1, 4-benzodiazepine compound targeting ANXA3 protein and medicinal application of 1, 4-benzodiazepine compound

Abstract

The invention belongs to the technical field of medicinal chemistry and medicine, and relates to a 1, 4-benzodiazepine compound targeting ANXA3 protein and medicinal application thereof. Specifically, the invention relates to an application of a 1, 4-benzodiazepine compound targeting ANXA3 protein, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a composition of a medicine taking the 1, 4-benzodiazepine compound as an effective active component in preparation of a medicine for preventing and treating breast cancer. The compounds can target ANXA3 protein, selectively induce degradation of the ANXA3 protein, well inhibit proliferation, cloning, migration, invasion and other functions of tumor cells in vivo and in vitro, and play an anti-tumor treatment effect. Breast cancer refers to breast cancer molecular subtypes such as triple negative, Luminal A type, Luminal B type, Her2 + type and the like. The small molecule compound targeting ANXA3 anti-tumor activity not only has structural innovation, but also shows a novel degradation mechanism for treating TNBC (tumor necrosis factor C) aiming at ANXA3, and provides novel treatment selectivity and a medication scheme for clinically treating breast cancer.

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and pharmaceutical technology, and particularly relates to 1,4-benzodiazepines targeting the ANXA3 protein. Class of compounds and their medicinal uses. Background Technology

[0002] Breast cancer is a type of malignant tumor that begins with the uncontrolled growth of breast cells. According to data released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2023, the incidence of breast cancer worldwide has far exceeded that of other cancers affecting women over the past decade, and it is increasing at a rate of 0.5% per year. In 2023, there were 2.26 million new cases of breast cancer, accounting for about 30% of all cancers affecting women, and the number of deaths from breast cancer among women was about 685,000, making it the leading cause of death among women worldwide (Journal of the National Cancer Center, 2024, 4(1)). It is evident that breast cancer not only seriously affects women's health, but the difficulty in improving its survival rate has also become a major challenge in the treatment of breast cancer.

[0003] Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer characterized by its highly aggressive and metastatic nature. A key feature is its negative expression of three biomarkers: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This makes its pathogenesis extremely complex and its precise clinical targets difficult to identify (Breast Cancer Research, 2011, 13(2):R22.). Currently, the main clinical treatment for TNBC patients is surgical resection, supplemented by chemotherapy and radiotherapy. However, this only provides temporary control for some TNBC patients. Most importantly, chemotherapy and radiotherapy have significant side effects and are prone to drug resistance. Furthermore, TNBC is incurable and easily leads to recurrence and metastasis (Cancers (Basel), 2022, 14(5):1253.). Although several monoclonal antibodies, such as pembrolizumab and atezolizumab, have emerged in recent years and have significantly improved patient survival, these biological antibody drugs have low response rates, benefit a limited number of women, and suffer from numerous drawbacks such as immunogenicity, drug resistance, and high cost. Therefore, it is urgent to explore new, highly effective small molecule drugs targeting breast cancer with novel targets and mechanisms.

[0004] According to literature research, annexin ANXA3 is a member of the annexin family. This family is a class of calcium-containing proteins. 2+ And phospholipid-binding proteins, which access Ca2+ through gating 2+Channels and energy-dependent pumps (located in organelle membranes and plasma membranes) and as regulators of Ca2+ 2+ Intracellular Ca in the buffer solution 2+ Binding proteins to strictly control intracellular calcium 2+ Concentration enables calcium ions to function as second messengers in various processes that couple extracellular signals with cellular responses. In recent years, clinical medical big data research has found that annexin ANXA3 is closely related to the occurrence and development of TNBC (Nature reviews Molecular cell biology, 2005, 6(6):449-461.). ANXA3 protein is now considered a novel angiogenic factor. Its high expression promotes the development and progression of various cancers, including upper urothelial carcinoma (UTUC), gastric cancer, hepatocellular carcinoma (HCC), breast cancer, and osteosarcoma. Among these, annexin ANXA3 is highly expressed in breast cancer tumor tissues, especially in TNBC samples, with a positive expression rate of 62%, significantly higher than the 10% positive expression rate in adjacent normal tissues. More importantly, high expression of ANXA3 can promote the proliferation, migration, and drug resistance of TNBC cells, and it has been shown to be a key pathogenic protein in the development and progression of TNBC (Oncologyreports, 2017, 37(1):388-398; Clinical breast cancer, 2018, 18(4):713-719; CellDeath&Disease, 2018, 9(2):1-11). However, to date, the number of small molecule ligands that interact with ANXA3 protein has been extremely limited. Only the Francoise research group has reported two 1,4-benzodiazepines. Small molecules BDA452 and BDA753 have affinities for ANXA3 of 34.8 μM and 147 μM, respectively. However, unfortunately, no reports have been made on their biological activity at the cellular or animal level, especially their anti-TNBC proliferative activity and further cellular function studies (Journal of Biological Chemistry, 1998, 273(5):2885-2894.). Until 2023, the inventors' team of this application screened a class of atrazomycin-1,4-benzodiazepines from a library of natural antitumor active compounds. Analogs, after retaining 1,4-benzodiazepines A series of compounds were obtained by modifying the core region and its side chain substituents. Structure-activity relationship studies first reported the first preferred lead compound (R)-SL18 with anti-TNBC proliferation activity. This not only elucidated the binding of this class of compounds to the ANXA3 protein but also revealed that they mediate the ubiquitination and degradation of the ANXA3 protein (Acta Pharmaceutica Sinica B, 2023, 13(4): 1686-1698; Patent application number: 202210680501.3). However, the compounds involved in the aforementioned published patents did not undergo systematic structural optimization and structure-activity relationship studies targeting the hydrophobic structural region of the ANXA3 protein, especially not targeting 1,4-benzodiazepines. By introducing different substituents on the nitrogen at position 1 of the parent nucleus, and / or by making diverse structural changes in the linker chain (X) connecting the parent nucleus and the side chain, it is hoped that novel compounds can be designed through undisclosed structural optimization to obtain lead compounds with drug-like properties such as improved binding activity against ANXA3 and anti-triple-negative breast cancer (TNBC) activity, as well as improved degradation selectivity and pharmacokinetics of ANXA3 protein. Summary of the Invention

[0005] The purpose of this invention is to focus on a structure optimization strategy aimed at improving the binding activity with ANXA3 and the anti-triple-negative breast cancer (TNBC) activity of the lead (R)-SL18, targeting its parent nucleus 1,4-benzodiazepine. By introducing different substituents at the nitrogen position 1 and by varying the linking chain groups (X) connecting the parent nucleus and the side chain, 1,4-benzodiazepine can be extended. The structure-activity relationship between the compounds targeting ANXA3 protein degradation and their anti-triple-negative breast cancer (TNBC) activity provides a class of 1,4-benzodiazepines targeting ANXA3 protein. Class of compounds and their pharmaceutical uses. Specifically, 1,4-benzodiazepines targeting the ANXA3 protein. Use of class compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof in the preparation of ANXA3 degrading agents and in the preparation of drugs for the prevention and treatment of cancer.

[0006] This invention uses (R)-SL18 as a lead to dock with the ANXA3 protein structure, focusing on 1,4-benzodiazepines. By rationally redesigning the core region at the nitrogen position 1 of the parent nucleus and linking regions (such as linker chain X), we can screen for anti-TNBC proliferation activity, ANXA3-targeting binding activity, and degradation activity. This will expand the structure-activity relationship studies of this type of structure in targeting ANXA3 protein degradation and anti-triple-negative breast cancer (TNBC) activity, and ultimately obtain a series of 1,4-benzodiazepine small molecule compounds with anti-TNBC pharmacological effects targeting ANXA3 that have not been reported in the literature.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The first objective of this invention :

[0009] Provide a 1,4-benzodiazepine targeting the ANXA3 protein The 1,4-benzodiazepine targeting the ANXA3 protein is a class of compounds, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof. The class of compounds is the compound shown in formula (I).

[0010]

[0011] in,

[0012] 1) When R 1 When X is selected from ethyl, isopropyl, isobutyl, or methylcyclopropane, X is a linking group, selected from:

[0013]

[0014] R 2 For side chains, selected from: Among them, R 3 R 4 It can be arbitrarily selected from: hydrogen, halogen, nitro, cyano, and R 3 R 4 It can be substituted individually or simultaneously in two ways;

[0015] 2) When R 1 When X is selected from methyl or isopropyl, X is a linking group, selected from:

[0016]

[0017]

[0018] R 2 Selected from: Among them, R 5 R 6 It can be arbitrarily selected from hydrogen, trifluoromethyl, nitro, cyano, or halogen, and R 5 R6 It can be monosubstituted or simultaneously disubstituted; R 7 It can be selected from hydrogen, nitro or cyano.

[0019] In some embodiments of the present invention, the 1,4-benzodiazepine The class of compounds is selected from the compounds shown in I-1 to I-9:

[0020]

[0021] In some embodiments of the present invention, the 1,4-benzodiazepine The specific compounds are selected from the compounds shown in I-10 to I-25 below:

[0022]

[0023]

[0024] In some embodiments of the present invention, the 1,4-benzodiazepine Stereoisomers of the compound class can be further described as having an R or S configuration at the C3 position, specifically selected from (R)- or (S)- compounds:

[0025]

[0026] In this invention, pharmaceutically acceptable salts refer to compounds whose salts, within a reliable pharmaceutical evaluation range, are suitable for contact with human or lower animal tissues without undue toxicity, irritation, or allergic reactions, possessing a reasonably reasonable benefit-risk ratio, and are typically water- or oil-soluble or dispersible, and can be effectively used for their intended purpose. Some compounds or their stereoisomers contain basic groups such as amines that can form salts with acids, can form acidic salts with inorganic and / or organic acids, and also include zwitterionic salts (internal salts), as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compound or its stereoisomers. Alternatively, they can be obtained by mixing the compound or its stereoisomers with an appropriate amount (e.g., an equimolar amount) of acid. These salts may form a precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or prepared by freeze-drying after reaction in an aqueous medium.

[0027] The pharmaceutically acceptable salts described in this invention include organic acid salts such as citrate, benzenesulfonate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate; inorganic acid salts such as hydrochloride, sulfate, hydrobromide, hydrofluoric acid, hydroiodide, hydrochloride, phosphate, etc.; or salts that can form glutamate or aspartate salts with amino acids such as glutamic acid or aspartic acid.

[0028] This invention relates to 1,4-benzodiazepines Solvents of the same type of compound are also within the scope of protection of this invention, and the solvents are preferably water, alcohol or alcohol-water mixtures, where alcohol refers to methanol or ethanol.

[0029] The second objective of this invention is:

[0030] Provides a 1,4-benzodiazepine targeting the ANXA3 protein as described in the first objective of this invention. Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of medicaments for the prevention and / or treatment of breast cancer.

[0031] In some embodiments of the present invention, a 1,4-benzodiazepine targeting the ANXA3 protein is provided as described in the first objective of the present invention. Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of ANXA3 degrading agents for the selective degradation of ANXA3.

[0032] Binding activity assays based on surface plasmon resonance indicate that the 1,4-benzodiazepine The compounds exhibit sub-micromolar levels of ANXA3 protein binding activity, and the activity results are shown in Table 1 of the examples.

[0033] Based on Western blot assays, the 1,4-benzodiazepines... The compound was able to induce ANXA3 protein degradation in TNBC cell lines MDA-MB-231 and MDA-MB-468, with activity at the micromolar level, as shown in Table 1.

[0034] Based on the in vitro antiproliferative activity test results against TNBC cell lines MDA-MB-231 and MDA-MB-468, the 1,4-benzodiazepine... The compounds exhibited micromolar-level antitumor activity, and the activity results are shown in Table 1.

[0035] Based on the results of the in vitro selective degradation activity assay of membrane-linked family proteins (ANXA1-11 and ANXA13) in the TNBC cell line MDA-MB-231, the 1,4-benzodiazepine... The preferred compound I-19 of this class of compounds exhibits good selectivity for ANXA3 family proteins, and its degradation activity results are as follows: Figure 1 As shown.

[0036] Based on the in vivo administration of the preferred compound I-19 to Balb / C mice via intraperitoneal administration, the metabolic test results show that the preferred compound I-19 has good drug metabolism properties in vivo, as shown in Table 2.

[0037] Based on the fact that the preferred compound I-19 can significantly induce apoptosis in breast cancer cells and inhibit cell cloning, migration, and invasion, the activity results are as follows: Figure 2 As shown;

[0038] Based on the results of the preferred compound I-19 in an in vivo subcutaneous xenograft model of breast cancer, it demonstrated effective therapeutic effects and induced the degradation of ANXA3 protein in tumor tissue. Figure 3 As shown.

[0039] Based on the good safety profile of the preferred compound I-19 in in vivo organ HE staining toxicity test in mice, the results are as follows: Figure 4 As shown.

