by compound 13 (111 mg, 0.38 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 30 / 1 to obtain compound 14 (82 mg, white solid, yield: 56%).

[0152] 3) Compound 14 (50 mg, 0.13 mmol), K 2 CO 3 (54mg, 0.39mmol), pd(dppf)Cl 2 (10 mg, 0.013 mmol), furan-2-boronic acid (22 mg, 0.2 mmol), Dioxane (4 ml) and H 2 O (2 ml). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP226 (19 mg, white solid, yield: 45%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.63–7.56(m,2H),7.54–7.49(m,1H),7.35–7.28(m,2H),6.91(m,1H),6.85–6.80(m,1H),6.72–6.67(m,1H) ,6.51–6.45(m,1H),6.20–6.14(m,1H),4.83–4.77(m,1H),3.29–3.20(m,1H),3.10–3.00(m,1H).HRMS(ESI)m / z calcd.for C 18 H 17 N 3 O 3 Na[M+Na] + 346.1270; found 346.1157.

[0153] 25. Synthesis of Compound NEP227

[0154]

[0155] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-8 (100 mg, 0.293 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 140 / 1 to obtain compound 15 (71 mg, white solid, yield: 73%).

[0156] 2) Compound 15 (71 mg, 0.21 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 16 (39 mg, transparent solid, yield: 75%).

[0157] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by compound 16 (39 mg, 0.16 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP227 (11 mg, transparent solid, yield: 21%). 1 H NMR (400 MHz, DMSO-d 6 )δ11.37(s,1H),8.07–8.00(m,1H),7.66–7.59(m,2H),7.58–7.52(m,2H),7.51(s,1H),7.46–7.37(m,3H),7.36–7.28(m,1H) ,7.09(s,1H),6.87–6.79(m,2H),6.09–6.03(m,1H),4.70–4.59(m,1H),3.15–3.06(m,1H),3.03–2.92(m,1H).HRMS(ESI)m / z calcd.for C 20 H 20 N 3 O 2 [M+H] + 334.1477; found 334.1545.

[0158] 26. Synthesis of Compound NEP228

[0159]

[0160] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-9 (300 mg, 1.0 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 17 (230 mg, white solid, yield: 77%).

[0161] 2) Compound 17 (230 mg, 0.51 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 18 (89 mg, transparent solid, yield: 89%).

[0162] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by compound 18 (50 mg, 0.25 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP228 (20 mg, white solid, yield: 29%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.18–7.08(m,4H),6.79(m,1H),6.73–6.67(m,1H),6.08–6.02(m,1H),4.68–4.65(m,1H),3.15–3.09(m,1H),2.95–2.84(m,1H).

[0163] 27. Synthesis of Compound NEP229

[0164]

[0165] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-10 (200 mg, 0.69 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound 19 (130 mg, white solid, yield: 65%).

[0166] 2) Compound 18 (130 mg, 0.45 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 20 (63 mg, transparent solid, yield: 74%).

[0167] 3) Compound 20 (50 mg, 0.33 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (46 mg, 0.42 mmol), HBTU (188 mg, 0.49 mmol) and DIPEA (286 μL, 1.65 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP229 (14 mg, white solid, yield: 15%). 1 H NMR (400 MHz, Methanol-d 4 )δ8.78–8.72(m,1H),8.48–8.41(m,1H),7.68–7.61(m,1H),7.58–7.46(m,2H),7. 33(s,1H),4.90–4.88(m,1H),3.41–3.35(m,1H),3.20–3.10(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 N 5 O 2 [M+H] + 284.1147; found 284.1148.

[0168] 28. Synthesis of Compound NEP230

[0169]

[0170] 1) Compound SM-11 (200 mg, 0.65 mmol), DMF (5 mL), NH 4 Cl (46 mg, 0.85 mmol), HBTU (324 mg, 1 mmol) and DIPEA (566 μL, 3.25 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), and the organic phases were combined. Solids were found to be precipitated, filtered, and the residue was washed three times with EA and dried. Compound 21 (123 mg, yellow solid, yield: 61%) was obtained.

[0171] 2) Compound 21 (123 mg, 0.39 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 22 (58 mg, transparent solid, yield: 72%).

[0172] 3) Compound 22 (58 mg, 0.27 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (36 mg, 0.32 mmol), HBTU (153 mg, 0.41 mmol) and DIPEA (234 μL, 1.35 mmol) were added to a 50 mL single-mouth bottle in sequence. The mixture was stirred at room temperature for 9 h. After the reaction was completed, solid precipitation was found. The reaction solution was filtered, and the residue was washed alternately with distilled water and EA three times and dried to obtain compound NEP230 (40 mg, white solid, yield: 49%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.96(s,1H),8.25–8.18(m,1H),8.16–8.09(d,2H),7.64(s,1H),7.57–7.51(d,2H),7.29–7. 22(m,2H),7.02(s,1H),4.77–4.67(m,1H),3.29–3.23(m,1H),3.21–3.14(m,1H).HRMS(ESI)m / z calcd.forC 13 H 14 N 5 O 4 [M+H] + 304.1046; found 304.1044.

[0173] 29. Synthesis of Compound NEP231

[0174]

[0175] Compound NEP230 (30 mg, 0.1 mmol), MeOH (3 mL), H 2 / Pd (6 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP231 (17 mg, white solid, yield: 64%). 1 H NMR (400 MHz, DMSO-d 6)δ12.99(s,1H),7.90–7.84(m,1H),7.53(m,1H),7.25(m,1H),7.15(m,1H),7.02(m,1H),6.95–6.82(d,2H ),6.45–6.39(d,2H),4.85(s,2H),4.56–4.46(m,1H),2.96–2.87(m,1H),2.87–2.77(m,1H).HRMS(ESI)m / z calcd.for C 13 H 16 N 5 O 2 [M+H] + 274.1304; found 274.1325.

[0176] 30. Synthesis of Compound NEP232

[0177]

[0178] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-12 (200 mg, 0.64 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 23 (194 mg, white solid, yield: 70%).

[0179] 2) Compound 23 (194 mg, 0.61 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 24 (92 mg, transparent solid, yield: 71%).

[0180] 3) Compound 24 (50 mg, 0.24 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (35 mg, 0.312 mmol), HBTU (119 mg, 0.312 mmol) and DIPEA (209 μL, 1.2 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, solid precipitation was found. The reaction solution was filtered, and the residue was washed alternately with distilled water and EA three times and dried to obtain compound NEP232 (10 mg, white solid, yield: 14%). 1 H NMR (400 MHz, DMSO-d 6)δ12.95(s,1H),8.15–8.09(m,1H),7.87–7.82(m,1H),7.82–7.75(m,2H),7.74(s,1H),7.67(s,1H),7.48–7.40 (m,3H),7.28–7.21(m,2H),7.00(s,1H),4.79–4.74(m,1H),3.31–3.24(m,1H),3.23–3.15(m,1H).HRMS(ESI)m / z calcd.for C 17 H 17 N 4 O 2 [M+H] + 309.1352; found 309.1376.

[0181] 31. Synthesis of Compound NEP233

[0182]

[0183] Compound SM-3 (40 mg, 0.23 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (26 mg, 0.23 mmol), HBTU (142 mg, 0.375 mmol) and DIPEA (217 μL, 1.25 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP233 (16 mg, transparent solid, yield: 26%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.50–7.38(m,2H),7.29(q,J=7.3Hz,1H),7.16–7.04(m,2H),7.00–6.91(m ,1H),4.85–4.82(m,1H),3.31–3.26(m,1H),3.14–3.04(m,1H).HRMS(ESI)m / z calcd.for C 13 H 14 FN 4 O 2 [M+H] + 277.1101; found 277.1117.

[0184] 32. Synthesis of Compound NEP234

[0185]

[0186] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by 2-thiophenecarboxylic acid (36 mg, 0.27 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP234 (23 mg, white solid, yield: 34%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.73–7.68(m,1H),7.67–7.61(m,1H),7.33–7.23(m,1H),7.15–7.04(m,3H),6.98– 6.88(m,1H),4.87–4.79(m,1H),3.31–3.25(m,1H),3.12–3.02(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 FN 2 O 2 S[M+H] + 293.0760; found 293.0761.

[0187] 33. Synthesis of Compound NEP235

[0188]

[0189] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-13 (200 mg, 0.74 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 25 (127 mg, white solid, yield: 60%).

[0190] 2) Compound 25 (127 mg, 0.44 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 26 (59 mg, white solid, yield: 73%).

[0191] 3) Prepared according to the preparation step of NEP229 (3), except that compound 20 was replaced by compound 26 (50 mg, 0.27 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP235 (13 mg, white solid, yield: 18%). 1H NMR (400 MHz, Methanol-d 4 )δ7.56(m,2H),4.69–4.61(m,1H),1.92–1.65(m,8H),1.51–1.42(m,1H),1.28–1.21(m,2H),1.07–0.95(m,2H).HRMS(ESI)m / z calcd.for C 13 H 21 N 4 O 2 [M+H] + 265.1664; found 265.1676.

[0192] 34. Synthesis of Compound NEP236

[0193]

[0194] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by 2-furancarboxylic acid (31 mg, 0.27 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound NEP236 (26 mg, white solid, yield: 41%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.66(s,1H),7.32–7.22(m,1H),7.14–7.08(m,2H),7.08–7.02(m,1H),6.98–6.89(m,1H) ,6.60–6.54(m,1H),4.85–4.81(m,1H),3.31–3.24(m,1H),3.12–3.02(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 FN 2 O 3 [M+H] + 277.0988; found 277.1010.

[0195] 35. Synthesis of Compound NEP237

[0196]

[0197] Compound SM-14 (50 mg, 0.32 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (36 mg, 0.32 mmol), HBTU (159 mg, 0.416 mmol) and DIPEA (279 μL, 1.35 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, solid precipitation was found. The reaction solution was filtered, and the residue was washed alternately with distilled water and EA three times and dried to obtain compound NEP237 (14 mg, white solid, yield: 20%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.16–8.03(m,1H),7.71(s,1H),7.38(s,2H),7.29(s,1H),4.46–4.38(m,1H),1.97 –1.86(m,1H),1.64–1.50(m,1H),1.26–1.11(m,1H),1.02–0.90(m,6H).HRMS(ESI)m / z calcd.for C 10 H 17 N 4 O 2 [M+H] + 225.1351; found 225.1353.

[0198] 36. Synthesis of Compound NEP238

[0199]

[0200] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-15 (200 mg, 0.72 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 27 (134 mg, white solid, yield: 67%).

[0201] 2) Compound 27 (134 mg, 0.48 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 28 (60 mg, transparent solid, yield: 70%).

[0202] 3) Prepared according to the preparation step of NEP229 (3), except that compound 20 was replaced by compound 28 (50 mg, 0.28 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP238 (15 mg, white solid, yield: 16%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.46(s,2H),7.31–7.20(m,4H),7.20–7.12(m,1H),4.64–4.56(m,1H),2.85–2.69(m,2H),2.32–2.19(m,1H),2.17–1.99(m,1H).HRMS(ESI)m / z calcd.for C 14 H 17 N 4 O 2 [M+H] + 273.1352; found 273.1368.

[0203] 37. Synthesis of Compound NEP239

[0204]

[0205] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by indole-6-carboxylic acid (41 mg, 0.27 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP239 (25 mg, white solid, yield: 34%). 1 H NMR (400 MHz, DMSO-d 6 )δ11.39(s,1H),8.41–8.35(m,1H),7.93(s,1H),7.60–7.53(m,2H),7.53–7.46(m,2H),7.35–7.25(m,1H),7.24–7.16(m,2H) ,7.13(s,1H),7.04–6.94(m,1H),6.51–6.45(m,1H),4.74–4.64(m,1H),3.20–3.11(m,1H),3.11–3.01(m,1H).HRMS(ESI)m / z calcd.for C 18 H 17 FN 3 O 2 [M+H] + 326.1305; found 326.1297.

