An hdac / pi3k dual-target inhibitor based on imidazopyridazine parent nucleus and application thereof

Abstract

The application discloses an HDAC / PI3K dual-target inhibitor based on an imidazopyridazine parent nucleus and application thereof, and belongs to the technical field of medicinal chemistry. In view of the technical defects that existing HDAC and PI3K single-target anti-tumor drugs are prone to drug resistance, the effect on solid tumors is limited, and existing dual-target inhibitors have insufficient target activity and poor drugability, the application provides a dual-target inhibitor shown in a structural general formula (I), including pharmaceutically acceptable salts, hydrates, solvates, crystal forms, metabolites or prodrugs thereof. The compound has good inhibitory activity on HDAC1 and PI3K alpha, has excellent in-vivo and in-vitro anti-tumor activity, excellent in-vivo tumor inhibition effect and good liver and kidney safety, has a simple and mild synthesis route, is easy to be industrially produced, and can be used for preparing anti-tumor drugs, and provides a candidate scheme with novel structure and excellent drugability for clinical treatment of malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to an HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core and its application. Background Technology

[0002] Histone deacetylases (HDACs) are key enzymes regulating cellular epigenetic modifications. They catalyze the removal of acetyl groups from lysine residues of histones, compressing chromatin structure and negatively regulating gene transcription. During tumorigenesis and development, HDACs are often overexpressed or abnormally activated, leading to tumor suppressor gene silencing and imbalances in cell cycle and apoptosis programming, making them a core driver of malignant tumor progression. HDAC inhibitors can reactivate tumor suppressor gene expression by increasing the acetylation levels of histones and non-histone substrates, inducing tumor cell cycle arrest and apoptosis. Several HDAC inhibitors have been approved for marketing, primarily for the treatment of hematologic malignancies such as T-cell lymphoma. However, their efficacy as monotherapy in solid tumors is limited, and they easily induce drug resistance in tumor cells, significantly limiting their clinical application.

[0003] PI3K belongs to the lipid kinase family and catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), thereby activating the downstream AKT / mTOR cascade signaling and precisely regulating the growth, metabolism, survival, invasion, and metastasis of tumor cells. This pathway is frequently and aberrantly activated in human solid tumors, mostly due to gain-of-function mutations in PIK3CA, deletion of the PTEN tumor suppressor gene, or persistent activation of upstream receptor tyrosine kinases. It is a core pathway for tumor proliferation, treatment resistance, and drug resistance. Currently, several subtype-selective or pan-PI3K inhibitors have been approved for the treatment of malignant tumors such as breast cancer and lymphoma. However, single PI3K pathway inhibition easily triggers feedback compensatory activation of tumor cells, leading to insufficient efficacy, rapid drug resistance, and difficulty in overcoming the treatment bottleneck of solid tumors.

[0004] In summary, although HDAC and PI3K inhibitors act on different cellular regulatory pathways, they both face the common dilemma of limited efficacy as monotherapy and easy induction of tumor drug resistance in the clinical treatment of solid tumors. The core cause of this dilemma lies in the multi-level cross-regulation and compensatory activation mechanisms between the two pathways. Existing research has confirmed that HDAC can regulate the activity and stability of key proteins in the PI3K pathway by modifying their acetylation state; while the PI3K / AKT pathway can indirectly regulate chromatin structure and the expression of tumor-related genes by phosphorylating substrates such as histone modifying enzymes and transcription factors. This close interactive regulation is the core adaptive mechanism for tumor cells to escape single-target drug inhibition, making simultaneous targeting of both HDAC and PI3K pathways an important direction for anti-tumor drug development: simultaneous dual-target inhibition can block the compensatory escape pathway of tumor cells, producing a synergistic anti-tumor effect, while showing significant advantages in inducing tumor cell apoptosis, inhibiting angiogenesis, and regulating the tumor immune microenvironment, and is expected to overcome the drug resistance defects of single-target drugs and improve the treatment effect of solid tumors.

[0005] Currently, several HDAC / PI3K dual-target inhibitors have entered the research and development stage. For example, Chinese patent CN117736192 A discloses a class of PI3K / HDAC dual inhibitors. These compounds exhibit strong inhibitory activity and subtype selectivity for PI3Kδ and HDAC6, and some compounds show certain anti-tumor cell proliferation effects. However, overall, the research and development of HDAC / PI3K dual-target inhibitors is still in its early stages. Existing compounds mostly focus on PI3Kδ and HDAC6 subtypes associated with hematological malignancies, and their inhibitory activity against PI3Kα and HDAC1 targets, which are highly expressed in solid tumors and closely related to tumor malignant progression, is insufficient to meet the clinical treatment needs of solid tumors. At the same time, the core structures of existing dual-target inhibitors are relatively simple, and the space for structural optimization is limited. They generally face the problem of difficulty in simultaneously achieving target activity, in vivo druggability, safety, and industrial production feasibility, and cannot provide clinical candidates that are highly effective, low in toxicity, and easy to industrialize.

[0006] Therefore, developing a novel HDAC / PI3K dual-target inhibitor that possesses potent concurrent inhibitory activity against solid tumor-related PI3Kα / HDAC1 targets, while also exhibiting excellent in vitro and in vivo antitumor efficacy, good in vivo safety, and feasibility for industrial production, is a pressing technical problem in this field and has significant scientific research value and clinical application prospects. Summary of the Invention

[0007] This invention aims to address the shortcomings of existing technologies by providing an HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core and its application. This type of compound can achieve simultaneous inhibition and synergistic anti-tumor effects of both HDAC and PI3K targets, possessing excellent in vitro and in vivo anti-tumor activity, good in vivo safety, and feasibility for industrial production. It can be widely used in the preparation of anti-tumor drugs, providing a new high-activity candidate for the clinical treatment of malignant tumors.

[0008] This invention is achieved as follows: This invention provides an HDAC / PI3K dual-target inhibitor based on an imidazopyridazine core, wherein the dual-target inhibitor is a compound represented by general structural formula (I), its pharmaceutically acceptable hydrate, solvate, crystal form, metabolite, or prodrug:

[0009] ;

[0010] Wherein, R1 is a linking group connecting the triazole ring and R2, selected from... , , , , , or n = 1~10;

[0011] R2 is a zinc ion-binding group, selected from... .

[0012] Preferably, the HDAC / PI3K dual-target inhibitor is selected from any of the following compounds:

[0013] .

[0014] Furthermore, the HDAC / PI3K dual-target inhibitor has inhibitory activity against both HDAC1 and PI3Kα; some preferred compounds have potent inhibitory effects on both targets and can produce a synergistic anti-tumor effect through the cross-regulation of the PI3K and HDAC pathways.

[0015] The present invention also provides the application of the above-mentioned HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core in the preparation of a drug for the treatment and / or prevention of tumors, inflammation or autoimmune diseases.

[0016] Furthermore, the tumors include, but are not limited to, colorectal cancer, breast cancer, cholangiocarcinoma, non-small cell lung cancer, osteosarcoma, cholangiocarcinoma, endometrial cancer, prostate cancer, glioblastoma multiforme, and lymphoma.

[0017] This application also protects a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core, and a pharmaceutically acceptable carrier or excipient.

[0018] Furthermore, the pharmaceutical composition further includes at least one other therapeutically active ingredient; in some preferred embodiments, the other therapeutically active ingredient is an antitumor chemotherapy drug.

[0019] The dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form, suitable for any pharmaceutically acceptable route of administration, including but not limited to oral (including sublingual, rectal, nasal, topical (including transdermal, oral mucosal, and inhalation), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal injection); the dosage form can be prepared using conventional methods known in the field of pharmaceutics.

[0020] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for the treatment and / or prevention of tumors, inflammations or autoimmune diseases.

[0021] Beneficial effects: 1. This application designs and provides a novel HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core. Through reasonable pharmacophore splicing and structural optimization, it achieves simultaneous and potent inhibition of both HDAC and PI3K targets. At the same time, it can generate a synergistic anti-tumor effect through the cross-regulation of the two pathways, providing a new idea for the structural design of dual-target anti-tumor drugs. 2. The compounds disclosed in this application exhibit excellent inhibitory effects on the proliferation of human colon cancer HCT116 cells, effectively blocking the proliferation process of tumor cells. Among them, several preferred compounds in the hydroxamic acid series have half-maximal inhibitory concentrations (IC50) of HCT116 cell proliferation that are all below 1 μM, and their activities are all superior to the positive control drug SAHA. All compounds in the o-phenylenediamine amide series show strong anti-tumor activity at the nanomolar level. Among them, ZWJ-A-4d, which has the best activity, has an IC50 concentration as low as 1 nM for HCT116 cells, and its activity far exceeds that of the positive control drug. At the same time, ZWJ-A-4d also has excellent broad-spectrum anti-tumor potential, and has a strong inhibitory effect on various clinically common solid tumor cells such as breast cancer and non-small cell lung cancer, and can be used for the treatment of various solid tumors. 3. The preferred compound ZWJ-A-4d disclosed in this application also exhibits excellent anti-tumor efficacy in in vivo models, with a tumor-suppressing effect significantly superior to capecitabine, a first-line chemotherapy drug for colorectal cancer. Furthermore, this compound can be used in combination with capecitabine to further enhance anti-tumor efficacy, with a tumor-suppressing effect significantly superior to either drug alone, demonstrating a clear synergistic effect. This type of compound can not only be used alone for the treatment of solid tumors, but can also be used in combination with conventional chemotherapy drugs to enhance efficacy, providing a new candidate for combination therapy in the clinical treatment of solid tumors. 4. The compound disclosed in this application has good in vivo safety and tolerability at effective therapeutic doses, and does not increase the risk of toxicity when used in combination with capecitabine; moreover, the compound does not cause significant damage to the liver and kidneys of mice at therapeutic doses, has no obvious target organ toxicity, and has excellent potential for drug development. 5. The synthetic route of the target compound in this application is reasonably designed. The starting materials used are all commercially available conventional chemical raw materials. They can be obtained by conventional organic synthesis reactions such as amide condensation and click cycloaddition. The reaction conditions are mild, the operation is simple, the product post-processing and purification methods are mature, the yield is stable, and it is easy to realize industrial-scale production, thus having good industrial application value. Attached Figure Description

