ANXA3 protein targeted degradation chimera, and preparation method and application thereof

Abstract

The present application relates to ANXA3 protein targeted degradation chimera and its preparation method and application. Specifically, the present application relates to ANXA3 protein targeted degradation chimera, or its pharmaceutically acceptable salt, or its stereoisomer, or a pharmaceutical composition composed of a medically acceptable carrier, and the use in preparing ANXA3 protein targeted degradation chimera and the use in preparing drugs for preventing and / or treating breast cancer. The present application utilizes the PROTAC technology to provide a kind of ANXA3 protein targeted degradation chimera, which can combine ANXA3 protein, selectively degrade ANXA3 protein, inhibit the proliferation of breast cancer cells in vitro, and play the role of anti-breast cancer. Breast cancer refers to breast cancer molecular subtypes such as triple negative, Luminal A type, Luminal B type, Her2+ type.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and in particular relates to an ANXA3 protein-targeted degradation chimera, its preparation method and application. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer in which the expression of ER, PgR and HER-2 is negative in immunohistochemical staining. It is characterized by high malignancy, rapid development and strong invasiveness. The five-year survival rate is less than 40%, the recurrence rate is high, and the three-month mortality rate after recurrence is as high as 75%. It is the most important molecular subtype causing breast cancer death (Clinical Cancer Research, 2007, 13(15): 4429-4434; Journal of Clinical Oncology, 2006, 24(36): 5652-5657).

[0003] Clinically, the main drug treatments for TNBC are chemotherapy and immunotherapy. Chemotherapy is the most important drug treatment, but long-term use has disadvantages such as large side effects, poor tolerability, and easy development of drug resistance, and it is difficult to cure the tumor and prevent metastasis (Clin Cancer Res, 2013, 19(19): 5533–5540). Currently approved monoclonal antibodies for immunotherapy, such as atezolizumab and pembrolizumab, have improved the survival of patients well (Cancerimmunology, immunotherapy: CII, 2020, 70(3): 607-617), but they have the disadvantage of low response rate in the population. Goxatuzumab, an antibody-drug conjugate targeting Trop-2, is mainly used for the second-line treatment of patients with advanced or metastatic TNBC. It has not been approved for the treatment of early TNBC, which has the disadvantage of a narrow target population (The New England Journal of Medicine, 2021, 384(16): 1529-1541).

[0004] Antibody drugs have drawbacks such as complex preparation processes, demanding storage conditions, strong immunogenicity, and high prices, which limit their use. Currently, only two small molecule targeted drugs for TNBC, the PARP inhibitors olaparib and talapazolidone, have been approved (The New England Journal of Medicine, 2017, 377(6):523-533; The New England Journal of Medicine, 2018, 379(8):753-763). However, their applicable patient population is extremely limited, and they have high rates of drug side effects, resulting in a significant unmet clinical need for TNBC patients. Consequently, their prognosis is extremely poor, with a median overall survival of only about 18 months or less and a 5-year survival rate of less than 20% (Nature Reviews Clinical Oncology, 2016, 13:674-690; Journal of Clinical Oncology, 2019, 37(15_suppl):e12549.). Therefore, the development of effective targeted small molecule drugs for TNBC is of great scientific significance and has become a global challenge that urgently needs to be addressed in clinical practice.

[0005] ANXA3 belongs to the Annexin A (ANXA) family, which contains 12 homologous proteins capable of using Ca2+ to... 2+ It binds to acidic phospholipids in a dependent manner and participates in a series of Ca2+ processes on the cell membrane surface. 2+ The physiological activities involved include vesicle transport, membrane fusion during exocytosis, signal transduction, and Ca2+. 2+ The formation of channels and the interaction between cytoskeletal proteins (Nature reviews Molecular cell biology, 2005, 6(6):449-461.). Studies have found that abnormally high expression of ANXA3 is closely related to the progression of TNBC, and reducing the expression level of ANXA3 can effectively inhibit the proliferation and metastasis of TNBC, which is a potential therapeutic target for TNBC (Oncology reports, 2017, 37(1):388-398; Clinical breast cancer, 2018, 18(4):713-719; Cell Death & Disease, 2018, 9(2):1-11).

