PROTAC Degrading Agent and Its Application

Abstract

The application discloses a PROTAC degrading agent and application thereof, and belongs to the technical field of medicines. The PROTAC degrading agent provided by the application comprises a compound shown in formula (1) or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, co-crystal or deuteride thereof.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to PROTAC degrading agents and their applications. Background Technology

[0002] Janus kinase (JAK) is a non-receptor tyrosine kinase. The JAK-STAT signaling pathway, consisting of JAK and signal transduction and transcription activating factors (STATs), influences cell proliferation, differentiation, apoptosis, and immune regulation. JAK and STAT proteins bind to the intracellular portions of type I / II cytokine receptors, which are responsible for recognizing soluble inflammatory mediators such as interleukins and interferons. Following binding of extracellular ligands to cytokine receptors, JAK proteins are activated and phosphorylate STAT proteins. STAT proteins then dimerize and translocate to the nucleus, activating downstream gene transcription. Four members of the JAK family (JAK1, JAK2, JAK3, and TYK2) and seven members of the STAT family selectively bind to different cytokine receptors in various combinations. These cytokines include interferon (IFN), interleukin (IL), and hormones [such as growth hormone (GH), erythropoietin (EPO), thrombopoietin (TPO), etc.] signal transduction, and cytokines have functions such as regulating autoimmunity, promoting cell growth and hematopoiesis, and repairing tissue damage.

[0003] Currently, several small-molecule JAK inhibitors are in Phase III clinical trials or have been approved for marketing, used to treat diseases such as rheumatoid arthritis (RA), psoriasis (PS), and atopic dermatitis. JAK inhibitors can be divided into first-generation non-selective inhibitors and second-generation selective inhibitors. The former targets two or more of the four subtypes of JAK—JAK1, JAK2, JAK3, and TYK2. However, traditional JAK inhibitors have several limitations, mainly in the following aspects: ① Insufficient targeting: Traditional JAK inhibitors often inhibit multiple JAK family members, leading to non-target effects and increased side effects; ② Drug resistance: Long-term use of JAK inhibitors may lead to target mutations, resulting in drug resistance; ③ Low bioavailability: Some JAK inhibitors have low bioavailability, requiring frequent dosing and affecting patient compliance.

[0004] Proteolytic targeting chimeras (PROTACs) are bifunctional molecules with two recruitment ligands linked together by a linker strand. One ligand recruits an E3 ubiquitin ligase, while the other specifically recruits a target protein (POI). During ternary complex formation, the PROTAC recruits the E3 ligase to the target protein, spatially favoring substrate polyubiquitination, leading to subsequent proteasome degradation of the POI. Compared to site-based pharmacology, PROTAC technology offers several advantages. First, due to its catalytic properties, PROTACs can achieve therapeutic effects comparable to site-based inhibitors at lower concentrations. Second, PROTACs can serve as a novel approach for targeting "undruggable" proteins, such as transcription factors, scaffold proteins, and non-enzymatic proteins. Third, PROTACs can selectively bind to targets that are difficult to reach with small molecules. Therefore, developing novel JAK degraders using PROTAC technology to achieve more precise and effective treatment has become a key technical challenge in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes PROTAC degrading agent and its applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a PROTAC degrading agent comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, solvate, hydrate, prodrug, stereoisomer, polymorph, eutectic, or deuterated product.

[0008] Equation (1);

[0009] Where L is , or R is H, , or G1 and G2 are independently bonds, CH2 or O, and G1 and G2 are not simultaneously bonds; n and m are independently 1, 2, 3 or 4.

[0010] Furthermore, L is , , , , , , , or .

[0011] Furthermore, the structural formula of the PROTAC degrading agent is as follows: , , or .

[0012] Furthermore, the structural formula of the PROTAC degrading agent is as follows:

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] .

[0019] This invention provides a method for preparing the PROTAC degrading agent as described above, comprising the following steps:

[0020] (1) Preparation of compound VI:

[0021] ;

[0022] a) The compound of formula I is mixed with N,N-dimethylformamide dimethyl acetal and reacted at 50-120°C for 10-14 h to obtain the compound of formula II;

[0023] b) Compound III is mixed with cyanamide and hydrochloric acid and reacted at 50-120°C for 8-12 hours to obtain compound IV;

[0024] c) Mix the compound of formula II obtained in step a) with the compound of formula IV obtained in step b) and react at 80-120℃ for 40-60 h to obtain compound V;

[0025] d) The compound of formula V obtained in step c) is subjected to hydrolysis at 20-80℃ for 1-8 hours to obtain compound of formula VI;

[0026] (2) Preparation of compound XI:

[0027] ;

[0028] e) Compounds VII and VIII are reacted at 50-100°C for 2-8 h in the presence of N,N-diisopropylethylamine to give compound IX;

[0029] f) React the compound of formula IX obtained in step e) with HCl-dioxane solution at 20-50℃ for 1-3 h to obtain compound of formula X;

[0030] g) React the compound X obtained in step f) with the compound VI obtained in step d) at 20-50℃ for 8-12 h to obtain the compound XI.

[0031] Or preparation of compound XV of formula (3):

[0032] ;

[0033] h) Mix the compound X obtained in step f) with the compound XII and react at 20-50°C for 8-12 h to obtain the compound XIII.

[0034] i) The compound of formula XIII obtained in step h) is mixed with HCl-dioxane solution and reacted at 20-50℃ for 1-3 h to obtain compound of formula XIV;

[0035] j) Mix the compound XIV obtained in step i) with the compound VI obtained in step d) and react them at 20-50℃ for 8-12 h to obtain compound XV.

[0036] Or, preparation of compound XXI of formula (4):

[0037] ;

[0038] k) Mix the compound of formula VI obtained in step d) with the compound of formula XVI and react at 20-50℃ for 10-14 h to obtain the compound of formula XVII.

[0039] i) The compound of formula XVII obtained in step k) is mixed with HCl-dioxane solution and reacted at 20-50℃ for 1-3 h to obtain compound of formula XVIII;

[0040] m) The compound of formula XIX was reacted at 20-50 °C for 2-4 h in the presence of a Desmond-Martin oxidant to obtain the compound of formula XX;

[0041] n) The compound of formula XX obtained in step m) and the compound of formula XVIII obtained in step i) are reacted with glacial acetic acid and sodium triacetoxyborohydride at 20-50°C for 14-18h to obtain compound of formula XXI.

[0042] In equations VIII, IX, X, XI, XIII, XIV, XV, XIX, XX, and XXI, R represents H. , or G1 and G2 are independently bonds, CH2 or O, and G1 and G2 are not simultaneously bonds; n and m are independently 1, 2, 3 or 4.

[0043] The present invention also provides a pharmaceutical composition comprising the PROTAC degrading agent described in the above technical solution and pharmaceutically acceptable excipients or excipients.

[0044] The present invention also provides the use of the PROTAC degrading agent or pharmaceutical composition as described above in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases, respiratory diseases, skin diseases, immune system diseases or colitis.

[0045] Furthermore, the inflammatory disease is an inflammatory skin disease; the respiratory disease is chronic obstructive pulmonary disease, colitis, or asthma; the skin disease is psoriasis or atopic dermatitis; and the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

[0046] The present invention also provides the use of the PROTAC degrading agent or pharmaceutical composition described above in the preparation of a medicament or JAK inhibitor for the prevention and / or treatment of JAK-mediated diseases.

[0047] Furthermore, the JAK is JAK1 or JAK2.

[0048] Compared with the prior art, the present invention has the following advantages and technical effects:

[0049] The PROTAC degrader provided by this invention has anti-inflammatory activity, can effectively alleviate inflammatory response, has strong selectivity for JAK1 / 2 subtypes, and can degrade JAK1 protein in a dose-dependent manner. Attached Figure Description

[0050] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0051] Figure 1 Screening results of the compounds on the degradation activity of JAK1 protein (A) and heatmap of JAK1 protein degradation rate (B);

[0052] Figure 2 The results of cytotoxicity experiments for compounds A8, C2, C4, and C6 are shown.

[0053] Figure 3 The left image shows the screening results (left) and degradation rate curve (right) of JAK1 protein degradation activity in RAW264.7 cells after treatment with compounds A8 (A), C2 (B), and C4 (C).

[0054] Figure 4 The anti-inflammatory effects of compounds A8, C2, and C4 in LPS-induced RAW264.7 cells are shown, where A represents NO production, B represents IL-6 level, and C represents TNF-α level.

[0055] Figure 5 The screening results (left) and degradation rate curves (right) of compound A8 on the degradation activity of JAK2 (A), JAK3 (B) and TYK2 (C);

[0056] Figure 6 The results show the ameliorative effect of compound A8 on symptoms of DSS-induced colitis in mice. In the figure, A is a schematic diagram of the animal experimental design, B is the change in body weight during the experiment, C is the disease activity index (DAI) score, D is the colon length, E is the spleen index, F is a representative image of the colon in each group, and G is a representative image of the spleen in each group. The values ​​are expressed as mean ± standard deviation (n=8). Compared with the model group, *P<0.05, **P<0.01, ***P<0.001;

[0057] Figure 7 The results of histopathological evaluation of intestinal tissue in DSS-induced colitis mice are shown in Figure A, where H&E staining and Alcianblue-PAS staining images of the colon are shown, and immunohistochemical staining results of colonic tight junction protein ZO-1 and Occludin are shown in Figure B.

[0058] Figure 8 The results show the levels of inflammatory factors, blood routine tests, and immunohistochemical analysis in DSS-induced colitis mice. A represents serum levels of IL-6, IL-10, IL-1β, and TNF-α (ELISA detection); B represents colon tissue levels of IL-6, IL-10, IL-1β, and TNF-α (ELISA detection); C represents peripheral blood immune cell subtype analysis, including total white blood cell count (WBCs), lymphocytes, monocytes, and neutrophils. Dashed lines indicate the upper and lower limits of normal ranges; D represents immunohistochemical staining of MPO and iNOS in colon tissue, with positive signals appearing in brown. Scale bar = 200 μm. Values ​​are expressed as mean ± standard deviation (n = 8). Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0060] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0061] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0062] Example 1: Synthesis of intermediates 3a-3f

[0063] The reaction route is as follows: ;

[0064] The preparation process is as follows:

[0065] (1) Synthesis of compounds 2a-2f

[0066] The reaction route is as follows: ;

[0067] 2.0 g of compound 1 (7.24 mmol, 1.0 equiv) was dissolved in 10.0 mL of N,N-dimethylformamide solution. An amine reagent (8.69 mmol, 1.2 equiv) and N,N-diisopropylethylamine (DIPEA, 14.48 mmol, 2.0 equiv) were then added to the solution. The reaction was heated at 90 °C for 4 h, and monitored by thin-layer chromatography. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to rapid column chromatography to obtain compounds 2a-2f.

[0068] The structural formula of compound 2a-2f is:

[0069]

[0070] ;

[0071] Synthesis of compound 2a: Using mono-Boc-ethylenediamine (CAS No.: 57260-73-8) as the amine reagent, compound 2a was obtained in 25% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.57 (dd, J = 8.5,7.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.05–6.95 (m, 2H), 6.71 (t, J = 6.0Hz, 1H), 5.05 (dd, J = 12.5, 5.5 Hz, 1H), 3.39–3.34 (m, 2H), 3.14–3.09 (m,2H), 2.92–2.85 (m, 1H), 2.63–2.51 (m, 2H), 2.04–1.99 (m, 1H), 1.36 (s, 9H).ESI-MS m / z: The calculated value is C 20 H 24 O6N4Na + [M + Na] + , 439.2; The measured value is 439.2.

[0072] Synthesis of compound 2b: N-Boc-1,3-propanediamine (CAS No.: 75178-96-0) was selected as the amine reagent to yield compound 2b in 25% yield. 1 H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.57 (dd, J =8.5, 7.0 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.91(t, J = 6.0 Hz, 1H), 6.66 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.5, 5.5 Hz,1H), 3.33–3.29 (m, 2H), 3.02–2.97 (m, 2H), 2.92–2.85 (m, 1H), 2.63–2.51 (m,2H), 2.05–2.00 (m, 1H), 1.69–1.63 (m, 2H), 1.37 (s, 9H). ESI-HRMS m / z: Calculated value is C 21 H 26 O6N4Na + [M + Na] + , 453.1745; The measured value is 453.1731.

[0073] Synthesis of compound 2c: Using tert-butyl (4-aminobutyl)carbamate (CAS No.: 68076-36-8) as the amine reagent, compound 2c was obtained in yield: 27%. 1 H NMR (500 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.57(dd, J = 8.5, 7.0 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz,1H), 6.82 (t, J = 6.0 Hz, 1H), 6.54 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 13.0,5.5 Hz, 1H), 3.31–3.26 (m, 2H), 2.98–2.92 (m, 2H), 2.91–2.84 (m, 1H), 2.62–2.51 (m, 2H), 2.05–2.00 (m, 1H), 1.57–1.51 (m, 2H), 1.48–1.41 (m, 2H), 1.36 (s, 9H). ESI-HRMS m / z: Calculated value is C 22 H 28 O6N4Na + [M + Na] + , 467.1901; The measured value is 467.1876.