[0040] In summary, the 1,4-benzodiazepine These compounds, or their pharmaceutically acceptable salts, or their stereoisomers, or their solvates, can target and degrade ANXA3 protein, thereby downregulating the expression level of ANXA3 protein and exerting an anti-breast cancer effect.

[0041] In some embodiments of the present invention, the breast cancer is selected from one of the molecular subtypes of breast cancer, such as triple-negative breast cancer, Luminal A, Luminal B, or Her2+.

[0042] In some embodiments of the present invention, a 1,4-benzodiazepine targeting the ANXA3 protein is provided as described in the first objective of the present invention. Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of medicaments for the prevention and / or treatment of triple-negative breast cancer.

[0043] In some embodiments of the present invention, 1,4-benzodiazepines related to the first objective of the present invention are provided. Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of medicaments targeting ANXA3 protein degradation for the prevention and / or treatment of triple-negative breast cancer.

[0044] In some embodiments of the present invention, the drug may also contain one or more pharmaceutically acceptable carriers, including conventional pharmaceutical diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorbents, lubricants, etc., and flavoring agents, sweeteners, etc. may be added if necessary.

[0045] The present invention also provides an antitumor pharmaceutical composition that exerts its antitumor effect by acting as an ANXA3 degrader, wherein the antitumor pharmaceutical composition uses 1,4-benzodiazepines that target the ANXA3 protein. The active ingredient is a class of compounds, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof.

[0046] In some embodiments of the present invention, an anti-breast cancer drug composition is also provided that exerts its anti-breast cancer tumor effect by acting as a target annexin ANXA3 degrader, wherein the anti-breast cancer drug composition is a tablet, capsule, pill, injection, sustained-release formulation, spray or nano-drug delivery system.

[0047] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0048] The 1,4-benzodiazepine provided by this invention These compounds, or their pharmaceutically acceptable salts, stereoisomers, or solvates, which have not been reported by SciFinder, represent a novel class of ANXA3 small-molecule degraders targeting the ANXA3 protein. Compared to the lead compound (R)-SL18, they exhibit comparable or better submicromolar ANXA3 binding activity and demonstrate significant ANXA3-degrading activity in both breast cancer cells and animal studies. Importantly, they significantly inhibit the proliferation, cloning, migration, and invasion of breast cancer cells in vitro, and demonstrated superior anti-breast cancer therapeutic effects in vivo using a subcutaneous xenograft tumor model of human MDA-MB-231 cells from female BALB / cnude nude mice. In particular, the preferred compound I-19 exhibits 10-fold higher in vitro ANXA3 binding activity than (R)-SL18 and superior inhibition of TNBC proliferation. It also demonstrates excellent ANXA3 protein degradation selectivity in vitro and in vivo, mediating ubiquitination and degradation of the ANXA3 protein, and good in vivo inhibitory activity (TGI = 96%) in a TNBC xenograft model, along with attractive pharmacokinetic properties and safety. In summary, the small molecule compounds of this invention targeting ANXA3 for antitumor activity not only possess structural innovation but also demonstrate a novel degradation mechanism for ANXA3 in the treatment of TNBC, providing new therapeutic selectivity and dosing regimens for clinical use in breast cancer treatment. Attached Figure Description

[0049] Figure 1 Results of the detection of the selective degradation activity of the preferred compound I-19 on the annexin family ANXA1-11 / 13;

[0050] Figure 2 The results of the detection of the effects of the preferred compound I-19 on the functions of breast cancer cells, such as apoptosis, cloning, migration, and invasion;

[0051] Figure 3 Results of in vivo detection of the efficacy of the preferred compound I-19 against TNBC and its ability to induce ANXA3 degradation in tumor tissue;

[0052] Figure 4 Results of in vivo detection of the organ toxicity of the preferred compound I-19 in mice. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, these embodiments are only used to further illustrate the present invention and do not change the scope of protection of the present invention.

[0054] Example 1

[0055] Preparation of 2-(3-(3-cyanopyridin-2-yl)ureo)-2-oxoethyl(1-ethyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-1)

[0056]

[0057] 1.1 Preparation of intermediate compound 10

[0058]

[0059] Weigh 10.0 g (133.2 mmol) benzyl carbamate 2 and 15.9 g (105.0 mmol) benzotriazole 3 and 150 mL toluene solvent, and dissolve them in a 250 mL round-bottom flask with stirring. Then add 15 mL (133.2 mmol) glyoxylic acid hydrate 1, place the system in an oil bath and heat to 80 °C. After reacting for 2 h, a solid precipitates. Filter the solid using a sieve funnel to obtain intermediate 4, 32.0 g of white solid, yield 97%; ESI-MS: m / z 349.0 [M+Na] + C 16 H 14 N4O4.

[0060] Weigh 10.0 g (30.0 mmol) of compound 4 into a 100 mL round-bottom flask, add 60 mL of tetrahydrofuran and stir to dissolve. After cooling to 0 °C in a cold trap, slowly add 4 mL (47.2 mmol) of oxalyl chloride to the reaction system, add 2-3 drops of catalytic DMF, and react for 2 h. Weigh 6.0 g (30.6 mmol) of 2-aminophenylbenzophenone and 7.3 mL (67.4 mmol) of N-methylmorpholine and dissolve in 40 mL of tetrahydrofuran. At 0 °C, slowly add the solution to the above reaction system 5 using a constant-pressure dropping funnel. The process takes about 20-30 min. After the addition is complete, heat the system to room temperature and continue the reaction for 2-4 h. The reaction solution was filtered through diatomaceous earth and washed with 40 mL of tetrahydrofuran. The organic phase was concentrated under vacuum, and the crude product was purified by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:2) to give intermediate 6, 7.6 g of yellow oil, yield 49%; ESI-MS: m / z 506.0 [M+H] + C 29 H 23 N5O4.

[0061] Weigh 5.0 g (9.9 mmol) of compound 6 into a 100 mL round-bottom flask, add 10 mL of methanol and stir to dissolve. Cool to 0 °C in a cold trap, then add 15 mL of ammonia-methanol solution. Slowly raise the system to room temperature and react for 16 h. Filter the precipitated solid, collect the organic phase and concentrate under vacuum. Add 10-20 mL of ethyl acetate to the organic phase, wash the organic phase twice with 1 N sodium hydroxide aqueous solution, then add sodium chloride aqueous solution and ethyl acetate. Collect the organic phase and dry it with sodium sulfate or magnesium sulfate. After concentrating the organic phase under vacuum, add 60 mL of acetic acid and 3.8 g (49.3 mmol) of ammonium acetate to the system, and react at room temperature for 12 h under inert gas (argon) protection. After concentrating the reaction solution under vacuum, add an organic solvent (ethyl acetate: ether = 1:3) and stir. Adjust the pH to above 8 with 1 N sodium hydroxide aqueous solution, and observe the precipitation of solid. The suspension was placed in a 0℃ refrigerator and allowed to stand for 30-60 minutes. While still cold, it was filtered through a partition funnel to obtain a pale yellow solid. The solid was washed three times with water and ether to obtain intermediate 8, 1.1 g of light white solid, with a yield of 30%. 1 H NMR (400MHz, DMSO-d6) δ10.87(s,1H),8.47(d,J=7.6Hz,1H),7.62(s,1H),7.54-7.15(m,13H),5.03(d,J=16.1Hz,3H).ESI-MS:m / z 386.0[M+H] + C 24 H 20 N2O3.

[0062] Weigh 5.0 g (13.0 mmol) of compound 8, 3.0 g (21.7 mmol) of anhydrous potassium carbonate, 2.5 g (17.6 mmol) of iodoethane, and 30 mL of ultra-dry DMF into a 100 mL round-bottom flask. The mixture was purged three times with an inert gas (argon) and reacted at room temperature for 5 h. The system was quenched with ice water and ethyl acetate, washed with saturated brine, extracted with ethyl acetate, and the organic phase was collected, dried over Na₂SO₄, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:3) to give intermediate 9, 3.0 g of gray solid, yield 58%. 1 H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.5Hz,1H),7.75-7.69(m,2H),7.51(d,J=7.1Hz,3H),7.49-7.43(m,2H),7.41-7.26( m,7H),5.13-5.03(m,3H),4.21(dt,J=14.4,7.2Hz,1H),3.79(dt,J=13.6,6.9Hz,1H),0.95(t,J=7.1Hz,3H).ESI-MS:m / z 436.20[M+Na] + C 25 H 23 N3O3.

[0063] Weigh 3.0 g (7.5 mmol) of compound 9 and 10 mL of acetic acid, place them in a 100 mL round-bottom flask, and add 30 mL of 33% hydrobromic acid solution to the reaction system using a needle. React at room temperature for 2-4 h. Adjust the pH of the reaction system to neutral with sodium bicarbonate solution, wash with saturated brine, extract with ethyl acetate, collect the organic phase, dry with Na2SO4, concentrate under vacuum, and purify the crude product by silica gel column chromatography (mobile phase: dichloromethane:methanol = 10:1) to give intermediate 10, 1.4 g of light blue oil, yield 60%. 1 H NMR (400MHz, DMSO-d6) δ7.69(d,J=4.0Hz,2H),7.52(d,J=7.0Hz,3H),7.47(d,J=6.8Hz,2H),7.33(dt,J=8.3,4.1Hz,1H) ,7.27(d,J=7.8Hz,1H),4.52(s,1H),4.19(dq,J=14.2,7.1Hz,1H),3.79(dq,J=14.1,7.0Hz,1H),0.97(t,J=7.0Hz,3H).

[0064] 1.2 Preparation of intermediate compound (14)

[0065]

[0066] Weigh 0.4 g (4.2 mmol) of chloroacetamide 12 and place it in a 100 mL round-bottom flask. Add 20 mL of 1,2-dichloroethane and stir to dissolve. Cool to 0 °C in a cold trap. Add 0.53 mL (6.3 mmol) of oxaloyl chloride 13 dropwise to the system and react at 0 °C for 30-60 min. Then, heat the reaction system to 90 °C and reflux for 5 h. After cooling the system to room temperature, slowly add 0.5 g (4.2 mmol) of 2-amino-3-cyanopyridine 11 to the system and react for 8 h. Filter the mixture using a diaphragm funnel to obtain intermediate 14, with a yield of 65%. 1 H NMR (400MHz, DMSO-d6) δ11.29(s,1H),10.61(s,1H),8.72(d,J=5.3Hz,1H),8.40(d,J=8.0Hz,1H),7.49(t,J=6.5Hz,1H),4.46(s,2H).

[0067] 1.3 Preparation of final product I-1

[0068]

[0069] Weigh 50.0 mg (0.2 mmol) of intermediate 10, 60 μL of triethylamine, and 50 μL of carbon disulfide into a 25 mL round-bottom flask. Add 5 mL of tetrahydrofuran solvent and react at room temperature for 2 h. Then add 40.2 mg (0.2 mmol) of intermediate 14 and continue reacting at room temperature for 6 h. Monitor the reaction on a TLC plate. Terminate the reaction when the starting materials 10 and 14 have essentially disappeared. Concentrate the reaction system under reduced pressure, extract with ethyl acetate and saturated brine, collect the organic phase, dry it with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether = 1:3) to give final product I-1, 30 mg of white solid, yield 20%, Mp 125.7-125.9℃. 1H NMR (400MHz, DMSO-d6) δ11.50(d,J=6.6Hz,1H),11.26(s,1H),10.68(s,1H),8.66(dd,J=4.9, 1.9Hz,1H),8.34(dd,J=7.9,1.8Hz,1H),7.74-7.70(m,2H),7.58-7.52(m,3H),7.49(d,J=7.4 Hz,2H),7.46-7.42(m,1H),7.37-7.33(m,1H),7.29(d,J=7.9Hz,1H),5.92(d,J=6.4Hz,1H),4 .29(s,2H),4.17(dq,J=14.1,6.9Hz,1H),3.80(dd,J=14.1,7.3Hz,1H),0.97(t,J=7.0Hz,3H). 13 CNMR(150MHz,DMSO)δ196.91,171.02,167.57,165.39,152.98,151.42,150.48,143.32,141.44,137.97,133.01,131.37,130.14,1 29.77,128.98,125.55,123.45,121.61,115.95,104.25,76.11,60.23,42.72,21.24,14.56,13.48.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 27 H 23 N7O3S2,558.1377; found,558.1398.

[0070] Example 2

[0071] Preparation of 2-oxo-2-(3-(pyridin-2-yl)ureo)ethyl(1-ethyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioide (I-2)

[0072]

[0073] 2.1 Preparation of intermediate compound 15

[0074]

[0075] Intermediate 15 was synthesized according to the method in step 1.2 of Example 1. 1H NMR (400MHz, DMSO-d6) δ11.21(s,1H),10.61(s,1H),8.34-8.31(m,1H),7.96-7.81(m,2H),7.20-7.15(m,1H),4.43(s,2H).