[0206] 38. Synthesis of Compound NEP240

[0207]

[0208] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by indole-7-carboxylic acid (41 mg, 0.27 mmol, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to give compound NEP240 (15 mg, orange solid, yield: 20%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.75(d,J=7.8Hz,1H),7.54(d,J=7.5Hz,1H),7.36–7.23(m,2H),7.19–7.04(m,3H),6.98–6.89(m,1H ),6.54–6.48(m,1H),4.96–4.91(m,1H),3.36–3.34(m,1H),3.18–3.08(m,1H).HRMS(ESI)m / zcalcd.for C 18 H 17 FN 3 O 2 [M+H] + 326.1305; found 326.1310.

[0209] 39. Synthesis of Compound NEP241

[0210]

[0211] 1) Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by 4-Boc-3-morpholinecarboxylic acid (68 mg, 0.29 mmol, and purified by column chromatography with a developing solvent ratio of DCM / MeOH=60 / 1 to give compound 29 (20 mg, transparent solid, yield: 18%).

[0212] 2) Compound 29 (20 mg, 0.051 mmol), DCM (2 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP241 (7 mg, transparent solid, yield: 47%). 1 H NMR (400 MHz, Methanol-d 4)δ7.37–7.27(m,1H),7.15–7.08(m,1H),7.08–7.02(m,1H),7.02–6.92(m,1H),4.72–4.64(m,1H),4.32–4.23(m,1 H),4.09–3.95(m,2H),3.80–3.61(m,2H),3.32–3.22(m,2H),3.22–3.15(m,1H),3.02–2.89(m,1H).HRMS(ESI)m / z calcd.for C 14 H 19 FN 3 O 3 [M+H] + 296.1411; found 296.1405.

[0213] 40. Synthesis of Compound NEP242

[0214]

[0215] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-16 (200 mg, 0.8 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 80 / 1 to obtain compound 30 (142 mg, white solid, yield: 71%).

[0216] 2) Compound 30 (142 mg, 0.56 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 31 (61 mg, transparent solid, yield: 73%).

[0217] 3) Prepared according to the preparation step of NEP229 (3), except that compound 20 was replaced by compound 31 (50 mg, 0.34 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 30 / 1 to obtain compound NEP242 (17 mg, transparent solid, yield: 22%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.68–7.62(m,2H),7.58–7.44(m,5H),5.69(s,1H).HRMS(ESI)m / z calcd.for C 12 H 13 N 4 O 2 [M+H]+ 245.1039; found 245.1079.

[0218] 41. Synthesis of Compound NEP243

[0219]

[0220] Compound SM-17 (50 mg, 0.21 mmol), acetonitrile (3 mL), imidazole-2-carboxylic acid (26 mg, 0.23 mmol), HATU (120 mg, 0.315 mmol) and DIPEA (183 μL, 1.05 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP243 (24 mg, white solid, yield: 40%). 1 H NMR (400 MHz, DMSO-d 6 )δ13.02(s,1H),10.85–10.80(m,1H),8.03–7.96(m,1H),7.66–7.59(m,2H),7.35–7.28(m,1H),7.26(s,1H),7.21(s ,1H),7.17–7.12(m,1H),7.10–7.00(m,2H),7.00–6.91(m,1H),4.73–4.63(m,1H),3.30–3.13(m,2H).HRMS(ESI)m / z calcd.for C 15 H 16 N 5 O 2 [M+H] + 298.1304; found 298.1313.

[0221] 42. Synthesis of Compound NEP244

[0222]

[0223] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by thiazole-5-carboxylic acid (33 mg, 0.253 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP244 (23 mg, brown solid, yield: 34%). 1 H NMR (400 MHz, DMSO-d6 )δ9.21(s,1H),8.89–8.82(m,1H),8.53(s,1H),7.64(s,1H),7.36–7.26(m,1H),7.23–7.13(m, 3H),7.08–6.96(m,1H),4.69–4.59(m,1H),3.18–3.12(m,1H),3.01–2.90(m,1H).HRMS(ESI)m / z calcd.forC 13 H 13 FN 3 O 2 S[M+H] + 294.0713; found 294.0713.

[0224] 43. Synthesis of Compound NEP245

[0225]

[0226] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by oxazole-5-carboxylic acid (29 mg, 0.253 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP245 (20 mg, transparent solid, yield: 32%). 1 H NMR (400 MHz, Methanol-d 4 )δ8.31(s,1H),7.71(s,1H),7.28–7.22(m,1H),7.14–7.09(m,1H),7.09–7.02(m,1H),6. 99–6.88(m,1H),4.90–4.81(m,1H),3.32–3.26(m,1H),3.10–3.01(m,1H).HRMS(ESI)m / z calcd.for C 13 H 13 FN 3 O 3 [M+H] + 278.0941; found 278.0938.

[0227] 44. Synthesis of Compound NEP246

[0228]

[0229] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-18 (200 mg, 0.74 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound 32 (152 mg, white solid, yield: 76%).

[0230] 2) Compound 32 (152 mg, 0.56 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound 33 (61 mg, transparent solid, yield: 79%).

[0231] 3) Prepared according to the preparation step of NEP229 (3), except that compound 20 was replaced by compound 33 (50 mg, 0.29 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 30 / 1 to obtain compound NEP246 (17 mg, transparent solid, yield: 27%). 1 H NMR (400 MHz, DMSO-d 6 )δ13.03(s,1H),8.18–8.12(m,1H),7.61(s,1H),7.33–7.28(m,1H),7.28–7.24(m,2H) ,7.03(s,1H),6.93–6.83(m,2H),4.65–4.56(m,1H),3.31–3.28(m,2H).HRMS(ESI)m / z calcd.for C 11 H 13 N 4 O 2 S[M+H] + 265.0681; found 265.0769.

[0232] 45. Synthesis of Compound NEP247

[0233]

[0234] Prepared according to the preparation steps of NEP233, except that imidazole-2-carboxylic acid was replaced by pyrazine-2-carboxylic acid (32 mg, 0.253 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP247 (28 mg, transparent solid, yield: 42%). 1 H NMR (400 MHz, Methanol-d 4)δ9.20–9.16(m,1H),8.81–8.76(m,1H),8.74–8.65(m,1H),7.31–7.21(m,1H),7.13–7.08(m,1H),7.08– 7.01(m,1H),7.00–6.88(m,1H),4.94–4.89(m,1H),3.31–3.22(m,1H),3.21–3.09(m,1H).HRMS(ESI)m / z calcd.for C 14 H 14 FN 4 O 2 [M+H] + 289.1023; found 289.1091.

[0235] 46. ​​Synthesis of Compound NEP248

[0236]

[0237] Compound SM-19 (62 mg, 0.31 mmol), CH 3 COOH (4 mL), phthalic anhydride (46 mg, 0.31 mmol), CH 3 COONa (50 mg, 0.37 mmol). Stir at 90°C overnight. After the reaction is complete, add 100 ml of ddH 2 O, the precipitated solid was filtered, and the residue was washed three times with water and dried to obtain compound NEP248 (35 mg, white solid, yield: 38%). 1 H NMR (400 MHz, DMSO-d 6 )δ7.80(m,4H),7.71(s,1H),7.32(s,1H),7.19–7.04(m,5H),4.97–4.88(m,1H),3.57–3.47(m,1H),3.37(m,1H).HRMS(ESI)m / zcalcd.for C 17 H 15 N 2 O 3 [M+H] + 295.1004; found 295.1081. 47. Synthesis of compound NEP249

[0238]

[0239] Compound SM-19 (30 mg, 0.148 mmol), toluene (3 mL), methyl 3-(bromomethyl)pyridine-2-carboxylate (38 mg, 0.165 mmol), K 2 CO 3 (57 mg, 0.41 mmol). Stir at 80°C overnight. After the reaction was completed, the reaction solution was concentrated to dryness and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP249 (15 mg, white solid, yield: 33%). 1 H NMR (400 MHz, DMSO-d 6 )δ8.68–8.62(m,1H),8.04–7.98(m,1H),7.55–7.48(m,1H),7.30–7.17(m,4H),7.17–7.07(m,1H), 5.03–4.96(m,1H),4.77(m,1H),4.36(m,1H),3.54–3.50(m,1H),3.11–3.00(m,1H).HRMS(ESI)m / z calcd.forC 16 H 16 N 3 O 2 [M+H] + 282.1164; found 282.1023.

[0240] 48. Synthesis of Compound NEP250

[0241]

[0242] Compound SM-19 (62 mg, 0.31 mmol), CH 3 COOH (4 mL), 2,3-pyridinedicarboxylic anhydride (48 mg, 0.31 mmol), CH 3 COONa (50 mg, 0.37 mmol). Reflux at 90°C and stir overnight. After the reaction is completed, the reaction solution is concentrated to dryness and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP250 (20 mg, white solid, yield: 22%). 1 H NMR (400 MHz, DMSO-d 6)δ8.98–8.92(m,1H),8.29–8.22(m,1H),7.80–7.73(m,1H),7.70(s,1H),7.33(s,1H),7.18–7.13( m,1H),7.13–7.03(m,4H),5.01–4.92(m,1H),3.58–3.49(m,1H),3.31–3.22(m,1H).HRMS(ESI)m / z calcd.forC 16 H 14 N 3 O 3 [M+H] + 296.0957; found 296.1044.

[0243] 49. Synthesis of Compound NEP251

[0244]

[0245] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-20 (400 mg, 1.40 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 100 / 1 to obtain compound 34 (262 mg, white solid, yield: 66%).

[0246] 2) Compound 34 (262 mg, 0.92 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound 35 (104 mg, transparent solid, yield: 74%).

[0247] 3) Prepared according to the preparation step of NEP229 (3), except that compound 20 was replaced by compound 35 (40 mg, 0.23 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP251 (19 mg, transparent solid, yield: 30%). 1 H NMR (400 MHz, DMSO-d 6 )δ13.01(s,1H),8.14–8.07(m,1H),7.62(s,1H),7.57–7.20(m,4H),7.19–6.94 (m,3H),4.82–4.64(m,1H),3.24–3.15(m,1H),3.10–3.00(m,1H).HRMS(ESI)m / z calcd.for C 13H 14 FN 4 O 2 [M+H] + 277.1023; found 277.1108.

[0248] 50. Synthesis of Compound NEP252

[0249]

[0250] (1) Compound SM-19 (62 mg, 0.31 mmol), CH 3 COOH (4 mL), 3-nitrophthalic anhydride (48 mg, 0.31 mmol), CH 3 COONa (50 mg, 0.37 mmol). Reflux at 90°C and stir overnight. After the reaction is completed, the reaction solution is concentrated to dryness and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 100 / 1 to obtain compound 36 (31 mg, yellow solid, yield: 30%).

[0251] (2) Compound 36 (31 mg, 0.091 mmol), MeOH (3 mL), and H 2 / Pd (6 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP252 (15 mg, yellow solid, yield: 54%). 1 H NMR(400MHz,MeOD)δ7.40–7.32(m,1H),7.20–7.08(m,5H),6.96–6.88(m,2H),5.05–5.00(m,1H),3.59–3.47(m,2H).HRMS(ESI)m / z calcd.for C 17 H 16 N 3 O 3 [M+H] + 310.1113; found 310.1180.