[0074] Synthesis of compound 2d: The amine reagent selected was tert-butyl N-(5-aminopentyl)carbamate (CAS No.: 51644-96-3), yielding compound 2d in 42% yield. 1H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.57(dd, J = 8.5, 7.0 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 7.0 Hz,1H), 6.77 (t, J = 6.0 Hz, 1H), 6.51 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 13.0,5.5 Hz, 1H), 3.29–3.25 (m, 2H), 2.94–2.90 (m, 2H), 2.89–2.84 (m, 1H), 2.62–2.51 (m, 2H), 2.06–2.00 (m, 1H), 1.59–1.53 (m, 2H), 1.43–1.38 (m, 2H), 1.36 (s, 9H), 1.33–1.28 (m, 2H). ESI-HRMS m / z: Calculated value is C 23 H 30 O6N4Na + [M + Na] + ,481.2058; The measured value is 481.2039.

[0075] Synthesis of compound 2e: Using tert-butyl (6-aminohexyl)carbamate (CAS No.: 51857-17-1) as the amine reagent, compound 2e was obtained in 47% yield. 1 H NMR (500 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.57(dd, J = 8.5, 7.0 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz,1H), 6.75 (t, J = 6.0 Hz, 1H), 6.52 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 13.0,5.5 Hz, 1H), 3.30–3.26 (m, 2H), 2.92–2.88 (m, 2H), 2.88–2.84 (m, 1H), 2.61–2.51 (m, 2H), 2.05–2.00 (m, 1H), 1.59–1.52 (m, 2H), 1.40–1.37 (m, 2H), 1.36 (s, 9H), 1.34–1.26 (m, 4H). ESI-HRMS m / z: Calculated value is C 24 H32 O6N4Na + [M + Na] + ,495.2214; The measured value is 495.2190.

[0076] Synthesis of compound 2f: Using tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (CAS No.: 153086-78-3) as the amine reagent, compound 2f was obtained in 41% yield. 1 H NMR (500 MHz, DMSO-d6) δ11.09 (s, 1H), 7.58 (dd, J = 8.5, 7.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 7.03(d, J = 7.0 Hz, 1H), 6.72 (t, J = 6.0 Hz, 1H), 6.60 (t, J = 6.0 Hz, 1H), 5.06 (dd, J = 13.0, 5.5 Hz, 1H), 3.62 (t, J = 5.5 Hz, 2H), 3.57–3.54 (m, 2H), 3.52–3.50 (m, 2H), 3.48–3.45 (m, 2H), 3.38 (t, J = 6.0 Hz, 2H), 3.08–3.03 (m, 2H), 2.92–2.84 (m, 1H), 2.62–2.51 (m, 2H), 2.05–2.00 (m, 1H), 1.35 (s, 9H). ESI-HRMS m / z: Calculated value is C 24 H 32 O8N4Na + [M + Na] + , 527.2112; The measured value is 527.2097.

[0077] (2) Synthesis of compounds 3a-3f

[0078] The reaction route is as follows: ;

[0079] 500 mg of compound 2a-2f obtained in step (1) was dissolved in 9.0 mL of DCM, and 3 mL of hydrogen chloride-1,4-dioxane solution was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the solid was collected by filtration and dried to obtain yellow-green solid 3a-3f with a yield of 75-89%.

[0080] Example 2 Synthesis of compound 5a

[0081] The reaction route is as follows:

[0082] ;

[0083] The preparation process is as follows:

[0084] (1) Synthesis of compound 4a

[0085] The reaction route is as follows: ;

[0086] 500 mg of compound 3f (1.1 mmol, 1.0 equiv) prepared in Example 1 and 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)acetic acid (CAS No.: 156478-71-6) (1.21 mmol, 1.1 equiv) were dissolved in 5.0 mL of N,N-dimethylformamide, and O-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.1 mmol, 1.0 equiv, CAS No.: 148893-10-1) and N,N-diisopropylethylamine (DIPEA, 5.5 mmol, 5.0 equiv) were added. The reaction was stirred at room temperature for 10 h, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated, and subjected to rapid column chromatography to obtain a yellow-green solid compound 4a, with a yield of 44%.

[0087] The obtained compound 4a was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results are as follows: 1H NMR (500MHz, DMSO-d6) δ 11.08 (s, 1H), 7.71 (t, J = 6.0 Hz, 1H), 7.58 (dd, J = 8.5,7.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.61 (t, J =6.0 Hz, 1H), 5.05 (dd, J = 13.0, 5.5 Hz, 1H), 3.62 (t, J = 5.5 Hz, 2H), 3.57–3.51 (m, 4H), 3.49–3.45 (m, 2H), 3.43 (t, J = 6.0 Hz, 2H), 3.32–3.29 (m, 4H), 3.26–3.22 (m, 2H), 2.91 (s, 2H), 2.89–2.84 (m, 1H), 2.62–2.51 (m, 2H), 2.35 (t, J = 5.5 Hz, 4H), 2.05–2.00 (m, 1H), 1.37 (s, 9H). ESI-HRMS m / z: Calculated value is C 30 H 43 O9N6 + [M + H] + , 631.3086; The measured value is 631.3058.

[0088] (2) Synthesis of compound 5a

[0089] The reaction route is as follows: ;

[0090] 500 mg of compound 4a prepared in step (1) was dissolved in 9.0 mL of DCM, and then 3 mL of hydrogen chloride-1,4-dioxane solution was added to the above solution. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the solid was collected by filtration and dried to obtain yellow-green solid 5a. Yield: 78%.

[0091] The obtained compound 5a was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results are as follows: 1H NMR(500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.89 (br, 1H), 8.61 (br, 1H), 7.59 (dd, J= 8.5, 7.0 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.60(br, 1H), 5.05 (dd, J = 13.0, 5.5 Hz, 1H), 3.91-3.80 (m, 2H), 3.63–3.61 (m,2H), 3.58–3.53 (m, 6H), 3.49–3.44 (m, 6H), 3.39–3.34 (m, 4H), 3.29–3.26 (m, 2H), 2.93–2.85 (m, 1H), 2.63–2.51 (m, 2H), 2.05–2.00 (m, 1H). ESI-HRMS m / z: calculated value is C 25 H 35 O7N6 + [M + H] + , 531.2562; The measured value is 531.2537.

[0092] Example 3 Synthesis of compounds 8a-8b

[0093] The reaction route is as follows: ;

[0094] The preparation process is as follows:

[0095] (1) Synthesis of compounds 7a-7b

[0096] The reaction route is as follows: ;

[0097] 1.0 g of compound 6 (3.65 mmol, 1.0 equiv) was dissolved in 5.0 mL of N,N-dimethylformamide, followed by the addition of 5-bromo-1-pentanol or 6-bromo-1-hexanol (7.30 mmol, 2.0 equiv), then NaHCO3 (14.60 mmol, 4.0 equiv) and KI (0.37 mmol, 0.1 equiv). The reaction was carried out at 80 °C for 8 h, monitored by thin-layer chromatography. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to rapid column chromatography to obtain white solid compounds 7a-7b.

[0098] The structural formulas of compounds 7a-7b are: ;

[0099] Synthesis of compound 7a: 5-bromo-1-pentanol was selected as the reactant to give compound 7a in 48% yield; 1 H NMR (500MHz, DMSO-d6) δ 11.11 (br, 1H), 7.80 (dd, J = 8.5, 7.0 Hz, 1H), 7.50 (d, J =8.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 5.08 (dd, J = 13.0, 5.5 Hz, 1H), 4.40(br, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.42–3.40 (m, 2H), 2.92–2.84 (m, 1H), 2.63–2.50 (m, 2H), 2.06–2.00 (m, 1H), 1.79–1.73 (m, 2H), 1.51–1.45 (m, 4H). ESI-MS m / z: Calculated value is C 18 H 21 O6N2 + [M + H] + , 361.1; The measured value is 361.1.

[0100] Synthesis of compound 7b: 6-bromo-1-hexanol was selected as the reactant to give compound 7b in 45% yield; 1 H NMR (500MHz, DMSO-d6) δ 11.10 (s, 1H), 7.80 (dd, J = 8.5, 7.0 Hz, 1H), 7.50 (d, J =8.5 Hz, 1H), 7.43 (d, J = 7.0 Hz, 1H), 5.08 (dd, J = 13.0, 5.5 Hz, 1H), 4.35(t, J = 5.5 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.42–3.38 (m, 2H), 2.92–2.84(m, 1H), 2.63–2.50 (m, 2H), 2.06–2.00 (m, 1H), 1.78–1.72 (m, 2H), 1.48–1.41(m, 4H), 1.38–1.33 (m, 2H). ESI-HRMS m / z: Calculated value is C 19 H 22 O6N2Na + [M + Na] +,397.1370; The measured value is 397.1350.

[0101] (2) Synthesis of compounds 8a-8b

[0102] The reaction route is as follows: ;

[0103] Compounds 7a-7b (2.0 mmol, 1.0 equiv) prepared in step (1) were dissolved in dichloromethane, and Dess-Martin Periodinane (CAS No.: 87413-09-0, 4.0 mmol, 2.0 equiv) was added. The mixture was stirred at room temperature for 3 h, and then saturated sodium bicarbonate and saturated sodium thiosulfate solutions were added. The mixture was stirred for 10 min, and the organic phase was collected. The aqueous phase was extracted with dichloromethane, and the organic phase was dried with anhydrous sodium sulfate. The organic phase was concentrated and subjected to rapid column chromatography to obtain white solid compounds 8a-8b.

[0104] The structural formulas of compounds 8a and 8b are: ;

[0105] Synthesis of compound 8a: Reactant 7a was selected to obtain compound 8a in 53% yield; 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.69 (t, J = 1.6 Hz, 1H), 7.81 (dd, J = 8.4, 7.2Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.21 (t, J = 6.0 Hz, 2H), 2.93–2.83 (m, 1H), 2.63–2.50 (m, 4H), 2.07–1.97 (m, 1H), 1.85–1.65 (m, 4H). ESI-HRMS m / z: Calculated value is C 18 H 19 O6N2 + [M+ H] + , 359.1238; The measured value is 359.1238.

[0106] Synthesis of compound 8b: Reactant 7b was selected to yield compound 8b in 55% yield; 1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.67 (t, J = 1.6 Hz, 1H), 7.80 (dd, J = 8.4, 7.2Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.08 (dd, J =12.8, 5.4 Hz, 1H), 4.19 (t, J = 6.4 Hz, 2H), 2.93–2.84 (m, 1H), 2.62–2.51 (m,2H), 2.46 (td, J = 7.2, 1.6 Hz, 2H), 2.06–1.98 (m, 1H), 1.80–1.72 (m, 2H), 1.65–1.56 (m, 2H), 1.49–1.42 (2H). ESI-HRMS m / z: Calculated value is C 19 H 21 O6N2 + [M + H] + ,373.1394; The measured value is 373.1395.

[0107] Example 4 Synthesis of compounds 14a-14d

[0108] The reaction route is as follows: ;

[0109] The preparation process is as follows:

[0110] (1) Synthesis of compound 10

[0111] The reaction route is as follows: ;

[0112] 4.0 g of compound 9 (22.4 mmol, 1.0 equiv) was dissolved in 20 mL of N,N-dimethylformamide dimethyl acetal (DMF-DMA), and the mixture was heated under reflux at 85 °C for 12 h. After the reaction was completed, the mixture was filtered to obtain 4.5 g of yellow solid compound 10, yield: 86%.

[0113] Compound 10 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows: 1H NMR (500 MHz, DMSO-d6) δ 8.00 (s, 4H), 7.76 (d, J = 12.0 Hz, 1H), 5.84 (d, J = 12.0 Hz, 1H), 3.87 (s, 3H), 3.16 (s, 3H), 2.93 (s, 3H). ESI-HRMS m / z: Calculated values ​​are C 13 H 16 O3N + [M + H] + , 234.1125; The measured value is 234.1124.

[0114] (2) Synthesis of compounds 12a-12d

[0115] The reaction route is as follows: ;

[0116] Amine reagent compound 11a-11d (20.0 mmol, 1.0 equiv) and cyanamide (CAS No.: 420-04-2, 60.0 mmol, 3.0 equiv) were dissolved in 30 mL of ethanol. 8 mL of concentrated hydrochloric acid was added, and the mixture was heated under reflux at 85 °C for 10 h. After the reaction was complete, the ethanol was removed by concentration, and the pH was adjusted to 12 by adding sodium carbonate aqueous solution. The crude product was then concentrated, and intermediate 12a-12d was obtained by rapid column chromatography.

[0117] The structural formulas of compounds 12a-12d are as follows:

[0118] ;

[0119] Synthesis of compound 12a: Reactant 11a was selected to obtain compound 12a, yield: 79%; 1 H NMR (500MHz, DMSO-d6) δ 6.85 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 2H), 6.58–5.49 (m, 4H), 3.72 (t, J = 4.5 Hz, 4H), 3.01 (t, J = 4.5 Hz, 4H). ESI-HRMS m / z: Calculated values ​​are C 11 H 17 ON4 + [M + H] + , 221.1397; The measured value is 221.1399.

[0120] Synthesis of compound 12b: Reactant 11b was selected to yield compound 12b in 67% yield.1 ¹H NMR (500MHz, Methanol-d⁴) δ 7.50–7.45 (m, 2H), 7.39–7.34 (m, 1H), 7.31–7.27 (m, 2H), 5.71 (s, 1H), NH₃ (3H, not observed). ESI-HRMS m / z: Calculated value is C₇H₂. 10 N3 + [M + H] + ,136.0869; The measured value is 136.0870.

[0121] Synthesis of compound 12c: Reactant 11c was selected to yield compound 12c, yield: 68%; 1 ¹H NMR (500MHz, Methanol-d⁴) δ 7.75–7.71 (m, 2H), 7.66–7.61 (m, 2H), 7.48–7.43 (m, 2H), 7.39–7.33 (m, 3H), NH₄⁺ (4H, not observed). ESI-HRMS m / z: Calculated values ​​are for C₁₀. 13 H 14 N3 + [M +H] + , 212.1182; The measured value is 212.1184.