[0076] 2.2 Preparation of final product I-2

[0077] Compound I-2 was synthesized according to step 1.3 of Example 1, with the starting material replaced by an equivalent amount of 15. 30 mg of a white solid was obtained, yield 20%, MPa 135.6-135.9 °C. 1 H NMR(400MHz,DMSO-d6)δ11.50(d,J=6.5Hz,1H),11.13(s,1H),10.69(s,1H),8.33-8.28( m,1H),7.95(d,J=8.4Hz,1H),7.84-7.78(m,1H),7.75-7.72(m,3H),7.60-7.58(m,1H),7. 57(d,J=1.7Hz,1H),7.52-7.48(m,3H),7.30(d,J=7.8Hz,1H),7.14-7.11(m,1H),5.93(d ,J=6.5Hz,1H),4.27(s,2H),4.21-4.14(m,1H),3.85-3.80(m,1H),0.97(t,J=7.2Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ170.90,167.55,165.40,150.70,148.73,141.46,139.04,137.96,133.01,132.82,131.38,129.91,1 29.76,129.59,128.98,128.95,128.83,123.44,120.13,113.52,76.08,66.54,42.71,13.48.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 26 H 24 N6O3S2,533.1424; found,533.1428.

[0078] Example 3

[0079] Preparation of 2-oxo-2-(3-(pyridin-2-yl)ureo)ethyl(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-3)

[0080]

[0081] 3.1 Preparation of intermediate compound 16

[0082]

[0083] The reaction method for the compound is the same as the synthesis method for intermediate 10, with iodoisopropane as the raw material. 1 H NMR (400MHz, DMSO-d6) δ7.62(t,J=7.7Hz,1H),7.57-7.51(m,3H),7.49-7.42(m,3H),7.33(t,J=7.5Hz,1H),7. 24(d,J=7.8Hz,1H),4.22(s,1H),4.04-4.00(m,1H),1.38(d,J=6.7Hz,3H),1.17(d,J=7.0Hz,3H).ESI-MS:m / z 294.17[M+H] + C 18 H 19 N3O.

[0084] 3.2 Preparation of final product I-3

[0085] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 15 and 16, yielding 30 mg of white solid, yield 20%, Mp 168.8-169.3℃; 1 H NMR(400MHz,DMSO-d6)δ11.45(d,J=6.6Hz,1H),11.13(s,1H),10.69(s,1H),8.31-8.30(m,1H),7 .95(d,J=8.4Hz,1H),7.84-7.79(m,1H),7.72-7.67(m,1H),7.65-7.60(m,3H),7.56(d,J=6.7Hz,1 H),7.51(t,J=7.6Hz,2H),7.39(t,J=7.4Hz,1H),7.30(d,J=7.9Hz,1H),7.17-7.10(m,1H),5.90( d,J=6.5Hz,1H),4.44(p,J=6.8Hz,1H),4.28(s,2H),1.40(d,J=6.6Hz,3H),1.22(d,J=6.9Hz,3H). 13C NMR(150MHz,DMSO-d6)δ196.94,170.92,167.35,166.27,151.30,150.70,148.70,141.14,139.03,137.70,132.35,131.42,1 30.64,129.81,129.68,129.05,126.18,124.84,120.13,113.52,76.43,52.42,21.82,20.56.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 27 H 26 N6O3S2,547.1581; found,547.1591.

[0086] Example 4

[0087] Preparation of 2-(3-(3-cyanopyridin-2-yl)ureido)-2-oxoethyl(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-4)

[0088]

[0089] 4.1 Preparation of final product I-4

[0090] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 14 and 16, yielding 30 mg of a light yellow solid, yield 20%, Mp 148.7-150.1℃; 1 H NMR(600MHz,DMSO-d6)δ11.45(d,J=6.4Hz,1H),11.26(s,1H),10.69(s,1H),8.67(dd,J=4.9,1.8Hz,1H), 8.35(dd,J=7.8,1.8Hz,1H),7.72-7.68(m,1H),7.64(d,J=8.2Hz,1H),7.62-7.60(m,2H),7.56(t,J=7.3Hz ,1H),7.50(t,J=7.5Hz,2H),7.45(dd,J=7.8,4.9Hz,1H),7.39(t,J=7.5Hz,1H),7.30(dd,J=7.8,1.3Hz,1H ),5.90(d,J=6.2Hz,1H),4.44(p,J=6.9Hz,1H),4.30(s,2H),1.40(d,J=6.7Hz,3H),1.21(d,J=6.9Hz,3H). 13C NMR (150MHz, DMSO) δ195.83,169.97,166.27,165.20,151.91,150.36,149.41,140.07,136.62,131.28,130.34,129.57,128.61,1 27.97,125.11,123.78,120.53,114.88,103.19,75.40,59.15,51.35,20.75,20.16,19.49,13.49.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 28 H 25 N7O3S2,572.1533; found,572.1538.

[0091] Example 5

[0092] Preparation of 2-(3-(6-chloro-3-nitropyridin-2-yl)ureo)-2-oxoethyl(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-5)

[0093]

[0094] 5.1 Preparation of intermediate compound 17

[0095]

[0096] Intermediate 17 was synthesized according to the method in step 1.2 of Example 1. 1 H NMR (400MHz, DMSO-d6) δ11.46 (s, 1H), 11.28 (s, 1H), 8.56 (dd, J = 8.5, 1.4Hz, 1H), 7.59-7.52 (m, 1H), 4.43 (s, 2H).

[0097] 5.1 Preparation of final product I-5

[0098] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 16 and 17, yielding 30 mg of a light yellow solid, yield 20%, Mp 148.2-148.6℃; 1H NMR (400MHz, DMSO-d6) δ11.56 (s, 1H), 11.47 (d, J = 6.7Hz, 1H), 11.28 (s, 1H), 8.51 (dd ,J=8.5,4.1Hz,1H),7.70-7.65(m,1H),7.64-7.57(m,3H),7.55-7.53(m,1H),7.51-7 .45(m,3H),7.39-7.34(m,1H),7.28(dd,J=7.9,1.6Hz,1H),5.87(d,J=6.6Hz,1H),4. 40(dd,J=13.9,6.6Hz,1H),4.26(s,2H),1.37(d,J=6.8Hz,3H),1.19(d,J=7.0Hz,3H). 13 C NMR (150MHz, DMSO) δ196.86,170.92,167.36,166.27,152.46,149.41,143.95,141.15,138.50,137.70,136.14,132.36,131.42 ,130.65,129.82,129.69,129.04,126.18,124.85,121.28,118.26,76.49,52.42,21.83,20.56.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 27 H 24 ClN7O5S2,626.1042; found,626.1054.

[0099] Example 6

[0100] Preparation of 2-oxo-2-(3-(pyridin-2-yl)ureo)ethyl(1-isobutyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-6)

[0101]

[0102] Preparation of intermediate compound 18

[0103]

[0104] The reaction method for the compound is the same as the synthesis method for intermediate 10, with iodoisobutane as the raw material. 1H NMR(400MHz,DMSO-d6)δ7.71-7.62(m,2H),7.52-7.42(m,5H),7.31-7.24(m,2H),4.27(s,1H),4.19-4.1 2(m,1H),3.59(dd,J=15.2,4.7Hz,1H),1.64-1.51(m,1H),0.71(d,J=6.6Hz,3H),0.49(d,J=6.4Hz,3H).

[0105] 6.2 Preparation of final product I-6

[0106] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 15 and 18, yielding 30 mg of white solid, yield 20%, Mp 140.2-140.7℃; 1 H NMR(400MHz,DMSO-d6)δ11.51(s,1H),11.13(s,1H),10.69(s,1H),8.32 -8.29(m,1H),7.95(d,J=8.4Hz,1H),7.85-7.78(m,2H),7.75-7.69(m,1H),7.62- 7.58(m,2H),7.55(d,J=6.7Hz,1H),7.53-7.47(m,2H),7.39-7.30(m,2H),7.16-7 .11(m,1H),5.97(s,1H),4.26(s,2H),4.15(dd,J=13.8,9.5Hz,1H),3.67(dd,J=1 3.8,5.3Hz,1H),1.64-1.55(m,1H),0.74(d,J=6.6Hz,3H),0.52(d,J=6.6Hz,3H). 13 CNMR(150MHz,DMSO)δ197.09,170.90,167.55,166.66,151.29,150.70,148.70,141.63,139.03,137.68,132.92,131.44,130.2 1,130.12,129.74,129.01,125.54,123.68,120.13,113.52,76.03,52.91,27.05,20.24,19.43.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 28 H 28 N6O3S2,561.1737; found,561.1746.

[0107] Example 7

[0108] Preparation of 2-(3-(3-cyanopyridin-2-yl)ureo)-2-oxoethyl(1-isobutyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-7)

[0109]

[0110] 7.1 Preparation of final product I-7

[0111] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 14 and 18, yielding 30 mg of yellow solid, yield 20%, Mp 121.0-121.4℃; 1 H NMR (600MHz, DMSO-d6) δ11.51(d,J=6.7Hz,1H),11.26(s,1H),10.69(s,1H),8.67(dd,J=4.9,1.6Hz,1H),8.35(dd,J=7.8,1 .6Hz,1H),7.80(d,J=8.3Hz,1H),7.74-7.70(m,1H),7.59(d,J=7.3Hz,2H),7.55(d,J=7.3Hz,1H),7.50(t,J=7.5Hz,2H),7.4 5(dd,J=7.8,4.9Hz,1H),7.36(t,J=7.4Hz,1H),7.34-7.30(m,1H),5.99(d,J=6.7Hz,1H),4.29(d,J=2.0Hz,2H),4.15(dd,J =13.8,9.5Hz,1H),3.67(dd,J=13.8,5.2Hz,1H),1.61(dq,J=12.9,6.6Hz,1H),0.74(d,J=6.7Hz,3H),0.52(d,J=6.6Hz,3H). 13 C NMR (150MHz, DMSO) δ195.86,169.94,166.47,165.57,151.90,149.40,142.24,140.56,136.60,131.84,130.35,129.14,128.66,127. 93,124.46,122.62,120.53,114.88,103.17,74.99,59.15,51.84,25.98,20.16,19.17,18.36,13.49.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 29 H 27N7O3S2,586.169; found,586.1694.

[0112] Example 8

[0113] Preparation of 2-oxo-2-(3-(pyridin-2-yl)ureo)ethyl(1-(cyclopropylmethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-8)

[0114]

[0115] 8.1 Preparation of intermediate compound 19

[0116]

[0117] The reaction method for the compound is the same as the synthesis method for intermediate 10, and the starting material is (iodomethyl)cyclopropane. 1 H NMR(400MHz,DMSO-d6)δ7.70-7.62(m,2H),7.53-7.49(m,2H),7.49-7.43(m,3H),7.32-7.22(m,2H),4.28(s,1H),4 .15(dd,J=14.2,6.9Hz,1H),3.61(dd,J=14.3,7.0Hz,1H),0.79-0.71(m,1H),0.25-0.13(m,2H),0.06-0.01(m,2H).

[0118] 8.1 Preparation of final product I-8

[0119] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 15 and 19, yielding 30 mg of white solid, yield 20%, Mp 131.9-132.5℃; 1H NMR (400MHz, DMSO-d6) δ11.50(s,1H),11.15(s,1H),10.71(s,1H),8.32-8.29(m,1H),7.95(d,J=8.4Hz,1H),7.84-7.81(m, 1H),7.80-7.76(m,1H),7.74-7.68(m,1H),7.61-7.58(m,2H),7.57-7.54(m,1H),7.50(dd,J=8.1,6.4Hz,2H),7.36(t,J=7.4 Hz,1H),7.32-7.28(m,1H),7.14-7.11(m,1H),5.96(s,1H),4.27(s,2H),4.15(dd,J=14.3,7.3Hz,1H),3.69(dd,J=14.2,6.9 Hz,1H),0.81-0.71(m,1H),0.32-0.24(m,1H),0.19(dt,J=8.6,4.3Hz,1H),0.04(dd,J=9.0,4.5Hz,1H),0.00--0.06(m,1H). 13 C NMR (150MHz, DMSO) δ196.91,170.90,167.67,165.80,151.29,150.70,148.70,141.82,139.03,138.03,132.79,131.33,130.3 5,130.00,129.73,128.97,125.67,124.21,120.14,113.52,75.97,51.27,10.23,4.34,2.93.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcdfor C 28 H 26 N6O3S2,559.1581; found,559.1599.