[0252] 51. Synthesis of Compound NEP253

[0253]

[0254] Compound SM-19 (62 mg, 0.31 mmol), CH 3COOH (4 mL), 3-nitrophthalic anhydride (48 mg, 0.31 mmol), CH 3 COONa (50 mg, 0.37 mmol). Reflux at 90°C and stir overnight. After the reaction is completed, the reaction solution is concentrated to dryness and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP253 (31 mg, yellow solid, yield: 45%). 1 H NMR(400MHz,DMSO)δ7.66(s,1H),7.60–7.52(m,1H),7.27(s,1H),7.21–7.06(m,7H),4.89–4.80(m,1H),3.52–3.45(m,2H).HRMS(ESI)m / zcalcd.for C 17 H 15 N 2 O 4 [M+H] + 311.0954; found 311.1043.

[0255] 52. Synthesis of Compound NEP254

[0256]

[0257] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by 1H-indole-2-carboxylic acid (60 mg, 0.37 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP254 (48 mg, white solid, yield: 50%). 1 H NMR(400MHz,DMSO)δ11.04(s,1H),7.66(s,1H),7.60–7.52(m,2H),7.27(s, 1H),7.22–7.05(m,9H),4.87–4.82(m,1H),3.51–3.45(m,2H).HRMS(ESI)m / z calcd.for C 18 H 18 N 3 O 2 [M+H] + 308.1321.; found 308.1400.

[0258] 53. Synthesis of Compound NEP255

[0259]

[0260] Compound SM-21 (50 mg, 0.31 mmol), DMF (3 mL), pyrrole-2-carboxylic acid (41 mg, 0.37 mmol), HATU (180 mg, 0.47 mmol) and DIPEA (280 μL, 1.6 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP255 (41 mg, white solid, yield: 52%). 1 H NMR (400MHz, DMSO) δ11.36(s,1H),8.03–7.97(m,1H),7.49(s,1H),7.35–7.29(m,2H),7.29–7.21(m,2H),7.20–7.12(m,1H),7. 11–7.06(m,1H),6.86–6.80(m,2H),6.10–6.04(m,1H),4.67–4.56(m,1H),3.13–3.04(m,1H),2.98–2.88(m,1H).HRMS(ESI)m / z calcd.for C 14 H 16 N 3 O 2 [M+H] + 258.1164.; found 258.1249.

[0261] 54. Synthesis of Compound NEP256

[0262]

[0263] Compound SM-22 (55 mg, 0.31 mmol), DMF (3 mL), pyrrole-2-carboxylic acid (41 mg, 0.37 mmol), HATU (180 mg, 0.47 mmol) and DIPEA (280 μL, 1.6 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP256 (38 mg, white solid, yield: 45%). 1H NMR (400 MHz, MeOD) δ7.25–7.16 (m, 5H), 6.90 (s, 1H), 6.46 (s, 1H), 6.18–6.12 (m, 1H), 5.33–5.08 (m, 1H), 3.39 (dd, J = 14.5, 4.9 Hz, 1H), 3.20 (dd, J = 15.1, 10.8 Hz, 1H), 3.11 (s, 3H). 55. Synthesis of Compound NEP257

[0264]

[0265] 1) In a 50 mL single-mouth bottle, dissolve compound SM-23 (100 mg, 0.56 mmol) in an appropriate amount of THF, then add 3-nitro-2-pyridone (94 mg, 0.67 mmol), PH 3 P (221 mg, 0.84 mmol), N 2 After protection, DIAD (142 μL, 0.84 mmol) was added at 0°C, and the reaction was stirred at 60°C for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of PE:EA=8:1 to obtain compound 37 (88 mg, transparent solid, yield: 52%).

[0266] 2) Compound 37 (88 mg, 0.29 mmol), LiOH·H 2 O (123 mg, 2.9 mmol), MeOH (6 mL), H 2 O (3 mL). Stir the reaction at room temperature for 5 h. After the reaction is completed, the pH is adjusted to neutral, and the product is concentrated under reduced pressure to remove methanol. The residue is washed with dilute hydrochloric acid and extracted three times with EA (5 mL). The organic phases are combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product is purified by reverse chromatography (MeOH / H 2 D, 0.1% TFA) to give compound 38 (60 mg, white solid, yield: 72%).

[0267] 3) Compound 38 (60 mg, 0.2 mmol), DMF (3 mL), NH 4Cl (14 mg, 0.26 mmol), HBTU (99 mg, 0.26 mmol) and DIPEA (174 μL, 1 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 200 / 1 to obtain compound 39 (43 mg, yellow solid, yield: 75%).

[0268] 4) Compound 39 (43 mg, 0.15 mmol), MeOH (4 mL), and H 2 / Pd (9 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP257 (20 mg, yellow solid, yield: 53%). 1 H NMR(400MHz,MeOD)δ7.46–7.38(m,1H),7.33–7.27(m,2H),7.27–7.21(m,2H),7.21–7.14(m,1H) ,6.99(dd,J=7.6,1.6Hz,1H),6.74(dd,J=7.6,4.9Hz,1H),5.64–5.57(m,1H),3.34–3.18(m,2H).

[0269] 56. Synthesis of Compound NEP258

[0270]

[0271] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by SM-24 (200 mg, 0.72 mmol), and purified by column chromatography with a developing solvent ratio of PE / EA = 1 / 1 to obtain compound 40 (103 mg, white solid, yield: 51%).

[0272] 2) Compound 40 (103 mg, 0.369 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain compound 41, which was directly carried out to the next step.

[0273] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by compound 41 (33 mg, 0.184 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP258 (27 mg, white solid, yield: 55%). 1 H NMR (400MHz, DMSO) δ11.37(s,1H),8.00–7.94(m,1H),7.50–7.46(m,1H),7.23–7.17(m,2H),7.10–7.02(m,3H) ,6.86–6.80(m,2H),6.10–6.04(m,1H),4.60–4.56(m,1H),3.06–3.00(m,1H),2.92–2.85(m,1H),2.23(s,3H).

[0274] 57. Synthesis of Compound NEP259

[0275]

[0276] 1) Prepared according to the preparation steps of NEP221 (1), except that SM-3 was replaced by compound SM-25 (200 mg, 0.72 mmol), and purified by column chromatography with a developing solvent ratio of PE / EA = 1 / 1 to obtain compound 42 (111 mg, white solid, yield: 55%).

[0277] 2) Compound 42 (111 mg, 0.39 mmol), DCM (6 mL) and trifluoroacetic acid (3 mL) were added to a 50 mL single-mouth bottle in sequence. The mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain compound 43, which was directly carried out to the next step.

[0278] 3) Prepared according to the preparation steps of NEP221 (3), except that compound 4 was replaced by compound 43 (45 mg, 0.251 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP259 (29 mg, white solid, yield: 43%). 1H NMR (400MHz, MeOD) δ7.18–7.10(m,2H),7.10–7.04(m,1H),7.04–6.97(m,1H),6.91(dd,J=2.6,1.4Hz,1H),6.81(dd,J=3.8 ,1.5Hz,1H),6.17(dd,J=3.8,2.5Hz,1H),4.77(dd,J=8.9,5.7Hz,1H),3.25–3.15(m,1H),3.05–2.95(m,1H),2.27(s,3H).

[0279] 58. Synthesis of Compound NEP260

[0280]

[0281] Prepared according to the preparation steps of NEP204, except that 1-naphthoic acid was replaced by isoquinoline-1-carboxylic acid (60 mg, 0.34 mmol), and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP260 (32 mg, white solid, yield: 36%). 1 H NMR(400MHz,MeOD)δ8.55–8.46(m,2H),8.00–7.90(m,2H),7.82–7.73(m,1H),7.66–7. 57(m,1H),7.38–7.20(m,5H),5.04–4.96(m,1H),3.40–3.35(m,1H),3.11–3.01(m,1H).

[0282] 59. Synthesis of Compound NEP261

[0283]

[0284] Prepared according to the preparation steps of NEP226 (3), except that furan-2-boronic acid was replaced by 1-methyl-1H-pyrazole-4-boronic acid (20 mg, 0.156 mmol), and prepared by high performance liquid chromatography (MeOH / H 2 After purification by HPLC-MS / MS, the compound NEP263 (10 mg, transparent solid, yield: 38%) was obtained. 1H NMR(400MHz,MeOD)δ7.91(s,1H),7.79(s,1H),7.45(dd,J=8.1,1.6Hz,2H),7.32–7.26(m,2H),6.94–6.88(m,1H ),6.85–6.79(m,1H),6.20–6.14(m,1H),4.82–4.77(m,1H),3.92(s,3H),3.27–3.20(m,1H),3.07–3.00(m,1H).

[0285] 60. Synthesis of Compound NEP262

[0286]

[0287] 1) Compound SM-1 (50 mg, 0.31 mmol), CH 3 COOH (4 mL), 3-bromofuran-2,5-dione (55 mg, 0.31 mmol), CH 3 COONa (51 mg, 0.37 mmol) was added and stirred at 90°C for overnight reflux. After the reaction was completed, the reaction solution was concentrated to dryness and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 160 / 1 to obtain compound 44 (78 mg, white solid, yield: 78%).

[0288] 2) Compound 44 (78 mg, 0.24 mmol), DMF (3 mL), aniline (23 mg, 0.24 mmol), Et 3 N (49 mg, 0.48 mmol). Stir at 60 ° C overnight. After the reaction is completed, the reaction solution is washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases are combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It is purified by column chromatography with a developing solvent ratio of PE: EA = 1: 2 to obtain compound NEP262 (36 mg, yellow solid, yield: 45%). 1 H NMR (400MHz, MeOD) δ7.41–7.36(m,2H),7.27–7.21(m,4H),7.20–7.11(m,4H),5.42(s,1H),4.95–4.91(m,1H),3.49–3.44(m,2H).

[0289] Example 2: PROTAC Synthesis

[0290] 1. Synthesis of Compound NEP216-1

[0291]

[0292] 1) Add compound NEP216 (50 mg, 0.16 mmol), DMF (3 mL), N (tert-butyloxycarbonyl)-2-aminoacetic acid (34 mg, 0.192 mmol), HATU (92 mg, 0.24 mmol), and DIPEA (139 μL, 0.8 mmol) to a 50 mL single-mouth bottle. Stir and react at room temperature for 9 h. After the reaction is complete, remove the reaction solution with saturated brine (100 mL).

[0293] The mixture was washed and extracted with EA (5 mL) three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP216-1-1 (20 mg, white solid, yield: 29%).

[0294] 2) Compound NEP216-1-1 (20 mg, 0.045 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain compound NEP216-1-2, which was directly used for the next step without purification.

[0295] 3) Compound NEP216-1-2 (15 mg, 0.044 mmol), DMF (3 mL), JQ1-COOH (19 mg, 0.044 mmol), HATU were added in sequence into a 50 mL single-necked bottle.

[0296] (26 mg, 0.066 mmol), DIPEA (57 μL, 0.22 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP216-1 (8 mg, light yellow solid, yield: 23%). 1 H NMR (400 MHz, Methanol-d 4)δ8.70(d,J=2.5Hz,1H),8.07(dd,J=8.7,2.5Hz,1H),7.85(d,J=8.5Hz,1H),7.37(d,J=8.2Hz,2H),7. 23(d,J=8.3Hz,2H),7.19–7.08(m,4H),7.08–7.01(m,1H),4.56(dd,J=8.8,5.4Hz,1H),4.06(d,J=16.8 Hz,1H),3.99(d,J=16.9Hz,1H),3.46(dd,J=15.3,8.9Hz,1H),3.33(dd,J=15.3,5.4Hz,1H),3.13(dd,J =13.8,5.7Hz,1H),2.99(dd,J=13.8,8.1Hz,1H),2.58(s,3H),2.31(s,3H),1.55(s,3H).HRMS(ESI)m / z calcd.for C 36 H 35 C1N 9 O 4 S[M+H] + 724.2143; found 724.2209.