[0122] Synthesis of compound 12d: Reactant 11d was selected to yield compound 12d, yield: 86%; 1 ¹H NMR (500MHz, DMSO-d⁶) δ 7.14 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 2.48–2.40 (m, 1H), 2.10–1.85 (m, 1H), 1.82–1.74 (m, 4H), 1.72–1.67 (m, 1H), 1.40–1.32 (m, 4H), NH₄⁺ (4H, not observed). ESI-HRMS m / z: Calculated values ​​are for C₁₀. 13 H 20 N3 + [M + H] + ,218.1652; The measured value is 218.1654.

[0123] (3) Synthesis of compounds 13a-13d

[0124] The reaction route is as follows: ;

[0125] 3.5 g of compound 10 (15.0 mmol, 1.0 equiv) prepared in step (1) and compound 12a-12d (15.0 mmol, 1.0 equiv) prepared in step (2) were added to 40 mL of acetonitrile and heated to reflux at 100 °C for 48 h. The reaction was detected by thin-layer chromatography. After the reaction was completed, the reaction solution was cooled to room temperature and a solid was formed. The crude product was obtained by filtration and finally intermediate 13a-13d was obtained by rapid column chromatography.

[0126] The structural formulas of compounds 13a-13d are as follows: ;

[0127] Synthesis of compound 13a: Reactant 12a was selected to yield compound 13a in 74% yield. 1 H NMR (400MHz, DMSO-d6) δ 9.55 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.30–8.26 (m, 2H), 8.13–8.08 (m, 2H), 7.69–7.64 (m, 2H), 7.40 (d, J = 5.2 Hz, 1H), 6.96–6.91 (m, 2H), 3.89 (s, 3H), 3.77–3.72 (m, 4H), 3.07–3.02 (m, 4H). ESI-HRMS m / z: Calculated value is C 22 H 23 O3N4 + [M + H] + , 391.1765; The measured value is 391.1762.

[0128] Synthesis of compound 13b: Reactant 12b was selected to yield compound 13b in 22% yield. 1 H NMR (500MHz, DMSO-d6) δ 9.76 (s, 1H), 8.61 (d, J = 5.0Hz, 1H), 8.32–8.27 (m, 2H), 8.15–8.09 (m, 2H), 7.83 (d, J = 7.5Hz, 2H), 7.48 (d, J = 5.0Hz, 1H), 7.35–7.29 (m, 2H), 6.98 (t, J = 7.5Hz, 1H), 3.90 (s, 3H). ESI-HRMS m / z: Calculated value is C 18 H 16 O2N3 +[M + H] + , 306.1237; The measured value is 306.1238.

[0129] Synthesis of compound 13c: Reactant 12c was selected to yield compound 13c, yield: 57%; 1 ¹H NMR (500MHz, DMSO-d⁶) δ 9.92 (s, 1H), 8.64 (d, J = 5.0 Hz, 1H), 8.35–8.31 (m, 2H), 8.16–8.11 (m, 2H), 7.97–7.92 (m, 2H), 7.68–7.64 (m, 4H), 7.51 (d, J = 5.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 2H), 7.34–7.30 (m, 1H), 3.90 (s, 3H). ESI-HRMS m / z: calculated values ​​are C 24 H 19 O2N3Na + [M + Na] + , 404.1369; The measured value is 404.1358.

[0130] Synthesis of compound 13d: Reactant 12d was selected to yield compound 13d in 76% yield. 1 H NMR (500MHz, DMSO-d6) δ 9.68 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.31–8.25 (m, 2H), 8.13–8.06 (m, 2H), 7.73–7.67 (m, 2H), 7.43 (d, J = 5.0 Hz, 1H), 7.15 (d, J =8.5 Hz, 2H), 3.89 (s, 3H), 2.47–2.38 (m, 1H), 1.78 (d, J = 9.5 Hz, 4H), 1.72–1.66 (m, 1H), 1.41–1.32 (m, 4H), 1.26–1.21 (m, 1H). ESI-HRMS m / z: Calculated value is C 24 H 25 O2N3Na + [M + Na] + , 410.1839; The measured value is 410.1857.

[0131] (4) Synthesis of compounds 14a-14d

[0132] The reaction route is as follows: ;

[0133] Weigh out 4.0 mmol (1.0 equiv) of compound 13a-13d prepared in step (3) and dissolve it in 20 mL of a 1:1 mixture of ethanol and water. Add 1.6 g of sodium hydroxide (40.0 mmol (10.0 equiv) and stir the mixture at 50 °C for 4 h. Monitor the reaction by TLC. After the reaction is complete, remove the ethanol by rotary evaporation and adjust the pH to 6 with dilute hydrochloric acid (1 mol / L). Filter the mixture to obtain a solid, and dry it to obtain a yellow solid compound 14a-14d.

[0134] The structural formulas of compounds 14a-14d are as follows:

[0135] ;

[0136] Synthesis of compound 14a: Reactant 13a was selected to obtain compound 14a, yield: 91%; 1 ¹H NMR (500MHz, DMSO-d⁶) δ 13.17 (br, 1H), 9.60 (s, 1H), 8.55 (d, J = 5.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 5.0 Hz, 1H), 7.05 (s, 2H), 3.79 (t, J = 4.5 Hz, 4H), 3.13 (s, 4H). ESI-HRMS m / z: Calculated value is C 21 H 21 O3N4 + [M + H] + , 377.1608; The measured value is 377.1605.

[0137] Synthesis of compound 14b: Reactant 13b was selected to yield compound 14b, yield: 79%; 1H NMR (500MHz, DMSO-d6) δ 13.18 (br, 1H), 9.75 (s, 1H), 8.60 (d, J = 5.0 Hz, 1H), 8.29–8.25 (m, 2H), 8.12–8.08 (m, 2H), 7.86–7.81 (m, 2H), 7.46 (d, J = 5.0 Hz, 1H), 7.36–7.29 (m, 2H), 7.00–6.95 (m, 1H). ESI-HRMS m / z: Calculated values ​​are C 17 H 14 O2N3 + [M + H] + , 292.1081; The measured value is 292.1077.

[0138] Synthesis of compound 14c: Reactant 13c was selected to obtain compound 14c, yield: 80%; 1 ¹H NMR (500MHz, DMSO-d⁶) δ 9.83 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.16–8.11 (m, 2H), 8.07–8.02 (m, 2H), 8.00–7.94 (m, 2H), 7.66 (td, J = 7.5, 7.0, 2.0 Hz, 4H), 7.46–7.41 (m, 3H), 7.33–7.28 (m, 1H), NH₄⁺ (1H, not observed). ESI-HRMS m / z: Calculated values ​​are C 23 H 18 O2N3 + [M + H] + , 368.1394; The measured value is 368.1392.

[0139] Synthesis of compound 14d: Compound 13d was selected as the reactant to yield compound 14d, yield: 65%; 1H NMR (500MHz, DMSO-d6) δ 13.16 (br, 1H), 9.64 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.29–8.24 (m, 2H), 8.12–8.07 (m, 2H), 7.74–7.69 (m, 2H), 7.43 (d, J = 5.0 Hz, 1H), 7.18–7.14 (m, 2H), 2.48–2.40 (m, 1H), 1.82–1.75 (m, 4H), 1.72–1.66 (m, 1H), 1.43–1.32 (m, 4H), 1.26–1.21 (m, 1H). ESI-HRMS m / z: The calculated value is C 23 H 24 O2N3 + [M + H] + , 374.1863; The measured value is 374.1859.

[0140] Example 5 Synthesis of compounds 16a-16d

[0141] The reaction route is as follows:

[0142] ;

[0143] The preparation process is as follows:

[0144] (1) Synthesis of compounds 15a-15d

[0145] The reaction route is as follows:

[0146] ;

[0147] Compounds 14a-14d (1.5 mmol, 1.0 equiv) prepared in Example 4 and piperazine-1-carboxylic acid tert-butyl ester (CAS No.: 57260-71-6, 1.65 mmol, 1.1 equiv) were dissolved in DCM. N-hydroxy-7-azabenzotriazole (CAS No.: 39968-33-7, HOAT, 1.65 mmol, 1.1 equiv), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.65 mmol, 1.1 equiv), and N-methylmorpholine (NMM, 1.65 mmol, 1.1 equiv) were added at room temperature, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the crude product was concentrated under vacuum and then subjected to rapid column chromatography to obtain the yellow solid intermediate 15a-15d.

[0148] The structural formulas of compounds 15a-15d are as follows:

[0149] ;

[0150] Synthesis of compound 15a: Reactant 14a was selected to obtain compound 15a, yield: 71%; 1 ¹H NMR (500MHz, DMSO-d⁶) δ 9.46 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.23–8.18 (m, 2H), 7.69–7.64 (m, 2H), 7.59–7.55 (m, 2H), 7.36 (d, J = 5.0 Hz, 1H), 6.95–6.91 (m, 2H), 3.76–3.72 (m, 4H), 3.68–3.52 (m, 2H), 3.49–3.26 (m, 6H), 3.06–3.02 (m, 4H), 1.41 (s, 9H). ESI-HRMS m / z: Calculated value is C 30 H 37 O4 N6 + [M + H] + , 545.2871; The measured value is 545.2872.

[0151] Synthesis of compound 15b: Reactant 14b was selected to yield compound 15b in 80% yield. 1 H NMR (500MHz, DMSO-d6) δ 9.71 (s, 1H), 8.58 (d, J = 5.0 Hz, 1H), 8.25–8.22 (m, 2H), 7.85–7.82 (m, 2H), 7.61–7.57 (m, 2H), 7.45 (d, J = 5.0 Hz, 1H), 7.34–7.30 (m, 2H), 6.97 (tt, J = 7.5, 1.0 Hz, 1H), 3.68–3.53 (s, 2H), 3.51–3.33 (m, 6H), 1.41 (s, 9H). ESI-HRMS m / z: Calculated value is C 26 H 30 O3N5 + [M + H] + , 460.2343; The measured value is 460.2378.

[0152] Synthesis of compound 15c: Reactant 14c was selected to yield compound 15c, yield: 75%; 1¹H NMR (500MHz, DMSO-d⁶) δ 9.86 (s, 1H), 8.61 (d, J = 5.0 Hz, 1H), 8.28–8.24 (m, 2H), 7.97–7.93 (m, 2H), 7.68–7.64 (m, 4H), 7.61–7.58 (m, 2H), 7.47 (d, J = 5.0 Hz, 1H), 7.46–7.41 (m, 2H), 7.33–7.29 (m, 1H), 3.68–3.35 (m, 8H), 1.41 (s, 9H). ESI-HRMS m / z: Calculated value is C 32 H 34 O3N5 + [M + H] + , 536.2656; The measured value is 536.2648.

[0153] Synthesis of compound 15d: Reactant 14d was selected to yield compound 15d in 85% yield. 1 H NMR (500MHz, DMSO-d6) δ 9.58 (s, 1H), 8.55 (d, J = 5.0 Hz, 1H), 8.24–8.19 (m, 2H), 7.74–7.69 (m, 2H), 7.60–7.55 (m, 2H), 7.40 (d, J = 5.0 Hz, 1H), 7.17–7.13 (m,2H), 3.66–3.33 (m, 8H), 2.44 (td, J = 10.0, 5.0 Hz, 1H), 1.81–1.74 (m, 4H), 1.73–1.66 (m, 1H), 1.41 (s, 9H), 1.39–1.30 (m, 4H), 1.27–1.21 (m, 1H). ESI-HRMS m / z: Calculated value is C 32 H 40 O3N5 + [M + H] + , 542.1326; The measured value is 542.3120.

[0154] (2) Synthesis of compounds 16a-16d

[0155] The reaction route is as follows:

[0156] ;

[0157] 500 mg of compound 15a-15d prepared in step (1) was dissolved in 9.0 mL of DCM, and 3 mL of hydrogen chloride-1,4-dioxane solution was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the solid was collected by filtration and dried to obtain yellow solid compound 16a-16d.

[0158] Example 6 Synthesis of compounds A1-A24

[0159] The reaction route is as follows:

[0160] ;

[0161] Compounds 14a-14d (0.2 mmol, 1.0 equiv) prepared in Example 4 and compounds 3a-3f (0.22 mmol, 1.1 equiv) prepared in Example 1 were dissolved in N,N-dimethylformamide, and HATU (0.22 mmol, 1.1 equiv) and DIPEA (1.0 mmol, 5.0 equiv) were added. The mixture was reacted at room temperature for 10 h. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to rapid column chromatography to obtain compounds A1-A24.

[0162] The structural formula of compounds A1-A24 is:

[0163]

[0164] .

[0165] Synthesis of compound A1: Compound 14a and compound 3a reacted to give compound A1, yield: 43%; 1H NMR(400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.51 (s, 1H), 8.86 (t, J = 5.2 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.23 ​​(d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H),7.67 (d, J = 8.4 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 5.2 Hz, 1H),7.26 (d, J = 8.8 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.93 (d, J = 8.4 Hz, 2H), 6.90–6.84 (m, 1H), 5.06 (dd, J = 12.8, 5.2 Hz, 1H), 3.80–3.70 (m, 4H), 3.59–3.48 (m, 4H), 3.09–2.99 (m, 4H), 2.93–2.83 (m, 1H), 2.63–2.52 (m, 2H), 2.05–1.98 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 168.8, 167.4, 166.3,162.6, 160.4, 159.3, 146.4, 146.3, 139.3, 136.3, 136.1, 132.9, 132.3, 127.8(2×C), 126.8 (2×C), 120.4 (2×C), 117.3, 115.7 (2×C), 110.6, 109.3, 107.6,66.2 (2×C), 49.3 (2×C), 48.6, 41.3, 39.0, 31.0, 22.2. ESI-HRMS m / z: Calculated value is C 36 H 34 O6N8Na + [M + Na] + , 697.2494; The measured value is 697.2500.