[0022] Figure 1 The ¹H-NMR spectrum of compound ZWJ-5b; Figure 2 The ¹H-NMR spectrum of compound ZWJ-5c; Figure 3 The ¹H-NMR spectrum of compound ZWJ-5d; Figure 4 The ¹H-NMR spectrum of compound ZWJ-A-4d; Figure 5 The in vivo antitumor effect of compound ZWJ-A-4d in the HCT116 human colon cancer xenograft model is shown in Figure 1. A is a physical image of the tumor tissue excised from each group after drug administration; B is the tumor volume growth curve of each group during drug administration; C is the mouse body weight change curve of each group during drug administration; and D is a scatter plot of tumor weight statistics of each group after drug administration. Figure 6 The hepatotoxicity and nephrotoxicity of compound ZWJ-A-4d and its combination regimens were evaluated, where A represents serum alanine aminotransferase (ALT) activity; B represents serum aspartate aminotransferase (AST) activity; C represents serum urea (UREA) concentration; and D represents serum uric acid (UA) concentration. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0024] Example 1: Synthesis of compounds ZWJ-5a ~ 5e

[0025] ; When n=4, the target compound is ZWJ-5a, and the specific synthetic steps of this compound are as follows:

[0026] 1) Methyl 5-bromopentanoate (2.00 g, 10.26 mmol), acetone / water mixed solvent (3:1, 20 mL, v / v), and sodium azide (1.33 g, 20.46 mmol) were added to a reaction flask, and the mixture was heated to 55 °C and refluxed with stirring for 8 h. After the reaction was completed, the acetone was removed by concentration under reduced pressure. 60 mL of water was added to the residue, and the mixture was extracted with dichloromethane (DCM). The organic phases were combined, washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 1.28 g of a transparent oily substance, which was ZWJ-1a, with a yield of 79.4%. The product did not require further purification and was directly added to the next reaction step.

[0027] 2) Add a methanol / water mixture (5:4, 45 mL, v / v) to crude ZWJ-1a, and add KOH (0.85 g, 15.15 mmol) in portions. Stir at room temperature for 12 h. After the reaction is complete, concentrate under reduced pressure to remove methanol. Add 20 mL of water to the residue, wash the aqueous phase with DCM, adjust the pH of the aqueous phase to 1-2 with 1 M HCl, extract with ethyl acetate, combine the organic phases, dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 1.00 g of transparent oil, namely ZWJ-2a, with a yield of 85.8%. The product does not require further purification and can be used directly in the next reaction.

[0028] 3) Under nitrogen protection, intermediate ZWJ-2a (1.00 g, 6.99 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 10 mL), cooled to -15 °C, and N-methylmorpholine (NMM, 2.12 g, 20.97 mmol) was added. The mixture was stirred for 5 min, followed by the addition of isobutyl chloroformate (IBCF, 0.95 g, 6.96 mmol), and stirring for 15 min. Subsequently, a THF solution (5 mL) of O-triphenylmethylhydroxylamine (NH2-O-Trityl, 8.70 g, 31.8 mmol) was added, and the mixture was stirred for another 20 min before being brought back to room temperature. The reaction was continued with stirring for 2 h. After the reaction was complete, 1 M HCl was added with stirring to adjust the pH of the system to neutral. The organic phase was separated by extraction with ethyl acetate and water, washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude product. The crude product was recrystallized from isopropanol to obtain 1.40 g of white solid, which is ZWJ-3a, with a yield of 50.1%.

[0029] 4) At room temperature and under nitrogen protection, intermediate ZWJ-3a (110.00 mg, 0.27 mmol) and intermediate I-6 (100.00 mg, 0.23 mmol; for the preparation of compound I-6, see pages 11-12 of Li C, Han Y, Wang Z, et al. Function-oriented synthesis of Imidazo[1,2-a]pyrazine and Imidazo[1,2-b]pyridazine derivatives as potent PI3K / mTOR dual inhibitors[J]. European Journal of Medicinal Chemistry, 2023, 247: 115030. DOI: 10.1016 / j.ejmech.2022.115030) were dissolved in dimethyl sulfoxide (DMSO, 9 mL), and CuI (22.00 mg, 0.12 mmol) was added. The mixture was stirred in the dark for 12 h. After the reaction was completed, 100 mL of DCM was added for dilution. The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (volume ratio 1:4), pure water, and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the product was filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane / methanol) to obtain 70.00 mg of pale yellow solid, which is ZWJ-4a, with a yield of 36.7%.

[0030] 5) Under ice bath conditions, intermediate ZWJ-4a (70 mg, 0.083 mmol) and triisopropylsilane (0.5 mL) were dissolved in DCM (5 mL), and trifluoroacetic acid (TFA, 0.5 mL) was added dropwise. The mixture was stirred for 60 min. After the reaction was completed, the solvent and excess trifluoroacetic acid were removed by concentration under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol) to give 6.00 mg of off-white solid, which was the target compound ZWJ-5a, with a yield of 12.0%.

[0031] The synthesis methods of compounds ZWJ-5b (n=5), ZWJ-5c (n=6), ZWJ-5d (n=7), and ZWJ-5e (n=8) are completely consistent with those of ZWJ-5a (n=4), except that the starting material methyl 5-bromopentanoate is replaced with methyl 5-bromohexanoate, methyl 5-bromoheptanoate, methyl 5-bromooctanoate, and methyl 5-bromononanoate, which have matching alkyl chain lengths. All other reaction conditions, feed equivalents, post-treatment, and purification methods remain unchanged. The yields of ZWJ-5b to 5e are 30.4%, 51.1%, 27.1%, and 18.0%, respectively.

[0032] The structural verification data for ZWJ-5a are as follows:

[0033] 1 H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 10.36 (s, 1H), 8.85 (d, J=15.5 Hz, 2H), 8.54 (s, 1H), 8.37 (d, J=9.5 Hz, 2H), 7.93 (d, J=9.6 Hz, 1H),7.79 (q, J=8.2 Hz, 1H), 7.58 (t, J=9.9 Hz, 1H), 7.21 (t, J=8.6 Hz, 1H), 4.55(t, J=6.7 Hz, 2H), 3.77 (s, 3H), 2.00 (t, J=6.8 Hz, 2H), 1.97–1.82 (m, 2H), 1.51 (dt, J=14.2, 7.5 Hz, 2H), 1.24 (s, 1H).

[0034] HRMS (ESI) m / z calcd. for C 25 H 23 F2N9O5S (M+H) + 600.1589 was found; 600.1584 was also found.

[0035] The structural confirmation data for ZWJ-5b are as follows (its ¹H-NMR spectrum is shown below). Figure 1 ):

[0036] 1 H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.90 (s, 1H), 8.72 (s, 1H), 8.48 (s, 1H), 8.32 – 8.14 (m, 2H), 7.79 (d, J=9.5 Hz, 2H), 7.20 (t, J=9.6 Hz,1H), 7.00 (t, J = 8.6 Hz, 1H), 4.56 (t, J=6.7 Hz, 2H), 3.85 (s, 3H), 2.28 (s,2H), 2.15 (s, 1H), 2.07–1.81 (m, 4H), 1.49 (dt, J=14.8, 8.0 Hz, 2H).

[0037] HRMS (ESI) m / z calcd. for C 26 H 25 F2N9O5S (M+H) + 614.1746, found 614.1738.

[0038] The structural confirmation data for ZWJ-5c are as follows (its ¹H-NMR spectrum is shown below). Figure 2 ):

[0039] 1 H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 10.30 (s, 1H), 8.83 (d, J=12.1 Hz, 2H), 8.64 (s, 1H), 8.57 (s, 1H), 8.36 (d, J=9.4 Hz, 1H), 7.92 (d, J=9.5 Hz, 1H), 7.80 (q, J=7.9 Hz, 1H), 7.57 (t, J=9.7 Hz, 1H), 7.22 (t, J=8.2Hz, 1H), 4.52 (t, J=6.9 Hz, 2H), 3.78 (s, 3H), 2.07–1.77 (m, 4H), 1.53–1.35(m, 2H), 1.27(s, 4H).

[0040] HRMS (ESI) m / z calcd. for C 27 H 27 F2N9O5S (M+H) +628.1902, found 628.1903.

[0041] The structural confirmation data for ZWJ-5d are as follows (its ¹H-NMR spectrum is shown below). Figure 3 ):

[0042] 1 H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.90 (s, 1H), 8.71 (s, 1H), 8.55 (s, 1H), 8.40 (s, 1H), 8.28 (d, J=10.6 Hz, 2H), 7.83 (dd, , 1.49–1.38 (m, 2H), 1.25 (s, 6H).

[0043] HRMS (ESI) m / z calcd. for C 28 H 29 F2N9O5S (M+H) + 642.2059, found 642.2096.

[0044] The structural verification data for ZWJ-5e are as follows:

[0045] 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 10.31 (s, 1H), 8.82 (d, J=10.3 Hz, 2H), 8.58 (s, 1H), 8.33 (s, 2H), 7.93 (d, J=9.0 Hz, 1H), 7.80 (s,1H), 7.57 (s, 1H), 7.23 (s, 1H), 4.53 (s, 2H), 3.78 (s, 3H), 1.91 (s, 4H),1.41 (d, J=11.6 Hz, 2H), 1.23 (d, J=29.7 Hz, 8H).

[0046] HRMS (ESI) m / z calcd. for C 29 H 31 F2N9O5S (M+H)+ 656.2215, found 656.2240.

[0047] Example 2: Synthesis of compound ZWJ-B-3

[0048] ;

[0049] The specific synthesis steps are as follows:

[0050] Ethyl 4-(bromomethyl)benzoate (2.20 g, 9.05 mmol), DMF (15 mL), and sodium azide (0.59 g, 9.08 mmol) were added to the reaction flask, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, 50 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a clear, oily crude product. The crude product was purified by silica gel column chromatography to obtain 2.00 g of intermediate ZWJ-B-1, which was used directly in the next step.

[0051] At room temperature and under nitrogen protection, intermediates ZWJ-B-1 (107.0 mg, 0.52 mmol) and I-6 (277.00 mg, 0.63 mmol) were dissolved in DMSO (5 mL), and CuI (50.00 mg, 0.26 mmol) was added. The mixture was stirred for 12 h. After the reaction was complete, 50 mL of DCM was added for dilution. The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 50 mL), pure water, and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol) to obtain 210.00 mg of intermediate ZWJ-B-2, with a yield of 62.1%.