[0297] 2. Synthesis of Compound NEP216-2

[0298]

[0299] 1) Compound NEP216 (40 mg, 0.14 mmol), DMF (3 mL), N (tert-butyloxycarbonyl) -4-aminobutyric acid (35 mg, 0.17 mmol), HATU (81 mg, 0.21 mmol), DIPEA (123 μL, 0.7 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 60 / 1 to obtain compound NEP216-2-1 (37 mg, white solid, yield: 57%).

[0300] 2) Compound NEP216-2-1 (37 mg, 0.079 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP216-2-2 (20 mg, transparent solid, yield: 69%).

[0301] 3) Compound NEP216-2-2 (20 mg, 0.054 mmol), DMF (3 mL), JQ1-COOH (22 mg, 0.054 mmol), HATU (31 mg, 0.081 mmol), and DIPEA (48 μL, 0.27 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP216-2 (8 mg, light yellow solid, yield: 20%). 1 H NMR (400 MHz, Methanol-d 4 )δ8.80–8.75(m,1H),8.22–8.15(m,1H),8.00–7.93(m,1H),7.48–7.42(m,2 H),7.41–7.35(m,2H),7.32–7.23(m,4H),7.23–7.15(m,1H),4.70–4.60(m,2 H),3.48–3.36(m,4H),3.29–3.23(m,1H),3.15–3.08(m,1H),2.70(s,3H),2. 58–2.49(m,2H),2.45(s,3H),2.02–1.93(m,2H),1.70(s,3H).HRMS(ESI)m / z calcd.for C 38 H 39 C1N 9 O 4 S[M+H] + 752.2534; found 752.2533.

[0302] 3. Synthesis of Compound NEP216-3

[0303]

[0304] 1) Compound NEP216 (40 mg, 0.14 mmol), DMF (3 mL), N (tert-butyloxycarbonyl)-5-aminopentanoic acid (46 mg, 0.17 mmol), HATU (81 mg, 0.21 mmol), and DIPEA (123 μL, 0.7 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP216-3-1 (32 mg, white solid, yield: 47%).

[0305] 2) Compound NEP216-3-1 (32 mg, 0.066 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP216-3-2 (20 mg, white solid, yield: 79%). 1 H NMR (400 MHz, Methanol-d 4 )δ8.83–8.78(m,1H),8.24–8.17(m,1H),8.02–7.95(m,1H),7.32–7.15(m,5H),3.31–3.19(m,1H) ,3.17–3.07(m,1H),3.03–2.88(m,2H),2.57–2.49(m,2H),1.87–1.75(m,3H),1.75–1.68(m,1H).

[0306] 3) Compound NEP216-3-2 (20 mg, 0.053 mmol), DMF (3 mL), JQ1-COOH (22 mg, 0.053 mmol), HATU (31 mg, 0.08 mmol), and DIPEA (48 μL, 0.27 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP216-3 (9 mg, light yellow solid, yield: 22%). 1 H NMR (400 MHz, Methanol-d 4)δ8.79–8.74(m,1H),8.21–8.14(m,1H),8.01–7.92(m,1H),7.47–7.41(m,2H),7.41 –7.35(m,2H),7.34–7.22(m,4H),7.22–7.15(m,1H),4.67–4.62(m,1H),4.55(s,1H), 3.48–3.38(m,2H),3.27–3.22(m,1H),3.15–3.08(m,1H),2.69(s,3H),2.52–2.46(m ,2H),2.42(s,3H),1.84–1.75(m,2H),1.66(s,3H),1.37–1.28(m,4H).HRMS(ESI)m / z calcd.for C 39 H 41 C1N 9 O 4 S[M+H] + 766.2691; found 766.2682.

[0307] 4. Synthesis of Compound NEP214-1

[0308]

[0309] 1) In a 50 mL single-mouth bottle, succinic anhydride (92 mg, 0.91 mmol) was dissolved in 5 ml DCM, and then the DCM solution of compound NEP214 (260 mg, 0.91 mmol) was slowly added and reacted at 90°C for 5 hours. After the reaction was completed, the reaction solution was filtered, and the residue was washed three times with DCM and dried to obtain compound TM-1 (220 mg, transparent solid, yield: 60%).

[0310] 2) Compound TM-1 (110 mg, 0.276 mmol), DMF (4 mL), mono-Boc-ethylenediamine (20 mg, 0.358 mmol), HATU (158 mg, 0.414 mmol), and DIPEA (240 μL, 1.38 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP214-1-1 (55 mg, white solid, yield: 56%).

[0311] 3) Compound NEP214-1-1 (55 mg, 0.13 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP214-1-2 (20 mg, transparent solid, yield: 53%).

[0312] 4) Compound NEP214-1-2 (20 mg, 0.067 mmol), DMF (3 mL), JQ1-COOH (26 mg, 0.067 mmol), HATU (38 mg, 0.1 mmol), and DIPEA (59 μL, 0.34 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain a crude product of compound Fb-20, which was prepared by high performance liquid chromatography (MeOH / H 2 After purification by HPLC-MS / MS, the compound NEP214-1 (7 mg, light yellow solid, yield: 13%) was obtained. 1 H NMR (400 MHz, Methanol-d 4 )δ8.31–8.24(m,1H),7.87–7.79(m,1H),7.75–7.68(m,1H),7.48–7.38(m,4H),7 .27–7.22(m,4H),7.20–7.16(m,1H),4.70–4.62(m,1H),3.50–3.45(m,1H),3.44– 3.40(m,2H),3.30–3.20(m,1H),3.11–3.01(m,1H),2.82–2.74(m,2H),2.70(s,3H ),2.61–2.53(m,2H),2.43(s,3H),1.69(s,3H),1.37–1.29(m,4H).HRMS(ESI)m / z calcd.for C 40 H 42 C1N 10 O 5 S[M+H] + 809.2749; found 809.2764.

[0313] 5. Synthesis of Compound NEP214-2

[0314]

[0315] 1) Compound TM-1 (25 mg, 0.062 mmol), DMF (3 mL), N-tert-butyloxycarbonyl-1,3-propylenediamine (14 mg, 0.074 mmol), HATU (36 mg, 0.093 mmol), and DIPEA (81 μL, 0.31 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP214-2-1 (26 mg, white solid, yield: 79%).

[0316] 2) Compound NEP214-2-1 (26 mg, 0.048 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP214-2-2 (14 mg, transparent solid, yield: 67%).

[0317] 3) Compound NEP214-2-2 (14 mg, 0.03 mmol), DMF (3 mL), JQ1-COOH (12 mg, 0.03 mmol), HATU (19 mg, 0.05 mmol), and DIPEA (26 μL, 0.15 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP214-2 (3 mg, light yellow solid, yield: 12%). 1 H NMR (400 MHz, Methanol-d 4)δ8.16–8.10(m,1H),7.76–7.68(m,1H),7.62–7.56(m,1H),7.35–7.24(m,4H), 7.17–7.07(m,4H),7.07–7.00(m,1H),4.72–4.49(m,2H),3.35–3.28(m,1H),3.2 4(s,2H),3.22–3.17(m,4H),3.00–2.90(m,1H),2.70–2.62(m,2H),2.57(s,3H) ,2.51–2.43(m,2H),2.30(s,3H),1.71–1.59(m,2H),1.55(s,3H).HRMS(ESI)m / z calcd.for C 41 H 44 C1N 10 O 5 S[M+H] + 823.2905; found 823.2893.

[0318] VI. Synthesis of Compound NEP219-1

[0319]

[0320] 1) Compound SM-1 (200 mg, 1 mmol), CH 3 CN (6 mL), 5-nitro-imidazole-2-carboxylic acid (160 mg, 1 mmol), BOP (784 mg, 1.5 mmol), DIPEA (872 μL, 5 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP219-1-1 (187 mg, white solid, yield: 47%).

[0321] 2) Compound NEP219-1-1 (187 mg, 0.55 mmol), MeOH (4 mL), and H 2 / Pd (40 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP219-1-2 (110 mg, yellow solid, yield: 65%)

[0322] 3) Compound NEP219-1-2 (48 mg, 0.156 mmol), CH 3 CN (3mL), 4-(tert-butyloxycarbonylamino)butyric acid (40mg, 0.188mmol), HATU (90mg, 0.234mmol), DIPEA (136μL, 0.78mmol). The reaction was stirred at room temperature for 9h. After the reaction was completed, the reaction solution was washed with saturated brine (100mL), extracted three times with EA (5mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP219-1-3 (48mg, white solid, yield: 63%).

[0323] 4) Compound NEP219-1-3 (48 mg, 0.096 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP219-1-4 (20 mg, transparent solid, yield: 55%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.38(s,1H),7.34–7.16(m,5H),4.83–4.75(m,1H),3.28–3.19(m,1H),3.11–2.99(m,3H),2.60–2.52(m,2H),2.07–1.96(m,2H).

[0324] 5) Compound NEP219-1-4 (10 mg, 0.051 mmol), DMF (3 mL), JQ1-COOH (21 mg, 0.051 mmol), HATU (26 mg, 0.066 mmol), and DIPEA (45 μL, 0.255 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP219-1 (5 mg, light yellow solid, yield: 14%). 1 H NMR (400 MHz, Methanol-d 4)δ7.48–7.45(m,2H),7.43–7.36(m,3H),7.28–7.18(m,5H),4.80–4.70(m,2H),3.50–3. 38(m,4H),2.75(s,3H),2.53–2.48(m,2H),2.46(s,3H),2.00–1.92(m,2H),1.70(s,3H).

[0325] VII. Synthesis of Compound NEP233-1

[0326]

[0327] 1) Compound SM-23 (225 mg, 1.22 mmol), MeOH (6 mL), and H 2 / Pd (45 mg, 20%, wet). The reaction was stirred at room temperature under 1 atm hydrogen pressure for 9 h. After the reaction was completed, the reaction solution was filtered and concentrated to dryness, and purified by column chromatography. The developing solvent ratio was DCM / MeOH = 20 / 1 to obtain compound TM-2 (127 mg, black solid, yield: 67%)

[0328] 2) Compound TM-2 (50 mg, 0.33 mmol), DMF (3 mL), SM-24 (78 mg, 0.36 mmol), HATU (189 mg, 0.49 mmol), and DIPEA (287 μL, 1.65 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 50 / 1 to obtain compound NEP233-1-1 (73 mg, white solid, yield: 63%).

[0329] 3) Compound NEP233-1-1 (73 mg, 0.2 mmol), LiOH·H 2 O (84 mg, 2 mmol), MeOH (6 mL), H 2 O (3 mL). Stir the reaction at room temperature for 5 h. After the reaction is completed, adjust the pH to neutral, concentrate under reduced pressure to remove methanol, wash the residue with dilute hydrochloric acid, extract three times with EA (5 mL), combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to dryness. Obtain the crude compound NEP233-1-2, which is directly carried out to the next step.

[0330] 4) Compound NEP233-1-2 (50 mg, 0.154 mmol), DMF (3 mL), S-33-2 (31 mg, 0.17 mmol), HATU (77 mg, 0.2 mmol), and DIPEA (134 μL, 0.77 mmol) were added to a 50 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP233-1-3 (16 mg, yellow solid, yield: 21%).