[0120] Example 9

[0121] Preparation of 2-(3-(3-cyanopyridin-2-yl)ureo)-2-oxoethyl(1-(cyclopropylmethyl)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-9)

[0122]

[0123] 9.1 Preparation of final product I-9

[0124] Synthesized according to step 1.3 of Example 1, replacing the raw materials with the same equivalent amounts of 14 and 19, yielding 30 mg of a light yellow solid, yield 20%, Mp 126.8-126.9℃; 1 H NMR (600MHz, DMSO-d6) δ11.50(d,J=6.6Hz,1H),11.26(s,1H),10.69(s,1H),8.67(dd,J=4.9,1.7Hz,1H),8.35(dd,J=7.8,1.7Hz,1H),7. 78(d,J=8.2Hz,1H),7.73-7.69(m,1H),7.59(d,J=7.2Hz,2H),7.55(t,J=7.3Hz,1H),7.50(t,J=7.5Hz,2H),7.45(dd,J=7.8,4.9Hz,1H), 7.36(t,J=7.5Hz,1H),7.32-7.29(m,1H),5.97(d,J=6.6Hz,1H),4.29(d,J=2.2Hz,2H),4.15(dd,J=14.3,7.3Hz,1H),3.69(dd,J=14.3,6 .9Hz,1H),0.76(tt,J=7.6,5.0Hz,1H),0.27(tt,J=8.9,4.9Hz,1H),0.18(tt,J=9.0,4.7Hz,1H),0.06-0.02(m,1H),-0.01--0.03(m,1H). 13 C NMR (150MHz, DMSO) δ169.95,169.73,166.59,164.72,150.36,149.41,142.25,140.75,136.96,131.72,130.24,129.28,128.93,128 .64,127.89,124.60,120.53,114.88,103.18,74.94,59.15,50.20,20.16,13.49,9.16,3.27,1.86.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 29 H 25 N7O3S2,584.1533; found,584.1548.

[0125] Example 10

[0126] Preparation of N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-5-(3-(2-nitrophenyl)ureo)pyrazine-2-carboxamide (I-10)

[0127]

[0128] 10.1 Preparation of intermediate compound 20

[0129]

[0130] Weigh 0.5 g (3.0 mmol) of ethyl 5-aminopyrazine-2-carboxylate and 0.7 mL (4.5 mmol) of triethylamine, dissolve them in a 100 mL round-bottom flask, add 20 mL of dichloromethane and stir to dissolve. Add 0.45 g (2.7 mmol) of 2-nitrophenol isobutyrate to the above system and react at room temperature for 12 h. A solid precipitates during the reaction, which is filtered through a sieve funnel. Add 20 mL of 2N NaOH:methanol = 1:1 solvent to the obtained solid compound and react at room temperature for 3 h. Adjust the pH to acidic by adding excess hydrochloric acid to the reaction system, concentrate the organic solvent under reduced pressure, extract three times with ethyl acetate and saturated brine, collect the organic phase, dry it with anhydrous sodium sulfate, concentrate it under reduced pressure, and purify the crude product by silica gel column chromatography (mobile phase: dichloromethane:methanol = 10:1). 1 H NMR(400MHz,DMSO-d6)δ13.38(s,1H),11.08(s,1H),10.97(s,1H),8.93(s,1H),8.87(s,1H) ,8.33(d,J=8.3Hz,1H),8.12(d,J=8.2Hz,1H),7.75(t,J=8.2Hz,1H),7.31(t,J=7.7Hz,1H).

[0131] 10.2 Preparation of intermediate compound 21

[0132]

[0133] The reaction method for the compound is the same as the synthesis method for intermediate 10, with iodomethane as the raw material. 1 H NMR (400MHz, DMSO-d6) δ7.69-7.56(m,2H),7.53-7.40(m,5H),7.31-7.23(m,2H),4.26(d,J=6.0Hz,1H),3.35(s,3H). 13 C NMR(150MHz,DMSO-d6)δ169.89,164.23,142.76,137.83,131.59,130.18,1 29.16,129.03,128.21,128.15,123.91,121.74,70.19,34.45.ESI-MS:m / z 266.0[M+H] + C16 H 15 N3O.

[0134] 10.3 Preparation of final product I-10

[0135]

[0136] Weigh 0.1 g (0.3 mmol) of intermediate compound 20, 0.1 g (0.4 mmol) of EDCI, and 0.1 g (0.5 mmol) of HOBT into a 100 mL round-bottom flask. Add 1 mL of N-methylmorpholine and 20 mL of dichloromethane. Stir at room temperature for 30 min. Then add 0.1 g (0.4 mmol) of intermediate compound 21 to the reaction system and react at room temperature for 12 h. After vacuum concentration, pass the reaction system through a column chromatography column to obtain the final product I-10, 40 mg of yellow solid, yield 40%, Mp 202.1-202.7℃. 1 H NMR (400MHz, DMSO-d6) δ11.09(s,2H),9.20(d,J=8.0Hz,1H),9.01(s,1H),8.89(s,1H),8.33(d,J=8.5Hz,1H),8.13(d,J=8.0Hz,1H) ,7.80-7.70(m,3H),7.58-7.52(m,3H),7.48-7.44(m,2H),7.40-7.39(m,2H),7.34-7.29(m,1H),5.47(d,J=8.0Hz,1H),3.44(s,3H). 13 C NMR(100MHz,DMSO-d6)δ167.47,167.28,162.73,151.95,151.70,143.00,141.39,139.53,137.95,137.79,135.34,134.23,133.65 ,132.80,131.32,130.16,129.89,128.88,128.59,125.89,125.21,124.17,122.89,67.62,35.41.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcdfor C 28 H 22 N8O5,551.1786; found,551.1781.

[0137] Example 11

[0138] Preparation of N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-5-(3-(2-(trifluoromethyl)phenyl)ureo)thiazolyl-2-carboxamide (I-11)

[0139]

[0140] 11.1 Preparation of intermediate compound 22

[0141]

[0142] The reaction method for the compound is the same as that for intermediate 20. The starting materials used were 0.5 g (2.9 mmol) of ethyl 2-aminothiazolium-5-carboxylate and 0.45 g (2.4 mmol) of m-trifluorotoluene isocyanate, yielding ethyl 5-(3-(2-(trifluoromethyl)phenyl)ureo)thiazolium-2-carboxylate without deethylation. 1 ¹H NMR (400MHz, Chloroform-d) δ 8.11–8.09 (m, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.32–7.29 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). 20 mL of a 1:1 mixture of 2N NaOH and methanol was added to the intermediate, and the reaction was carried out at room temperature for 2–3 h. Excess hydrochloric acid was added to adjust the pH to acidic, and the organic phase was concentrated under reduced pressure. The mixture was then extracted three times with saturated brine and ethyl acetate. The collected organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give intermediate 22, 0.3 g of white solid, yield 50%. ESI-MS: m / z 354.11 [M+Na] + C 12 H8F3N3O3S.

[0143] 11.2 Preparation of final product I-11

[0144] The mixture was synthesized according to step 10.3 of Example 10, with the raw material replaced by the same equivalent of 22, yielding 30 mg of white solid, yield 20%, Mp 218.7-219.1℃; 1H NMR (400MHz, DMSO-d6) δ11.46(s,1H),9.69(d,J=7.7Hz,1H),8.66(s,1H),8.45(s,1H),7.92(d,J=8.3Hz,1H),7.77-7.65(m, 4H),7.63-7.57(m,2H),7.53(d,J=7.1Hz,1H),7.47(t,J=7.5Hz,2H),7.40-7.31(m,3H),5.50(d,J=7.7Hz,1H),3.40(s,3H). 13 C NMR(100MHz,DMSO)δ167.59,166.86,160.84,142.81,137.50,134.89,133 .14,132.26,130.76,129.67,129.44,128.35,128.04,126.30,126.15(d,J C,F =5Hz), 126.07(d,J C,F =6Hz),125.09,125.03,124.43,122.38,122.09,120.99(d,J C,F =29Hz),68.55,34.84.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 28 H 21 F3N6O3S,579.1421; found,579.1402.

[0145] Example 12

[0146] Preparation of N3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-N1-(2-nitrophenyl)azacyclobutane-1,3-dicarboxamide (I-12)

[0147]

[0148] 12.1 Preparation of intermediate compound 23

[0149]

[0150] The reaction method for the compound is the same as that used in the synthesis of intermediate 20. The starting materials were 0.5 g (3.9 mmol) ethyl 3-azapyridine-carboxylate and 0.45 g (2.7 mmol) 2-nitrophenol isobutyrate, with a yield of 30%. The undeethylated 1-((2-nitrophenyl)carbamoyl)azacyclobutane-3-carboxylic acid was obtained. 1¹H NMR (400MHz, DMSO-d6) δ 9.17 (s, 1H), 7.98–7.96 (m, 1H), 7.87–7.88 (m, 1H), 7.66–7.65 (m, 1H), 7.22–7.21 (m, 1H), 4.24–4.03 (m, 6H), 3.57–3.50 (m, 1H), 1.22 (t, J = 7.3Hz, 3H). The method for deethylating this intermediate is shown in Figure 20. ESI-MS: m / z 266.10 [M+H] + C 11 H 11 N3O 5.

[0151] 12.2 Preparation of final product I-12

[0152] Synthesized according to step 10.3 of Example 10, replacing the raw material with the same equivalent of 23, yielding 30 mg of yellow solid, yield 20%, Mp 237.5-237.7℃; 1 H NMR (400MHz, DMSO-d6) δ9.41(d,J=7.9Hz,1H),9.17(d,J=2.5Hz,1H),7.96(d,J=8 .2Hz,1H),7.89(d,J=8.2Hz,1H),7.76-7.70(m,1H),7.68-7.62(m,2H),7.56-7.5 1(m,3H),7.49-7.43(m,2H),7.36-7.30(m,2H),7.19(t,J=7.8Hz,1H),5.34(d,J= 7.8Hz,1H),4.19-4.10(m,2H),4.07-3.99(m,2H),3.70-3.66(m,1H),3.38(s,3H). 13 C NMR (150MHz, DMSO) δ172.12,167.85,167.28,155.59,143.24,140.01,137.97,134.94,134.54,132.74,131.27,130.10,129.85,12 8.87,128.52,125.64,124.98,123.55,123.32,122.65,68.57,60.24,35.32,32.22,21.23,14.55.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 27 H 24 N6O5,513.1881; found,513.1864.

[0153] Example 13

[0154] Preparation of 4-(2-((1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)amino)-2-oxoethyl)-N-(2-(trifluoromethyl)phenyl)piperazine-1-carboxamide (I-13)

[0155]

[0156] 13.1 Preparation of intermediate compound 24

[0157]

[0158] The reaction method for the compound is the same as that used in the synthesis of intermediate 20, with 0.5 g (2.9 mmol) of 1-(ethoxycarbonylmethyl)piperazine as the starting material, yielding 25%. The result was ethyl 2-(4-((2-(trifluoromethyl)phenyl)carbamoyl)piperazin-1-yl)acetate, which was not deethylated. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.19 (s, 1H), 7.67 (d, J = 7.9Hz, 1H), 7.62 (t, J = 7.8Hz, 1H), 7.39 (dd, J = 12.9, 7.4Hz, 2H), 4.08 (q, J = 7.1Hz, 2H), 3.42–3.40 (m, 4H), 3.26 (s, 2H), 2.54–2.51 (m, 4H), 1.23–1.15 (m, 3H). The method for deethylating this intermediate is shown in Figure 20. ESI-MS: m / z 354.17 [M+Na] + C 14 H 16 F3N3O3.

[0159] 13.2 Preparation of final product I-13

[0160] Synthesized according to step 10.3 of Example 10, replacing the raw material with the same equivalent of 24, yielding 30 mg of a light yellow solid, yield 20%, Mp 149.2-149.5℃; 1H NMR (400MHz, DMSO-d6) δ8.87(d,J=8.2Hz,1H),8.29(s,1H),7.77-7.73(m,1H),7.69(t,J=6.5Hz,2H),7.63(d,J=8.2Hz,1H),7.54(t,J=6.7Hz,3H), 7.48(d,J=7.1Hz,2H),7.45-7.40(m,2H),7.39-7.34(m,2H),5.30(d,J=8. 2Hz,1H),3.52-3.49(m,4H),3.41(s,3H),3.35(s,2H),2.61-2.55(m,4H). 13 C NMR (150MHz, DMSO) δ170.81,169.82,167.90,167.14,156.31,143.09,137.96,137.87(d,J C,F =1Hz), 132.93(d,J C,F =43Hz),131.26,131.08,130.09,129.81,128.88,128.58,126.70(d,J C,F =3Hz), 126.44, 126.12(d,J C,F =20Hz),125.24,125.08,123.43,122.76,67.44,61.11,60.22,52.97,44.46,35.35,21.23,14.56.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 30 H 29 F3N6O3,579.2326; found,579.2340.