[0052] ZWJ-B-2 (210.00 mg, 0.32 mmol) was dissolved in a dichloromethane-methanol mixture (1:1, 6 mL, v / v) and cooled to 0°C in an ice bath. A 10 wt% aqueous solution of hydroxylamine (320.00 mg, containing 9.70 mmol of pure hydroxylamine) was added, and the mixture was stirred at 0°C for 10 min. NaOH (130.00 mg, 3.25 mmol) was then added, and the mixture was stirred at 0°C for 30 min. The ice bath was removed, and stirring continued at room temperature for 3 h. The reaction mixture was dried under reduced pressure. The residue was dispersed in 2 mL of deionized water, and the pH was adjusted to 8 by adding 1 mol / L hydrochloric acid dropwise in an ice bath. A large amount of yellow solid precipitated. This solid was filtered, dried, and yielded 106.00 mg of compound ZWJ-B-3, with a yield of 51.5%.

[0053] The structural verification data for ZWJ-B-3 is as follows:

[0054] 1 H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 10.54 (s, 1H), 8.98 (s,1H), 8.86 (s, 1H), 8.53 (s, 1H), 7.99 (s, 1H), 7.89–7.62 (m, 4H), 7.55 (t, J=9.9 Hz, 1H), 7.45 (d, J=7.4 Hz, 3H), 7.19 (t, J=8.6 Hz, 1H), 5.85 (s, 2H), 3.89 (s, 3H).

[0055] HRMS (ESI) m / z calcd. for C 28 H 21 F2N9O5S (M+H) + 634.1433, found 634.1378.

[0056] Example 3: Synthesis of compound ZWJ-B-5b

[0057] ;

[0058] The specific synthesis steps are as follows:

[0059] 5-Azidevallic acid (300.00 mg, 2.10 mmol) was dissolved in DMF (1 mL). Under nitrogen atmosphere and an ice bath, 2 mL of DMF solution of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 2.00 g, 5.25 mmol), 2 mL of DMF solution of N,N-diisopropylethylamine (DIPEA, 1.49 g, 11.52 mmol), ethyl 4-(aminomethyl)benzoate hydrochloride (540.00 mg, 2.50 mmol), and 2 mL of DMF solution were added sequentially. After stirring at 0 °C for 5 min, DIPEA (0.61 g, 4.72 mmol) was added, and the reaction mixture was heated to ambient temperature and stirred for 16 h. Add 50 mL of water to the reaction solution, extract with ethyl acetate (3 × 50 mL), combine the organic phases, wash with pure water and saturated sodium chloride solution, dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude product is purified by silica gel column chromatography (EA / PE gradient: 10-80%) to give 600.00 mg of white solid ZWJ-B-1b, yield 94.1%.

[0060] A methanol / water mixture (5:4, 45 mL, v / v) was added to ZWJ-B-1b (600.00 mg, 2.0 mmol), followed by the addition of KOH (224.0 mg, 4.0 mmol) in portions. The mixture was stirred at room temperature for 8 h. After the reaction was complete, the methanol was removed by concentration under reduced pressure. 60 mL of water was added to the residue, and the aqueous phase was washed with dichloromethane (2 × 20 mL). The pH of the aqueous phase was adjusted to 1–2 with 1 mol / L hydrochloric acid, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 450.00 mg of a clear oil, ZWJ-B-2b, in 82.6% yield. The product did not require further purification and was directly used in the next reaction step.

[0061] Under nitrogen protection, intermediate ZWJ-B-2b (100.00 mg, 0.40 mmol) was dissolved in THF (3 mL), cooled to -15°C, and NMM (109.9 mg, 1.09 mmol) was added. After stirring for 5 min, IBCF (49.5 mg, 0.36 mmol) was slowly added dropwise, and the reaction was stirred at -15°C for 15 min. Subsequently, a THF solution of O-triphenylmethylhydroxylamine (99.7 mg, 0.36 mmol) (2 mL) was added, and the mixture was kept at this temperature and stirred for 20 min. Then, the temperature was slowly restored to room temperature, and the reaction was continued with stirring for 12 h. After the reaction was completed, 1 mol / L hydrochloric acid was added dropwise under stirring to adjust the pH of the system to neutral. Ethyl acetate (50 mL) was added for extraction, and the organic phase was separated. The organic phase was washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was recrystallized from isopropanol to give 40.00 mg of white solid ZWJ-B-3b, with a yield of 20.7%.

[0062] Under nitrogen protection at room temperature, intermediates ZWJ-B-3b (40.0 mg, 0.08 mmol) and I-6 (27.6 mg, 0.06 mmol) were dissolved in DMSO (5 mL), and CuI (6 mg, 0.03 mmol) was added. The mixture was stirred in the dark for 12 h. After the reaction was complete, DCM (10 mL) was added for dilution. The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 50 mL), pure water, and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradients from 100:1 to 40:1) to give 25.0 mg of the yellow product ZWJ-B-4b, with a yield of 34.2%.

[0063] Under ice bath conditions, intermediate ZWJ-B-4b (25.0 mg, 0.08 mmol) and triisopropylsilane (0.5 mL) were dissolved in DCM (5 mL), and TFA (0.5 mL) was slowly added dropwise. The reaction was maintained at this temperature with stirring for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and the crude product was purified by reversed-phase column chromatography (elution system: 0.1% TFA aqueous solution / acetonitrile gradient elution) to give 6 mg of off-white solid ZWJ-B-5b, with a yield of 31.9%.

[0064] The structural verification data for ZWJ-B-5b is as follows:

[0065] 1 H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 10.53 (s, 1H), 8.85 (d, J=10.7 Hz, 2H), 8.54 (s, 1H), 8.37 (d, J=10.0 Hz, 2H), 7.95 (d, J=9.3 Hz, 1H), 7.79 (q, J=8.2 Hz, 1H), 7.67 (d, J=7.8 Hz, 2H), 7.57 (t, J=9.9 Hz, 1H), 7.33–7.23 (m, 2H), 7.20 (t, J=8.3 Hz, 1H), 4.56 (t, J=6.5 Hz, 2H), 4.27 (d, J=5.7Hz, 2H), 3.77 (s, 3H), 2.21 (t, J=7.2 Hz, 2H), 2.02–1.87 (m, 2H), 1.55 (dt, J=14.1, 6.4 Hz, 2H), 1.33–1.11 (m, 1H).

[0066] HRMS (ESI) m / z calcd. for C 33 H 30 F2N 10 O6S (M+H) + 733.2117, found733.2096.

[0067] Example 4: Synthesis of compound ZWJ-B-7c

[0068] ;

[0069] The specific synthesis steps are as follows:

[0070] 4.07 mL (41.14 mmol) of n-butylamine was added to a 20 mL THF solution of ethyl 4-(bromomethyl)benzoate (2.00 g, 8.23 ​​mmol), and the mixture was stirred at room temperature for 3 h. After the reaction was complete, 20 mL of chloroform and 20 mL of saturated sodium bicarbonate solution were added to the reaction mixture. The mixture was extracted by separation, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 2.00 g of a brownish-red oily substance, ZWJ-B-1c. This product was directly used in the next reaction step.

[0071] 5-Bromovalerate (543.00 mg, 3.00 mmol) was dissolved in a dichloromethane / N,N-dimethylformamide mixed solvent (DCM / DMF, 1:1, 10 mL, v / v) and cooled to 0°C in an ice bath. O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 1.45 g, 4.50 mmol) and DIPEA (0.78 mL, 4.50 mmol) were dissolved in DMF (1.5 mL) and slowly added dropwise to the 5-bromovalerate solution at 0°C. After the addition was complete, the mixture was stirred at 0°C for 15 min. Subsequently, a DMF solution (1.5 mL) of ZWJ-B-1c (706.00 mg, 3.00 mmol) was added to the reaction system at 0°C, and the mixture was slowly restored to room temperature and stirred for 3.5 h. After the reaction was complete, 30 mL of pure water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with pure water and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:10) to give 0.90 g of a yellow oil ZWJ-B-2c, with a yield of 75.5%.

[0072] An acetone-water mixture (3:1, 20 mL, v / v) and sodium azide (0.28 g, 4.34 mmol) were added to ZWJ-B-2c (0.90 g, 2.17 mmol), and the mixture was heated to 50 °C and stirred for 8 h. After the reaction was complete, the acetone was removed by concentration under reduced pressure. 30 mL of water was added to the residue, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 0.40 g of ZWJ-B-3c, with a yield of 51.0%.

[0073] A methanol-water mixture (5:4, 9 mL, v / v) was added to ZWJ-B-3c (260.0 mg, 0.7 mmol), followed by the addition of KOH (81.0 mg, 1.44 mmol) in portions. The mixture was stirred at room temperature for 8 h. After the reaction was complete, the methanol was removed by concentration under reduced pressure. 30 mL of pure water was added to the residue, and the aqueous phase was washed with dichloromethane (2 × 15 mL) to remove neutral impurities. The pH of the aqueous phase was adjusted to 1–2 with 1 mol / L hydrochloric acid under ice bath conditions. The phase was extracted with ethyl acetate (3 × 20 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 210.0 mg of a transparent oil, ZWJ-B-4c, in 87.6% yield. The product did not require further purification and was directly added to the next reaction step.

[0074] Under nitrogen protection, intermediate ZWJ-B-4c (210.0 mg, 0.6 mmol) was dissolved in THF (3 mL), cooled to -15°C and kept at that temperature. NMM (210.0 mg, 2.08 mmol) was added, and the mixture was stirred for 5 min. Then, IBCF (82.0 mg, 0.6 mmol) was slowly added dropwise, and the mixture was stirred at -15°C for 15 min. Subsequently, a THF solution of O-triphenylmethylhydroxylamine (174.0 mg, 0.63 mmol) (2 mL) was added, and the mixture was stirred and kept at that temperature for 20 min. The temperature was then slowly lowered to room temperature, and the reaction was continued for 12 h. After the reaction was completed, 1 mol / L hydrochloric acid was added dropwise under stirring to adjust the pH of the system to neutral. Ethyl acetate (30 mL) was added for extraction and separation. The organic phase was separated and washed successively with pure water and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was recrystallized from isopropanol to give 177.0 mg of white solid ZWJ-B-5c, with a yield of 47.5%.