[0166] Synthesis of compound A2: Compound 14a and compound 3b reacted to give compound A2, yield: 56%; 1H NMR(400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.51 (s, 1H), 8.71 (t, J = 5.6 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.27–8.21 (m, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.71–7.64 (m, 2H), 7.58 (dd, J = 8.4, 7.2 Hz, 1H), 7.39 (d, J = 5.2 Hz, 1H), 7.12(d, J = 8.8 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.97–6.89 (m, 2H), 6.79 (t, J= 6.0 Hz, 1H), 5.06 (dd, J = 12.8, 5.2 Hz, 1H), 3.78–3.71 (m, 4H), 3.45–3.38(m, 4H), 3.08–3.01 (m, 4H), 2.93–2.91 (m, 1H), 2.62–2.51 (m, 2H), 2.07–1.98(m, 1H), 1.88–1.78 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 168.9,167.4, 165.9, 162.7, 160.4, 159.3, 146.3, 146.3, 139.2, 136.4, 136.3, 132.9,132.3, 127.8 (2×C), 126.8 (2×C), 120.4 (2×C), 117.2, 115.7 (2×C), 110.5,109.2, 107.6, 66.2 (2×C), 49.3 (2×C), 48.6, 39.0, 36.8, 31.0, 28.7, 22.2. ESI-HRMS m / z: Calculated value is C 37 H 36 O6N8Na + [M + Na] + , 711.2650; The measured value is 711.2653.

[0167] Synthesis of compound A3: Compound 14a and compound 3c reacted to give compound A3, yield: 51%; 1H NMR(500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.47 (s, 1H), 8.62 (t, J = 5.5 Hz, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.22 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.57 (dd, J = 8.5, 7.0 Hz, 1H), 7.38 (d, J = 5.0Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.93 (d, J = 9.0Hz, 2H), 6.58 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.5, 5.5 Hz, 1H), 3.78–3.71(m, 4H), 3.44–3.29 (m, 4H), 3.09–3.29 (m, 4H), 2.94–2.82 (m, 1H), 2.63–2.51 (m, 2H), 2.06–1.99 (m, 1H), 1.70–1.58 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ173.0, 170.2, 169.0, 167.4, 165.7, 162.7, 160.4, 159.3, 146.5, 146.3, 139.2,136.5, 136.4, 133.0, 132.3, 127.8 (2×C), 126.8 (2×C), 120.4 (2×C), 117.3,115.7 (2×C), 110.5, 109.1, 107.6, 66.3 (2×C), 49.3 (2×C), 48.6, 41.6,39.0, 31.0, 26.6, 26.3, 22.2. ESI-HRMS m / z: Calculated value is C 38 H 38 O6N8Na + [M + Na] + ,725.2807; The measured value is 724.2803.

[0168] Synthesis of compound A4: Compound 14a and compound 3d reacted to give compound A4, yield: 40%; 1H NMR(500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.47 (s, 1H), 8.59 (t, J = 5.5 Hz, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.24–8.18 (m, 2H), 8.01–7.95 (m, 2H), 7.70–7.63 (m,2H), 7.57 (dd, J = 8.5, 7.0 Hz, 1H), 7.38 (d, J = 5.0 Hz, 1H), 7.11 (d, J =8.5 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.96–6.89 (m, 2H), 6.55 (t, J = 6.0Hz, 1H), 5.04 (dd, J = 12.5, 5.5 Hz, 1H), 3.77–3.71 (m, 4H), 3.32–3.28 (m,4H), 3.08–3.00 (m, 4H), 2.91–2.83 (m, 1H), 2.61–2.51 (m, 2H), 2.04–1.99 (m,1H), 1.66–1.56 (m, 4H), 1.46–1.39 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 169.0, 167.4, 165.6, 162.7, 160.4, 159.3, 146.5, 146.3, 139.1, 136.5,136.4, 132.9, 132.3, 127.7 (2×C), 126.8 (2×C), 120.4 (2×C), 117.3, 115.7(2×C), 110.5, 109.0, 107.6, 66.3 (2×C), 49.3 (2×C), 48.6, 41.9, 39.0,31.0, 28.9, 28.5, 23.9, 22.2. ESI-HRMS m / z: Calculated value is C 39 H 40 O6N8Na + [M + Na] + ,739.2963; The measured value is 739.2969.

[0169] Synthesis of compound A5: Compound 14a and compound 3e react to give compound A5, yield: 60%; 1H NMR(500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.47 (s, 1H), 8.58 (t, J = 5.5 Hz, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.24–8.18 (m, 2H), 8.01–7.95 (m, 2H), 7.69– 7.64(m, 2H), 7.57 (dd, J = 8.5, 7.0 Hz, 1H), 7.38 (d, J = 5.0 Hz, 1H), 7.08 (d, J= 8.5 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.96–6.90 (m, 2H), 6.53 (t, J = 6.0Hz, 1H), 5.05 (dd, J = 12.5, 5.5 Hz, 1H), 3.77–3.70 (m, 4H), 3.31–3.25 (m,4H), 3.07–3.01 (m, 4H), 2.92–2.83 (m, 1H), 2.61–2.51 (m, 2H), 2.06–1.98 (m,1H), 1.62–1.52 (m, 4H), 1.42–1.34 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 169.0, 167.4, 165.6, 162.7, 160.4, 159.3, 146.5, 146.2, 139.1, 136.5,136.3, 132.9, 132.2, 127.7 (2×C), 126.8 (2×C), 120.3 (2×C), 117.2, 115.7(2×C), 110.4, 109.0, 107.6, 66.2 (2×C), 49.3 (2×C), 48.6, 41.8, 39.0,31.0, 29.1, 28.7, 26.3, 26.1, 22.2. ESI-HRMS m / z: Calculated value is C 40 H 42 O6N8Na + [M + Na] + , 753.3120; The measured value is 753.3126.

[0170] Synthesis of compound A6: Compound 14a and compound 3f reacted to give compound A6, yield: 43%; 1H NMR(400 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.50 (s, 1H), 8.65 (t, J = 5.2 Hz, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.21 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H),7.70–7.63 (m, 2H), 7.54 (dd, J = 8.4, 7.2 Hz, 1H), 7.37 (d, J = 4.8 Hz, 1H),7.10 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.96–6.89 (m, 2H), 6.59(t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.77–3.71 (m, 4H), 3.61(t, J = 5.2 Hz, 2H), 3.60–3.53 (m, 6H), 3.47–3.41 (m, 4H), 3.08–3.00 (m, 4H), 2.92–2.82 (m, 1H), 2.62–2.51 (m, 2H), 2.06–1.96 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.3, 165.8, 162.6, 160.4, 159.3, 146.4,146.3, 139.2, 136.3, 136.2, 132.9, 132.1, 127.7 (2×C), 126.8 (2×C), 120.4(2×C), 117.4, 115.7 (2×C), 110.7, 109.3, 107.6, 69.7 (2×C), 68.9 (2×C),66.2 (2×C), 49.3 (2×C), 48.6, 41.7, 39.0, 31.0, 22.2. ESI-HRMS m / z: Calculated value is C 40 H 42 O8N8Na + [M + Na] + , 785.3018; The measured value is 785.3034.

[0171] Synthesis of compound A7: Compound 14b reacts with compound 3a to give compound A7, yield: 60%;1 H NMR(500 MHz, DMSO-d6) δ 11.09 (br, 1H), 9.73 (s, 1H), 9.00 (t, J = 5.5 Hz, 1H), 8.59 (d, J = 5.0 Hz, 1H), 8.27–8.23 (m, 2H), 8.05–8.01 (m, 2H), 7.86–7.81 (m,2H), 7.59 (dd, J = 8.5, 7.0 Hz, 1H), 7.48 (d, J = 5.0 Hz, 1H), 7.34–7.27 (m,3H), 7.02 (d, J = 7.0 Hz, 1H), 6.97 (tt, J = 7.5, 1.0 Hz, 1H), 6.86 (t, J =6.0 Hz, 1H), 5.05 (dd, J = 12.5, 5.5 Hz, 1H), 3.57–3.48 (m, 4H), 2.92–2.84 (m, 1H), 2.61–2.51 (m, 2H), 2.04–1.99 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 173.0, 170.2, 168.8, 167.4, 166.3, 162.8, 160.3, 159.4, 146.4, 140.6, 139.2, 136.3, 136.2, 132.3, 128.6 (2×C), 127.9 (2×C), 126.9 (2×C), 121.6, 119.0 (2×C), 117.4, 110.7, 109.3, 108.4, 48.6, 41.4, 39.0, 31.0, 22.2. ESI-HRMS m / z: Calculated values ​​are for C 32 H 28 O5N7 + [M + H] + , 590.2146; The measured value is 590.2141.

[0172] Synthesis of compound A8: Compound 14b and compound 3b reacted to give compound A8, yield: 54%; 1H NMR(400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.75 (s, 1H), 8.73 (t, J = 5.6 Hz, 1H), 8.59 (d, J = 5.2 Hz, 1H), 8.29–8.23 (m, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.88–7.82 (m, 2H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.35–7.29 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 7.00–6.94(m, 1H), 6.79 (t, J = 6.0 Hz, 1H), 5.07 (dd, J = 12.8, 5.2 Hz, 1H), 3.44–3.38(m, 4H), 2.93–2.83 (m, 1H), 2.63–2.51 (m, 2H), 2.06–1.99 (m, 1H), 1.88–1.80(m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 168.9, 167.4, 165.9, 162.8, 160.2, 159.3, 146.3, 140.6, 139.1, 136.5, 136.3, 132.3, 128.6 (2×C), 127.8 (2×C), 126.8 (2×C), 121.5, 119.0 (2×C), 117.2, 110.5, 109.2, 108.3, 48.6, 39.0, 36.8, 31.0, 28.7, 22.2. ESI-HRMS m / z: Calculated values ​​are for C 33 H 30 O5N7 + [M + H] + ,604.2303; The measured value is 604.2299.

[0173] Synthesis of compound A9: Compound 14b and compound 3c reacted to give compound A9, yield: 44%; 1H NMR(500 MHz, DMSO-d6) δ 11.09 (br, 1H), 9.73 (s, 1H), 8.78 (t, J = 5.5 Hz, 1H), 8.58 (d, J = 5.0 Hz, 1H), 8.26–8.21 (m, 2H), 8.05–8.01 (m, 2H), 7.86–7.81 (m,2H), 7.56 (dd, J = 8.5, 7.0 Hz, 1H), 7.47 (d, J = 5.0 Hz, 1H), 7.34–7.29 (m,2H), 7.13 (d, J = 8.5 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 6.97 (tt, J = 7.0,1.0 Hz, 1H), 6.58 (t, J = 6.0 Hz, 1H), 5.04 (dd, J = 13.0, 5.5 Hz, 1H), 3.35–3.31 (m, 4H), 2.92–2.84 (m, 1H), 2.60–2.51 (m, 2H), 2.05–1.99 (m, 1H), 1.67–1.59 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 173.0, 170.2, 169.0, 167.4, 165.6,162.8, 160.2, 159.4, 146.5, 140.6, 139.0, 136.5, 136.4, 132.3, 128.6 (2×C),127.9 (2×C), 126.8 (2×C), 121.5, 119.0 (2×C), 117.4, 110.5, 109.0, 108.4,48.6, 41.6, 38.9, 31.0, 26.5, 26.3, 22.2. ESI-HRMS m / z: The calculated value is C 34 H 32 O5N7 + [M+ H] + , 618.2459; The measured value is 618.2453.

[0174] Synthesis of compound A10: Compound 14b and compound 3d reacted to give compound A10, yield: 42%; 1HNMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.74 (s, 1H), 8.65 (t, J = 5.6 Hz,1H), 8.59 (d, J = 5.2 Hz, 1H), 8.27–8.20 (m, 2H), 8.02–7.97 (m, 2H), 7.87–7.81 (m, 2H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.35–7.29 (m, 2H), 7.11 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.97 (tt, J = 7.2, 1.2 Hz, 1H), 6.55 (t, J = 6.0 Hz, 1H), 5.04 (dd, J = 12.8, 5.4 Hz, 1H), 3.34–3.27 (t, 4H), 2.92–2.81 (m, 1H), 2.60–2.53 (m, 2H), 2.03–1.98 (m,1H), 1.65–1.55 (m, 4H), 1.44–1.38 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 169.0, 167.4, 165.6, 162.8, 160.3, 159.4, 146.5, 140.6, 139.0, 136.6,136.4, 132.3, 128.6 (2×C), 127.8 (2×C), 126.8 (2×C), 121.6, 119.0 (2×C), 117.3, 110.5, 109.0, 108.4, 48.6, 41.9, 39.0, 31.0, 28.9, 28.5, 23.9, 22.2.ESI-HRMS m / z: Calculated value is C 35 H 34 O5N7 + [M + H] + , 632.2616; The measured value is 632.2616.