[0161] Example 14

[0162] Preparation of 2-(4-(2-nitrophenyl)piperazin-1-yl)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-14)

[0163]

[0164] 14.1 Preparation of intermediate compound 25

[0165]

[0166] Weigh 0.3 g (1.5 mmol) of 1-(2-nitrophenyl)piperazine, dissolve it in 5 mL of dichloromethane, and add 0.6 mL (4.0 mmol) of triethylamine. Then, add 0.2 mL (1.6 mmol) of chloroacetyl chloride dropwise to the above system at 0-5 °C and react at room temperature for 1-2 h. Wash the resulting system three times with sodium bicarbonate, collect the organic phase, and purify the crude product by silica gel column chromatography (mobile phase: dichloromethane:methanol = 20:1), with a yield of 30%. 1 H NMR(400MHz,DMSO-d6)δ7.83(d,J=8.1Hz,1H),7.64-7.57(m,1H),7.34(d,J=8.4H z,1H),7.17(t,J=7.9Hz,1H),4.42(s,2H),3.60-3.55(m,4H),3.06-2.97(m,4H).

[0167] 14.2 Preparation of final product I-14

[0168]

[0169] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 25 and 21, yielding 30 mg of yellow solid, yield 20%, Mp 128.8-129.1℃; 1 H NMR (400MHz, DMSO-d6) δ11.39(d,J=6.3Hz,1H),7.83(d,J=7.9Hz,1H),7.76-7.66 (m,2H),7.58(d,J=7.9Hz,3H),7.53(d,J=6.7Hz,1H),7.47(t,J=7.6Hz,2H),7.36 -7.31(m,3H),7.16(t,J=7.7Hz,1H),5.96(d,J=5.6Hz,1H),4.27(s,2H),3.67-3. 66(m,2H),3.58-3.57(m,2H),3.38(s,3H),3.06-3.05(m,2H),2.98-2.97(m,2H). 13C NMR (100MHz, DMSO) δ196.77,167.04,166.05,165.97,144.92,143.16,142.67,137.38,133.84,132.41,130.87,129.72,129.47,128.3 7,128.02,125.37,124.55,122.54,122.22,121.86,75.21,51.35,50.94,45.58,41.79,37.27,34.87.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 29 H 28 N6O4S2,589.1686; found,589.1675.

[0170] Example 15

[0171] Preparation of 2-(4-(2-cyanophenyl)piperazin-1-yl)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-15)

[0172]

[0173] 15.1 Preparation of intermediate compound 26

[0174]

[0175] The reaction method for the compound is the same as that for intermediate 25, using 0.3 g (1.6 mmol) of 1-(2-benzonitrile)piperazine as the starting material, with a yield of 30%. 1 H NMR (400MHz, DMSO-d6) δ7.73 (d, J = 8.1Hz, 1H), 7.62-7.59 (m, 1H), 7.18-7.11 (m, 2H), 4.44 (s, 2H), 3.70-3.61 (m, 4H), 3.18-3.13 (m, 4H).

[0176] 15.2 Preparation of final product I-15

[0177] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 26 and 21, yielding 30 mg of white solid, yield 20%, Mp 133.6-133.9℃; 1H NMR(400MHz,DMSO-d6)δ11.39(s,1H),7.74-7.69(m,3H),7.62-7.58(m,3H),7 .55(d,J=7.4Hz,1H),7.47(d,J=7.5Hz,2H),7.35-7.31(m,2H),7.19(d,J=8.2 Hz,1H),7.13(t,J=7.6Hz,1H),5.98(d,J=6.4Hz,1H),4.31(s,2H),3.75-3.72 (m,2H),3.66-3.62(m,2H),3.40(s,3H),3.22-3.19(m,2H),3.12-3.10(m,2H). 13 C NMR (100MHz, DMSO) δ196.77,167.04,166.00,154.82,142.67,137.38,134.37,134.20,132.41,130.88,129.72,129.46,128.37,128.0 3,124.55,122.44,122.22,119.38,118.09,105.05,75.21,51.35,50.86,45.68,41.80,37.27,34.87.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 30 H 28 N6O2S2,569.1788; found,569.1770.

[0178] Example 16

[0179] Preparation of 2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-16)

[0180]

[0181] 16.1 Preparation of intermediate compound 27

[0182]

[0183] The reaction method for the compound is the same as that for intermediate 25, using 0.3 g (1.8 mmol) of 1-(2-pyridyl)piperazine as the starting material, with a yield of 35%. 1H NMR (400MHz, DMSO-d6) δ8.16-8.10(m,1H),7.56-7.52(m,1H),6.85(d,J=7.8Hz,1H),6.68-6.65(m,1H),4.44(s,2H),3.55-3.46(m,8H).

[0184] 16.2 Preparation of final product I-16

[0185] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 27 and 21, yielding 30 mg of white solid, yield 20%, Mp 128.9-129.1℃; 1 H NMR(400MHz,DMSO-d6)δ11.39(d,J=6.5Hz,1H),8.11(dd,J=5.0,2.0Hz,1H),7.75 -7.67(m,2H),7.59-7.53(m,4H),7.47(t,J=7.4Hz,2H),7.37-7.29(m,2H),6.84(d ,J=8.6Hz,1H),6.66(dd,J=7.1,4.9Hz,1H),5.97(d,J=5.9Hz,1H),4.29(d,J=3.5 Hz,2H),3.66-3.64(m,2H),3.57-3.56(m,4H),3.48(t,J=5.3Hz,2H),3.38(s,3H). 13 C NMR (150MHz, DMSO) δ197.27,167.55,166.98,166.55,166.42,159.12,148.01,143.15,138.15,137.85,132.93,131.40,130.21,129.96,1 28.88,128.50,125.07,122.72,113.82,107.78,75.70,60.24,45.62,44.79,37.75,35.37,21.23,14.55.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 28 H 28 N6O2S2,545.1788; found,545.1792.

[0186] Example 17

[0187] Preparation of 2-(4-(3-nitropyridin-2-yl)piperazin-1-yl)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-17)

[0188]

[0189] 17.1 Preparation of intermediate compound 28

[0190]

[0191] The reaction method for the compound is the same as that for intermediate 25. The starting material used is 0.3 g (1.4 mmol) of 1-(3-nitropyridin-2-yl)piperazine, with a yield of 35%. 1 H NMR (400MHz, DMSO-d6) δ8.47-8.43(m,1H),8.32-8.28(m,1H),6.99-6.94(m,1H),4.44(s,2H),3.64-3.56(m,4H),3.46-3.39(m,4H).

[0192] 17.2 Preparation of final product I-17

[0193] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 28 and 21, yielding 30 mg of white solid, yield 20%, Mp 125.3-125.7℃; 1 H NMR (400MHz, DMSO-d6) δ11.40(s,1H),8.43(d,J=4.4Hz,1H),8.28(d,J=8.1H z,1H),7.73-7.68(m,2H),7.59(d,J=7.5Hz,2H),7.54(d,J=7.2Hz,1H),7.49- 7.46(m,2H),7.34-7.31(m,2H),7.00-6.92(m,1H),5.97(s,1H),4.27(s,2H) ,3.72-3.67(m,2H),3.62-3.57(m,2H),3.49-3.43(m,2H),3.39-3.36(m,5H). 13CNMR(150MHz,DMSO)δ197.27,167.54,166.59,152.60,152.44,143.17,137.87,136.39,133.12,132.92,131.39,130.22,129.97,128.88, 128.51,125.05,122.73,114.71,114.68,75.71,60.23,47.42,45.19,41.80,37.81,35.37,21.24,14.56.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 28 H 27 N7O4S2,590.1639; found,590.1657.

[0194] Example 18

[0195] Preparation of 2-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-18)

[0196]

[0197] 18.1 Preparation of intermediate compound 29

[0198]

[0199] The reaction method for the compound is the same as that for intermediate 25, using 0.3 g (1.6 mmol) of 2-piperazine nicotinamide as the starting material, with a yield of 35%. 1 H NMR(400MHz,d6)δ8.44(d,J=2.8Hz,1H),8.19-8.02(m,1H),6.97(d,J=7.5Hz,1H),4.43(s,2H),3.64-3.59(m,8H).

[0200] 18.2 Preparation of final product I-18

[0201] Synthesized according to step 1.3 of Example 1, replacing the raw materials with equivalent amounts of 29 and 21, yielding 30 mg of white solid, yield 20%, Mp 131.5-131.7℃; 1H NMR (600MHz, DMSO-d6) δ11.38(d,J=6.7Hz,1H),8.43(dd,J=4.8,1.9Hz,1H),8.10(dd,J=7.7,1. 9Hz,1H),7.74(ddd,J=8.5,6.8,1.9Hz,1H),7.70(dd,J=8.4,1.3Hz,1H),7.61-7.58(m,2H),7.57 -7.54(m,1H),7.50-7.47(m,2H),7.36-7.31(m,2H),6.96(dd,J=7.6,4.8Hz,1H),5.98(d,J=6.7 Hz,1H),4.34-4.26(m,2H)3.76-3.71(m,2H),3.70-3.68(m,2H),3.62-3.61(m,4H),3.40(s,3H). 13 CNMR(150MHz,DMSO)δ197.29,167.53,166.55,160.57,152.53,144.82,143.17,137.87,132.91,131.38,130.23,129.97,128.87, 128.51,125.04,122.73,118.23,115.48,95.08,75.71,48.24,47.82,45.54,41.91,37.81,35.37.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 29 H 27 N7O2S2,570.174; found,570.1746.

[0202] Example 19

[0203] Preparation of 2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-19)

[0204]

[0205] 19.1 Preparation of final product I-19

[0206] The synthesis was carried out according to step 1.3 of Example 1, with the raw materials replaced by equivalent amounts of 27 and 16, yielding 50 mg of white solid, yield 35%, Mp 198.7-199.3℃; 1H NMR (400MHz, DMSO-d6) δ11.34(d,J=6.5Hz,1H),8.12(dd,J=5.0,1.9Hz,1H),7.72-7.66(m,1H),7.63(d,J=8.1Hz,1 H),7.61-7.58(m,2H),7.54(td,J=7.9,6.4,3.6Hz,2H),7.49(t,J=7.4Hz,2H),7.38(t,J=7.5Hz,1H),7.29(d,J=7.7 Hz,1H),6.84(d,J=8.6Hz,1H),6.66(dd,J=7.1,4.9Hz,1H),5.88(d,J=6.5Hz,1H),4.42(p,J=6.9Hz,1H),4.34-4.2 0(m,2H),3.66(t,J=4.9Hz,2H),3.57-3.55(m,4H),3.52-3.45(m,2H),1.39(d,J=6.7Hz,3H),1.21(d,J=6.9Hz,3H). 13 CNMR(150MHz,DMSO)δ197.13,167.38,166.55,166.25,159.08,147.95,141.10,138.24,137.62,132.39,131.47,130.62,129.75,129.63, 129.06,126.22,124.82,113.89,107.84,76.19,52.49,45.62,45.12,44.80,41.89,37.60,21.77,20.51.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 30 H 32 N6O2S2,573.2101; found,573.2118.

[0207] Example 20

[0208] Preparation of 2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl(R)-(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester ((R)-I-19)

[0209]

[0210] Synthesized according to the method in Example 19. Given the seven-membered nitrogen in the structure of compound I-19. The ring contains one chiral C atom, and it is a mixture with a pair of enantiomers. We performed chiral column separation on I-19 and obtained an optically pure compound (R)-I-19. The separation method is as follows:

[0211] Column: CHIRALPAK IA (IA00CE-OB011), Column size: 0.46cm ID×25cm L,

[0212] Injection: 1μL, Mobile phase: MeOH / DCM=70 / 30 (V / V), Flow rate: 1.0mL / min,

[0213] Wavelength: UV 254nm, Temperature: 35℃, HPLC equipment: Shimadzu LC-20ATCP-HPLC-06.

[0214] Compound (R)-I-19 was prepared. ee value > 98%. Mp 113.5-113.9℃; 1 H NMR(400MHz,DMSO-d6)δ11.36(s,1H),8.15-8.10(m,1H),7.71-7.68(m,1 H),7.67-7.47(m,7H),7.41-7.37(m,1H),7.32-7.29(m,1H),6.88-6.84( m,1H),6.69-6.65(m,1H),5.90(d,J=5.1Hz,1H),4.45-4.40(m,1H),4.30 (s,2H),3.74-3.43(m,8H),1.40(d,J=5.5Hz,3H),1.23(d,J=5.7Hz,3H). 13 CNMR(150MHz,DMSO)δ197.13,167.30,166.43,166.24,159.11,148.01,141.17,138.14,137.69,132.36,131.42,130.63,129.81,129.67, 129.05,126.17,124.84,113.81,107.78,76.23,52.47,45.60,45.11,44.78,41.85,37.76,21.80,20.55.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 30 H 32N6O2S2,573.2101; found,573.2114.