[0075] Under nitrogen protection and in the dark, intermediates ZWJ-B-5c (48.0 mg, 0.08 mmol) and I-6 (30.0 mg, 0.07 mmol) were dissolved in DMSO (5 mL) at room temperature, under nitrogen protection, and with the mixture stirred for 12 h. After the reaction was complete, the reaction solution was diluted with dichloromethane (30 mL). The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 30 mL), pure water, and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradients from 100:1 to 40:1) to obtain 60 mg of ZWJ-B-6c, with a yield of 53.5%.

[0076] Under ice bath conditions, intermediate ZWJ-B-6c (60.0 mg, 0.08 mmol) and triisopropylsilane (0.5 mL) were dissolved in DCM (5 mL), and TFA (0.5 mL) was slowly added dropwise. The reaction was maintained at this temperature with stirring for 1 h. After the reaction was completed, the mixture was concentrated under reduced pressure, and the crude product was purified by reversed-phase column chromatography (elution system: 0.1% TFA aqueous solution / acetonitrile gradient elution) to give 20.0 mg of the target compound ZWJ-B-7c, with a yield of 43.6%.

[0077] The structural confirmation data for ZWJ-B-7c is as follows:

[0078] 1 H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 10.53 (s, 1H), 8.84 (d, J=16.2 Hz, 2H), 8.56 (d, J=12.4 Hz, 1H), 8.38 (s, 2H), 7.97 (dd, J=9.3, 4.5 Hz,1H), 7.78 (p, J=7.2, 6.8 Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.66 (d, J=7.8 Hz,1H), 7.62–7.50 (m, 1H), 7.22 (d, J=7.4 Hz,3H), 4.64–4.54 (m, 2H), 4.51 (d, J=12.8 Hz, 2H), 3.77 (s,3H), 2.38 (dt, J=43.3, 7.2 Hz, 2H), 1.97 (dt, J=34.8,8.1 Hz, 2H), 1.67–1.47 (m,2H), 1.37 (tt, J=16.2, 7.4 Hz, 2H), 1.14 (h, J=8.0Hz, 2H), 0.77 (q, J=7.8 Hz, 2H).

[0079] HRMS (ESI) m / z calcd. for C 37 H 38 F2N 10 O6S (M+H) + 789.2743, found 789.2758.

[0080] Example 5: Synthesis of compound ZWJ-B-6d

[0081] ;

[0082] The specific synthesis steps are as follows:

[0083] N-bromosuccinimide (NBS, 217.00 mg, 1.22 mmol) and benzoyl peroxide (5.90 mg, 0.02 mmol) were added to an anhydrous CCl4 (10 mL) solution of methyl 4-ethylbenzoate (200.00 mg, 1.22 mmol). The mixture was stirred and refluxed at 80 °C for 8 h. The mixture was filtered and concentrated, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give 250.00 mg of colorless oil ZWJ-B-1d, with a yield of 84.8%.

[0084] ZWJ-B-1d (250.00 mg, 1.03 mmol), acetone-water mixed solvent (3:1, 4 mL, v / v), and sodium azide (100.00 mg, 1.54 mmol) were added to the reaction flask, and the mixture was heated to 55 °C and refluxed with stirring for 8 h. After the reaction was completed, the acetone was removed by concentration under reduced pressure. 15 mL of pure water was added to the residue, and the mixture was extracted with dichloromethane. The organic phases were combined, washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 15:1) to give 190.00 mg of clear oil ZWJ-B-2d, with a yield of 89.7%.

[0085] Under nitrogen protection at room temperature, intermediates ZWJ-B-2d (130.00 mg, 0.63 mmol) and I-6 (140.00 mg, 0.32 mmol) were dissolved in DMSO (5 mL), and CuI (30.00 mg, 0.16 mmol) was added. The mixture was stirred for 12 h. After the reaction was complete, the reaction solution was diluted with DCM (50 mL). The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 50 mL), pure water, and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the solution was filtered and concentrated. The solution was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradient elution from 100:1 to 50:1) to obtain 189.00 mg of pale yellow solid ZWJ-B-3d, with a yield of 71.3%.

[0086] ZWJ-B-3d (50.00 mg, 0.07 mmol) was added to a reaction flask, followed by a methanol-water mixture (5:4, 9 mL, v / v). KOH (8.70 mg, 0.16 mmol) was added in portions, and the mixture was stirred at room temperature for 8 h. After the reaction was complete, the methanol was removed by concentration under reduced pressure. 15 mL of pure water was added to the residue, and the aqueous phase was washed with dichloromethane (2 × 15 mL) to remove neutral impurities. The pH of the aqueous phase was adjusted to 1-2 with 1 mol / L hydrochloric acid under ice bath conditions. The phase was extracted with ethyl acetate (3 × 15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 50.00 mg of a transparent oil, ZWJ-B-4d, with a yield of nearly 100%. The product could be used directly in the next reaction without further purification.

[0087] ZWJ-B-4d (50.00 mg, 0.07 mmol) was dissolved in DCM (5 mL), and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (14.00 mg, 0.12 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 346.00 mg, 1.80 mmol), and 1-hydroxybenzotriazole (HOBT, 21.00 mg, 0.16 mmol) were added sequentially. Finally, DIPEA (51.00 mg, 0.40 mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, DCM (20 mL) and saturated sodium bicarbonate aqueous solution (20 mL) were added to the reaction solution for extraction and separation. The organic phase was separated and washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative thin-layer chromatography (PTLC) (developing solvent: dichloromethane / methanol = 20:1) to give 20.00 mg of white solid ZWJ-B-5d, with a yield of 34.6%.

[0088] ZWJ-B-5d (20.00 mg, 0.025 mmol) was dissolved in methanol (3 mL), and 1 mol / L hydrochloric acid-methanol solution (0.14 mL, 0.14 mmol) was added. The mixture was stirred at room temperature for 1 h. After the reaction was complete, the methanol was removed by concentration under reduced pressure. The residue was extracted with ethyl acetate (15 mL) and saturated sodium bicarbonate aqueous solution (15 mL), and the organic phase was separated. The organic phase was washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was recrystallized from dichloromethane / n-hexane to give 6.00 mg of the target compound ZWJ-B-6d as a white solid, with a yield of 33.1%.

[0089] The structural confirmation data for ZWJ-B-6d are as follows:

[0090] 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 10.52 (s, 1H), 9.03 (s,1H), 8.90 (s, 1H), 8.80 (s, 1H), 8.53 (s, 1H), 8.35 (d, J=12.3 Hz, 2H), 7.92(d, J=9.9 Hz, 1H), 7.73 (d, J=7.9 Hz, 2H), 7.55 (d, J=9.5 Hz, 1H), 7.49 (d, J=7.4 Hz, 2H), 7.23–7.11 (m, 1H), 6.25–6.15 (m, 1H), 5.76 (s, 1H), 3.76 (s,3H), 2.05 (d, J=6.9 Hz, 3H).

[0091] HRMS (ESI) m / z calcd. for C 29 H 23 F2N9O5S (M+H) + 648.1589, found 648.1532.

[0092] Example 6: Synthesis of compound ZWJ-O-3a / 3b / 3c / 3d / 3e / 3f / 3g

[0093] ;

[0094] The specific synthesis steps of ZWJ-O-3a are as follows:

[0095] Under nitrogen protection, 2-(2-azidoethoxy)acetic acid (100.00 mg, 0.69 mmol) was dissolved in THF (3 mL), cooled to -15°C and kept at that temperature, NMM (209.00 mg, 2.07 mmol) was added, and the mixture was stirred for 5 min. Then, IBCF (94.00 mg, 0.69 mmol) was slowly added dropwise, and the mixture was stirred at -15°C for 15 min. Subsequently, a THF (3 mL) solution of O-triphenylmethylhydroxylamine (190.00 mg, 0.69 mmol) was added, and the mixture was stirred and kept at that temperature for 20 min. The mixture was then slowly restored to room temperature, and the reaction was continued with stirring for 12 h. After the reaction was completed, 1 mol / L hydrochloric acid was added dropwise under stirring to adjust the pH of the system to neutral. Ethyl acetate (30 mL) and pure water (20 mL) were added for extraction and separation. The organic phase was separated and washed successively with pure water and saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the product was filtered and concentrated under reduced pressure to obtain the crude product, yielding 200.00 mg of white solid ZWJ-O-1a, with a yield of 72.1%.

[0096] Under nitrogen protection at room temperature, intermediates ZWJ-O-1a (200.00 mg, 0.50 mmol) and I-6 (127.60 mg, 0.21 mmol) were dissolved in DMSO (5 mL), and CuI (24.00 mg, 0.12 mmol) was added. The mixture was stirred for 12 h. After the reaction was complete, the reaction solution was diluted with DCM (30 mL). The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 30 mL), pure water, and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradient elution of 100:1 to 50:1) to give 100.00 mg ZWJ-O-2a, with a yield of 82.6%.

[0097] Under ice bath conditions, intermediate ZWJ-O-2a (100.00 mg, 0.11 mmol) and triisopropylsilane (0.5 mL) were dissolved in DCM (5 mL), and TFA (0.5 mL) was added dropwise. The mixture was stirred and kept at this temperature for 60 min. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase silica gel column chromatography (elution system: 0.1% TFA aqueous solution / acetonitrile gradient elution) to give 5.00 mg of the target compound ZWJ-O-3a as a yellow solid, with a yield of 7.0%.

[0098] The preparation process of the PEG acetic acid series (ZWJ-O-1a / 1c / 1e, ZWJ-O-2a / 2c / 2e, ZWJ-O-3a / 3c / 3e, n=1~3) compounds: The synthesis operation and reaction type of ZWJ-O-1c (n=2) and ZWJ-O-1e (n=3) are completely consistent with those of ZWJ-O-1a, except that the starting material 2-(2-azidoethoxy)acetic acid is replaced with equimolar amounts of azido-PEG2-acetic acid and azido-PEG3-acetic acid with matching oligoethylene glycol chain lengths; the reaction conditions, feed equivalents, post-treatment and purification methods of the subsequent Huisgen-Click cycloaddition reaction and triphenylmethyl protecting group removal reaction are consistent with the corresponding synthesis steps of ZWJ-O-2a and ZWJ-O-3a. The yields of ZWJ-O-3c / 3e are 45.8% and 36.3%, respectively.