[0175] Synthesis of compound A11: Compound 14b reacted with compound 3e to give compound A11, yield: 47%; 1HNMR (400 MHz, DMSO-d6) δ 11.10 (br, 1H), 9.75 (s, 1H), 8.68 (t, J = 5.6 Hz,1H), 8.58 (d, J = 5.2 Hz, 1H), 8.27–8.20 (m, 2H), 8.04–7.99 (m, 2H), 7.87–7.81 (m, 2H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.35–7.28 (m, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.99–6.94(m, 1H), 6.55 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.33–3.25(m, 4H), 2.92–2.82 (m, 1H), 2.62–2.52 (m, 2H), 2.06–1.99 (m, 1H), 1.62–1.52(m, 4H), 1.42–1.33 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0,167.4, 165.5, 162.8, 160.2, 159.3, 146.5, 140.6, 138.9, 136.6, 136.4, 132.2,128.6 (2×C), 127.8 (2×C), 126.8 (2×C), 121.5, 119.0 (2×C), 117.3, 110.4,109.0, 108.3, 48.6, 41.8, 39.0, 31.0, 29.1, 28.7, 26.3, 26.1, 22.2. ESI-HRMSm / z: Calculated value is C 36 H 36 O5N7 + [M + H] + , 646.2772; The measured value is 646.2768.

[0176] Synthesis of compound A12: Compound 14b reacted with compound 3f to give compound A12, yield: 42%; 1HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.75 (s, 1H), 8.66 (t, J = 5.6 Hz,1H), 8.58 (d, J = 5.2 Hz, 1H), 8.26–8.20 (m, 2H), 8.03–7.98 (m, 2H), 7.87–7.80 (m, 2H), 7.54 (dd, J = 8.4, 7.0 Hz, 1H), 7.46 (d, J = 5.2 Hz, 1H), 7.36–7.28 (m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.97 (tt, J = 7.2, 1.2 Hz, 1H), 6.59 (t, J = 5.6 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.61 (t, J = 5.2 Hz, 2H), 3.60–3.54 (m, 6H), 3.48–3.41 (m, 4H), 2.92–2.81 (m, 1H), 2.62–2.51 (m, 2H), 2.05–1.97 (m, 1H). 13 C NMR (101 MHz, DMSO-d6)δ 172.9, 170.2, 169.0, 167.3, 165.7, 162.7, 160.2, 159.3, 146.4, 140.6,139.1, 136.3, 136.3, 132.1, 128.6 (2×C), 127.8 (2×C), 126.8 (2×C), 121.5,119.0 (2×C), 117.4, 110.7, 109.3, 108.3, 69.7 (2×C), 68.9 (2×C), 48.6,41.7, 39.0, 31.0, 22.2. ESI-HRMS m / z: Calculated value is C 36 H 36 O7N7 + [M + H] + , 678.2671; The measured value is 678.2671.

[0177] Synthesis of compound A13: Compound 14c reacted with compound 3a to give compound A13, yield: 46%; 1HNMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.87 (s, 1H), 8.84 (t, J = 5.5 Hz,1H), 8.62 (d, J = 5.0 Hz, 1H), 8.30–8.26 (m, 2H), 8.03–7.99 (m, 2H), 7.97–7.94 (m, 2H), 7.68–7.64 (m, 4H), 7.59 (dd, J = 8.5, 7.0 Hz, 1H), 7.51 (d, J =5.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 2H), 7.33–7.30 (m, 1H), 7.27 (d, J = 8.5Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.87 (t, J = 6.0 Hz, 1H), 5.06 (dd, J =12.5, 5.5 Hz, 1H), 3.57–3.48 (m, 4H), 2.92–2.83 (m, 1H), 2.62–2.51 (m, 2H), 2.05–1.99 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 173.0, 170.2, 168.8, 167.4,166.4, 162.9, 160.2, 159.4, 146.4, 140.1, 140.0, 139.2, 136.3, 136.3, 133.2,132.3, 129.0 (2×C), 127.9 (2×C), 127.0 (2×C), 126.9 (2×C), 126.8, 126.2(2×C), 119.3 (2×C), 117.3, 110.7, 109.4, 108.6, 48.6, 41.4, 39.0, 31.1, 22.2. ESI-HRMS m / z: Calculated value is C 38 H 32 O5N7 + [M + H] + , 666.2459; The measured value is 666.2449.

[0178] Synthesis of compound A14: Compound 14c reacted with compound 3b to give compound A14, yield: 47%; 1HNMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.91 (s, 1H), 8.73 (t, J = 5.6 Hz,1H), 8.62 (d, J = 5.2 Hz, 1H), 8.32–8.25 (m, 2H), 8.05–8.00 (m, 2H), 7.98–7.93 (m, 2H), 7.69–7.63 (m, 4H), 7.58 (dd, J = 8.4, 7.2 Hz, 1H), 7.51 (d, J =5.2 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 7.34–7.29 (m, 1H), 7.13 (d, J = 8.4Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.80 (t, J = 6.0 Hz, 1H), 5.06 (dd, J =12.8, 5.2 Hz, 1H), 3.44–3.38 (m, 4H), 2.93–2.83 (m, 1H), 2.62–2.51 (m, 2H), 2.06–1.98 (m, 1H), 1.89–1.78 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 168.9, 167.4, 165.9, 162.9, 160.2, 159.4, 146.3, 140.1, 140.0, 139.0,136.5, 136.3, 133.1, 132.3, 128.9 (2×C), 127.8 (2×C), 126.9 (2×C), 126.8(2×C), 126.7, 126.1 (2×C), 119.2 (2×C), 117.2, 110.5, 109.2, 108.5, 48.6,39.0, 36.8, 31.0, 28.7, 22.2. ESI-HRMS m / z: Calculated value is C 39 H 34 O5N7 + [M + H] + ,680.2616; The measured value is 680.2612.

[0179] Synthesis of compound A15: Compound 14c reacted with compound 3c to give compound A15, yield: 34%; 1HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.90 (s, 1H), 8.67 (t, J = 5.6 Hz,1H), 8.62 (d, J = 5.2 Hz, 1H), 8.31–8.24 (m, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.97–7.93 (m, 2H), 7.67 (dd, J = 5.2, 1.6 Hz, 2H), 7.65–7.64 (m, 1H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.50 (d, J = 5.2 Hz, 1H), 7.44 (t, J = 7.6 Hz,2H), 7.34–7.29 (m, 1H), 7.28–7.25 (m, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.01 (d,J = 7.2 Hz, 1H), 6.60 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H),3.36–3.31 (m, 4H), 2.93–2.82 (m, 1H), 2.62–2.51 (m, 2H), 2.06–1.98 (m, 1H),1.68–1.59 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.4,165.6, 162.9, 160.2, 159.5, 159.4, 146.4, 140.1, 140.0, 139.0, 136.7, 136.6,136.3, 133.1, 132.3, 130.2, 128.9 (2×C), 127.8, 126.9, 126.8, 126.7, 126.1(2×C), 120.0, 119.2, 117.3, 110.4, 109.0, 108.5, 48.6, 41.6, 39.0, 31.0, 26.6, 26.3, 22.2. ESI-HRMS m / z: Calculated value is C 40 H 36 O5N7 + [M + H] + , 694.2772; The measured value is 694.2767.

[0180] Synthesis of compound A16: Compound 14c and compound 3d reacted to give compound A16, yield: 35%; 1 HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.90 (s, 1H), 8.63 (d, J = 5.2 Hz,2H), 8.30–8.24 (m, 2H), 8.02–7.99 (m, 2H), 7.96 (dd, J = 8.8, 2.0 Hz, 2H),7.67 (d, J = 1.2 Hz, 1H), 7.66–7.64 (m, 2H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H),7.50 (d, J = 5.2 Hz, 1H), 7.47–7.42 (m, 2H), 7.34–7.30 (m, 1H), 7.30–7.24 (m,1H), 7.10 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.56 (t, J = 6.0 Hz,1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 3.34–3.27 (m, 4H), 2.93–2.82 (m, 1H), 2.62–2.51 (m, 2H), 2.05–1.96 (m, 1H), 1.67–1.55 (m, 4H), 1.46–1.36 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.4, 165.6, 162.9, 160.2,159.5, 159.4, 146.4, 140.1, 140.0, 138.9, 136.6, 136.3, 133.1, 132.2, 130.2,128.9 (2×C), 127.8, 126.9, 126.8, 126.7, 126.1 (2×C), 120.0, 119.2 (2×C),117.3, 110.4, 109.0, 108.5, 48.6, 41.8, 39.0, 31.0, 28.9, 28.5, 23.9, 22.2. ESI-HRMS m / z: Calculated value is C 41 H 38 O5N7 + [M + H] +, 708.2929; The measured value is 708.2962.

[0181] Synthesis of compound A17: Compound 14c reacted with compound 3e to give compound A17, yield: 59%; 1 HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.90 (s, 1H), 8.62 (d, J = 5.2 Hz,2H), 8.27 (d, J = 8.4 Hz, 2H), 8.04–7.99 (m, 2H), 7.98–7.93 (m, 2H), 7.69–7.62 (m, 4H), 7.56 (dd, J = 8.4, 7.2 Hz, 1H), 7.50 (d, J = 5.2 Hz, 1H), 7.44(t, J = 7.6 Hz, 2H), 7.34–7.28 (m, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.55 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.4 Hz, 1H), 3.33–3.24 (m, 4H), 2.93–2.83 (m, 1H), 2.63–2.51 (m, 2H), 2.06–1.99 (m, 1H), 1.63–1.52 (m, 4H), 1.42–1.34 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 169.0, 167.4, 165.6, 162.9, 160.2, 159.3, 146.4, 140.1, 140.0, 138.9,136.6, 136.3, 133.1, 132.2, 128.9 (2×C), 127.8 (2×C), 126.9 (2×C), 126.8(2×C), 126.7, 126.1 (2×C), 119.2 (2×C), 117.2, 110.4, 109.0, 108.5, 48.6,41.8, 39.0, 31.0, 29.1, 28.7, 26.3, 26.2, 22.2. ESI-HRMS m / z: Calculated value is C 42 H 40 O5N7 + [M + H] +, 722.3085; The measured value is 722.3084.

[0182] Synthesis of compound A18: Compound 14c reacted with compound 3f to give compound A18, yield: 54%; 1 HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.90 (s, 1H), 8.67 (t, J = 5.6 Hz,1H), 8.61 (d, J = 5.2 Hz, 1H), 8.28–8.22 (m, 2H), 8.05–7.99 (m, 2H), 7.98–7.92 (m, 2H), 7.69–7.63 (m, 4H), 7.54 (dd, J = 8.4, 7.2 Hz, 1H), 7.48 (d, J =5.2 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 7.33–7.28 (m, 1H), 7.10 (d, J = 8.4Hz, 1H), 7.00 (d, J = 7.2 Hz, 1H), 6.59 (t, J = 6.0 Hz, 1H), 5.05 (dd, J =12.8, 5.4 Hz, 1H), 3.61 (t, J = 5.4 Hz, 2H), 3.60–3.55 (m, 6H), 3.48–3.42 (m,4H), 2.93–2.80 (m, 1H), 2.61–2.51 (m, 2H), 2.04–1.97 (m, 1H). 13 C NMR (101MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.4, 165.8, 162.8, 160.2, 159.4,146.4, 140.1, 140.0, 139.1, 136.3, 136.2, 133.1, 132.1, 128.9 (2×C), 127.8(2×C), 126.9 (2×C), 126.8 (2×C), 126.7, 126.1 (2×C), 119.2 (2×C), 117.5,110.7, 109.3, 108.5, 69.7 (2×C), 68.9 (2×C), 48.6, 41.7, 39.0, 31.0, 22.2. ESI-HRMS m / z: Calculated value is C 42 H 40 O7N7 +[M + H] + , 754.2984; The measured value is 754.2981.

[0183] Synthesis of compound A19: Compound 14d reacted with compound 3a to give compound A19, yield: 60%; 1 HNMR (500 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.63 (s, 1H), 8.84 (t, J = 5.5 Hz, 1H), 8.56 (d, J = 5.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz,2H), 7.71 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 5.0 Hz,1H), 7.26 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 7.0 Hz,1H), 6.89 (d, J = 6.0 Hz, 1H), 5.06 (dd, J = 12.5, 5.5 Hz, 1H), 3.58–3.47 (m,4H), 2.94–2.93 (m, 1H), 2.62-2.52 (m, 2H), 2.46–2.39 (m, 1H), 2.07–1.98 (m,1H), 1.78 (d, J = 10.0 Hz, 4H), 1.72–1.66 (m, 1H), 1.41–1.31 (m, 4H), 1.26–1.19 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 173.0, 170.2, 168.8, 167.4, 166.4,162.7, 160.4, 159.4, 146.5, 141.0, 139.3, 138.3, 136.4, 136.2, 132.3, 127.8(2×C), 126.9 (2×C), 126.7 (2×C), 119.2 (2×C), 117.3, 110.7, 109.4, 108.1,48.6, 43.3, 41.4, 39.0, 34.3 (2×C), 31.1, 26.5 (2×C), 25.8, 22.3. ESI-HRMSm / z: Calculated value is C 38 H38 O5N7 + [M + H] + , 672.2929; The measured value is 672.2924.