[0215] Example 21

[0216] Preparation of 2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl(S)-(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester ((S)-I-19)

[0217]

[0218] Compound (S)-I-19 was prepared and resolved according to the steps of Example 20. ee value > 98%. Mp 113.1-114.6℃; 1 H NMR(400MHz,DMSO-d6)δ11.36(d,J=6.8Hz,1H),8.14-8.11(m,1H),7.69(d, J=7.2Hz,1H),7.67-7.46(m,7H),7.39(s,1H),7.31(d,J=7.5Hz,1H),6.85(d ,J=8.0Hz,1H),6.70-6.64(m,1H),5.90(d,J=5.3Hz,1H),4.45-4.40(m,1H) ,4.30(s,2H),3.58(t,J=34.3Hz,8H),1.43-1.37(m,3H),1.24-1.20(m,3H). 13 C NMR (150MHz, DMSO) δ197.13,167.30,166.43,166.24,159.10,148.00,141.17,138.15,137.69,132.36,131.42,130.63,129.81,129.67, 129.05,126.17,124.84,113.81,107.78,76.23,52.47,45.60,45.11,44.78,41.85,37.76,21.80,20.54.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 30 H 32 N6O2S2,573.2101; found,573.2112.

[0219] Example 22

[0220] Preparation of N-(1-isopropyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-4-oxo-4-(4-(pyridin-2-yl)piperazin-1-yl)butyramide (I-20)

[0221]

[0222] 22.1 Preparation of intermediate compound 30

[0223]

[0224] The reaction method for the compound is the same as that for intermediate 31, using 0.3 g (1.8 mmol) of 1-(2-pyridyl)piperazine as the starting material, with a yield of 20%. 1 H NMR (400MHz, DMSO-d6) δ8.13(dd,J=4.9,1.9Hz,1H),7.55(ddd,J=8.9,7.1,2.0Hz,1H),6.84(d,J=8.6 Hz,1H),6.66(dd,J=7.1,4.9Hz,1H),3.59-3.43(m,8H),2.58(t,J=6.5Hz,2H),2.43(t,J=6.5Hz,2H).

[0225] 22.2 Preparation of final product I-20

[0226] Synthesized according to step 10.3 of Example 10, replacing the raw materials with the same equivalent amounts of 30 and 21, yielding 30 mg of white solid, yield 20%, Mp 218.7-218.9℃; 1 H NMR (400MHz, DMSO-d6) δ9.04(d,J=8.2Hz,1H),8.04(d,J=4.9Hz,1H),7.61(t,J=7.7Hz,1H),7.54(d,J=8 .2Hz,1H),7.47(t,J=8.6Hz,4H),7.40(t,J=7.3Hz,2H),7.31(t,J=7.6Hz,1H),7.21(d,J=7.8Hz,1H),6.7 5(d,J=8.6Hz,1H),6.58(t,J=6.0Hz,1H),5.20(d,J=8.2Hz,1H),4.34(p,J=7.0Hz,1H),3.45(q,J=7.2,6 .0Hz,6H),3.37(d,J=5.2Hz,2H),3.27(m,2H),2.43(m,2H),1.31(d,J=6.7Hz,3H),1.12(d,J=6.8Hz,3H). 13C NMR (150MHz, DMSO) δ172.25,170.50,167.71,166.75,159.20,148.03,141.26,138.07,137.89,132.09,131.20,130.72,129.60,129.49, 129.01,126.12,124.84,113.70,107.69,68.51,52.21,45.12,44.85,41.24,30.68,28.06,21.83,20.64.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 31 H 34 N6O3,539.2765; found,539.2776.

[0227] Example 23

[0228] Preparation of N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-2-(2-(2-(trifluoromethyl)phenyl)acetamide)-1H-imidazol-5-carboxamide (I-21)

[0229]

[0230] 23.1 Preparation of intermediate compound 31

[0231]

[0232] Weigh 0.3 g (1.3 mmol) of compound 2-(trifluoromethyl)phenylacetic acid, 0.3 g (1.5 mmol) of EDCI, and 0.2 g (1.5 mmol) of HOBT into a 100 mL round-bottom flask. Add 1 mL of N-methylmorpholine and 20 mL of dichloromethane. Stir at room temperature for 30 min, then add 0.2 g (1.3 mmol) of compound 2-amino-1H-imidazolium-5-carboxylic acid ethyl ester to the reaction system and react at room temperature for 12 h. Concentrate the reaction system under reduced pressure and purify by silica gel column chromatography to obtain 0.3 g of intermediate, 50% yield. The obtained undeethylated 2-(2-(2-(trifluoromethyl)phenyl)acetamido)-1H-imidazolium-5-carboxylic acid ethyl ester is: 1¹H NMR (400MHz, Chloroform-d) δ 7.73 (d, J = 7.9Hz, 1H), 7.60 (t, J = 7.7Hz, 1H), 7.50–7.46 (m, 3H), 7.29–7.27 (m, 2H), 4.36–4.28 (m, 2H), 4.07 (s, 2H), 1.36–1.31 (m, 3H). The method for deethylating this intermediate is shown in Figure 20. 1 H NMR (400MHz, DMSO-d6) δ11.92(s,1H),7.72(d,J=7.7Hz,1H),7.65(d,J=6.4Hz,1H),7.58-7.40(m,4H),4.02(s,2H).

[0233] 23.2 Preparation of final product I-21

[0234] Synthesized according to step 10.3 of Example 10, replacing the raw materials with the same equivalent amounts of 31 and 21, yielding 30 mg of white solid, yield 20%, Mp 154.8-155.6℃; 1 H NMR (400MHz, DMSO-d6) δ12.04(s,1H),11.70(s,1H),8.45(d,J=8.3Hz,1H),7.73(t,J=5.3Hz,2H),7.67(t,J=8.5Hz,2H),7 .56-7.50(m,4H),7.47-7.43(m,3H),7.37(d,J=5.5Hz,2H),7.31(m,1H),5.39(d,J=8.2Hz,1H),3.99(s,2H),3.41(s,3H). 13 C NMR (100MHz, DMSO) δ171.44,169.23,167.73,166.93,161.95,143.06,141.08,138.06,133.98,133.64(d,J C,F =8Hz), 132.74(d,J C,F =7Hz),131.92,131.22,130.08,129.81,128.87,128.65,128.03,127.45,126.16(d,J C,F =6Hz), 124.01(d,J C,F =230Hz),117.22,66.97,35.37,29.44.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd forC 29 H 23F3N6O3,561.1857; found,561.1840.

[0235] Example 24

[0236] Preparation of N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)-1-(2-(2-nitrophenyl)acetyl)piperidine-4-carboxamide (I-22)

[0237]

[0238] 24.1 Preparation of intermediate compound 32

[0239]

[0240] The compound was synthesized using the same method as intermediate 31, with 0.3 g (1.9 mmol) of ethyl 4-piperidinecarboxylate and 0.3 g (1.7 mmol) of o-nitrophenylacetic acid, yielding 40%. Undeethylated 1-(2-(2-nitrophenyl)acetyl)piperidine-4-carboxylic acid ethyl ester was obtained: ESI-MS: m / z 321.25 [M+H] + C 16 H 20 N₂O₅. The method for deethylating this intermediate is shown in Figure 20. ESI-MS: m / z 315.10 [M+Na] + C 14 H 16 N2O5.

[0241] 24.2 Preparation of final product I-22

[0242] The mixture was synthesized according to step 10.3 of Example 10, with the raw materials replaced by equivalent amounts of 32 and 21, yielding 30 mg of white solid, yield 20%, Mp 198.1-199.0℃; 1 H NMR (400MHz, DMSO-d6) δ9.18(d,J=8.2Hz,1H),8.03(d,J=8.1Hz,1H),7.73(t,J =7.7Hz,1H),7.68-7.65(m,2H),7.56-7.50(m,4H),7.48-7.45(m,3H),7.36-7.3 0(m,2H),5.31(d,J=8.1Hz,1H),4.30-4.27(m,1H),4.16-4.06(m,2H),3.38(s,3 H),3.23-3.06(m,2H),2.83-2.60(m,2H),1.91-1.70(m,2H),1.59-1.39(m,2H).13 C NMR (150MHz, DMSO) δ174.79,168.12,167.40,167.11,149.69,143.31,138.01,133.97,133.84,132.66,132.39,131.21,130.07,129.83, 128.86,128.55,124.96,124.90,122.62,68.15,45.06,41.49,38.38,35.27,29.39,28.99,28.86,28.46.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 30 H 29 N5O5,540.2241; found,540.2244.

[0243] Example 25

[0244] Preparation of 2-(4-(5-chloro-2-nitrobenzoyl)piperazin-1-yl)-N-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)acetamide (I-23)

[0245]

[0246] 25.1 Preparation of intermediate compound 33

[0247]

[0248] The reaction method for the compound is the same as that for intermediate 31, using 0.3 g (1.5 mmol) of 5-chloro-2-nitrobenzoic acid and 0.3 g (1.7 mmol) of 1-(ethoxycarbonylmethyl)piperazine, with a yield of 40%. 1 H NMR (400MHz, DMSO-d6) δ8.26 (d, J = 8.9Hz, 1H), 7.87-7.78 (m, 2H), 4.20 (s, 2H), 3.94-3.92 (m, 4H), 3.63-3.56 (m, 4H).

[0249] 25.2 Preparation of final product I-23

[0250] Synthesized according to step 10.3 of Example 10, replacing the raw materials with the same equivalent amounts of 33 and 21, yielding 30 mg of yellow solid, yield 20%, Mp 155.7-156.3℃; 1H NMR (400MHz, DMSO-d6) δ8.89(d,J=8.2Hz,1H),8.24(d,J=8.7Hz,1H),7.81-7.76(m,2H),7.73(d,J=7.2Hz,1H),7.67(d,J=8.1 Hz,1H),7.55-7.53(m,3H),7.47(d,J=7.3Hz,2H),7.37-7.32(m,2H),5.28(d,J=8.1Hz,1H),3.39(s,3H),3.34-3.26(m,10H). 13 C NMR (100MHz, DMSO) δ169.24,167.38,166.65,163.84,143.82,142.58,139.70,137.46,134.14,132.21,130.75,130.17,129.59,129.3 1,128.36,128.08,127.87,126.83,124.57,122.24,66.95,60.41,52.19,51.84,46.51,41.39,34.83.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 29 H 27 ClN6O5,575.1804; found,575.1794.

[0251] Example 26

[0252] Preparation of 2-(4-(5-chloro-2-nitrobenzoyl)piperazin-1-yl)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminodithioester (I-24)

[0253]

[0254] 26.1 Preparation of intermediate compound 34

[0255]

[0256] Weigh 0.5 g (2.7 mmol) of compound 5-chloro-2-nitrobenzoic acid, 0.6 g (3.3 mmol) of EDCI, and 0.4 g (3.3 mmol) of HOBT into a 100 mL round-bottom flask. Add 1 mL of N-methylmorpholine and 20 mL of dichloromethane, and stir at room temperature for 30 min. Add 0.5 g (2.7 mmol) of compound 1-Boc-piperazine to the above reaction system, and react at room temperature for 12 h. After purification by silica gel column chromatography, concentrate the organic phase under reduced pressure, add 5 mL of DCM and 2 mL of TFA to the organic phase, and react at room temperature for 8–12 h. Adjust the pH to neutral by adding sodium bicarbonate to the system, extract with DCM and saturated brine, collect the organic phase, dry it with anhydrous sodium sulfate, concentrate it under reduced pressure, and purify it by silica gel column chromatography to give intermediate 30, 0.2 g of a pale yellow solid, in 40% yield. 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.21 (d, J = 8.7Hz, 1H), 7.76 (d, J = 8.9Hz, 1H), 7.72 (s, 1H), 3.60–3.39 (m, 2H), 3.17–3.05 (m, 2H), 2.82–2.68 (m, 2H), 2.65–2.56 (m, 2H). 0.2 g (0.7 mmol) of intermediate 30 was weighed and placed in a 100 mL round-bottom flask. 5 mL of dichloromethane was added to dissolve it. 0.1 g (0.8 mmol) of chloroacetyl chloride was slowly added dropwise to the above system, followed by 0.2 g (2.0 mmol) of triethylamine. The reaction was carried out in an ice-water bath at 5 °C for 1 h. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain intermediate 31, with a yield of 50%. 1 H NMR (400MHz, DMSO-d6) δ8.25(d,J=8.6Hz,1H),7.80(t,J=4.6Hz,2H),4.43(s,2H),3.25-3.13(m,4H),2.40-2.29(m,4H).

[0257] 26.2 Preparation of final product I-24

[0258] Synthesized according to step 1.3 of Example 1, replacing the raw materials with the same equivalent amounts of 34 and 21, yielding 30 mg of white solid, yield 20%, Mp 165.8-166.0℃; 1H NMR (400MHz, DMSO-d6) δ11.38(s,1H),8.24(d,J=7.8Hz,1H),7.80-7.70(m,4H),7.60-7.56(m,5H),7.33-7.31(m ,2H),5.96(s,1H),4.28(d,J=27.2Hz,2H),3.74-3.65(m,2H),3.60-3.55(m,2H),3.38(s,3H),3.30-3.26(m,4H). 13 C NMR (100MHz, DMSO) δ167.04,166.02,164.14,164.08,143.79,142.67,139.78,137.37,133.88,132.42,130.88,130.28,129.72,129.46,1 28.37,128.02,127.93,126.88,124.55,122.22,75.26,67.00,56.00,41.48,37.31,34.86,25.10,18.53.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 30 H 27 ClN6O5S2,651.1246; found,651.1244.