[0099] The preparation process of PEG propionic acid series compounds (ZWJ-O-1b / 1d / 1f / 1g, ZWJ-O-2b / 2d / 2f / 2g, ZWJ-O-3b / 3d / 3f / 3g, n=1~4) is as follows: The synthesis operation and reaction type of ZWJ-O-1b (n=1), ZWJ-O-1d (n=2), ZWJ-O-1f (n=3), and ZWJ-O-1g (n=4) are completely consistent with those of ZWJ-O-1a, except that the starting material 2-(2-azidoethoxy) is changed. Acetic acid was replaced with equimolar amounts of azido-PEG1-propionic acid, azido-PEG2-propionic acid, azido-PEG3-propionic acid, and azido-PEG4-propionic acid, matching the chain length of the oligoethylene glycol. The reaction conditions, feed equivalences, post-treatment, and purification methods for the subsequent Huisgen-Click cycloaddition reaction and the removal of the triphenylmethyl protecting group were consistent with the corresponding synthesis steps of ZWJ-O-2a and ZWJ-O-3a. The yields of ZWJ-O-3b / 3d / 3f / 3g were 34.8%, 100%, 71.1%, and 6.2%, respectively.

[0100] The structural confirmation data for ZWJ-O-3a are as follows:

[0101] 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.88 (d, J=6.1 Hz, 2H), 8.49 (s, 1H), 8.43 (d, J=3.5 Hz, 1H), 8.40 (s, 1H), 8.01 (d, J=9.5 Hz, 1H), 7.80 (q, J=8.2 Hz, 1H), 7.58 (t, J=9.5 Hz, 1H), 7.21 (t, J=8.3 Hz, 1H), 4.81–4.75 (m, 2H), 3.99 (t, J=4.6 Hz, 2H), 3.91 (s, 2H), 3.76 (s, 3H).

[0102] HRMS (ESI) m / z calcd. for C 24 H 21 F2N9O6S (M+H) + 602.1382 was found; 602.1392 was also found.

[0103] The structural verification data for ZWJ-O-3b are as follows:

[0104] 11H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 10.36 (s, 1H), 8.88 (s,1H), 8.82 (s, 1H), 8.50 (s, 1H), 8.40 (d, J=9.1 Hz, 2H), 7.99 (d, J=9.4 Hz,1H), 7.79 (q, J=8.0 Hz, 1H), 7.58 (t, J=9.6 Hz, 1H), 7.21 (t, J=8.1 Hz, 1H),4.71 (s, 2H), 3.91 (t, J=4.5 Hz, 2H), 3.75 (s, 3H), 3.66 (t, J=6.2 Hz, 2H),2.17 (t, J=6.2 Hz, 2H).

[0105] HRMS (ESI) m / z calcd. for C 25 H 23 F2N9O6S (M+H) + 616.1538, found 616.1591.

[0106] The confirmation data of ZWJ-O-3c are as follows:

[0107] 1 1H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 10.41 (s, 1H), 8.86 (s,1H), 8.83 (s, 1H), 8.49 (s, 1H), 8.39 (d, J=9.9 Hz, 2H), 7.97 (d, J=9.3 Hz,1H), 7.79 (q, J=7.8 Hz, 1H), 7.58 (t, J=9.1 Hz, 1H), 7.21 (t, J=8.1 Hz, 1H),4.73 (d, J=5.5 Hz, 2H), 3.96 (s, 3H), 3.76 (d, J=9.6 Hz, 5H), 3.65–3.58 (m,2H), 3.52 (t, J=4.5 Hz, 2H).

[0108] HRMS (ESI) m / z calcd. for C 26 H 25 F2N9O7S (M+H) + 646.1644, found 646.1689.

[0109] The verification data for the ZWJ-O-3d structure are as follows:

[0110] 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 10.36 (s, 1H), 8.87 (s,1H), 8.82 (s, 1H), 8.50 (s, 1H), 8.40 (d, J=10.7 Hz, 2H), 7.98 (d, J=9.5 Hz,1H), 7.80 (q, J=8.1 Hz, 1H), 7.59 (t, J=9.5 Hz, 1H), 7.22 (t, J=8.0 Hz, 1H), 4.73 (t, J=4.4 Hz, 2H), 4.01–3.88 (m, 2H), 3.77 (s, 3H), 3.59–3.53 (m, 2H), 3.51 (t, J=6.1 Hz, 2H), 3.48–3.40 (m, 2H), 2.10 (t, J=6.0 Hz, 2H).

[0111] HRMS (ESI) m / z calcd. for C 27 H 27 F2N9O7S (M+H) + 660.1800, found 660.1790.

[0112] The structural verification data for ZWJ-O-3e are as follows:

[0113] 1 H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 10.41 (s, 1H), 8.85 (d, J=15.6 Hz, 2H), 8.50 (s, 1H), 8.41 (d, J=10.8 Hz, 2H), 7.99 (d, J=9.4 Hz, 1H),7.80 (q, J=7.8 Hz, 1H), 7.58 (t, J=9.4 Hz, 1H), 7.21 (t, J=8.1 Hz, 1H), 4.74(t, J=4.6 Hz, 2H), 4.01–3.92 (m, 2H), 3.76 (s, 5H), 3.62–3.55 (m, 2H), 3.51–3.46 (m, 2H), 3.43 (s, 4H).

[0114] HRMS (ESI) m / z calcd. for C 28 H 29 F2N9O8S (M+H) + 690.1901, found 690.2048.

[0115] The verification data for the ZWJ-O-3f structure are as follows:

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.88 (s, 1H), 8.84 (s, 1H), 8.51 (s, 2H), 8.45 (d, J=9.4 Hz, 1H), 8.07 (d, J=9.4 Hz, 1H), 7.80 (q, J=8.0Hz, 1H), 7.58 (t, J=9.3 Hz, 1H), 7.24–7.19 (m, 1H), 4.80–4.66 (m, 2H), 4.03–3.88 (m, 2H), 3.77 (s, 3H), 3.63–3.54 (m, 2H), 3.51–3.42 (m, 4H), 3.40–3.35(m, 2H), 3.35–3.29 (m, 2H), 2.11 (t, J= 6.0 Hz, 2H).

[0117] HRMS (ESI) m / z calcd. for C 29 H 31 F2N9O8S (M+H) + 704.2063, found 704.2034.

[0118] The structural confirmation data for ZWJ-O-3g are as follows:

[0119] 1H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.86 (s, 1H), 8.82 (s, 1H), 8.50 (s, 1H), 8.40 (d, J=8.9 Hz, 2H), 7.97 (d, J=9.3 Hz, 1H), 7.79 (q, J=7.7Hz, 1H), 7.58 (t, J=9.6 Hz, 1H), 7.22 (q, J=9.9, 7.9 Hz, 1H), 4.77–4.69 (m,2H), 3.76 (s, 3H), 3.59–3.55 (m, 2H), 3.52 (t, J=5.9 Hz, 2H), 3.48–3.44 (m,2H), 3.34 (d, J=11.9 Hz, 10H), 2.15 (t, J=5.7 Hz, 2H).

[0120] HRMS (ESI) m / z calcd. for C 31 H 35 F2N9O9S (M+H) + 748.2325, found 748.2290.

[0121] Example 7: Synthesis of compound ZWJ-A-4a / 4b / 4c / 4d / 4e / 4j / 4k

[0122] ;

[0123] The specific synthesis steps of ZWJ-A-1a are as follows:

[0124] 1.30 g (6.24 mmol) of tert-butyl(2-aminophenyl)carbamate was added to a 5 mL solution of DCM containing 5-bromopentanoic acid (1.00 g, 5.52 mmol), triethylamine (Et3N, 1.12 g, 11.07 mmol), and HATU (3.20 g, 8.35 mmol). The mixture was stirred at room temperature for 16 h. After the reaction was complete, the reaction solution was diluted with DCM, and the organic phase was washed successively with pure water and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with a gradient of petroleum ether / ethyl acetate = 5:1 to 2:1) to give 2.00 g of pale yellow solid ZWJ-A-1a, with a yield of 93.8%.

[0125] ZWJ-A-1a (100.00 mg, 0.27 mmol) was dissolved in DMF (5 mL), and sodium azide (26.00 mg, 0.41 mmol) was added with stirring. The mixture was heated to 45 °C and stirred for 8 h. After the reaction was complete, DCM and a large amount of pure water were added for extraction and separation. The organic phase was separated and washed successively with pure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 100.00 mg of colorless, transparent oil ZWJ-A-2a, with a yield of nearly 100%. The product did not require further purification and was directly added to the next reaction.

[0126] Under nitrogen protection at room temperature, intermediates ZWJ-A-2a (100.00 mg, 0.30 mmol) and I-6 (66.00 mg, 0.13 mmol) were dissolved in DMSO (5 mL), and CuI (14 mg, 0.08 mmol) was added. The mixture was stirred for 12 h. After the reaction was complete, the reaction solution was diluted with DCM (30 mL). The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 30 mL), pure water, and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradient elution of 100:1 to 50:1) to obtain 60.00 mg of product ZWJ-A-3a, with a yield of 52.5%.

[0127] Under ice bath conditions, intermediate ZWJ-A-3a (60.00 mg, 0.076 mmol) was dissolved in DCM (5 mL), and TFA (0.30 mL, 3.81 mmol) was added dropwise. The mixture was stirred and kept at this temperature for 60 min. After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase silica gel column chromatography (elution system: 0.1% TFA aqueous solution / acetonitrile gradient elution) to give 8.00 mg of the target compound ZWJ-A-4a as a yellow solid, with a yield of 15.3%.

[0128] The synthesis method of ZWJ-A-1b / 1c / 1d / 1e / 1j / 1k (n=5~10) is completely consistent with that of ZWJ-A-1a, except that the starting material 5-bromopentanoic acid is replaced with a bromocarboxylic acid of the corresponding alkyl chain length. The subsequent azido substitution reaction, Click cyclization reaction, Boc deprotection reaction reaction conditions, feed equivalents, post-treatment and purification methods are all consistent with those of ZWJ-A-2a, ZWJ-A-3a and ZWJ-A-4a, finally yielding compounds ZWJ-A-4b, ZWJ-A-4c, ZWJ-A-4d, ZWJ-A-4e, ZWJ-A-4j and ZWJ-A-4k, with final yields of 25.5%, 56.9%, 45.7%, 97.5%, 78.1% and 84.9%, respectively.