[0184] Synthesis of compound A20: Compound 14d reacted with compound 3b to give compound A20, yield: 65%; 1 HNMR (500 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.63 (s, 1H), 8.71 (t, J = 5.5 Hz, 1H), 8.56 (d, J = 5.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.01 (d, J = 8.0 Hz,2H), 7.72 (d, J = 8.0 Hz, 2H), 7.58 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 5.0 Hz,1H), 7.18-7.11 (m, 3H), 7.03 (d, J = 7.0 Hz, 1H), 6.78 (t, J = 6.0 Hz, 1H),5.06 (dd, J = 13.0, 5.5 Hz, 1H), 3.45–3,37 (m, 4H), 2.92–2.83 (m, 1H), 2.61–2.52 (m, 2H), 2.46–2.39 (m, 1H), 2.05–1.99 (m, 1H), 1.88–1.81 (m, 2H), 1.78 (d, J = 10.0 Hz, 4H), 1.71–1.66 (m, 1H), 1.42–1.31 (m, 4H), 1.27–1.21 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 168.9, 167.4, 165.9, 162.7,160.3, 159.3, 146.3, 140.9, 139.1, 138.3, 136.4, 136.3, 132.3, 127.8 (2×C),126.8 (2×C), 126.6 (2×C), 119.2 (2×C), 117.2, 110.5, 109.2, 108.0, 48.6,43.2, 39.0, 36.8, 34.2 (2×C), 31.0, 28.7, 26.5 (2×C), 25.7, 22.2. ESI-HRMSm / z: Calculated value is C39 H 40 O5N7 + [M + H] + , 686.3085; The measured value is 686.3081.

[0185] Synthesis of compound A21: Compound 14d reacted with compound 3c to give compound A21, yield: 42%; 1 HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.64 (s, 1H), 8.65 (t, J = 5.6 Hz, 1H), 8.56 (d, J = 5.2 Hz, 1H), 8.23 ​​(d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz,2H), 7.71 (d, J = 8.0 Hz, 2H), 7.57 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 5.2 Hz,1H), 7.16 (d, J = 8.2 Hz, 2H), 7.14–6.98 (m, 2H), 6.60 (t, J = 6.4 Hz, 1H),5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.36–3.31 (m, 4H), 2.94–2.81 (m, 1H), 2.62–2.51 (m, 2H), 2.47–2.40 (m, 1H), 2.06–1.98 (m, 1H), 1.78 (d, J = 9.6 Hz, 4H), 1.73–1.68 (m, 1H), 1.67–1.57 (m, 4H), 1.44–1.31 (m, 4H), 1.26–1.22 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.4, 165.7, 162.8, 160.3,159.3, 146.4, 140.9, 139.1, 138.3, 136.5, 136.3, 132.3, 127.8 (2×C), 126.8(2×C), 126.7 (2×C), 119.2 (2×C), 117.3, 110.5, 109.1, 108.0, 48.6, 43.2,41.6, 39.0, 34.2 (2×C), 31.0, 26.6, 26.5 (2×C), 26.3, 25.7, 22.2. ESI-HRMSm / z: Calculated value is C 40 H 42 O5N7 + [M + H] + , 700.3242; The measured value is 700.3235.

[0186] Synthesis of compound A22: Compound 14d and compound 3d reacted to give compound A22, yield: 37%; 1 HNMR (500 MHz, DMSO-d6) δ 11.11 (s, 1H), 9.63 (s, 1H), 8.66–8.52 (m, 2H), 8.23(d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.57(t, J = 8.0 Hz, 1H), 7.46–7.38 (m, 1H), 7.15 (d, J = 8.5 Hz, 2H), 7.10 (d, J= 8.5 Hz, 1H), 7.01 (d, J = 6.5 Hz, 1H), 6.60–6.48 (m, 1H), 5.05 (dd, J =13.0, 5.5 Hz, 1H), 3.33–3.23 (m, 4H), 2.91–2.82 (m, 1H), 2.64–2.52 (m, 2H), 2.45–2.39 (m, 1H), 2.05–1.96 (m, 1H), 1.78 (d, J = 10.0 Hz, 4H), 1.71–1.67 (m, 1H), 1.65–1.53 (m, 4H), 1.44–1.31 (m, 6H), 1.27–1.21 (m, 1H).13 C NMR (101MHz, DMSO-d6) δ 173.0, 170.3, 169.1, 167.4, 165.7, 162.8, 160.4, 159.4,146.5, 141.0, 139.1, 138.3, 136.6, 136.4, 132.3, 127.8 (2×C), 126.9 (2×C),126.7 (2×C), 119.3 (2×C), 117.3, 110.5, 109.1, 108.1, 48.6, 43.3, 41.9,39.0, 34.3 (2×C), 31.1, 28.9, 28.5, 26.5 (2×C), 25.8, 23.9, 22.3. ESI-HRMSm / z: Calculated value is C. 41 H 44 O5N7 + [M + H] + , 714.3398; The measured value is 714.3392.

[0187] Synthesis of compound A23: Compound 14d reacted with compound 3e to give compound A23, yield: 51%; 1HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.64 (s, 1H), 8.60 (t, J = 5.6 Hz,1H), 8.55 (d, J = 5.2 Hz, 1H), 8.25–8.20 (m, 2H), 8.00 (s, 1H), 7.98 (d, J =1.6 Hz, 1H), 7.74–7.69 (m, 2H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.43 (d, J =5.2 Hz, 1H), 7.18–7.13 (m, 2H), 7.08 (d, J = 8.8 Hz, 1H), 7.01 (d, J = 7.2Hz, 1H), 6.55 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.32–3.25(m, 4H), 2.92–2.82 (m, 1H), 2.62–2.51 (m, 2H), 2.47–2.40 (m, 1H), 2.06–1.98 (m, 1H), 1.78 (d, J = 9.2 Hz, 4H), 1.72–1.66 (m, 1H), 1.62–1.52 (m, 4H), 1.43–1.32 (m, 8H), 1.28–1.21 (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9,170.2, 169.0, 167.4, 165.6, 162.8, 160.3, 159.3, 146.5, 140.9, 139.0, 138.3,136.6, 136.4, 132.2, 127.8 (2×C), 126.8 (2×C), 126.6 (2×C), 119.2 (2×C), 117.2, 110.4, 109.0, 108.0, 48.6, 43.2, 41.8, 39.0, 34.2 (2×C), 31.0, 29.1,28.7, 26.5 (2×C), 26.3, 26.2, 25.7, 22.2. ESI-HRMS m / z: Calculated value is C. 42 H 46 O5N7 + [M+ H] + , 728.3555; The measured value is 728.3549.

[0188] Synthesis of compound A24: Compound 14d reacted with compound 3f to give compound A24, yield: 51%; 1 HNMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.63 (s, 1H), 8.65 (t, J = 5.6 Hz,1H), 8.55 (d, J = 5.2 Hz, 1H), 8.22 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.71 (dd, J = 8.8, 2.8 Hz, 2H), 7.54 (dd, J = 8.4, 7.2 Hz, 1H), 7.42 (d,J = 5.2 Hz, 1H), 7.18–7.12 (m, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (d, J =7.2 Hz, 1H), 6.59 (d, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.61(t, J = 5.2 Hz, 2H), 3.59–3.54 (m, 6H), 3.47–3.41 (m, 4H), 2.91–2.82 (m, 1H), 2.61–2.51 (m, 2H), 2.46–2.39 (m, 1H), 2.05–1.97 (m, 1H), 1.77 (d, J = 9.4 Hz,4H), 1.72–1.65 (m, 1H), 1.42–1.29 (m, 4H), 1.27–1.20 (m, 1H). 13 C NMR (101MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 167.3, 165.8, 162.7, 160.3, 159.3,146.4, 140.9, 139.1, 138.3, 136.3, 132.1, 127.8 (2×C), 126.8 (2×C), 126.7(2×C), 119.2 (2×C), 117.4, 114.7, 110.7, 109.3, 108.0, 69.7 (2×C), 68.9 (2×C), 48.6, 43.2, 41.7, 39.0, 34.2 (2×C), 31.0, 26.5 (2×C), 25.7, 22.2. ESI-HRMS m / z: Calculated value is C 42H 46 O7N7 + [M + H] + , 760.3453; The measured value is 760.3449.

[0189] Example 7 Synthesis of compounds B1-B4

[0190] The reaction route is as follows:

[0191] ;

[0192] Compounds 14a-14d (0.2 mmol, 1.0 equiv) prepared in Example 4 and compound 5a (0.22 mmol, 1.1 equiv) prepared in Example 2 were dissolved in N,N-dimethylformamide. HATU (0.22 mmol, 1.1 equiv) and DIPEA (1.0 mmol, 5.0 equiv) were added, and the mixture was reacted at room temperature for 10 h. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and subjected to rapid column chromatography to obtain compounds B1-B4.

[0193] The structural formulas of compounds B1-B4 are:

[0194] .

[0195] Synthesis of compound B1: Compound 14a and compound 5a reacted to give compound B1, yield: 56%; 11H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.48 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.22–8.14 (m, 2H), 7.78 (t, J = 6.0 Hz, 1H), 7.69–7.63 (m, 2H), 7.55 (dd, J = 8.4, 7.2 Hz, 1H), 7.53–7.49 (m, 2H), 7.33 (d, J = 4.8 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.95–6.90 (m, 2H), 6.58 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.76–3.71 (m, 4H), 3.71–3.61 (m, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.55–3.50 (m, 4H), 3.45–3.41 (m, 4H), 3.38–3.37 (m, 4H), 3.27–3.22 (m, 2H), 3.07–3.01 (m, 4H), 2.95 (s, 2H), 2.92–2.82 (m, 1H), 2.61–2.52 (m, 2H), 2.45–2.35 (m, 2H), 2.04–1.97 (m, 1H). 13 13C NMR (101MHz, DMSO-d6) δ 172.9, 170.2, 169.1, 169.0, 168.5, 167.4, 162.7, 160.4, 159.3, 146.4, 146.2, 137.9, 137.8, 136.3, 132.9, 132.1, 127.5 (2×C), 127.0 (2×C), 120.3 (2×C), 117.4, 115.7 (2×C), 110.8, 109.3, 107.4, 69.8, 69.6, 69.1, 68.9, 66.2 (2×C), 60.9, 53.0, 52.4, 49.3 (2×C), 48.6, 47.2, 41.7, 41.6, 38.2, 31.0, 22.2. ESI-HRMS m / z: Calculated for C 46 H 52 O9N 10 Na+ [M + Na] + ,911.3811; The measured value is 911.3817.

[0196] Synthesis of compound B2: Compound 14b reacted with compound 5a to give compound B2, yield: 49%; 1 H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.70 (s, 1H), 8.57 (d, J = 5.0 Hz, 1H), 8.23–8.18 (m, 2H), 7.85–7.81 (m, 2H), 7.79 (t, J = 6.0 Hz, 1H), 7.57–7.54 (m,1H), 7.53 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 5.0 Hz, 1H), 7.34–7.29 (m, 2H), 7.10 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.97 (tt, J = 7.5, 1.0Hz, 1H), 6.57 (t, J = 6.0 Hz, 1H), 5.04 (dd, J = 13.0, 5.5 Hz, 1H), 3.73–3.61(m, 2H), 3.59 (t, J = 5.5 Hz, 2H), 3.56–3.50 (m, 4H), 3.46–3.37 (m, 6H), 3.35–3.32 (m, 2H), 3.27–3.22 (m, 2H), 2.96 (s, 2H), 2.92–2.83 (m, 1H), 2.61–2.52 (m, 2H), 2.46–2.37 (s, 2H), 2.04–1.99 (m, 1H). 13C NMR (101 MHz, DMSO-d6)δ 172.9, 170.2, 169.1, 169.0, 168.4, 167.4, 162.8, 160.2, 159.4, 146.4,140.6, 138.1, 137.6, 136.3, 132.1, 128.6 (2×C), 127.6 (2×C), 127.0 (2×C),121.5, 119.0 (2×C), 117.5, 110.8, 109.3, 108.2, 69.8, 69.6, 69.0, 68.9,60.9, 53.9, 52.3, 48.6, 47.1, 41.7, 41.5, 38.2, 31.0, 22.2. ESI-HRMS m / z: Calculated value is C 42 H 46 O8N9 + [M + H] + , 804.3464; The measured value is 804.3458.

[0197] Synthesis of compound B3: Compound 14c reacted with compound 5a to give compound B3, yield: 38%; 11H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.88 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.26–8.21 (m, 2H), 7.97–7.92 (m, 2H), 7.79 (t, J = 6.0 Hz, 1H), 7.70–7.61 (m, 4H), 7.58–7.52 (m, 3H), 7.48–7.39 (m, 3H), 7.34–7.28 (m, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.58 (t, J = 5.8 Hz, 1H), 5.05 (dd, J = 12.8, 5.2 Hz, 1H), 3.74 - 3.61 (m, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.56–3.49 (m, 4H), 3.43 (t, J = 5.6 Hz, 6H), 3.37–3.33 (m, 2H), 3.25 (q, J = 5.6 Hz, 2H), 2.96 (s, 2H), 2.92–2.81 (m, 1H), 2.61–2.52 (m, 2H), 2.46–2.37 (m, 2H), 2.04–1.97 (m, 1H). 13 13C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.1, 169.0, 168.4, 167.3, 162.9, 160.2, 159.4, 146.4, 140.1, 140.0, 138.1, 137.6, 136.3, 133.1, 132.1, 128.9 (2×C), 127.6 (2×C), 127.1 (2×C), 126.8 (2×C), 126.7, 126.1 (2×C), 119.2 (2×C), 117.4, 110.8, 109.3, 108.3, 69.8, 69.6, 69.0, 68.8, 60.9, 52.9, 52.4, 48.6, 47.2, 41.6, 41.5, 38.2, 31.0, 22.2. ESI-HRMS m / z: Calculated for C 48 H 50 O8N9 + [M + H] +, 880.3777; The measured value is 880.3777.