[0331] 5) Compound NEP233-1-3 (16 mg, 0.03 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP233-1-4 (10 mg, transparent solid, yield: 62%).

[0332] 6) Compound NEP233-1-4 (10 mg, 0.026 mmol), DMF (3 mL), JQ1-COOH (11 mg, 0.026 mmol), HATU (15 mg, 0.039 mmol), and DIPEA (23 μL, 0.13 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (50 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP233-1 (5 mg, light yellow solid, yield: 25%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.53–7.35(m,5H),7.34–7.24(m,1H),7.12–6.98(m,2H),6.98–6.90(m,1H),4.71–4.65(m,2H),3.47–3.39(m,1H),3.38–3.34(m,1H) ,3.23–3.15(m,3H),2.92–2.85(m,1H),2.74(s,3H),2.47(s,3H),2.27–2.19(m,2H),1.71(s,3H),1.61–1.55(m,2H),1.49–1.42(m,2H).

[0333] 8. Synthesis of Compound NEP215-1

[0334]

[0335] 1) Compound NEP215 (114 mg, 0.4 mmol), MeOH (4 mL), tert-butyl 2-(methylamino)ethylcarbamate (84 mg, 0.48 mmol), and NaBH were added to a 50 mL single-necked bottle. 3 CN (63 mg, 1 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP215-1-1 (148 mg, transparent solid, yield: 84%).

[0336] 2) Compound NEP215-1-1 (148 mg, 0.33 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-1-2 (58 mg, transparent solid, yield: 52%).

[0337] 3) Compound NEP215-1-2 (20 mg, 0.06 mmol), DMF (3 mL), JQ1-COOH (24 mg, 0.06 mmol), HATU (34 mg, 0.09 mmol), and DIPEA (52 μL, 0.3 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-1 (8 mg, light yellow solid, yield: 19%). 1 H NMR (400 MHz, Methanol-d 4)δ7.47–7.41(d,J=8.4Hz,2H),7.40–7.33(d,J=8.2Hz,2H),7.27–7.20(m,4H), 7.19–7.11(m,1H),6.77–6.71(m,1H),6.11–6.05(m,1H),4.80–4.72(m,1H),4.7 1–4.63(m,1H),3.66(s,2H),3.52–3.35(m,6H),3.25–3.15(m,1H),3.05–2.95( m,1H),2.72–2.60(m,5H),2.41(s,3H),2.30(s,3H),1.66(s,3H).HRMS(ESI)m / z calcd.for C 37 H 41 C1N 9 O 3 S[M+H] + 726.2663; found 726.2732.

[0338] IX. Synthesis of Compound NEP215-2

[0339]

[0340] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), tert-butyl 2-(methylamino)propylcarbamate (41 μL, 0.194 mmol), and NaBH were added to a 50 mL single-necked bottle. 3 CN (28 mg, 0.44 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-2-1 (56 mg, transparent solid, yield: 58%).

[0341] 2) Compound NEP215-2-1 (56 mg, 0.1 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-2-2 (25 mg, transparent solid, yield: 56%). 1 H NMR (400 MHz, Methanol-d 4)δ7.34–7.29(m,2H),7.29–7.21(m,2H),7.21–7.14(m,1H),6.86(d,J=3.9Hz,1H),6.43(d,J=3.8Hz,1H) ,5.01(s,2H),4.83–4.75(m,1H),3.38–3.17(m,4H),3.05–2.97(m,2H),2.78(s,3H),2.20–2.08(m,2H).

[0342] 3) Compound NEP215-2-2 (25 mg, 0.055 mmol), DMF (3 mL), JQ1-COOH (23 mg, 0.055 mmol), HATU (33 mg, 0.08 mmol), and DIPEA (49 μL, 0.275 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-2 (9 mg, light yellow solid, yield: 23%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.35–7.24(m,4H),7.20–7.07(m,4H),7.07–6.99(m,1H),6.65–6.60(m,1H), 5.99–5.93(m,1H),4.67–4.53(m,2H),3.54–3.42(m,2H),3.32–3.25(m,3H),3.1 9–3.14(m,1H),3.12–3.01(m,1H),2.90–2.80(m,1H),2.54(s,3H),2.51–2.39( m,2H),2.31(s,3H),2.12(s,3H),1.73–1.61(m,2H),1.53(s,3H).HRMS(ESI)m / z calcd.for C 38 H 43 C1N 9 O 3 S[M+H] + 740.2898; found 740.2877.

[0343] 10. Synthesis of Compound NEP215-3

[0344]

[0345] 1) Compound SM-25 (400 mg, 1.98 mmol), dioxane (6 mL), H 2 O (3mL), Fmoc-Osu (802mg, 2.376mmol), Na 2 CO 3 (630 mg, 5.94 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the pH was adjusted to neutral, the dioxane was concentrated under reduced pressure and dried, the residue was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of PE / EA = 8 / 1 to obtain compound TM-3 (396 mg, white solid, yield: 47%).

[0346] 2) Compound TM-3 (396 mg, 0.93 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound TM-4 (250 mg, transparent solid, yield: 83%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.78–7.72(m,2H),7.64–7.58(m,2H),7.40–7.24(m,4H),4.35–4.29(m,2H),4.18–4.10(m, 1H),3.17–3.10(m,2H),3.00–2.92(m,2H),2.65(s,3H),1.71–1.63(m,2H),1.60–1.50(m,2H).

[0347] 3) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), TM-4 (58 mg, 0.194 mmol), NaBH 3 CN (28 mg, 0.44 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-3-1 (25 mg, transparent solid, yield: 24%).

[0348] 4) Compound NEP215-3-1 (25 mg, 0.043 mmol), CH 3CN (4 mL) and piperidine (41 μL). The mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and the crude product was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-3-2 (15 mg, transparent solid, yield: 93%).

[0349] 5) Compound NEP215-3-2 (15 mg, 0.027 mmol), DMF (3 mL), JQ1-COOH (11 mg, 0.027 mmol), HATU (56 mg, 0.041 mmol), and DIPEA (24 μL, 0.135 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-3 (4 mg, light yellow solid, yield: 20%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.49–7.42(m,4H),7.31–7.23(m,4H),7.22–7.14(m,1H),6.88(d,J=3.8Hz,1 H),6.41(d,J=3.8Hz,1H),4.72–4.66(m,1H),3.55–3.48(m,2H),3.41–3.36(m,1 H),3.27–3.12(m,4H),3.06–2.99(m,1H),2.91(s,2H),2.75(s,3H),2.62(s,3H) ,2.45(s,3H),1.86–1.81(m,2H),1.69(s,3H),1.67–1.62(m,2H).HRMS(ESI)m / z calcd.for C 39 H 43 C1N 9 O 3 S[MH] - 752.2976; found 752.2973.

[0350] XI. Synthesis of Compound NEP215-4

[0351]

[0352] (1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), tert-butyl (5-(methylamino)pentyl)carbamate (38 mg, 0.19 mmol), and NaBH were added in sequence into a 50 mL single-necked bottle. 3 CN (28 mg, 0.44 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-4-1 (68 mg, transparent solid, yield: 80%).

[0353] (2) Compound NEP215-4-1 (68 mg, 0.16 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-4-2 (25 mg, transparent solid, yield: 41%).

[0354] (3) Compound NEP215-4-2 (25 mg, 0.064 mmol), DMF (3 mL), JQ1-COOH (25 mg, 0.064 mmol), HATU (37 mg, 0.096 mmol), and DIPEA (56 μL, 0.32 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 3 / 1 to obtain a crude product of compound NEP215-6. The crude product was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-4 (19 mg, light yellow solid, yield: 38%). 1H NMR(400MHz,MeOD)δ7.51–7.40(m,4H),7.33–7.28(m,2H),7.28–7.22(m,2H),7.22–7.13(m,1H),6.86(d,J=3 .8Hz,1H),6.42(d,J=3.8Hz,1H),4.83–4.75(m,1H),4.74–4.66(m,1H),4.44–4.36(m,1H),4.30–4.22(m,1H), 3.44–3.39(m,2H),3.38–3.35(m,1H),3.28–3.20(m,2H),3.20–3.16(m,1H),3.08–2.98(m,2H),2.79(s,3H),2 .72(s,3H),2.46(s,3H),1.85–1.73(m,2H),1.70(s,3H),1.68–1.56(m,2H),1.50–1.38(m,2H).HRMS(ESI)m / z calcd.forC 40 H 47 C1N 9 O 3 S[M+H] + 768.3133; found 768.3209.

[0355] 12. Synthesis of Compound NEP215-5

[0356]

[0357] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), tert-butyl N-{2-[2-(methylamino)ethoxy]ethyl}carbamate (43 mg, 0.19 mmol), and NaBH were added to a 50 mL single-necked bottle. 3 CN (34 mg, 0.528 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 15 / 1 to obtain compound NEP215-5-1 (77 mg, transparent solid, yield: 90%).

[0358] 2) Compound NEP215-5-1 (70 mg, 0.144 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain the crude product NEP215-5-2, which was directly used for the next step without purification.

[0359] 3) Compound NEP215-5-2 (27 mg, 0.071 mmol), DMF (3 mL), JQ1-COOH (28 mg, 0.071 mmol), HATU (36 mg, 0.093 mmol), and DIPEA (61 μL, 0.355 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain a crude product of compound NEP215-5, which was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-5 (13 mg, light yellow solid, yield: 24%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.48–7.42(m,4H),7.30–7.17(m,5H),6.87–6.81(d,J=3.8Hz,1H),6.44– 6.38(d,J=3.8Hz,1H),4.80–4.69(m,2H),4.38(s,2H),3.90–3.80(m,2H),3. 70–3.57(m,4H),3.49–3.37(m,4H),3.25–3.19(m,1H),3.04–2.97(m,1H),2 .84(s,3H),2.70(s,3H),2.44(s,3H),1.67(s,3H).HRMS(ESI)m / zcalcd.for C 39 H 45 C1N 9 O 4 S[M+H] + 770.2925; found 770.3110.

[0360] 13. Synthesis of Compound NEP215-6

[0361]

[0362] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), tert-butyl N-(5-aminopentyl)carbamate (40 mg, 0.19 mmol), and NaBH were added to a 50 mL single-necked bottle. 3CN (28 mg, 0.44 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-6-1 (59 mg, transparent solid, yield: 71%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.20–7.11(m,4H),7.09–7.03(m,1H),6.72–6.67(m,1H),6.23–6.17(m,1H),4.69–4.66(m,1H),4.00(s,2H),3.19–3.08(m,1H) ,2.98–2.92(m,2H),2.92–2.85(m,1H),2.85–2.77(m,2H),1.61–1.49(m,2H),1.41–1.36(m,2H),1.32(s,9H),1.30–1.22(m,2H).

[0363] 2) Compound NEP215-6-1 (59 mg, 0.14 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-6-2 (23 mg, transparent solid, yield: 45%).

[0364] 3) Compound NEP215-6-2 (23 mg, 0.063 mmol), DMF (3 mL), JQ1-COOH (25 mg, 0.063 mmol), HATU (36 mg, 0.095 mmol), and DIPEA (56 μL, 0.32 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 3 / 1 to obtain a crude product of compound NEP215-6, which was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-6 (8 mg, light yellow solid, yield: 17%). 1 H NMR (400 MHz, Methanol-d 4)δ7.51–7.40(m,4H),7.32–7.13(m,6H),6.85–6.80(m,1H),6.38–6.33(m,1H),4.83 –4.74(m,1H),4.72–4.64(m,1H),4.22(s,2H),3.42–3.35(m,4H),3.27–3.23(m,1H), 3.22–3.16(m,1H),3.06–3.02(m,1H),3.02–2.99(m,1H),2.70(s,3H),2.46(s,3H), 1.71(s,3H),1.65–1.55(m,2H),1.53–1.43(m,2H),1.38–1.28(m,2H).HRMS(ESI)m / z calcd.for C 39 H 45 C1N 9 O 3 S[M+H] + 754.3055; found 754.3079.