[0129] The structural verification data for ZWJ-A-4a are as follows:

[0130] 1 H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.86 (s, 1H), 8.75 (s, 1H), 8.54 (s, 1H), 8.34 (d, J=10.8 Hz, 2H), 7.89 (d, J=9.1 Hz, 1H), 7.83 – 7.75(m, 1H), 7.50 (s, 1H), 7.19–7.07 (m, 2H), 6.86 (t, J=8.2 Hz, 1H), 6.68 (d, J=8.4 Hz, 1H), 6.49 (t, J=8.0 Hz, 1H), 4.60 (t, J=6.9 Hz, 2H), 3.78 (s, 3H), 3.17 (d, J=4.9 Hz, 2H), 2.37 (t, J=7.1 Hz, 2H), 2.04–1.95 (m, 2H), 1.65–1.55 (m, 2H).

[0131] HRMS (ESI) m / z calcd. for C 31 H 28 F2N 10 O4S (M+H) + 675.2062, found 675.2089.

[0132] The structural verification data for ZWJ-A-4b is as follows:

[0133] 11H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 8.85 (d, J = 8.8 Hz, 1H), 8.56 (s, 1H), 8.37 (d, J = 13.0 Hz, 2H), 7.94 (d, J = 9.9 Hz, 1H), 7.84–7.74 (m, 1H), 7.57 (t, J = 10.9 Hz, 1H), 7.24–7.16 (m, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.03–6.93 (m, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 4.60–4.52 (m, 2H), 3.77 (s, 3H), 2.54 (s, 2H), 2.39–2.24 (m, 2H), 2.04–1.92 (m, 2H), 1.72–1.54 (m, 2H), 1.44–1.28 (m, 2H).

[0134] HRMS (ESI) m / z calcd. for C 32 H 30 F2N 10 O4S (M + H) + 689.2219, found 689.2194.

[0135] The structure confirmation data of ZWJ-A-4c are as follows:

[0136] 1 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.87 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 8.31 (s, 2H), 7.88 (d, J = 9.6 Hz, 1H), 7.81 (q, J = 7.6 Hz, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 6.87 (t, J = 7.2 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 6.51 (t, J = 7.1 Hz, 1H), 4.57 (s, 2H), 3.80 (s, 3H), 2.51 (s, 2H), 2.29 (t, J = 6.8 Hz, 2H), 1.94 (d, J = 9.6 Hz, 2H), 1.64–1.45 (m, 2H), 1.34 (s, 4H).

[0137] HRMS (ESI) m / z calcd. for C 33 H 32 F2N 10 O4S (M+H) + 703.2375, found 703.2372

[0138] The structural confirmation data for ZWJ-A-4d are as follows (its ¹H-NMR spectrum is shown below). Figure 4 ):

[0139] 1 H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.85 (s, 1H), 8.71 (s, 1H), 8.58 (s, 1H), 8.33 (d, J=8.2 Hz, 2H), 7.90 (d, J=9.6 Hz, 1H), 7.81 (q, J=7.8Hz, 1H), 7.50 (t, J=9.7 Hz, 1H), 7.23–7.09 (m, 2H), 6.87 (t, J=7.4 Hz, 1H), 6.70 (d, J=7.9 Hz, 1H), 6.52 (t, J=7.5 Hz, 1H), 4.55 (t, J=6.6 Hz, 2H), 3.79(s, 3H), 2.51 (s, 2H), 2.28 (t, J=7.1 Hz, 2H), 2.05–1.83 (m, 2H), 1.63–1.48(m, 2H), 1.27 (d, J=33.0 Hz, 6H).

[0140] HRMS (ESI) m / z calcd. for C 34 H 34 F2N 10 O4S (M+H) + 717.2532, found 717.2581.

[0141] The structural verification data for ZWJ-A-4e are as follows:

[0142] 1H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.82 (d, J=5.8 Hz, 2H), 8.58 (s, 1H), 8.39–8.29 (m, 2H), 7.92 (d, J=9.6 Hz, 1H), 7.80 (q, J=8.3 Hz, 1H),7.55 (t, J=9.9 Hz, 1H), 7.20 (t, J=8.5 Hz, 1H), 7.12 (d, J=7.7 Hz, 1H), 6.87(t, J=7.5 Hz, 1H), 6.69 (d, J=8.0 Hz, 1H), 6.51 (t, J=7.5 Hz, 1H), 4.53 (t, J=6.9 Hz, 2H), 4.10 (q, J=5.2 Hz, 1H), 3.78 (s, 3H), 3.17 (d, J=4.8 Hz, 2H), 2.25 (t, J=7.2 Hz, 2H), 2.00–1.86 (m, 2H), 1.59–1.46 (m, 2H), 1.27 (d, J=13.6Hz, 7H).

[0143] HRMS (ESI) m / z calcd. for C 35 H 36 F2N 10 O4S (M+H) + 731.2688, found 731.2682.

[0144] The confirmation data for the ZWJ-A-4j structure are as follows:

[0145] 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 8.90–8.76 (m, 2H), 8.59 (s,1H), 8.38 (s, 1H), 8.04–7.94 (m, 1H), 7.88–7.76 (m, 1H), 7.57 (t, J=9.4 Hz,1H), 7.38 (d, J=50.0 Hz, 1H), 7.23 (t, J=8.0 Hz, 1H), 7.16 (d, J=7.5 Hz, 1H), 6.89 (t, J=7.1 Hz, 1H), 6.72 (d, J=7.6 Hz, 1H), 6.54 (t, J=7.1 Hz, 1H), 4.54(t, J=5.7 Hz, 2H), 3.79 (s, 3H), 3.58 (s, 1H), 3.19 (s, 1H), 2.27 (t, J=6.6Hz, 2H), 1.93 (s, 2H), 1.53 (s, 2H), 1.26 (d, J=21.7 Hz, 10H).

[0146] HRMS (ESI) m / z calcd. for C 36 H 38 F2N 10 O4S (M+H) + 745.2845, found 745.2832.

[0147] The ZWJ-A-4k structure verification data are as follows:

[0148] 1H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.85 (s, 1H), 8.59 (d, J=19.5 Hz, 2H), 8.35 (s, 2H), 7.91 (s, 1H), 7.81 (s, 1H), 7.48–7.38 (m, 1H),7.21–7.05 (m, 2H), 6.94–6.78 (m, 1H), 6.70 (d, J=6.7 Hz, 1H), 6.57–6.43 (m,1H), 4.61–4.46 (m, 2H), 3.80 (s, 3H), 3.16 (s, 2H), 3.00 (s, 1H), 2.55 (s,1H), 2.26 (s, 2H), 1.90 (s, 2H), 1.65 (s, 1H), 1.59–1.46 (m, 2H), 1.19 (d, J=15.1 Hz, 10H).

[0149] HRMS (ESI) m / z calcd. for C 37 H 40 F2N 10 O4S (M+H) + 759.3001, found 759.2996.

[0150] Example 8: Synthesis of compound ZWJ-A-5f

[0151] ;

[0152] The specific synthesis steps are as follows:

[0153] ZWJ-B-1 (the intermediate obtained in Example 2, 50.00 mg, 0.26 mmol), methanol-water mixed solvent (5:4, 9 mL, v / v), and KOH (29.37 mg, 0.52 mmol) were added in portions to a reaction flask, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, 30 mL of pure water was added to the reaction solution, and the aqueous phase was washed with dichloromethane to remove neutral impurities. The pH of the aqueous phase was adjusted to 2-3 with 1 mol / L hydrochloric acid, extracted with ethyl acetate (3 × 15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 50.00 mg of a transparent oil ZWJ-A-2f. The product did not require further purification and was directly added to the next reaction step.

[0154] tert-butyl(2-aminophenyl)carbamate (65.00 mg, 0.31 mmol) was added to a 5 mL solution of DCM containing ZWJ-A-2f (50.00 mg, 0.28 mmol), Et3N (57.15 mg, 0.56 mmol), and HATU (0.16 g, 0.42 mmol). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the solution was diluted with DCM, and the organic phase was washed successively with pure water and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1) to give 66.00 mg of pale yellow solid ZWJ-A-3f, with a yield of 63.7%.

[0155] Under nitrogen protection at room temperature, intermediates ZWJ-A-3f (66.00 mg, 0.18 mmol) and I-6 (40.00 mg, 0.09 mmol) were dissolved in DMSO (5 mL), and CuI (9.00 mg, 0.05 mmol) was added. The mixture was stirred for 12 h. After the reaction was complete, the reaction solution was diluted with DCM (30 mL). The organic phase was washed successively with ammonia-saturated ammonium chloride aqueous solution (1:4, 30 mL), pure water, and saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with dichloromethane / methanol gradient elution of 100:1 to 50:1) to give 50.00 mg of ZWJ-A-4f, with a yield of 68.2%.

[0156] Under ice bath conditions, intermediate ZWJ-A-4f (50.00 mg, 0.062 mmol) was dissolved in DCM (5 mL), and TFA (1 mL) was added dropwise. The mixture was stirred and kept at this temperature for 60 min. After the reaction was complete, the organic solvent and excess TFA were removed by concentration under reduced pressure. The residue was purified by preparative thin-layer chromatography (PTLC) (developing solvent: dichloromethane / methanol = 20:1) to give 8.00 mg of the target compound ZWJ-A-5f as a yellow solid, with a yield of 45.7%.

[0157] The structural verification data for ZWJ-A-5f is as follows:

[0158] 1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 9.10 (s, 1H), 8.55 (s, 1H), 8.43 (s, 1H), 8.35–8.23 (m, 2H), 7.95 (d, J=7.9 Hz, 2H), 7.85 (d, J=9.6 Hz, 1H), 7.76 (q, J=7.9 Hz, 1H), 7.54 (d, J=7.9 Hz, 2H), 7.28 (t, J=9.9 Hz, 1H), 7.14 (d, J=7.9 Hz, 1H), 7.04 (t, J=8.8 Hz, 1H), 6.96 (t, J=7.7 Hz, 1H), 6.76(d, J=8.0 Hz, 1H), 6.58 (t, J=7.6 Hz, 1H), 5.92 (s, 2H), 3.82 (s, 3H), 2.51(s, 2H).

[0159] HRMS (ESI) m / z calcd. for C 34 H 26 F2N 10 O4S (M+H) + 709.1906, found 709.1914.