[0006] Literature reports that (R)-SL18 is a lead compound with ANXA3 binding activity and an in vivo and in vitro anti-TNBC phenotype, exhibiting ubiquitination and degradation of ANXA3. The binding activity of (R)-SL18 to ANXA3 is 0.58 μM; its antiproliferative activities against TNBC cell lines MDA-MB-468 and MDA-MB-231 are 1.56 μM and 2.35 μM, respectively; and its degradation activity against ANXA3 in the MDA-MB-231 cell line is DC. 50 The concentration was 3.17 μM; in the TNBC patient PDX model, the tumor inhibition rate reached 62% after treatment with 20 mg / kg of (R)-SL18 (Acta Pharmaceutica Sinica B, 2023, 13(4): 1686-1698). However, (R)-SL18 also has a certain binding and degradation ability to the ANXA3 homolog ANXA6, among which K d，ANXA6 Treatment of MDA-MB-231 cell lines with 0.57 μM and 5 μM for 24 h resulted in a 75% degradation of ANXA6 protein. Since ANXA6 has a certain inhibitory effect on TNBC (Oncotarget 2019, 10(2):133-151; Cells, 2020, 9(8):1855), the degradation of ANXA6 may promote TNBC proliferation. Therefore, improving the selective degradation of ANXA3 / ANXA6 by (R)-SL18 is of great significance. The structural formula of (R)-SL18 is shown below:

[0007]

[0008] Protein-targeting chimeras (PROTACs) are heterobifunctional molecules that can simultaneously bind to an E3 ubiquitin ligase and a target protein (POI). Once a ternary complex (POI-PROTACs-E3) is formed, the lysine residues exposed on the POI are ubiquitinated by the E3 ubiquitin ligase, thereby degrading the POI. On the one hand, PROTACs rely on the mechanism of temporarily recruiting the target protein for degradation to drive the phenotype and lead to the pharmacological effect, known as "event-driven" pharmacology. This allows PROTACs to achieve selective degradation of the target protein without requiring high concentrations, high ligand affinity, or long binding times. On the other hand, by designing different PROTACs to adjust the conformation of the POI-PROTACs-E3 complex, selective degradation of the POI can be achieved. Summary of the Invention

[0009] Given the current lack of effective selective degradation of ANXA3 protein in existing technologies, this invention provides an ANXA3 protein-targeted degradation chimera, its preparation method, and its applications. Specifically, this invention relates to an ANXA3 protein-targeted degradation chimera, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical composition thereof consisting of a chimera and a medically acceptable carrier, as well as its use in the preparation of the ANXA3 protein-targeted degradation chimera and its use in the preparation of drugs for the prevention and / or treatment of breast cancer.

[0010] This invention employs PROTAC technology to structurally modify (R)-SL18, linking the ANXA3 ligand and the E3 ubiquitin ligase CRBN ligand (lenalidomide, pomalidomide, or their derivatives) through a series of linkers to obtain chimeric PROTACs capable of degrading ANXA3 protein, thereby achieving anti-TNBC proliferation activity and selective ANXA3 degradation capability. This protein-targeting degradation chimeric is a heterobifunctional small molecule capable of targeting and degrading ANXA3 protein, inducing death in TNBC cell lines, and therefore can be used for the treatment of TNBC.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] The first objective of this invention :

[0013] A chimeric ANXA3 protein targeting degradation, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, is provided; wherein the chimeric ANXA3 protein targeting degradation is a compound of formula (I).

[0014]

[0015] Wherein, R1 is -H or -NO2;

[0016] L is a connector, selected from one of the following structures:

[0017] Among them, R2 is selected from C 1-7 Straight-chain alkyl or alkoxy, R3 is selected from Furthermore, the triazole group or amide group is attached to the 3-position of the benzene ring, and R3 is attached to the 3- or 4-position of the benzene ring. It is linked to the CRBN ligand at the 4'- or 5'-position;

[0018] X is CH2 or C=O.

[0019] Furthermore, the compound is specifically selected from the following compounds:

[0020] In some implementations, when R1 = H, L is When linked to a CRBN ligand at the 4'- or 5'- position, the ANXA3 protein-targeting degradation chimera and its pharmaceutically acceptable salt can be further described as formula (Ia).

[0021]

[0022] In some more specific embodiments, formula (Ia) is further selected as one of the following structures Ia-1 to Ia-12:

[0023]

[0024]

[0025] In some implementations, when R1 = H or NO2, L is When the CRBN ligand is attached at the 4'-position, the ANXA3 protein-targeting degradation chimera and its pharmaceutically acceptable salt can be further described as formula (Ib).

[0026]

[0027] In some more specific implementations, formula (Ib) is further selected as the following structure Ib-1 or Ib2.

[0028]

[0029] In some implementations, when R1 = H, L is When the CRBN ligand is attached at the 4'-position, the ANXA3 protein-targeting degradation chimera and its pharmaceutically acceptable salt can be further described as formula (Ic).

[0030]

[0031] In some more specific embodiments, formula (Ic) is further selected as one of the following structures Ic-1 to Ic-4:

[0032]

[0033]

[0034] The solvates of the ANXA3 protein-targeted degradation chimeras of this invention are also within the scope of protection of this invention, with water, ethanol or methanol being the preferred solvents.

[0035] Pharmaceutically acceptable salts are those compounds that, 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 risk-benefit ratio, and are typically water- or oil-soluble or dispersible, and can be effectively used for their intended purpose. Some compounds or stereoisomers of this invention contain basic groups such as amino groups 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 alkyl ammonium 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.