[0198] Synthesis of compound B4: Compound 14d reacted with compound 5a to give compound B4, yield: 37%; 1 H NMR(400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.62 (s, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.22–8.17 (m, 2H), 7.78 (t, J = 5.6 Hz, 1H), 7.74–7.69 (m, 2H), 7.58–7.51 (m,3H), 7.38 (d, J = 5.2 Hz, 1H), 7.17–7.13 (m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.59 (t, J = 5.6 Hz, 1H), 5.05 (dd, J = 12.8, 5.2Hz, 1H), 3.74–3.61 (m, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.56–3.50 (m, 4H), 3.48–3.38 (m, 6H), 3.37–3.31 (m, 2H), 3.25 (q, J = 5.6 Hz, 2H), 2.95 (s, 2H),2.92–2.82 (m, 1H), 2.61–2.52 (m, 2H), 2.47–2.38 (m, 3H), 2.05–1.97 (m, 1H),1.77 (d, J = 9.2 Hz, 4H), 1.72–1.65 (m, 1H), 1.43–1.29 (m, 4H), 1.26–1.20 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 172.9, 170.2, 169.0, 169.0, 168.4, 167.3,162.8, 160.3, 159.3, 146.4, 140.9, 138.3, 138.0, 137.7, 136.3, 132.1, 127.5(2×C), 127.0 (2×C), 126.7 (2×C), 119.1 (2×C), 117.4, 110.8, 109.3, 107.8,69.8, 69.6, 69.1, 68.8, 60.9, 53.1, 52.3, 48.6, 47.1, 43.2, 41.6, 41.5, 38.2, 34.2 (2×C), 31.0, 26.5 (2×C), 25.7, 22.2. ESI-HRMS m / z: Calculated value is C 48 H 56 O8N9 + [M + H] + , 886.4246; The measured value is 886.4240.

[0199] Example 8 Synthesis of compounds C1-C8

[0200] The reaction route is as follows:

[0201] ;

[0202] Compounds 8a-8b (0.2 mmol, 1.0 equiv) prepared in Example 3 were dissolved in acetonitrile, and compounds 16a-16d (0.2 mmol, 1.0 equiv) prepared in Example 5 and glacial acetic acid were added. The mixture was reacted at room temperature for 1 h, and then sodium triacetoxyborohydride (NaBH(OAc)3, 0.6 mmol, 3.0 equiv) was added. The mixture was reacted at room temperature for 16 h. After the reaction was completed, the mixture was concentrated under vacuum to obtain the crude product, and then the white solid compounds C1-C8 were obtained by rapid column chromatography.

[0203] The structural formulas of compounds C1-C8 are as follows:

[0204]

[0205] .

[0206] Synthesis of compound C1: Compound 16a and compound 8a reacted to give compound C1, yield: 30%; 1H NMR(400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.23–8.16 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.71–7.63 (m, 2H), 7.56–7.49 (m, 3H), 7.43 (d, J = 7.2 Hz, 1H), 7.36 (d, J = 5.2 Hz, 1H), 6.97–6.89(m, 2H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.76–3.71(m, 4H), 3.70–3.56 (m, 2H), 3.35–3.29 (s, 2H), 3.09–2.98 (m, 4H), 2.93–2.83(m, 1H), 2.62–2.51 (m, 2H), 2.49–2.41 (m, 2H), 2.40–2.24 (m, 4H), 2.05–1.97 (m, 1H), 1.81–1.73 (m, 2H), 1.56–1.42 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ172.9, 170.0, 168.4, 166.9, 165.4, 162.7, 160.4, 159.3, 156.0, 146.2, 138.0,137.8, 137.1, 133.3, 132.9, 127.5 (2×C), 127.0 (2×C), 120.3 (2×C), 119.8,116.2, 115.7 (2×C), 115.2, 107.4, 68.8, 66.2 (2×C), 57.5, 53.0, 52.4, 49.3(2×C), 48.8, 47.2, 41.5, 31.0, 28.3, 25.8, 23.3, 22.0. ESI-HRMS m / z: Calculated value is C. 43 H 47 O7N8 + [M + H] + , 787.3562; The measured value is 787.3545.

[0207] Synthesis of compound C2: Compound 16a and compound 8b reacted to give compound C2, yield: 36%;1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.50 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.22–8.18 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.69–7.64 (m, 2H), 7.55–7.52 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.36 (d, J= 5.2 Hz, 1H), 6.95–6.91 (m, 2H), 5.08 (dd, J = 12.8, 5.2 Hz, 1H), 4.19 (t, J= 6.4 Hz, 2H), 3.76–3.72 (m, 4H), 3.65–3.59 (m, 2H), 3.34–3.28 (m, 2H), 3.06–3.02 (m, 4H), 2.92–2.83 (m, 1H), 1.37–1.32 (m, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.0, 168.3, 166.9, 165.4,162.7, 160.4, 159.3, 156.1, 146.2, 138.1, 137.7, 137.1, 133.3, 132.9, 127.5(2×C), 127.0 (2×C), 120.3 (2×C), 119.8, 116.2, 115.7 (2×C), 115.2, 107.4,68.8, 66.2 (2×C), 57.6, 53.0, 52.5, 49.3 (2×C), 48.8, 47.2, 41.8, 31.0, 28.4, 26.6, 26.2, 25.3, 22.0. ESI-HRMS m / z: Calculated value is C. 44 H 49 O7N8 + [M + H] + ,801.3719; The measured value is 801.3722.

[0208] Synthesis of compound C3: Compound 16b reacted with compound 8a to give compound C3, yield: 37%; 1 H NMR(400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.74 (br, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.24–8.19 (m, 2H), 7.86–7.82 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.57–7.53 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 6.0, 4.0 Hz, 2H), 7.35–7.29 (m, 2H), 6.97 (tt, J = 7.2, 1.2 Hz, 1H), 5.08 (dd, J = 12.8, 5.2 Hz,1H), 4.19 (t, J = 6.4 Hz, 2H), 3.70–3.56 (m, 2H), 3.36–3.28 (m, 2H), 2.93–2.82 (m, 1H), 2.61–2.51 (m, 2H), 2.46–2.38 (m, 2H), 2.37–2.26 (m, 4H), 2.05–1.98 (m, 1H), 1.80–1.72 (m, 2H), 1.54–1.43 (m, 4H). 13 C NMR (101 MHz, DMSO-d6)δ 172.9, 170.1, 168.4, 166.9, 165.4, 162.9, 160.3, 159.3, 156.1, 140.6,138.2, 137.6, 137.1, 133.3, 128.6 (2×C), 127.6 (2×C), 127.1 (2×C), 121.5,119.8, 119.0 (2×C), 116.2, 115.2, 108.2, 68.8, 57.6, 53.0, 52.5, 48.8, 47.3,41.6, 31.0, 28.3, 25.8, 23.3, 22.1. ESI-HRMS m / z: Calculated value is C 39 H 40 O6N7 + [M + H] + ,702.3035; The measured value is 702.3030.

[0209] Synthesis of compound C4: Compound 16b and compound 8b reacted to give compound C4, yield: 33%; 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.73 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.24–8.20 (m, 2H), 7.86–7.82 (m, 2H), 7.82–7.77 (m, 1H), 7.56–7.53 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.44 (dd, J = 6.0, 4.0 Hz, 2H), 7.35–7.28 (m, 2H), 6.97 (tt, J = 7.2, 1.2 Hz, 1H), 5.07 (dd, J = 12.8, 5.2 Hz, 1H), 4.18 (t, J =6.3 Hz, 2H), 3.65–3.59 (m, 2H), 3.35–3.27 (m, 2H), 2.93–2.83 (m, 1H), 2.62–2.51 (m, 2H), 2.46–2.38 (m, 2H), 2.35–2.25 (m, 4H), 2.05–1.98 (m, 1H), 1.78–1.71 (m, 2H), 1.49–1.41 (m, 4H), 1.37–1.31 (m, 2H). 13 C NMR (101 MHz, DMSO-d6)δ 172.9, 170.1, 168.4, 166.9, 165.4, 162.9, 160.3, 159.4, 156.1, 140.6,138.2, 137.6, 137.1, 133.3, 128.6 (2×C), 127.6 (2×C), 127.1 (2×C), 121.5,119.8, 119.0 (2×C), 116.2, 115.2, 108.2, 68.8, 57.7, 53.0, 52.5, 48.8, 47.3,41.6, 31.0, 28.4, 26.6, 26.2, 25.3, 22.1. ESI-HRMS m / z: Calculated value is C 40 H 42 O6N7 + [M+ H] + , 716.3191; The measured value is 716.3191.

[0210] Synthesis of compound C5: Compound 16c reacted with compound 8a to give compound C5, yield: 35%; 1 H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.86 (s, 1H), 8.61 (d, J = 5.0 Hz, 1H), 8.27–8.22 (m, 2H), 7.97–7.93 (m, 2H), 7.80 (dd, J = 8.5, 7.0 Hz, 1H), 7.68–7.63 (m, 4H), 7.58–7.54 (m, 2H), 7.50 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 5.0Hz, 1H), 7.46–7.42 (m, 3H), 7.33–7.29 (m, 1H), 5.08 (dd, J = 13.0, 5.5 Hz,1H), 4.20 (t, J = 6.5 Hz, 2H), 3.68–3.36 (m, 4H), 2.93–2.83 (m, 1H), 2.62–2.51 (m, 2H), 2.48–2.39 (m, 2H), 2.38–2.28 (m, 4H), 2.05–1.99 (m, 1H), 1.81–1.73 (m, 2H), 1.54–1.42 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.1,168.3, 166.9, 165.4, 162.9, 160.2, 159.4, 156.0, 140.1, 140.0, 138.2, 137.6,137.1, 133.3, 133.1, 128.9 (2×C), 127.6 (2×C), 127.1 (2×C), 126.8 (2×C), 126.7, 126.1 (2×C), 119.8, 119.2 (2×C), 116.2, 115.2, 108.3, 68.8, 57.6,53.1, 52.5, 48.8, 47.2, 41.6, 31.0, 28.3, 25.8, 23.3, 22.0. ESI-HRMS m / z: Calculated value is C 45 H 44 O6N7 + [M + H] + , 778.3348; The measured value is 778.3342.

[0211] Synthesis of compound C6: Compound 16c reacted with compound 8b to give compound C6, yield: 37%; 1 H NMR(400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.89 (s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 8.27–8.23 (m, 2H), 7.97–7.93 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.68–7.63 (m, 4H), 7.58–7.54 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.47 (d, J = 5.2Hz, 1H), 7.46–7.41 (m, 3H), 7.33–7.28 (m, 1H), 5.07 (dd, J = 12.8, 5.2 Hz,1H), 4.18 (t, J = 6.4 Hz, 2H), 3.70–3.54 (m, 2H), 3.36–3.29 (m, 2H), 2.93–2.81 (m, 1H), 2.62–2.51 (m, 2H), 2.46–2.37 (m, 2H), 2.36–2.24 (m, 4H), 2.05–1.97 (m, 1H), 1.79–1.69 (m, 2H), 1.50–1.40 (m, 4H), 1.38–1.29 (m, 2H). 13 C NMR(101 MHz, DMSO-d6) δ 172.9, 170.1, 168.3, 166.9, 165.4, 162.9, 160.2, 159.4,156.1, 140.1, 140.0, 138.2, 137.6, 137.1, 133.3, 133.1, 128.9 (2×C), 127.6(2×C), 127.1 (2×C), 126.8 (2×C), 126.7, 126.1 (2×C), 119.8, 119.2 (2×C),116.2, 115.2, 108.3, 68.8, 57.7, 53.3, 52.5, 48.8, 47.2, 41.7, 31.0, 28.4, 26.6, 26.2, 25.3, 22.1. ESI-HRMS m / z: Calculated value is C 46 H 46 O6N7 +[M + H] + , 792.3504; The measured value is 792.3504.

[0212] Synthesis of compound C7: Compound 16d reacted with compound 8a to give compound C7, yield: 36%; 1 H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.63 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.25–8.18 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.73–7.69 (m, 2H), 7.56–7.52 (m, 2H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.41 (d, J= 5.2 Hz, 1H), 7.18–7.13 (m, 2H), 5.08 (dd, J = 12.8, 5.2 Hz, 1H), 4.20 (t, J= 6.4 Hz, 2H), 3.68–3.57 (m, 2H), 3.34–3.29 (m, 2H), 2.92–2.82 (m, 1H), 2.62–2.52 (m, 2H), 2.47–2.39 (m, 3H), 2.37–2.27 (m, 4H), 2.04–1.98 (m, 1H), 1.78 (d, J = 8.8 Hz, 6H), 1.72–1.66 (m, 1H), 1.54–1.44 (m, 4H), 1.41–1.32 (m, 4H),1.29–1.23 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 172.9, 170.1, 168.4, 166.9,165.4, 162.8, 160.3, 159.3, 156.1, 140.9, 138.3, 138.1, 137.7, 137.1, 133.3,127.5 (2×C), 127.0 (2×C), 126.7 (2×C), 119.8, 119.1 (2×C), 116.2, 115.2,107.9, 68.8, 57.6, 53.1, 52.5, 48.8, 47.3, 43.2, 41.8, 34.2 (2×C), 31.0, 28.3, 26.5; (2×C), 25.8, 25.7, 23.3, 22.1. ESI-HRMS m / z: Calculated value is C. 45 H 50 O6N7 + [M+ H] + , 784.3817; The measured value is 784.3813.