[0160] Example 9: Synthesis of compound ZWJ-A-6g

[0161] ;

[0162] The synthesis method of the target compound ZWJ-A-6g is generally consistent with that of ZWJ-A-5f, except that the substrate ZWJ-A-1f in the hydrolysis step of the ZWJ-A-5f synthesis is replaced with an equimolar amount of ZWJ-B-2d (the intermediate obtained in Example 5). The reaction conditions, feed equivalents, post-treatment and purification methods of the remaining hydrolysis reaction, amide condensation reaction, Huisgen-Click cycloaddition reaction and Boc protecting group removal reaction are consistent with the synthesis process of ZWJ-A-5f. Finally, the target compound ZWJ-A-6g was obtained with a yield of 33%.

[0163] The structural confirmation data for ZWJ-A-6g are as follows:

[0164] 1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 9.02 (s, 1H), 8.50 (s, 1H), 8.29 (s, 1H), 8.23 ​​(d, J=9.6 Hz, 1H), 8.18 (s, 1H), 7.92 (d, J=7.9 Hz, 2H), 7.78 (dd, J=14.0, 8.6 Hz, 2H), 7.61 (d, J=7.9 Hz, 2H), 7.16 (dd, J=13.3, 8.8Hz, 2H), 6.95 (t, J=7.7 Hz, 2H), 6.76 (d, J=7.9 Hz, 1H), 6.57 (t, J=7.4 Hz,1H), 6.33 (q, J=7.1 Hz, 1H), 4.88 (s, 2H), 3.85 (s, 3H), 2.08 (d, J=6.9 Hz,3H).

[0165] HRMS (ESI) m / z calcd. for C 35 H 28 F2N 10 O4S (M+H) + 723.2062, found 723.2119.

[0166] Example 10: Synthesis of compound ZWJ-A-5h

[0167] ;

[0168] The synthesis method of the target compound ZWJ-A-5h is generally consistent with that of ZWJ-A-5f, except that the reaction substrate ZWJ-A-2f in the amide condensation step of the ZWJ-A-5f synthesis is replaced with an equimolar amount of ZWJ-B-2b (the intermediate obtained in Example 3). The reaction conditions, feed equivalents, post-treatment and purification methods of the remaining amide condensation reaction, Huisgen-Click cycloaddition reaction and Boc protecting group removal reaction are consistent with the synthesis process of ZWJ-A-5f. Finally, the target compound ZWJ-A-5h was obtained with a yield of 56%.

[0169] The structural confirmation data for ZWJ-A-5h are as follows:

[0170] 1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.93 (s, 1H), 8.50 (s, 1H), 8.41 (t, J=5.5 Hz, 1H), 8.27 (s, 1H), 8.22 (d, J=9.6 Hz, 1H), 8.17 (s, 1H),7.90 (d, J=7.6 Hz, 2H), 7.77 (dd, J=14.6, 8.5 Hz, 2H), 7.33 (d, J=7.8 Hz,2H), 7.18 (t, J=8.4 Hz, 2H), 6.96 (d, J=7.8 Hz, 2H), 6.78 (d, J=7.9 Hz, 1H),6.59 (t, J=7.1 Hz, 1H), 4.91 (s, 2H), 4.61 (t, J=6.4 Hz, 2H), 4.30 (d, J=5.4Hz, 2H), 3.85 (s, 3H), 2.23 (t, J=6.9 Hz, 2H), 2.02–1.89 (m, 2H), 1.58–1.48(m, 2H).

[0171] HRMS (ESI) m / z calcd. for C 39 H 35 F2N 11 O5S (M+H) + 808.2590, found 808.2600.

[0172] Example 11: Synthesis of compound ZWJ-A-7i

[0173] ;

[0174] The synthesis method of the target compound ZWJ-A-7i was consistent with that of ZWJ-A-5h, except that the initial raw material was replaced by an equimolar amount of ZWJ-B-4c (the intermediate obtained in Example 4). The reaction conditions, feed equivalence, post-treatment and purification methods of the remaining amide condensation reaction, Huisgen-Click cycloaddition reaction and Boc protecting group removal reaction were consistent with those of ZWJ-A-5h. Finally, the target compound ZWJ-A-7i was obtained with a yield of 86%.

[0175] The structural verification data for ZWJ-A-7i is as follows:

[0176] 1H NMR (400 MHz, DMSO-d6) δ 9.65 (d, J=11.3 Hz, 1H), 8.85 (d, J=9.7Hz, 2H), 8.56 (d, J=12.5 Hz, 1H), 8.36 (s, 2H), 7.92 (dd, J=15.8, 6.5 Hz,3H), 7.85–7.74 (m, 1H), 7.61–7.49 (m, 1H), 7.27 (t, J=8.9 Hz, 2H), 7.22–7.09(m, 2H), 6.97 (t, J=7.0 Hz, 1H), 6.78 (d, J=7.6 Hz, 1H), 6.60 (t, J=7.0 Hz,1H), 4.57 (d, J=20.8 Hz, 4H), 3.77 (s, 3H), 3.21–3.14 (m, 2H), 2.40 (d, J=41.2 Hz, 2H), 2.10–1.88 (m, 2H), 1.59 (s, 2H), 1.40 (dt, J=15.4, 7.4 Hz, 2H), 1.28–1.04 (m, 3H), 0.78 (q, J=8.1, 7.4 Hz, 3H).

[0177] HRMS (ESI) m / z calcd. for C 43 H 43 F2N 11 O5S (M+H) + 864.3216, found 864.3223.

[0178] Related activity tests

[0179] 1. In vitro kinase inhibitory activity test

[0180] This experiment used in vitro enzyme activity assays to evaluate the inhibitory activity of the compounds on two target sites, as follows:

[0181] PI3Kα kinase inhibitory activity assay: The KinaseGlo chemiluminescence assay was used. The test compound was dissolved in DMSO and serially diluted to the set concentration using a semi-logarithmic gradient method. The solution was then added to a white opaque 96-well plate. PI3Kα kinase solution and reaction buffer were added, mixed, and incubated at room temperature in the dark for 1 h. After incubation, the kinase reaction was terminated by adding KinaseGlo reagent. After standing at room temperature in the dark for 10 min, the chemiluminescence value of each well was detected using a microplate reader, and the kinase inhibition rate of the compound at the set concentration was calculated.

[0182] HDAC1 enzyme inhibitory activity assay: The assay was performed using a fluorescent substrate method. The test compound was dissolved in DMSO and serially diluted to the set concentration using a semi-logarithmic gradient method, then added to a 96-well plate. HDAC1 enzyme solution and fluorescent substrate were then added, mixed, and incubated at 37°C for 1 h. After incubation, stop solution was added, and the plate was allowed to stand at room temperature for 10 min. The fluorescence intensity of each well was then measured using a microplate reader, and the enzyme inhibition rate of the compound was calculated.

[0183] The experiment included a blank control group and an enzyme-free control group. SAHA (vorinostat) was used as a positive control for HDAC1, and compound 42, a PI3K / mTOR dual-target inhibitor reported in the literature (Li C, et al. European Journal of Medicinal Chemistry, 2023, 246:115030), was used as a positive control for PI3Kα.

[0184] The inhibitory activities of the target compounds against PI3Kα and HDAC1 are shown in Table 1. The results show that the compounds of this invention exhibit significant inhibitory activity against PI3Kα. At a concentration of 1 nM, most compounds showed inhibition rates higher than 70% against PI3Kα, all superior to compound 42. Among them, ZWJ-5c and ZWJ-5b showed inhibition rates of 90.7% and 88.0%, respectively, demonstrating potent PI3Kα kinase inhibitory activity. Simultaneously, some compounds also showed excellent inhibitory effects against HDAC1. At a concentration of 100 nM, ZWJ-5c and ZWJ-5b showed inhibition rates of 99.2% and 97.7% against HDAC1, respectively, superior to SAHA. ZWJ-5d, ZWJ-B-3, and ZWJ-B-6d also showed good inhibitory activity against HDAC1, with inhibition rates all exceeding 75%.

[0185] Table 1. Inhibitory activity of compounds against PI3Kα and HDAC1 kinases

[0186] ;

[0187] - Indicates "Not detected"

[0188] 2. In vitro anti-tumor cell proliferation experiment

[0189] The in vitro proliferation inhibitory activity of the target compound of this invention against human colon cancer HCT116 cells was detected using the CCK-8 assay. The specific experimental method is as follows:

[0190] Human HCT116 colon cancer cells in logarithmic growth phase were collected and a cell suspension was prepared using RPMI 1640 complete medium containing 10% fetal bovine serum. Cells were then cultured at a density of 5 × 10⁶ cells per well. 3Cells were seeded at a density of 1,000 cells per well in 96-well cell culture plates and incubated overnight at 37°C. The next day, the old culture medium in the wells was discarded. Fresh culture medium containing a series of semi-logarithmic concentration gradients of the test compounds was added to the drug treatment group. At the same time, a blank control group (no cells, only complete culture medium) and a solvent control group (containing cells, with the same volume of DMSO solvent as the drug treatment group, without the test compounds) were set up. Each group was set up in 3 replicates and incubated for another 72 h.

[0191] After incubation, discard the culture medium in the wells, add 100 μL of fresh complete culture medium and 10 μL of CCK-8 reagent to each well, mix well and incubate in the dark for 2 h; use a microplate reader to detect the absorbance value (OD value) of each well at a wavelength of 450 nm.

[0192] With the relative cell viability of the solvent control group set at 100%, the relative cell viability of each drug-treated group was calculated using the formula: Relative cell viability (%) = (OD value of drug-treated group - OD value of blank control group) / (OD value of solvent control group - OD value of blank control group) × 100%. Nonlinear fitting was performed on the data using software to calculate the half-maximal inhibitory concentration (IC50) of each compound on HCT116 cell proliferation. 50 The results are shown in Table 2.

[0193] The results showed that the compounds in this application series all exhibited varying degrees of inhibitory activity against human colon cancer HCT116 cells, with some compounds demonstrating potent antitumor effects at the nanomolar level.

[0194] Among the hydroxamic acid series compounds, six compounds—ZWJ-5b, ZWJ-5c, ZWJ-5d, ZWJ-5e, ZWJ-B-3, and ZWJ-B-6d—exhibited excellent anti-proliferative activity, with an IC50 value of [missing information]. 50 All values ​​were below 1 μM, outperforming SAHA; among them, ZWJ-5d exhibited the most outstanding activity, with an IC50 concentration of [missing value]. 50 At a concentration as low as 0.020 μM, it exhibits nearly 95 times the activity of SAHA and is superior to compound 42, perfectly matching the potent dual-target inhibitory activity of that compound.