[0365] 14. Synthesis of Compound NEP215-7

[0366]

[0367] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), N-tert-butyloxycarbonyl-2-(2-aminoethoxy)ethylamine (43 mg, 0.21 mmol), and NaBH were added to a 50 mL single-mouth bottle. 3 CN (28 mg, 0.44 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 20 / 1 to obtain compound NEP215-7-1 (54 mg, transparent solid, yield: 66%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.20–7.08(m,5H),6.72–6.63(m,1H),6.13–6.06(m,1H),4.71–4.67(m,1H),3.82(s,2H),3.51–3.47(m,2H) ,3.41–3.36(m,2H),3.19–3.15(m,1H),3.15–3.11(m,2H),3.00–2.90(m,1H),2.82–2.75(m,2H),1.33(s,9H).

[0368] 2) Compound NEP215-7-1 (54 mg, 0.1 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-7-2 (19 mg, transparent solid, yield: 51%).

[0369] 3) Compound NEP215-7-2 (19 mg, 0.05 mmol), DMF (3 mL), JQ1-COOH (20 mg, 0.05 mmol), HATU (33 mg, 0.075 mmol), and DIPEA (49 μL, 0.25 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain a crude product of compound NEP215-7, which was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-7 (8 mg, light yellow solid, yield: 22%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.51–7.42(m,5H),7.28–7.22(m,4H),6.82–6.78(m,1H),6.37–6.31(m,1H),4.78–4.69(m,2H),4.27(s,2H),3.77–3.73(m,2 H),3.61(s,2H),3.49–3.38(m,2H),3.27–3.18(m,4H),3.06–2.95(m,2H),2.69(s,3H),2.43(s,3H),1.67(s,3H).HRMS(ESI)m / z calcd.forC 38 H 43 C1N 9 O 4 S[M+H] + 756.2847; found 756.2855.

[0370] 15. Synthesis of Compound NEP215-8

[0371]

[0372] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), 1-(N-Boc-aminoethyl)piperazine (45 mg, 0.194 mmol), and NaBH were added to a 50 mL single-necked bottle. 3 CN (34 mg, 0.528 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 15 / 1 to obtain compound NEP215-8-1 (56 mg, transparent solid, yield: 64%).

[0373] 2) Compound NEP215-8-1 (52 mg, 0.104 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain the crude product of NEP215-8-2, which was directly carried out to the next step without purification.

[0374] 3) Compound NEP215-8-2 (30 mg, 0.075 mmol), DMF (3 mL), JQ1-COOH (30 mg, 0.075 mmol), HATU (38 mg, 0.098 mmol), and DIPEA (65 μL, 0.375 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 6 / 1 to obtain a crude product of compound NEP215-5, which was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to obtain compound NEP215-8 (15 mg, light yellow solid, yield: 26%). 1 H NMR (400 MHz, Methanol-d 4)δ7.48–7.42(m,4H),7.31–7.23(m,4H),7.22–7.14(m,1H),6.80–6.74(d,J=3.7Hz, 1H),6.15–6.12(d,J=3.7Hz,1H),4.81–4.76(m,1H),4.66–4.61(m,1H),3.67(s,2H), 3.56–3.46(m,2H),3.45–3.37(m,4H),3.27–3.21(m,1H),3.06–3.00(m,1H),2.75–2 .72(m,2H),2.71(s,3H),2.70–2.62(m,6H),2.46(s,3H),1.71(s,3H).HRMS(ESI)m / z calcd.for C 40 H 46 C1N 10 O 3 S[M+H] + 781.3085; found 781.3145.

[0375] 16. Synthesis of Compound NEP215-9

[0376]

[0377] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), 3-(Boc-aminomethyl)piperidine (42 mg, 0.19 mmol), and NaBH were added to a 50 mL single-necked bottle. 3 CN (34 mg, 0.528 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 15 / 1 to obtain compound NEP215-9-1 (50 mg, transparent solid, yield: 90%).

[0378] 2) Compound NEP215-9-1 (50 mg, 0.1 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain the crude product of NEP215-9-2, which was directly carried out to the next step without purification.

[0379] 3) Compound NEP215-9-2 (28 mg, 0.074 mmol), DMF (3 mL), JQ1-COOH (30 mg, 0.074 mmol), HATU (36 mg, 0.098 mmol), and DIPEA (61 μL, 0.375 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain a crude product of compound NEP215-9, which was prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to give compound NEP215-9 (15 mg, light yellow solid, yield: 27%). 1 H NMR (400 MHz, Methanol-d 4 )δ7.51–7.40(m,4H),7.33–7.22(m,4H),7.22–7.14(m,1H),6.85–6.80(m,1H),6.37 –6.27(m,1H),4.82–4.74(m,1H),4.72–4.63(m,1H),4.20–4.05(m,2H),3.52–3.34( m,4H),3.29–3.19(m,2H),3.13–2.99(m,2H),2.74–2.68(m,3H),2.46(s,3H),2.05– 2.01(m,1H),1.95–1.81(m,2H),1.72–1.69(m,3H),1.39–1.14(m,4H).HRMS(ESI)m / z calcd.for C 40 H 45 C1N 9 O 3 S[M+H] + 766.2976; found 766.3035.

[0380] 17. Synthesis of Compound NEP215-10

[0381]

[0382] 1) Compound NEP215 (50 mg, 0.176 mmol), MeOH (3 mL), 4-(Boc-aminomethyl)piperidine (42 mg, 0.19 mmol), and NaBH were added to a 50 mL single-necked bottle. 3CN (34 mg, 0.528 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 15 / 1 to obtain compound NEP215-10-1 (50 mg, transparent solid, yield: 90%).

[0383] 2) Compound NEP215-10-1 (50 mg, 0.1 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness to obtain the crude product of NEP215-10-2, which was directly carried out to the next step without purification.

[0384] 3) Compound NEP215-10-2 (28 mg, 0.074 mmol), DMF (3 mL), JQ1-COOH (30 mg, 0.074 mmol), HATU (36 mg, 0.098 mmol), and DIPEA (61 μL, 0.375 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude product was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain a crude product of compound NEP215-10, which was prepared by high performance liquid chromatography (MeOH / H 2 D, 0.1% TFA) to give compound NEP215-10 (15 mg, light yellow solid, yield: 27%). 1 H NMR(400MHz,MeOD)δ7.48–7.41(m,4H),7.32–7.29(m,2H),7.27–7.22(m,2H),7.20–7.15(m,1 H),6.90–6.84(m,1H),6.43–6.37(m,1H),4.84–4.76(m,1H),4.73–4.65(m,1H),4.30(s,2H), 3.53–3.39(m,4H),3.31–3.22(m,2H),3.15–2.99(m,2H),2.97–2.86(m,2H),2.73(s,3H),2.4 6(s,3H),2.06–1.98(m,2H),1.91–1.75(m,1H),1.70(s,3H),1.54–1.42(m,2H).HRMS(ESI)m / z calcd.forC 40 H 45 C1N 9 O 3 S[M+H]+ 766.2976; found 766.3065.

[0385] 18. Synthesis of Compound NEP215-11

[0386]

[0387] 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid was synthesized according to a published synthetic route, Xiao, L., Parolia, A., Qiao, Y. et al. Targeting SWI / SNF ATPases in enhancer-addicted prostate cancer. Nature 601, 434–439 (2022).

[0388]

[0389] Compound NEP215-2-2 (25 mg, 0.055 mmol), DMF (3 mL), 2-(4-(3-amino-6-(2-hydroxyphenyl)pyridazin-4-yl)piperazin-1-yl)acetic acid (21 mg, 0.055 mmol), HATU (33 mg, 0.08 mmol), and DIPEA (49 μL, 0.275 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-11 (11 mg, light yellow solid, yield: 30%). 1 H NMR (400 MHz, Methanol-d 4)δ7.83–7.76(m,1H),7.48(s,1H),7.31–7.19(m,5H),7.19–7.11(m,1H),6.99–6.91(m,2 H),6.82–6.76(m,1H),6.16–6.11(m,1H),4.81–4.75(m,1H),3.66(s,2H),3.25–3.20(m,1 H),3.19–3.12(m,4H),3.10(s,2H),3.07–3.02(m,1H),3.02–2.99(m,1H),2.90–2.81(m, 1H),2.74–2.65(m,4H),2.57–2.49(m,2H),2.32(s,3H),1.85–1.73(m,2H).HRMS(ESI)m / z calcd.for C 35 H 43 N 10 O 4 [MH] - 667.3547; found 667.3539.

[0390] 19. Synthesis of Compound NEP215-12

[0391]

[0392] According to the synthesis steps of NEP215-11, compound NEP215-12 (21 mg, white solid, yield: 47%) was obtained through synthesis and purification. 1 H NMR(400MHz,MeOD)δ7.80–7.73(m,1H),7.34(s,1H),7.32–7.24(m,3H),7.24–7.17(m,2H) ,7.17–7.09(m,1H),7.01–6.93(m,2H),6.73(d,J=3.7Hz,1H),6.04(d,J=3.7Hz,1H),4.78– 4.72(m,1H),3.69–3.62(m,4H),3.62–3.56(m,2H),3.55–3.49(m,2H),3.24–3.18(m,1H),3 .09(s,2H),3.07–2.99(m,4H),2.97(m,1H),2.73–2.60(m,6H),2.29(s,3H).HRMS(ESI)m / z calcd.for C 36 H 45 N 10 O 5 [MH] -697.3653; found 697.3678.XX. Synthesis of compound NEP215-13

[0393]

[0394] According to the synthesis steps of NEP215-11, compound NEP215-13 (18 mg, white solid, yield: 40%) was obtained by synthesis and purification. 1 H NMR(400MHz,MeOD)δ7.72(s,1H),7.68–7.61(m,1H),7.50–7.41(m,1H),7.33–7.22(m,4H),7.22–7.14(m ,1H),7.14–7.02(m,2H),6.86(d,J=3.8Hz,1H),6.40(d,J=3.8Hz,1H),4.82–4.78(m,1H),4.29(s,2H),4. 00(s,2H),3.67(m,4H),3.62–3.55(m,4H),3.54–3.46(m,2H),3.29–3.24(m,1H),3.24–3.19(m,2H),3.07 –3.00(m,1H),2.97–2.89(m,2H),2.04–1.95(m,2H),1.84–1.80(m,1H),1.56–1.42(m,2H).HRMS(ESI)m / z calcd.forC 37 H 47 N 10 O 4 [M+H] + 695.3704; found 695.3849.

[0395] 21. Synthesis of Compound NEP215-14

[0396]

[0397] 2-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)acetic acid was synthesized according to a published synthetic route. O'Boyle, NM, Pollock, JK et al. β-Lactam estrogen receptor antagonists and a dual-targeting estrogen receptor / tubulin ligand. Journal of medicinal chemistry, 57(22), 9370–9382 (2014).