[0259] Example 27

[0260] Preparation of 2-((5-chloro-2-nitrophenyl)amino)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza-3-yl)aminomethyl dithioester (I-24)

[0261]

[0262] 27.1 Preparation of intermediate compound 35

[0263]

[0264] Compound reaction methods, such as the synthesis method of intermediate 14. 1 H NMR (400MHz, DMSO-d6) δ10.82(s,1H),8.22-7.90(m,2H),7.60-7.43(m,1H),4.45(s,2H).

[0265] 27.2 Preparation of final product I-25

[0266] Synthesized according to step 1.3 of Example 1, replacing the raw materials with the same equivalent amounts of 35 and 21, yielding 30 mg of a light yellow solid, yield 20%, Mp 196.1-196.6℃; 1 H NMR (400MHz, DMSO-d6) δ11.54(d,J=6.7Hz,1H),10.72(s,1H),8.08(d,J=8 .9Hz,1H),8.04(d,J=2.3Hz,1H),7.77-7.69(m,2H),7.63-7.58(m,2H),7.5 7-7.53(m,1H),7.49(dd,J=8.2,6.6Hz,2H),7.44(dd,J=8.9,2.4Hz,1H),7. 38-7.31(m,2H),5.97(d,J=6.7Hz,1H),4.25(d,J=4.4Hz,2H),3.40(s,3H). 13 CNMR(100MHz,DMSO)δ196.72,167.58,167.42,166.47,143.16,139.49,139.24,137.85,133.58,132.93,131.39,130 .22,129.97,128.87,128.49,127.63,125.21,125.06,123.79,122.73,75.98,35.37.HRMS(ESI-Orbitrap)m / z:[M+H] + :calcd for C 25 H 20 ClN5O4S2,554.0718; found,554.0727.

[0267] Example 28

[0268] The 1,4-benzodiazepine described in this invention Tests on the binding activity of similar compounds to the ANXA3 protein.

[0269] Experimental methods: The binding activity of different compounds in Table 1 to ANXA3 protein was determined by surface plasmon resonance (SPR).

[0270] Specific methods:

[0271] Main reagents used in the experiment: recombinant ANXA3 protein (expressed and purified by the E. coli BL21 system), ethanolamine (Cytiva, USA), CM5 chip (Cytiva, USA), amino coupling kit (Cytiva, USA), Biacore T200 biomacromolecule interaction analyzer (GE, USA).

[0272] Experimental Procedure: The experiment was conducted using a Biacore T200 instrument. 50 μg / mL of ANXA3 protein was coupled to a CM5 sensor chip. PBS (pH 7.4) was used as the mobile phase. Solutions of different compounds at varying concentration gradients were flowed through the chip surface at a flow rate of 30 μL / min. The binding time was 30 s, dissociation time was 45 s, regeneration time was 30 s, and the injection interval was 60 s. Signal changes were recorded. The data were analyzed using Biacore T200 software to perform dual-state fitting and curve analysis to calculate the affinity (K0) between different compounds and ANXA3 protein. D The experimental results are shown in Table 1.

[0273] Structure-activity relationship studies showed that, compared with the positive compound (R)-SL18, the different compounds in Table 1 all exhibited better or comparable levels of binding activity with ANXA3 protein. Among them, the preferred compound with the best activity was I-19, whose binding activity with ANXA3 protein was 10 times that of the positive control (R)-SL18.

[0274] Example 29

[0275] Table 1 shows the in vitro anti-proliferation experiments of different compounds on TNBC tumor cells.

[0276] Experimental methods: The effect of the target compound on the proliferation of MDA-MB-231 and MDA-MB-468 cells was determined by the CCK-8 assay. The IC50 was calculated. 50 Values ​​were used to evaluate the in vitro antitumor proliferative activity of the compounds.

[0277] Specific methods:

[0278] Cell culture: The culture conditions for the two cell lines MDA-MB-468 and MDA-MB-231 were as follows: 1) Complete culture medium preparation: 10% fetal bovine serum, 1% penicillin and streptomycin and high glucose DMEM medium; 2) Culture environment: 37℃, saturated humidity, 5% CO2 incubator.

[0279] Main reagents used in the experiment: CCK8 (Meilun Biotechnology Co., Ltd.), fetal bovine serum (Meilun Biotechnology Co., Ltd.), DMEM culture medium (Meilun Biotechnology Co., Ltd.).

[0280] Experimental Procedure: 7000-9000 tumor cells were seeded into each well of a 96-well plate. After 12 hours, the culture medium was aspirated, and medium containing compound I was added as the experimental group. Medium containing 0.1% DMSO was used as the control group. Culture medium was added to wells without cell seeding as the blank group. After incubation for 72 hours, 100 μL of freshly prepared 10% CCK8 solution was added to each well of the experimental group, and the plates were incubated at 37°C and 5% CO2 for 2 hours. The absorbance (OD) of the 96-well plate at 450 nm was measured using a Thermo Fisher microplate reader. The inhibition rate of the target compound at this concentration was calculated as 1 - [(OD of the experimental group of the compound)]. 450 Value - Mean OD of blank group 450 (value) / (mean OD of negative control group) 450 Value - Mean OD of blank group 450 [Value] × 100%, IC 50 The values ​​were calculated using GraphPad Prism 8 software. The antiproliferative assays for each compound were independently repeated three times, and the mean and standard deviation were calculated. The experimental results are shown in Table 1.

[0281] Structure-activity relationship studies showed that, compared with the positive compound (R)-SL18, the target compounds exhibited comparable or better anti-proliferative activity against TNBC cell lines MDA-MB-231 and MDA-MB-468. Among them, the preferred compound with the best activity was I-19, whose anti-TNBC activity was 3 to 4 times that of the positive control (R)-SL18.

[0282] Example 30

[0283] The 1,4-benzodiazepine described in this invention Tests on the degradation activity of similar compounds on ANXA3 protein.

[0284] Experimental methods: Western blotting (WB) was used to determine the degradation activity of different compounds in Table 1 on ANXA3 protein.

[0285] Specific methods:

[0286] Cell culture: The cell culture method is the same as in Example 29.

[0287] Main reagents used in the experiment: RIPA lysis buffer, protease inhibitor PMSF, BeyoColor TMColor prestained protein molecular weight standards (10-170kD), ECL chromogenic solution, horseradish peroxidase-labeled goat anti-mouse IgG (H+L), and β-Tubulin Mouse Monoclonal Antibody were all purchased from Beyotime Biotechnology; ANXA3 monoclonal antibody was purchased from Santa Cruz Biotechnology.

[0288] Experimental procedure: Plate 6-well plates, with 3 × 10⁶ cells per well. 5 Cells were transferred to an incubator and cultured overnight. The next day, the culture medium was aspirated, and cultured for another 24 hours with culture medium containing different drug concentrations. The supernatant was then aspirated, and the cells were washed three times with PBS. RIPA (medium) lysis buffer containing 1% protease inhibitor PMSF was added to each well, and the cells were placed on ice for 10 minutes for lysis. The lysed cells were then transferred to 1.5 mL EP tubes and swirled for 20-30 seconds, then placed on ice for 10 minutes. This process was repeated three times. After the last swirling, the cells were centrifuged at 10,000 rpm for 10 minutes. 120 μL of the supernatant was collected and mixed with 30 μL of 5× Loading (protein denaturant). The mixture was then incubated at 100°C for 10 minutes and placed on ice for subsequent experiments. For short-term storage, the cells were stored at -20°C. Simultaneously, 1.5 mm 12% stacking and separating gels, 1× electrophoresis buffer, and electrotransfer buffer (200 mL + 800 mL anhydrous ethanol + transfer powder) were prepared.

[0289] Adjust the loading volume according to the test concentration to ensure a total protein loading of 50 μg. Set the electrophoresis voltage to 100V and run for 60-70 minutes. Trim off excess portions of the upper and lower gels. Immerse the separating gel in pre-cooled transfer buffer at 4°C. Cut the required size PVDF membrane and activate it in methanol solution for 2-3 minutes. Lay out the membrane in the following order from bottom to top: foam pad, 3 layers of thick filter paper, separating gel, PVDF membrane, 3 layers of thick filter paper, foam pad. Clamp the transfer clamps and place the membrane in pre-cooled transfer buffer. Then, place the entire transfer apparatus in ice water and transfer at 110V. The membrane was incubated for 90 min, then the PVDF membrane was placed in blocking solution for 60 min. The PVDF membrane was then removed and incubated overnight at 4°C with the primary antibody on a shaker. The next day, it was washed three times with 1×TBST washing solution for 10 min each time. After that, horseradish peroxidase-labeled IgG secondary antibody was added and incubated on a shaker at room temperature for 60 min. Then, it was washed three times with 1×TBST washing solution for 10 min each time. ECL chromogenic solution was evenly dropped onto the PVDF membrane, and the membrane was developed and photographed on a chemiluminescence imaging system. The grayscale value was analyzed using ImageJ software. The experimental results are shown in Table 1.

[0290] Table 1. Affinity, degradation activity, and inhibitory activity of compound formula I against ANXA3 and TNBC tumor cell lines.

[0291]

[0292]

[0293] Degradation activity DC 50 :*: 1-10μM, **: <1μM.

[0294] Structure-activity relationship studies showed that the different compounds in Table 1 exhibited micromolar degradation activities against ANXA3 protein, comparable to the positive control (R)-SL18.

[0295] Example 31

[0296] The 1,4-benzodiazepine described in this invention Degradation selectivity test of preferred compound I-19 for the annexin family.

[0297] Experimental methods: Western blotting (WB) was used to determine the degradation selectivity of the preferred compound I-19 for the annexin family (ANXA1-11 and ANXA13).

[0298] The specific method is the same as in Example 30.

[0299] The main reagents used in the experiment were: ANXA1 (mouse monoclonal antibody), ANXA2 (mouse monoclonal antibody), ANXA4 (mouse monoclonal antibody), ANXA5 (mouse monoclonal antibody), ANXA6 (mouse monoclonal antibody), ANXA7 (rabbit polyclonal antibody), ANXA9 (rabbit polyclonal antibody), ANXA10 (mouse monoclonal antibody), ANXA11 (rabbit polyclonal antibody), and ANXA13 (rabbit polyclonal antibody), all purchased from Proteintech; ANXA3 (mouse monoclonal antibody) purchased from Santa Cruz Biotechnology; and ANXA8 (rabbit polyclonal antibody) purchased from Signalway Antibody.

[0300] The experimental results are shown in Figure 1 .

[0301] The results showed that treatment with a high concentration of 6 μM resulted in a 70% degradation of ANXA3 protein; no degradation was observed in ANXA1, ANXA2, ANXA5, ANXA6, ANXA7, and ANXA8 proteins; and approximately 20%-30% degradation was observed in ANXA4, ANXA10, and ANXA13 proteins, with no significant degradation effect. ANXA9 protein expression was very low in these cells with no significant change; while ANXA11 showed a more significant decrease of approximately 50%. This preferred compound, I-19, primarily targets the degradation of ANXA3 protein and exhibits good family selectivity in vitro, which is beneficial for improving drug activity and avoiding adverse reactions or side effects.

[0302] Example 32

[0303] The preferred compound I-19 described in this invention was used in an in vitro experiment to induce apoptosis in TNBC tumor cells.

[0304] Specific methods:

[0305] Cell culture: Same as in Example 29.

[0306] Main reagents used in the experiment: FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen).

[0307] Plan the cells into 6-well plates, with 1.5 × 10⁶ cells per well. 5 Cells were transferred to an incubator and cultured for 24 hours. The next day, the supernatant was aspirated, and 2 mL of complete culture medium containing the target compound at high, medium, and low concentrations was added to each well. For the negative control group, 2 mL of complete culture medium containing 0.1% DMSO was added, and the cells were cultured for another 48 hours. The supernatant was not discarded. Six 5 mL sterile EP tubes were used, and the supernatant was aspirated into the tubes. The cells were washed twice with PBS, and the PBS used for washing was also aspirated into the EP tubes. During the third wash, 1 mL of PBS was added, the cells were mixed, and then transferred to 5 mL EP tubes. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded. 100 μL of Binding Buffer was added to each well containing cell pellet to resuspend the cells. Then, 5 μL of 0.5 mg / mL PI and 5 μL of Annexin V-FITC were added and mixed thoroughly. 400 μL of 1× Binding Buffer was added and gently mixed. After incubation at room temperature in the dark for 15 min, flow cytometry was performed within 1 hour to stimulate cell detection. The experimental results are shown in the figure below. Figure 2 .