[0213] Synthesis of compound C8: Compound 16d and compound 8b reacted to give compound C8, yield: 38%; 1H NMR (400 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.63 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.23–8.19 (m, 2H), 7.80 (dd, J = 8.4, 7.2 Hz, 1H), 7.73–7.70 (m, 2H), 7.56–7.53 (m, 2H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.41 (d, J= 5.2 Hz, 1H), 7.17–7.14 (m, 2H), 5.08 (dd, J = 12.8, 5.2 Hz, 1H), 4.19 (t, J= 6.4 Hz, 2H), 3.65–3.59 (m, 2H), 3.34–3.27 (m, 2H), 2.93–2.82 (m, 1H), 2.62–2.51 (m, 2H), 2.46–2.38 (m, 3H), 2.34–2.26 (m, 4H), 2.05–1.99 (m, 1H), 1.78 (d, J = 9.6 Hz, 6H), 1.71–1.67 (m, 1H), 1.48–1.42 (m, 4H), 1.39–1.32 (m, 6H),1.29–1.24 (s, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 172.9, 170.0, 168.3, 166.9,165.4, 162.8, 160.3, 159.3, 156.1, 140.9, 138.3, 138.1, 137.7, 137.1, 133.3,127.5 (2×C), 127.0 (2×C), 126.7 (2×C), 119.8, 119.1 (2×C), 116.2, 115.2,107.9, 68.8, 57.6, 53.0. 52.5, 48.8, 47.3, 43.2, 41.6, 34.2 (2×C), 31.0, 28.4, 26.6, 26.5; (2×C), 26.2, 25.7, 25.3, 22.0. ESI-HRMS m / z: Calculated value is C. 46 H 52 O6N7 + [M + H] + , 798.3974; The measured value is 798.3968.

[0214] Performance testing

[0215] 1. Evaluation of the in vitro JAK1 degradation activity of the compound

[0216] Experimental methods:

[0217] (1) Raw264.7 macrophages were selected and placed in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2.

[0218] (2) Drug treatment: After overnight culture and cell adhesion, cells were pre-treated with different compounds (3 μM) for 1 h and stimulated with LPS (1 μg / mL) for 6 h.

[0219] (3) Detection of JAK1 protein degradation: After 6 h of induction with LPS (1 μg / mL), the changes in JAK1 protein levels were detected by Western blot. The results are shown in […]. Figure 1 .

[0220] Figure 1 The results of screening for the degradation activity of each compound on JAK1 protein (A) and the heatmap of JAK1 protein degradation rate (B) are shown. In B, the color represents the degradation rate range, where blue indicates a lower degree of degradation and red indicates a higher degree of degradation. Figure 1 The results showed that compounds A8, C2, C4 and C6 had good degradation effects on JAK1 protein, so these four compounds were selected for subsequent activity detection and screening.

[0221] 2. Cytotoxicity assay of the compound:

[0222] Experimental methods:

[0223] (1) Raw264.7 macrophages were selected and placed in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2.

[0224] (2) Drug treatment: After the cells were seeded and cultured overnight, different compounds (0.47-60 μM) were administered for 24 h.

[0225] (3) Cytotoxicity assay: 24 h after drug administration, cell viability was detected using the CCK-8 assay, with absorbance measured at 450 nm. Cell viability was calculated, and the results are shown in […]. Figure 2 .

[0226] Figure 2 Results of cytotoxicity assays for compounds A8, C2, C4, and C6 are presented as a percentage of the control group. Data are expressed as mean ± standard deviation (n=3), ***P<0.001 compared with the control group. Figure 2The results showed that compounds A8, C2 and C4 had no significant toxicity to RAW264.7 cells (survival rate >90%) within the experimental concentration range.

[0227] 3. Compound DC 50 Detection

[0228] Experimental methods:

[0229] (1) Raw264.7 macrophages were selected and placed in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2.

[0230] (2) Drug treatment: After overnight culture and cell adhesion, cells were pre-treated with different compounds (0.47-60 μM) for 1 h and stimulated with LPS (1 μg / mL) for 6 h.

[0231] (3) Detection of JAK1 protein degradation: After 6 h of induction with LPS (1 μg / mL), the changes in JAK1 protein levels were detected by Western blot. The results are shown in […]. Figure 3 .

[0232] Figure 3 The left image shows the screening results (left) and degradation rate curve (right) of JAK1 protein degradation activity in RAW264.7 cells after treatment with compounds A8 (A), C2 (B), and C4 (C). Figure 3 The results showed that compounds A8, C2, and C4 could all reduce JAK1 levels in a dose-dependent manner. Specifically, the degradation of JAK1 by A8, C2, and C4 was dose-dependent, and their DC... 50 The values ​​were 1.40 μM, 4.0 μM, and 0.85 μM, respectively.

[0233] 4. Detection of the in vitro anti-inflammatory activity of the compound:

[0234] Experimental methods:

[0235] (1) Raw264.7 macrophages were selected and placed in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2.

[0236] (2) Drug treatment: After overnight culture and cell adhesion, different compounds were pre-administered for 1 h and then stimulated with LPS (1 μg / mL) for 24 h.

[0237] (3) Detection of inflammatory factors: After 24 hours of stimulation with LPS (1 μg / mL), the levels of NO (Griess method) and IL-6 and TNF-α (ELISA method) were detected. The results are shown in the figure. Figure 4 .

[0238] Figure 4The anti-inflammatory effects of compounds A8, C2, and C4 in LPS-induced RAW264.7 cells were evaluated. A represents NO production, B represents IL-6 level, and C represents TNF-α level. Cell treatment concentrations ranged from 0.05 to 60 μM, and treatment time was 24 h. Data are expressed as mean ± standard deviation (n=3). *P<0.05, ***P<0.001 compared with the LPS group. Figure 4 The results showed that compounds A8, C2, and C4 exhibited dose-dependent inhibitory effects on NO production, with A8 significantly reducing NO production even at concentrations as low as 0.469 μM. ELISA results further indicated that A8 possessed the strongest anti-inflammatory activity, with an IC50 value for IL-6 and TNF-α. 50 The values ​​were 12.89 μM and 17.17 μM, respectively; the IC50 values ​​of C2 and C4 for IL-6 were... 50 The values ​​were 18.51 μM and 33.44 μM, respectively, with IC50 values ​​for TNF-α. 50 The values ​​were greater than 60 μM and 21.05 μM, respectively.

[0239] 5. Degradation selectivity of compound A8 against JAK isoenzymes:

[0240] Experimental methods:

[0241] (1) Raw264.7 macrophages were selected and placed in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2.

[0242] (2) Drug treatment: After overnight culture and cell adhesion, different compounds were pre-administered for 1 hour and stimulated with LPS (1 μg / mL).

[0243] (3) Detection of JAKs protein degradation: After 6 h of induction with LPS (1 μg / mL), Western blot was used to detect changes in the protein levels of JAK2, JAK3, and TYK2. The results are shown in […]. Figure 5 .

[0244] Figure 5 The left image shows the screening results (left) and degradation rate curves (right) of compound A8 on the degradation activities of JAK2 (A), JAK3 (B), and TYK2 (C). Figure 5 The results showed that A8 exhibited a dose-dependent degradation effect on JAK2, DC 50 The value was 0.92 μM, with no significant effect on JAK3 and TYK2 (DC). 50 The values ​​were all greater than 60 μM, indicating that it has strong selectivity for the JAK1 / 2 subtype.

[0245] 6. Animal efficacy evaluation

[0246] Objective: To evaluate the anti-inflammatory effect of compound A8 in a mouse model of DSS-induced acute colitis.

[0247] Animal grouping: C57BL / 6J mice were randomly divided into blank control group, model group, A8 low-dose group (10 mg / kg), medium-dose group (20 mg / kg), high-dose group (40 mg / kg), and positive control group (5-aminosalicylic acid, 5-ASA).

[0248] Modeling and drug administration: Except for the control group, the other mice were allowed to drink 2.5% DSS solution freely for 7 days, and were also given the drug by gavage daily.

[0249] General indicators: monitor weight, record disease activity index (DAI), colon length, and spleen index.

[0250] Histological and inflammatory analysis: Colon samples were collected for H&E and Ab-PAS staining to assess mucosal structure, goblet cell number, and mucus secretion; IHC was used to detect MPO and iNOS expression; ELISA was used to detect serum and colon tissue levels of IL-6, IL-1β, TNF-α, and IL-10. Results are shown in the table below. Figure 6-8 .

[0251] Figure 6 The results show the ameliorative effect of compound A8 on symptoms of DSS-induced colitis in mice. In the figure, A is a schematic diagram of the animal experimental design, B is the change in body weight during the experiment, C is the disease activity index (DAI) score, D is the colon length, E is the spleen index, F is a representative image of the colon in each group, and G is a representative image of the spleen in each group. The values ​​are expressed as mean ± standard deviation (n=8). Compared with the model group, *P<0.05, **P<0.01, ***P<0.001. Figure 6 The results showed that in the DSS-induced colitis model group, mice exhibited significant weight loss, increased Disease Activity Index (DAI) scores, significantly shortened colon length, and increased spleen index accompanied by splenomegaly, indicating that the acute colitis model was successfully established. Compared with the model group, mice in the A8 treatment group showed varying degrees of improvement in all indicators, including maintaining body weight, reducing DAI scores, restoring colon length, and reducing spleen index. Notably, the medium- and high-dose A8 groups (20 mg / kg and 40 mg / kg) showed more significant efficacy, comparable to or even better than the 5-ASA group (positive control).

[0252] Figure 7The images show the histopathological evaluation results of intestinal tissue in DSS-induced colitis mice. Image A shows H&E and Alcianblue-PAS staining of the colon, and image B shows the immunohistochemical staining results of the colonic tight junction proteins ZO-1 and Occludin. Scale bar = 200 μm. H&E staining results showed significant pathological changes in the colonic tissue of DSS-treated mice, including epithelial desquamation, crypt damage, and extensive inflammatory cell infiltration. After A8 treatment, these pathological damages were alleviated to varying degrees, manifested as more intact mucosal structure and reduced inflammatory infiltration. To further evaluate mucosal barrier function, Alcianblue-PAS (Ab-PAS) staining was used to visualize goblet cells and mucus secretion. The results showed that the number of goblet cells was significantly reduced and mucus staining was weakened in the model group, indicating impaired intestinal barrier function; while the A8 treatment group effectively restored the number of goblet cells and improved mucus secretion. IHC results showed that the brown positive staining of tight junction proteins ZO-1 and Occludin in the colon tissue of the model group was significantly reduced and unevenly distributed, suggesting that DSS-induced colitis leads to damage to tight junction structures and decreased barrier function. However, after A8 treatment, the expression of both ZO-1 and Occludin was enhanced and showed a more continuous distribution, indicating that A8 can restore the expression of tight junction proteins to some extent and improve intestinal barrier integrity.

[0253] Figure 8 The results show the levels of inflammatory factors, complete blood count, and immunohistochemical analysis in DSS-induced colitis mice. A represents serum IL-6, IL-10, IL-1β, and TNF-α levels (ELISA detection); B represents colon tissue IL-6, IL-10, IL-1β, and TNF-α levels (ELISA detection); C represents peripheral blood immune cell subtype analysis, including total white blood cell count (WBCs), lymphocytes, monocytes, and neutrophils (dashed lines indicate the upper and lower limits of normal range); D represents immunohistochemical staining of MPO and iNOS in colon tissue (positive signals are shown in brown). Scale bar = 200 μm. Values ​​are expressed as mean ± standard deviation (n = 8). Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. ELISA results showed that the serum of DSS-induced colitis mice (… Figure 8 A) and colon tissue ( Figure 8In sample B), the levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly elevated, while the level of anti-inflammatory cytokine IL-10 was significantly decreased, indicating a strong systemic and local inflammatory response. Compared with the model group, the A8 treatment group showed varying degrees of anti-inflammatory effects, mainly manifested in decreased levels of pro-inflammatory cytokines and restored IL-10 expression. Peripheral immune cell analysis showed that the number of peripheral blood leukocytes (WBCs), lymphocytes, monocytes, and neutrophils in the model group was significantly increased, suggesting that DSS-induced colonic injury triggered systemic immune activation. After A8 treatment, the number of these cells decreased significantly, suggesting that it has an effective systemic anti-inflammatory effect. Myeloperoxidase (MPO) is a commonly used marker of neutrophil infiltration and acute inflammation; inducible nitric oxide synthase (iNOS) is expressed by activated macrophages and epithelial cells during inflammation and can lead to tissue damage. Immunohistochemical results showed that the expression of MPO and iNOS in the colon tissue of DSS-treated mice was significantly enhanced, indicating increased inflammatory activity; while the A8 treatment group significantly reduced the expression of these two markers, suggesting that it can effectively alleviate the inflammatory response.

[0254] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A PROTAC degrader, characterized in that, which is a compound as depicted in A8 or a pharmaceutically acceptable salt thereof, .

2. A pharmaceutical composition, characterized by, The PROTAC degrader of claim 1 and a pharmaceutically acceptable adjuvant.

3. Use of the PROTAC degrader of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for preventing and / or treating an inflammatory disease, a respiratory disease, a skin disease or an immune system disease.

4. Use according to claim 3, characterized in that, The inflammatory disease is an inflammatory skin disease; the respiratory disease is chronic obstructive pulmonary disease or asthma; the skin disease is psoriasis or atopic dermatitis; the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

5. Use of the PROTAC degrader of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for preventing and / or treating colitis.

6. Use of the PROTAC degrader of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for preventing and / or treating a JAK2-mediated disease.

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