[0398]

[0399] Compound NEP215-2-2 (25 mg, 0.055 mmol), DMF (3 mL), 2-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)acetic acid (20 mg, 0.055 mmol), HATU (33 mg, 0.08 mmol), and DIPEA (49 μL, 0.275 mmol) were added to a 25 mL single-mouth bottle in sequence. The reaction was stirred at room temperature for 9 h. After the reaction was completed, the reaction solution was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain compound NEP215-14 (15 mg, light yellow solid, yield: 39%). 1 H NMR(400MHz,MeOD)δ7.39–7.32(m,2H),7.32–7.07(m,13H),6.90–6.78(m,3H),6 .64(d,J=8.2Hz,2H),6.40(d,J=3.7Hz,1H),4.84–4.77(m,1H),4.41(s,2H),4.3 5–4.18(m,2H),3.28–3.20(m,2H),3.20–3.07(m,2H),3.06–2.96(m,2H),2.74(s ,3H),2.51–2.41(m,2H),1.97–1.89(m,2H),0.92(t,J=7.3Hz,3H).HRMS(ESI)m / z calcd.for C 43 H 48 N 5 O 4 [M+H] + 698.3628; found 698.3723.

[0400] 22. Synthesis of Compound NEP215-15

[0401]

[0402] According to the synthesis steps of NEP215-14, compound NEP215-15 (16 mg, light yellow solid, yield: 44%) was obtained by synthesis and purification. 1H NMR(400MHz,MeOD)δ7.32–7.28(m,1H),7.25–7.20(m,3H),7.19–7.13(m,4H),7.12–7.03(m,6H),6. 96–6.89(m,2H),6.84–6.72(m,3H),6.59–6.54(m,1H),6.38–6.30(m,1H),4.78–4.72(m,1H),4.42– 4.33(m,2H),4.33–4.14(m,2H),3.76–3.63(m,2H),3.61–3.38(m,4H),3.22–3.17(m,1H),3.17–2.9 9(m,2H),2.99–2.93(m,1H),2.75–2.67(m,3H),2.48–2.34(m,2H),0.89–0.83(m,3H).HRMS(ESI)m / z calcd.for C 44 H 50 N 5 O 5 [M+H] + 728.3734; found 728.3812.

[0403] 23. Synthesis of Compound NEP215-16

[0404]

[0405] 1) Compound NEP215 (0.1 mg, 0.35 mmol), MeOH (5 mL), 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester (115 mg, 0.39 mmol), NaBH 3 CN (56 mg, 0.875 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the concentrate was concentrated under reduced pressure, and the concentrate was purified by silica gel column with a developing solvent ratio of DCM / MeOH = 10 / 1 to obtain compound NEP215-16-1 (130 mg, transparent solid, yield: 66%).

[0406] 2) Compound NEP215-16-1 (130 mg, 0.234 mmol), Dioxane (4 mL), H 2 O (2mL), Fmoc-Osu (119mg, 0.35mmol), Na 2 CO 3(75 mg, 0.7 mmol). The reaction was stirred at room temperature for 9 h. After the reaction was completed, the pH was adjusted to neutral, the Dioxane was concentrated under reduced pressure and dried, the residue was washed with saturated brine (100 mL), extracted three times with EA (5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. It was purified by column chromatography with a developing solvent ratio of DCM / MeOH = 40 / 1 to obtain compound NEP215-16-2 (120 mg, white solid, yield: 65%).

[0407] 3) Compound NEP215-16-2 (120 mg, 0.15 mmol), DCM (4 mL) and trifluoroacetic acid (2 mL) were added to a 50 mL single-mouth bottle in sequence. The reaction was allowed to react at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and purified by a silica gel column with a developing solvent ratio of DCM / MeOH = 5 / 1 to obtain a crude compound NEP215-16-3, which was directly carried out to the next step without purification.

[0408] 4) Prepared according to the preparation steps of NEP215-14, except that NEP215-2-2 was replaced by NEP215-16-3 (34 mg, 0.05 mmol), and purified by column chromatography to obtain compound NEP215-16-4 (30 mg, white solid, yield: 61%).

[0409] 5) Compound NEP215-16-4 (30 mg, 0.03 mmol), CH 3 CN (4 mL) and piperidine (30 μL). The mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated to dryness and the crude product was purified by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to give compound NEP215-16 (16 mg, transparent solid, yield: 69%). 1H NMR(400MHz,MeOD)δ7.39–7.34(m,1H),7.30–7.09(m,13H),7.03–6.94(m,2H),6.90–6.80(m,3H), 6.67–6.61(m,1H),6.39–6.32(m,1H),4.85–4.76(m,1H),4.56(s,1H),4.39(s,1H),4.27–4.21(m,2 H),3.73–3.66(m,2H),3.65–3.53(m,8H),3.52–3.43(m,2H),3.43–3.35(m,2H),3.25(dd,J=13.9, 5.3Hz,1H),3.21–3.13(m,2H),3.02(dd,J=13.9,9.4Hz,1H),2.55–2.41(m,2H),0.97–0.87(m,3H).

[0410] 24. Synthesis of Compound NEP215-17

[0411]

[0412] 1) Prepared according to the preparation steps of NEP215-16 (1), except that 13-amino-5,8,11-trioxa-2-azatridecanoic acid was replaced by N-tert-butyloxycarbonyl-2-(2-aminoethoxy)ethylamine, and purified by column chromatography to obtain compound NEP215-17-1 (110 mg, transparent solid, yield: 60%).

[0413] 2) Prepared according to the preparation steps of NEP215-16 (2), except that NEP215-16-1 was replaced by NEP215-17-1, and purified by column chromatography to obtain compound NEP215-17-2 (90 mg, white solid, yield: 58%).

[0414] 3) Prepared according to the preparation steps of NEP215-16 (3), except that NEP215-16-2 was replaced by NEP215-17-2 to obtain crude NEP215-17-3, which was directly subjected to the next step reaction.

[0415] 4) Prepared according to the preparation steps of NEP215-16 (4), except that NEP215-16-3 was replaced by NEP215-17-3, and purified by column chromatography to obtain compound NEP215-17-4 (28 mg, transparent solid, yield: 53%).

[0416] 5) Prepared according to the preparation steps of NEP215-16 (5), except that NEP215-16-4 was replaced by NEP215-17-4, and prepared by high performance liquid chromatography (MeOH / H 2 O, 0.1% TFA) to give compound NEP215-17 (130 mg, transparent solid, yield: 66%) (13 mg, transparent solid, yield: 61%). 1 H NMR(400MHz,MeOD)δ7.40–7.32(m,2H),7.31–7.07(m,13H),6.87–6.79(m,3H),6.67–6.61( m,2H),6.36–6.31(m,1H),4.80(dd,J=9.3,5.4Hz,1H),4.38(s,2H),4.22(s,2H),3.72–3.65 (m,2H),3.60(s,4H),3.58–3.51(m,2H),3.46–3.39(m,2H),3.25(dd,J=13.9,5.4Hz,1H),3. 18–3.11(m,2H),3.02(dd,J=13.8,9.3Hz,1H),2.46(q,J=7.4Hz,2H),0.92(t,J=7.4Hz,3H).

[0417] Example 3: Isothermal titration calorimetry determination of the binding ability of the compounds of the present invention to SCF-FBXO31 protein

[0418] The binding ability of the compounds of the present invention to the E3 ubiquitin ligase SCF-FBXO31 was measured. The measurement was performed using isothermal titration calorimetry (ITC), and the activity was expressed as the dissociation constant K D (nM) indicates.

[0419] Isothermal titration calorimetry (ITC) measurements were performed at 16°C using a MicroCal PEAQ-ITC instrument (Malven PANalytical).

[0420] The FBXO31 fragment (amino acid residues 66-539) and SKP1 (amino acid residues 1-37-163 (38-43del, 70-81del)) were cloned into the pNIC-CH (Addgene plasmid #26117) vector and purified for ITC detection. Before the assay, 70 μl of the test compound at a concentration of 1 mM and 320 μl of FBXO31 protein at a concentration of 50 μM were prepared. Both the protein and the compound were prepared and stored in 20 mM Tris-HCl pH 7.5, 150 mM NaCl solution. Before titration, the solution was centrifuged at 12000 rpm at 4°C for 30 min to remove bubbles and precipitates. In each experiment, the test compound was titrated into the protein for a total of 15 times, with an interval of 90 s between each titration. The first drop of the compound was titrated with 0.5 μL, and the remaining 14 drops were titrated with 1.5 μL each. The entire titration process was carried out at 16°C. After the titration, MicroCal PEAQ-ITC analysis software was used to analyze the interaction between the compound and the protein, and the fitting model used one set of sites.

[0421] The results of isothermal titration calorimetry are shown in Table 1 below; when the dissociation constant K D When the dissociation constant K is less than or equal to 1000 nM, the activity of the compound is marked as "++++"; when the dissociation constant K is less than or equal to 1000 nM, the activity of the compound is marked as "++++"; D When the activity of the compound is greater than 1000 nM and less than 5000 nM, it is marked as "+++"; when the dissociation constant K D When the activity of the compound is greater than 5000 nM and less than 10000 nM, the activity of the compound is marked as "++"; when the dissociation constant K D Compounds with activity greater than 10000 nM were marked as “+”.

[0422] Table 1: Binding ability of compounds to SCF-FBXO31 protein, ITC assay

[0423]

[0424]

[0425] Experimental data show that more than half of the compounds of the present invention bind to SCF-FBXO31. D The value is less than 1000 nM, indicating that the compound of the present invention has good affinity with the SCF-FBXO31 protein and may become a drug for treating diseases related to SCF-FBXO31 (such as mental retardation, autosomal recessive non-syndromic intellectual disability, cerebral palsy, breast cancer, ovarian cancer, hepatocellular carcinoma, prostate cancer, pancreatic cancer, etc.). In addition, the compound of the present invention may also become a ligand or intermediate compound for developing a protein degradation bifunctional drug molecule based on SCF-FBXO31.

[0426] Example 4: Determination of the effect of the compounds of the present invention on target protein degradation

[0427] Western blot experiment: Lung cancer cells (H1299) or human malignant melanoma cells (A375) or human breast cancer cells (MCF7) were inoculated into 6-well plates at a concentration of 2*105 cells / mL, 2 mL was added to each well, and incubated for 24 hours.

[0428] The cells were observed under a microscope the next day to confirm the cell status. Prepare the compound, weigh a certain amount of the compound of the present invention or the control drug DMSO, first use pure DMSO to prepare a higher concentration of the mother solution to prepare the compound concentration: 20mM solution, shake it to make it fully mixed, and adopt the gradient dilution method to add different amounts of solution according to the required concentration of each well. After incubation for a certain period of time, take out the cells, add 1ml PBS and shake it slightly, repeat the washing 3 times, suck up the residual liquid, add 1ml pre-cooled PBS, scrape the cells from the wall of the culture dish with a cell scraper, suck them into a 1.5ml centrifuge tube, and centrifuge at 4000rpm for 4min to precipitate the cells. Carefully suck away the residual liquid. Add 160μL of Cell lysis lysis solution premixed with 1% PMSF and 1% PI, blow to make the lysis solution fully contact with the cells, and incubate on ice for 20min. Centrifuge at 12000rpm for 10min, aspirate the supernatant into a clean 1.5ml EP tube, and use the Bradford protein quantification kit to determine and homogenize the sample loading amount.

[0429] Take a 1.5ml EP tube, do 2 replicates for each sample, prepare the standard curve sample with the working solution and BSA standard, add 250μl of Coomassie Brilliant Blue G-250 working solution to each tube, add 2μl of sample, mix well, take 200μl to a 96-well ELISA plate, measure the A595 nm absorbance value with an ELISA reader, and substitute it into the protein standard curve formula to obtain the sample concentration. According to the prepared volume, prepare the sample to 1μg / μl according to the calculated value, and place it in a 95℃ metal bath for 5min to denature the protein.