[0308] Experimental results showed that the preferred compound I-19 could induce late apoptosis and early apoptosis in MDA-MB-468 and MDA-MB-231 cells in a concentration-dependent manner, respectively. The overall apoptosis rate of both cells was significantly increased, and both were superior to the positive control compound (R)-SL18.

[0309] Example 33

[0310] The preferred compound I-19 of this invention was used in an in vitro experiment to inhibit the clonogenic formation of TNBC tumor cells.

[0311] Specific methods:

[0312] Cell culture: Same as in Example 29.

[0313] Main reagents used in the experiment: 4% paraformaldehyde (Meilun Biotechnology Co., Ltd.), crystal violet (Beyotime Biotechnology Co., Ltd.).

[0314] Experimental Procedure: 1000 tumor cells were seeded into 6-well plates. After cell attachment, the culture medium was aspirated. Different concentrations of compound I-19 solution dissolved in the culture medium were added to the cells (2 mL / well) as experimental groups. Culture medium containing 0.1% DMSO was added to the cells (2 mL / well) as the control group. The culture medium containing the compound was changed every 3 days, and culture was continued for 2–3 weeks. Frequent observation was performed. When visible clones appeared in the control group, culture was terminated. The supernatant was discarded, and the cells were carefully washed twice with PBS. Cells were fixed with 4% paraformaldehyde for 30 min. The fixative was then aspirated, and the cells were stained with 0.1% crystal violet for 30 min. The staining solution was then slowly washed away with running water. After drying, the cells were photographed, and the number of clones was counted directly with the naked eye. Finally, the colony formation rate was calculated. Colony formation rate = (number of clones / number of seeded cells) × 100%. The experimental results are shown in […]. Figure 2 .

[0315] Experimental results showed that the preferred compound I-19 had better clonal activity against TNBC cell lines (MDA-MB-468 and MDA-MB-231) than the positive control (R)-SL18, especially at high concentrations against MDA-MB-468 cells, where the preferred compound was twice as effective as the positive control.

[0316] Example 34

[0317] Experiments on the ability of the preferred compound I-19 of this invention to inhibit the migration of TNBC tumor cells in vitro.

[0318] Specific methods:

[0319] Cell culture: Same as in Example 29.

[0320] Main reagents used in the experiment: Transwell chambers (BD Biocoat), crystal violet (Beyotime Biotechnology Co., Ltd.).

[0321] Experimental procedure: Add 680 μL of serum-containing culture medium to the lower chamber of the Transwell plate (i.e., the 24-well plate), and place the chamber into the wells containing the culture medium. Add 3 × 10⁻⁶ μL of serum-containing culture medium to the upper chamber of each well. 4Tumor cells were used as experimental groups. Cells were resuspended in 300 μL of serum-free culture medium containing different concentrations of compound I-19 as the culture medium resuspending solution. Cells were also resuspended in 300 μL of serum-free culture medium containing 0.1% DMSO as the culture medium control group. Before addition, the cells and serum-free culture medium were mixed thoroughly and added vertically to the upper chamber; incubation was performed for 24 h. Subsequently, the chamber was removed, the culture medium and cells in the upper chamber were discarded, and the remaining culture medium and cells in the upper chamber were gently wiped away with a cotton swab soaked in PBS. The chamber was then inverted and air-dried for 2 min, followed by fixation in 680 μL of 4% paraformaldehyde for 30 min. The chamber was then removed and air-dried for 2 min, followed by staining in 680 μL of 0.1% crystal violet solution for 30 min. The crystal violet in the upper chamber was gently wiped away with a cotton swab soaked in PBS. The lower chamber could be gently rinsed in PBS, taking care not to let the cotton swab touch the cells in the lower chamber. After rinsing, the chamber was inverted and air-dried. Take photos and count 3-5 random fields of view under a microscope. The experimental results are shown below. Figure 2 .

[0322] Experimental results showed that, at low, medium and high concentrations, the preferred compound I-19 reduced cell migration more effectively than the positive control (R)-SL18, indicating that the preferred compound's ability to reduce cell migration at low, medium and high concentrations was far superior to that of the positive control.

[0323] Example 35

[0324] Experimental study on the ability of the preferred compound I-19 of this invention to inhibit the invasion of TNBC tumor cells in vitro.

[0325] Specific methods:

[0326] Cell culture: Same as in Example 29.

[0327] The main reagents used in the experiment were the same as those in Example 34.

[0328] Experimental procedure: Except for pre-laying Matrigel in the upper chamber of the Transwell and changing the incubation time from 24h to 36h, the other operations were the same as in Example 34. The experimental results are shown in […]. Figure 2 .

[0329] Experimental results showed that the preferred compound I-19 reduced the invasive ability of the cell line by much more than the positive control (R)-SL18 at low, medium and high concentrations, indicating that the preferred compound has better anti-TNBC cell line invasion activity than the positive control.

[0330] Example 36

[0331] In vivo pharmacokinetic studies of the preferred compound I-19 described in this invention.

[0332] Specific methods:

[0333] Experimental Procedure: I-19 was dissolved in physiological saline containing 10% DMSO and 10% Tween-80 to prepare a 20 mg / kg dosing solution, which was administered intraperitoneally to 6-8 week old female Balb / C mice. Blood samples were collected via the orbital sinus at 0.083, 0.17, 0.25, 0.5, 1, 2, and 4 hours after administration. Blood drug concentrations were determined by LC-MS / MS (AB 4000Q-TRAP instrument, column size: Agilent, Zorbax sl-phenyl 3.5 μm, 3.0 × 150 mm, mobile phase: acetonitrile / water = 95% / 5%, flow rate: 0.9 mL / min, column temperature: 40℃). Compound I-16 was used as an internal standard at concentrations of 5, 20, 50, 100, 200, 500, and 1000 ng / mL to prepare a standard curve (R = 0.9962). The experimental results are shown in Table 2.

[0334] Table 2. In vivo PK properties of compound I-19

[0335] #imgpt98#

[0336] Experimental results show that the area under the concentration-time curve (AUC) of the preferred compound I-19 is [missing information]. (0-∞) The concentration was 1063.89 h·μg / L. The maximum plasma concentration (C0) was... max The concentration was 628.67 μg / L. The half-life (T) was... 1 / 2 The duration is 1.04h.

[0337] Example 37

[0338] In vivo pharmacodynamic studies of the preferred compound I-19 against TNBC.

[0339] Specific methods:

[0340] Cell culture: Same as in Example 29.

[0341] The main reagents used in the experiment were: Tween 80 and sterile physiological saline (0.9% NaCl), both purchased from Beyotime Biotechnology; and female Nude BALB / c nude mice (4-6 weeks old), purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0342] Experimental procedure: MDA-MB-231 cells were cultured in 15cm dishes to the logarithmic growth phase. The total number of cells in the cell suspension was determined using a cell counting chamber. 200μL of cell suspension was collected subcutaneously on the right side of the back of each female nude mouse, for a total of 9 × 10⁹ cells. 6 Each cell was monitored daily to observe tumor growth until the tumor reached 40-90 mm. 3 (Length × Width) 2 / 2) Nude mice were randomly divided into 6 groups of 6 mice each: blank solvent group, docetaxel DOC group (10 mg / kg), positive control (R)-SL18 group (20 mg / kg), I-19 group (20 mg / kg), I-19 group (10 mg / kg), and I-19 group (5 mg / kg). The drug preparation solvent was 10% DMSO + 10% Tween 80 + 80% sterile saline. The first day of drug administration was recorded. The positive control group and I-19 group were administered intraperitoneally daily, while the DOC group was administered intraperitoneally once every 5 days. The weight and tumor volume of the nude mice were measured and recorded every two days. After 21 days of continuous drug administration, the experiment was terminated. The nude mice were sacrificed and dissected. The heart, liver, spleen, lung, kidney organs and tumor tissue of each nude mouse were collected, photographed and weighed, and the TGI was calculated. A portion of organs and tumors were fixed in 4% paraformaldehyde. For group I-19 (20 mg / kg), three tumor samples were flash-frozen in liquid nitrogen and stored at -80°C. TGI = (1 - average tumor weight in experimental group / average tumor weight in control group) × 100%. Approximately 10 mg of tumor tissue was taken, added to 300 μL of IRA lysis buffer, homogenized, lysed, centrifuged, and the supernatant was collected. Protein quantification was performed using the BCA method. Subsequent procedures were performed according to the Western blot assay. Experimental results are shown in [Figure number missing]. Figure 3 .

[0343] The experimental results showed that the preferred compound I-19, when administered at low (5 mg / kg) and medium (10 mg / kg) doses, significantly increased the tumor inhibition rate (TGI = 96%) compared to the control group, with the tumor volume being smaller. Similarly, the high dose of I-19 (20 mg / kg) was also significantly better than the positive control group (R)-SL18 administered at 20 mg / kg, with statistically significant differences.

[0344] Example 38

[0345] The mouse organ HE staining toxicity test of the preferred compound I-19 for in vivo administration according to the present invention.

[0346] Specific methods:

[0347] Experimental Procedure: The heart, liver, spleen, lung, and kidney organs fixed with 4% paraformaldehyde were rinsed in running water until the formaldehyde odor disappeared. They were then placed in tissue embedding frames and soaked in 75% ethanol for 1 hour, followed by 85% ethanol for 15 minutes, then 95% ethanol for 45 minutes (repeated twice), and finally 100% ethanol for 40 minutes (repeated three times). The tissues were then removed and soaked in xylene for 30 minutes, followed by benzene wax (xylene:paraffin = 1:1) for 1 hour. Finally, they were soaked in paraffin I at 65°C for 1 hour, and then in paraffin II at 65°C overnight. The next day, the embedding frames were removed and air-dried for 30 minutes until completely dry, followed by sectioning. The sections were soaked in xylene for 15 minutes (repeated three times), and then subsequently soaked twice in the following solvents in sequence: anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 75% ethanol for 5 minutes, and double-distilled water for 5 minutes. Then, after staining with hematoxylin for 2 minutes, rinse with running water and soak in PBS for 5 minutes. Next, stain with eosin for 1 minute, rinse with running water, and soak in anhydrous ethanol and xylene for 5 minutes each, respectively. Finally, mount with neutral resin, air dry, and observe and photograph using an Olympus inverted phase-contrast fluorescence microscope. The experimental results are shown below. Figure 4 .

[0348] Experimental results showed that daily administration of the preferred compound I-19 for 21 days did not cause significant damage or inflammation to the organs of mice.

Claims

1. A 1,4-benzodiazepine targeting ANXA3 protein The compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, is characterized in that, The 1,4-benzodiazepine The class of compounds is the compound shown in formula (I). in, 1) When R 1 When X is selected from ethyl, isopropyl, isobutyl, or methylcyclopropane, X is a linking group, selected from: R 2 For side chains, selected from: Among them, R 3 R 4 It can be arbitrarily selected from hydrogen, halogen, nitro or cyano, and R 3 R 4 Single substitution or simultaneous dual substitution is permitted; 2) When R 1 When X is selected from methyl or isopropyl, X is a linking group, selected from: R 2 Selected from: Among them, R 5 R 6 Arbitrarily selected from hydrogen, trifluoromethyl, nitro, cyano, or halogen, and R 5 R 6 Allowed for either single substitution or simultaneous di substitution; R 7 It is selected from hydrogen, nitro or cyano.

2. The 1,4-benzodiazepine according to claim 1 The compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, is characterized in that, The 1,4-benzodiazepine The class of compounds is selected from one of the following structures I-1 to I-9:

3. The 1,4-benzodiazepine according to claim 1 The compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, is characterized in that, The 1,4-benzodiazepine The class of compounds is selected from the compounds shown in I-10 to I-25 below:

4. The 1,4-benzodiazepine according to claim 1, 2, or 3 Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of medicaments for the prevention and / or treatment of breast cancer.

5. The use according to claim 4, characterized in that, The breast cancer is selected from one of the molecular subtypes of triple-negative breast cancer, Luminal A, Luminal B, or Her2+ breast cancer.

6. The use according to claim 4, characterized in that, The 1,4-benzodiazepine Use of class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of ANXA3 protein degrading agents.

7. The use according to claim 4, characterized in that, The 1,4-benzodiazepine Use of a class of compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, in the preparation of medicaments targeting ANXA3 protein degradation for the prevention and / or treatment of triple-negative breast cancer.

8. The use according to claim 4, characterized in that, The drug contains one or more pharmaceutically acceptable carriers, including one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, flavorings, or sweeteners.

9. An antitumor pharmaceutical composition that exerts its antitumor effect by acting as an ANXA3 degrading agent, characterized in that, The antitumor drug composition comprises 1,4-benzodiazepine as described in claim 1, 2, or 3. The active ingredient is a compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof.

10. The antitumor pharmaceutical composition according to claim 9, which exerts its antitumor effect by acting as an ANXA3 degrading agent, characterized in that, The antitumor drug composition is in the form of tablets, capsules, pills, injections, sustained-release formulations, sprays, or nano-drug delivery systems.

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