[0195] The entire series of o-phenylenediamine amide compounds exhibited potent antitumor proliferative activity at the nanomolar level, IC50. 50 The antiproliferative activity ranged from 0.001 μM to 0.390 μM, all of which were significantly superior to SAHA; among them, ZWJ-A-4d exhibited the strongest antiproliferative activity, with an IC50 concentration of 100 mg / L. 50 At a concentration as low as 0.001 μM, it exhibits 1900 times higher activity than SAHA and 540 times higher activity than compound 42, demonstrating extremely strong tumor cell killing ability.

[0196] It is noteworthy that although ZWJ-A-4d exhibits weaker inhibitory activity against HDAC1 at the pure enzyme level than the dual-target preferred compounds ZWJ-5b and ZWJ-5c of this invention, it demonstrates extremely strong inhibitory activity against proliferation at the level of human colon cancer HCT116 cells. The core mechanism of this phenomenon is inferred to be: First, these compounds possess potent inhibitory activity against PI3Kα, directly blocking the core PI3K / Akt / mTOR proliferation pathway of tumor cells, thus exerting a basic anti-tumor effect; Second, these compounds possess excellent tumor cell membrane penetration and intracellular accumulation capabilities, achieving an effective intracellular concentration of HDAC target within tumor cells. Simultaneously, the blocking of the PI3K pathway significantly enhances the sensitivity of tumor cells to HDAC inhibition, resulting in a significant synergistic anti-tumor effect, ultimately achieving an extremely strong inhibitory effect on tumor cell proliferation.

[0197] The above results demonstrate that the compounds of this invention exhibit excellent in vitro antiproliferative activity against human colon cancer HCT116 cells, with the o-phenylenediamine amide series showing particularly outstanding activity. These compounds exert potent antitumor effects through synergistic regulation of dual-target pathways. Compound ZWJ-A-4d exhibited the most prominent cellular-level antitumor activity, and this compound was selected for subsequent systematic evaluation of its broad-spectrum antitumor activity, in vivo efficacy, and safety.

[0198] Table 2. Half-maximal inhibitory concentrations (IC50) of the compounds on the proliferation of human colon cancer HCT116 cells. 50 )

[0199] ;

[0200] 3. Broad-spectrum anti-tumor cell proliferation experiment of ZWJ-A-4d

[0201] The experimental method was the same as the aforementioned CCK-8 assay. The test cell lines were: human breast cancer MCF-7 cells, human non-small cell lung cancer A549 cells, human colorectal cancer HT-29 cells, and human osteosarcoma SJSA cells.

[0202] Table 3. In vitro proliferation inhibitory activity of compound ZWJ-A-4d against different human solid tumor cells.

[0203] ;

[0204] Table 3 shows that compound ZWJ-A-4d exhibited potent inhibitory activity at the nanomolar level against human solid tumor cells from four different tissue sources, with IC50 values ​​of [missing information]. 50 The effective concentration ranged from 0.050 μM to 0.176 μM, all below 0.2 μM. Among them, ZWJ-A-4d showed the strongest inhibitory activity against human non-small cell lung cancer A549 cells, with an IC50 concentration of [missing value]. 50The concentration was as low as 0.050 μM; it showed excellent inhibitory effects on epithelial-derived breast cancer, lung cancer, colorectal cancer cells, and mesenchymal-derived osteosarcoma cells, demonstrating that the compound has broad-spectrum inhibitory activity against solid tumors, and further verifying the anti-tumor development potential of the dual-target inhibitor of this invention.

[0205] 4. In vivo antitumor pharmacodynamic experiments

[0206] This experiment used SPF-grade BALB / c nude mice to construct a human colon cancer xenograft model of HCT116: HCT116 human colon cancer cells in the logarithmic growth phase were collected and resuspended in serum-free culture medium to a cell concentration of 5 × 10⁶ cells / mL. 7 A human colon cancer xenograft model was established by subcutaneously injecting 0.1 mL of cell suspension into the right axilla of nude mice at a density of [number] cells / mL. The tumor volume was increased to 100-150 mm. 3 At that time, the tumor-bearing mice were randomly divided into 4 groups (n=6):

[0207] ① Solvent control group (Control): An equal volume of blank solvent was injected intraperitoneally;

[0208] ② Positive control group (CAP): capecitabine, 150 mg / kg, administered by gavage (po);

[0209] ③ZWJ-A-4d monotherapy group (4d): 10 mg / kg, administered via intraperitoneal injection (ip);

[0210] ④ Combination group (4d+CAP): ZWJ-A-4d 10 mg / kg (ip) + capecitabine 150 mg / kg (po).

[0211] All groups were administered the drug once daily for 20 consecutive days. During the administration period, tumor-bearing mice were non-invasively measured in vivo every 5 days using calipers to record the long and short diameters of the tumor. The tumor volume was calculated using a formula, and a tumor growth curve was plotted. Mouse weight was also recorded every 5 days to monitor drug tolerance. After 20 days of continuous administration, all mice were euthanized, the subcutaneous tumor tissue was completely dissected, tumor morphology was photographed, and the tumor weight was measured using an analytical balance to calculate the tumor growth inhibition rate (TGI). Experimental results are shown below. Figure 5 .

[0212] The results showed that compound ZWJ-A-4d exhibited excellent antitumor activity and good tolerability in vivo:

[0213] Monotherapy efficacy: After 20 days of ZWJ-A-4d monotherapy, the tumor growth inhibition rate reached 59.25%, significantly better than the positive control drug capecitabine (TGI=28.49%); compared with the control group, the tumor volume and tumor weight in the monotherapy group were significantly lower. Figure 5Both A and B in the sample were significantly reduced, which can effectively inhibit the growth of human colon cancer xenograft tumors in vivo.

[0214] Synergistic effect of combination therapy: The combination of ZWJ-A-4d and capecitabine further enhanced the anti-tumor efficacy, with a TGI of up to 75.64%. Figure 5 The D group was significantly superior to the two single-drug groups, demonstrating a clear synergistic effect and providing a new candidate for combination therapy in colorectal cancer.

[0215] Furthermore, mouse weight monitoring during administration showed that mice in the ZWJ-A-4d single-drug group experienced a slight decrease in weight at the beginning of administration, which gradually recovered with the extension of administration time. No mice died during the entire administration period, and the final weight of the mice after administration was not significantly different from that of the control group. The weight fluctuation of mice in the combination group was not significantly different from that in the single-drug group, and no serious adverse reactions occurred. Figure 5 (C in the text). The results showed that ZWJ-A-4d could strongly inhibit tumor growth in vivo and had good in vivo tolerability. To further clarify its long-term in vivo safety, a systematic target organ toxicity evaluation was conducted.

[0216] 5. Evaluation of in vivo hepatotoxicity and nephrotoxicity

[0217] After drug administration, blood was collected from the orbital veins of mice in each group. Serum was separated by centrifugation after standing at room temperature. A fully automated biochemical analyzer was used to detect key indicators of liver and kidney function: liver function indicators included ALT and AST activities; kidney function indicators included UREA and UA concentrations. Experimental results are shown below. Figure 6 .

[0218] Experimental results showed that at a therapeutic dose of 10 mg / kg, the serum ALT and AST activities in mice treated with ZWJ-A-4d monotherapy were not significantly different from those in the solvent control group, and no abnormalities were observed in liver injury-related indicators. Simultaneously, serum UREA and UA concentrations were comparable to those in the control group, with no elevation in kidney function-related indicators, demonstrating that ZWJ-A-4d has no significant toxic effects on liver and kidney function in mice at the therapeutic dose. In mice treated with a combination of ZWJ-A-4d and capecitabine, serum ALT, AST, UREA, and UA levels were not significantly different from those in the control group, and no abnormalities in liver and kidney function were observed, demonstrating the good in vivo safety and feasibility of the combined treatment.

[0219] In summary, the HDAC / PI3K dual-target inhibitors designed in this invention can effectively inhibit PI3Kα kinase activity at the molecular level, and some preferred compounds also possess potent inhibitory activity against both PI3Kα and HDAC1. At the cellular level, they exhibit excellent anti-tumor proliferation activity, with compound ZWJ-A-4d demonstrating potent, broad-spectrum inhibition of solid tumors at the nanomolar level. In vivo, they can significantly inhibit the growth of human colon cancer xenografts, showing superior efficacy compared to the first-line clinical chemotherapy drug capecitabine, and exhibiting no significant hepatotoxicity or nephrotoxicity. They possess good potential for drug development and provide novel, highly active dual-target inhibitor candidate molecules for the treatment of malignant tumors.

[0220] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the protection scope of the present invention.

Claims

1. A dual-target HDAC / PI3K inhibitor based on the imidazopyridazine core, characterized in that, The HDAC / PI3K dual-target inhibitor is a compound of general structural formula (I), its pharmaceutically acceptable hydrate, solvate, crystal form, metabolite, or prodrug: ； Wherein, R1 is a linking group connecting the triazole ring and R2, selected from... , , , , , or n = 1~10; R2 is a zinc ion-binding group, selected from... or .

2. The HDAC / PI3K dual-target inhibitor based on imidazopyridazine core as described in claim 1, characterized in that, The HDAC / PI3K dual-target inhibitor is selected from any of the following compounds: 。 3. The HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core as described in claim 1, characterized in that, The HDAC / PI3K dual-target inhibitor exhibits inhibitory activity against both HDAC1 and PI3Kα.

4. The use of the HDAC / PI3K dual-target inhibitor based on the imidazopyridazine core as described in any one of claims 1-3 in the preparation of a drug, characterized in that, The drug is used to treat and / or prevent tumors, inflammation, or autoimmune diseases.

5. The application as described in claim 4, characterized in that, The tumors mentioned are colorectal cancer, breast cancer, cholangiocarcinoma, non-small cell lung cancer, osteosarcoma, cholangiocarcinoma, endometrial cancer, prostate cancer, glioblastoma multiforme, and lymphoma.

6. A pharmaceutical composition, characterized in that, The invention comprises a therapeutically effective amount of any one of claims 1-3 of an HDAC / PI3K dual-target inhibitor based on an imidazopyridazine core, and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, characterized in that, It also includes at least one other therapeutically active ingredient; the dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form.

8. The pharmaceutical composition according to claim 6, characterized in that, The other therapeutically active ingredients are anti-tumor chemotherapy drugs.

9. The use of the pharmaceutical composition of claim 7 in the preparation of a medicament for the treatment and / or prevention of tumors, inflammations, or autoimmune diseases.

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