[0430] Take the prepared 5% and 10% gel and fix them on the gel rack, add electrophoresis solution to the tank, remove the comb, load the marker and protein sample, and adjust the voltage to 120V after 80V electrophoresis for 60 minutes. Stop electrophoresis when the marker band reaches the forefront of the gel. Take out the gel block and cut it to the appropriate size. Cut the PVDF membrane and place it in methanol for activation. After activation, place the PVDF membrane in the transfer balance solution for 1 minute. Place the sponge pad, filter paper, PVDF membrane, gel, filter paper, and sponge pad in the order of the splint and put it in the transfer tank for rapid wet transfer. Remove the membrane and place it in 5% skim milk powder dissolved in 1xTBST for 1 hour. Cut the membrane according to the molecular weight of the target protein and the internal reference protein, place it in the primary antibody diluted in 1xTBST, and shake it overnight at 4°C.

[0431] The next day, the strips were transferred to 1xTBST and washed three times, and then the secondary antibody diluted in a specific ratio was added and incubated with shaking for 1 hour. The strips were transferred to 1xTBST again and washed three times, and then the ECL color developing solution was prepared. The membrane was placed in an imaging system, the surface was covered with ECL color developing solution, and the membrane was photographed using the imaging system.

[0432] Results Figure 1-7 From the above results, it can be seen that compounds NEP215-1, NEP215-2, NEP215-3, NEP215-6, NEP215-7, NEP215-9, and NEP215-11 have better degradation effects.

[0433] In the A375 cell line, the cells were treated with 1 μM of different compounds for 4 h, and it was found that NEP215-2 degradation was better ( Figure 1 ).

[0434] In the A375 cell line, the cells were treated with 1 μM of different compounds for 8 h, and it was found that NEP215-1 and NEP215-2 had better degradation effects ( Figure 2 ).

[0435] In the A375 cell line, cells were treated with 5 μM of different compounds for 6 h, and it was found that NEP215-2 and NEP215-7 had better degradation effects ( Figure 3 ).

[0436] In the A375 cell line, cells were treated with 1 μM NEP215-1 for different time periods. At 4 h, NEP215-1 showed a degradation trend towards BRD4, and the degradation was most obvious at 8 h. The degradation effect was maintained until 12 h ( Figure 4 ).

[0437] In the A375 cell line, cells were treated with different concentrations of NEP215-2 and NEP215-6 for 8 h. At a concentration of 1 μM, both NEP215-2 and NEP215-6 significantly degraded BRD4 ( Figure 5 ). In the A375 cell line, cells were treated with different concentrations of NEP215-1 for 12 hours. BRD4 began to degrade at a concentration of 0.2 μM, and the degradation of BRD4 was most obvious at a concentration of 0.5 μM. In the A375 cell line, cells were treated with different concentrations of NEP215-2 for 12 hours. BRD4 began to degrade at a concentration of 0.1 μM, and the degradation of BRD4 was most obvious at a concentration of 0.5 μM ( Figure 6 ).

[0438] In the H1299 cell line, the cells were treated with 1 μM NEP215-11 for 8 hours, and it was found that NEP215-11 had a good effect on SMARCA2 degradation. H1299 cells were treated with 5 μM different compounds, and it was found that NEP215-2, NEP215-3, and NEP215-9 had a significant effect on BRD4 degradation; H1299 cells were treated with 1 μM different compounds for 8 hours, and it was found that NEP215-4, NEP215-3, NEP215-5, and NEP215-9 had a BRD4 degradation effect ( Figure 7 ).

[0439] In the MCF7 cell line, cells were treated with 10 μM NEP215-16 and NEP215-17 for 12 h, and it was found that NEP215-16 had a better effect on ERα degradation ( Figure 8 ).

[0440] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A compound capable of binding to SCF-FBXO31 protein, characterized in that: The compound is shown in formula (I): In formula (I): m is any natural number from 0 to 3; R1 is selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon, benzene ring, 5-6 membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, 4-7 membered carbon heteromonocycle, 5-10 membered carbon heterocyclic ring, 4-7 membered monocyclic alkyl, 5-10 membered cyclic alkyl, 7-10 membered bridged cycloalkyl; R2 is selected from -XR4(R5), X is selected from CH, N, preferably X is N; Y is selected from CH2, C=O, preferably Y is C=O; Z is selected from CH2, NH or O; Ring A is selected from 5-6 membered aromatic ring, 5-6 membered aromatic heterocycle, benzene ring and condensed ring formed by 5-6 membered aromatic heterocycle, 4-7 membered carbon heteromonocycle, 5-10 membered carbon heterocyclic ring, 4-7 membered monocyclic alkyl, 5-10 membered cyclic alkyl, 7-10 membered bridged cycloalkyl; R3 is selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered carbon heterocyclic ring, 7-10-membered bridged cycloalkyl; R4 is selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon, -COR7, -CONR8(R9); R5, R6, R7, R8 and R9 are selected from H, C1-C8 saturated or unsaturated straight or branched hydrocarbon, benzene ring, 5-6 membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6 membered aromatic heterocycle, 4-7 membered carbon heteromonocycle, 5-10 membered carbon heterocyclic ring, 4-7 membered monocycloalkyl, 5-10 membered cycloalkyl and 7-10 membered bridged cycloalkyl.

2. The compound capable of binding to SCF-FBXO31 protein according to claim 1, characterized in that: The C1-C8 saturated or unsaturated straight or branched hydrocarbon group, benzene ring, aromatic heterocycle, aromatic condensed ring, aromatic condensed heterocycle, monocyclic alkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocyclic ring, carbon heterocyclic ring in R1, R3, R4, R5, R6, R7, R8 and / or R9 may be further optionally and independently replaced by 0 to 3 halogens, -OH, -COOH, -CN, -NO2, -CH3, -CHO, -NH2, -SH, -SCH3, -CONH2, -NHCOR 10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH2) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is optionally selected from the natural number 1-5; The aromatic ring, aromatic heterocycle, aromatic condensed ring, aromatic condensed heterocycle, monocyclic alkyl, cycloalkyl, bridged cycloalkyl, carbon heteromonocyclic ring, and carbon heterocyclic ring in the A ring may be further optionally and independently replaced by 0 to 3 halogens, -OH, -COOH, -CN, -NO2, -CH3, -CHO, -NH2, -SH, -SCH3, -CONH2, -NHCOR 10 、-NHCOCH=CR 10 (R 11 ), -NHCOCH=CR 10 (R 11 )、-(CH2) p OR 10 , C1-4 straight or branched alkyl, halogen-substituted C1-4 straight or branched alkyl, hydroxy-substituted C1-4 straight or branched alkyl, alkoxy-substituted C1-4 straight or branched alkyl, amino-substituted C1-4 straight or branched alkyl, benzene ring, 5-6-membered aromatic heterocycle, condensed ring formed by benzene ring and 5-6-membered aromatic heterocycle, 4-7-membered carbon heteromonocycle, 5-10-membered carbon heterocyclic ring, 4-7-membered monocyclic alkyl, 5-10-membered cycloalkyl, 7-10-membered bridged cycloalkyl; p is selected from the natural number 1-5; R 10 , R 11 They are independently selected from H, C1-C4 straight chain or branched alkane, C1-C4 straight chain or branched olefin, C1-C4 straight chain or branched alkyne, halogen-substituted C1-4 straight chain or branched alkyl, hydroxy-substituted C1-4 straight chain or branched alkyl, alkoxy-substituted C1-4 straight chain or branched alkyl, amino-substituted C1-4 straight chain or branched alkyl, benzene ring, 5-6 membered aromatic heterocycle, and a condensed ring formed by a benzene ring and a 5-6 membered aromatic heterocycle.

3. The compound capable of binding to SCF-FBXO31 protein according to claim 1 or 2, characterized in that: The aromatic heterocycle, aromatic fused heterocycle, carbon heteromonocycle, and carbon heterocyclic ring contain 1 to 4 heteroatoms selected from O, S, and N.

4. The compound capable of binding to SCF-FBXO31 protein according to claim 1, characterized in that The compound of formula (I) is specifically the following structure:

5. A pharmaceutically acceptable salt of the compound capable of binding to SCF-FBXO31 protein according to any one of claims 1 to 4, wherein the pharmaceutically acceptable salt is a salt prepared by adding a non-toxic acid or base to the parent compound.

6. A protein degradation agent, characterized in that The protein degradation agent is: target protein ligand + linker + SCF-FBXO31 protein conjugate (ligand); the protein degradation agent is shown in formula (II): In Formula II, L1 of the Linker substructure is connected to C, N, O or S to which R2 is connected; The SCF-FBXO31 conjugate (ligand) is a compound according to any one of claims 1-4.

7. The protein degradation agent according to claim 6, characterized in that The target protein ligand can be selected from the following: One or more of .

8. The protein degradation agent according to claim 6, characterized in that The substructures L1, L3 and L5 of the Linker may be selected from one or more of the following: Among them, R a One or more selected from H, alkyl, SO2(alyl) or SO2(aryl); The substructures L2 and L4 of the Linker may be selected from one or more of the following:

9. The protein degradation agent according to claim 6, characterized in that The protein degradation agent of formula (II) has the following structure:

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable diluent or carrier.

11. A pharmaceutical composition for targeted protein degradation, characterized in that: The pharmaceutical composition comprises a combination of the protein degradation agent of formula (II) or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 9 and a pharmaceutically acceptable diluent or carrier.

12. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 10 in the preparation of a drug, characterized in that: The drug is a drug for treating SCF-FBXO31 related diseases.

13. The use according to claim 12, characterized in that The SCF-FBXO31-related diseases include mental retardation, autosomal recessive non-syndromic intellectual disability, cerebral palsy, spastic-dystonic cerebral palsy syndrome, glioma, melanoma, breast cancer, liver cancer, gastric cancer, ovarian cancer, prostate cancer, colon cancer, esophageal cancer, esophageal squamous cell carcinoma, lung cancer, cervical cancer, pancreatic cancer, diabetes, cardiac hypertrophy or heart failure.

14. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, the protein degrading agent of formula (II) or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 9, the pharmaceutical composition according to claim 10, or the pharmaceutical composition for targeted protein degradation according to claim 11 in the preparation of a drug, characterized in that: The medicine is a medicine for treating tumors and / or neurodegenerative diseases.

15. The use according to claim 14, characterized in that The tumors include melanoma, renal cancer, adrenocortical carcinoma, bladder cancer, prostate cancer, ovarian cancer, breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, uterine cancer, testicular cancer, fallopian tube tumors, gastrointestinal cancer, colorectal cancer, lymphoma, Hodgkin lymphoma, liver cancer, intrahepatic bile duct cancer, non-small cell lung cancer, small cell lung cancer, head and neck cancer, esophageal squamous cell carcinoma, neuroendocrine tumors, gliomas, astrocytomas, oligodendrogliomas, glioblastomas, neuroblastomas, gangliocytomas, gangliogliomas, medulloblastomas, neurofibromas, neurotheliomas, peripheral neuroepitheliomas, brain cancer, meningiomas, meningiosarcomas or ependymomas.

16. The use according to claim 14, characterized in that The neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, Huntington's disease, bovine spongiform encephalopathy, multiple sclerosis, cerebral ischemia, amyotrophic lateral sclerosis, ataxia telangiectasia, Creutzfeldt-Jakob disease, cerebellar atrophy, primary lateral sclerosis, spastic paraplegia or myasthenia gravis.

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