[0036] 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.

[0037] The second objective of this invention is:

[0038] The invention provides the use of the ANXA3 protein-targeting degradation chimera, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, as described in the first objective of this invention, in the preparation of medicaments for the prevention and / or treatment of breast cancer.

[0039] In some embodiments of the present invention, the use of the ANXA3 protein-targeting degradation chimera, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, which are involved in the first objective of the present invention, in the preparation of selective ANXA3 degrading agents is provided.

[0040] Based on the in vitro antiproliferative activity assays of TNBC cell lines MDA-MB-231 and MDA-MB-468, the IC50 of the ANXA3 protein-targeted degradation chimera was demonstrated. 50 The activity values ​​are shown in Table 1.

[0041] Based on the in vitro ANXA3 and ANXA6 protein degradation activity results, the degradation percentages of ANXA3 and ANXA6 proteins after treatment of the MDA-MB-231 cell line at a concentration of 5 μM for 24 h by the ANXA3 protein targeted degradation chimera are shown in Table 2.

[0042] 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.

[0043] The ANXA3 protein-targeting degradation chimera, or its pharmaceutically acceptable salt, or its stereoisomer, or its solvate, can degrade ANXA3 protein, downregulate ANXA3 protein expression levels, and exert an anti-breast cancer effect.

[0044] 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+.

[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 the ANXA3 protein-targeting degradation chimera, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof as the active ingredient.

[0046] In some embodiments of the present invention, the antitumor drug composition is a tablet, capsule, pill, injection, sustained-release formulation, spray, or nano-drug delivery system.

[0047] The third objective of this invention is:

[0048] The present invention provides a method for preparing the ANXA3 protein-targeted degradation chimera as described in the first objective, comprising the following steps:

[0049] Synthesis of key intermediates (III and IV) for S1 and ANXA3 ligands (see Scheme 1 and Scheme 2)

[0050]

[0051] The preparation method of Scheme 1 includes the following steps:

[0052] Step 1: 2-Bromoacetamide (I) reacts with oxalyl chloride in 1,2-dichloroethane at 90°C, and then undergoes a nucleophilic addition reaction with aniline compound II to give a compound containing a terminal alkyne as shown in formula III;

[0053] The compound shown in intermediate IV was synthesized according to the method described in ACS Medicinal Chemistry Letters, 2015, 6(5): 523-527.

[0054] Synthesis of S2, CRBN ligand-linker-azide compound (Formula VIII) (see Scheme 2)

[0055]

[0056] The preparation method of Scheme 2 includes the following steps:

[0057] Step 2: The compound shown in Formula V undergoes a substitution reaction with the bromoacyl chloride shown in Formula VI in tetrahydrofuran to give the compound shown in Formula VII;

[0058] Step 3: The compound shown in Formula VII undergoes a nucleophilic substitution reaction with sodium azide in the presence of a catalyst (such as sodium iodide, potassium iodide, or tetra-n-butylammonium iodide) in a solvent (such as N,N-dimethylformamide or dimethyl sulfoxide) to give the compound shown in Formula VIII.

[0059] S3. Synthesis of the target compound of formula (Ia) (see Scheme 3)

[0060]

[0061] The Scheme 3 preparation method includes the following steps:

[0062] Step 4: The compounds shown in Formula III-1 and Formula VIII undergo a Click reaction in a solvent (such as N,N-dimethylformamide, tetrahydrofuran, and / or water) under the action of a catalyst (such as a combination of cuprous iodide, copper sulfate pentahydrate, and sodium ascorbate) to obtain the compound shown in Formula IX.

[0063] Step 5: The compound shown in Formula IV undergoes an addition reaction with carbon disulfide in a base (such as potassium phosphate, sodium phosphate, triethylamine or diisopropylethylamine) in a solvent (such as N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran or acetonitrile), followed by a nucleophilic substitution reaction with the compound shown in Formula IX in the same reaction system to obtain the target compound Ia.

[0064] S4. The synthesis of target compound (Ib) is similar to that of target compound (Ia). However, unlike target compound (Ia), the starting material is the compound shown in formula III-2, and the benzene ring substitution site of the compound shown in formula VIII is only at the 4'- position.

[0065] S5. Synthesis of compounds of general formula (Ic) (see Scheme 4)

[0066]

[0067] The Scheme 4 preparation method includes the following steps:

[0068] Step 6: The compound shown in Formula V and the compound shown in Formula X undergo an acid-amine condensation reaction in a solvent (such as N,N,N',N'-tetramethylchlorourea hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole or carbonyl diimidazole) and a base (such as diisopropylethylamine, triethylamine or N-methylimidazole) in a solvent (such as N,N-dimethylformamide, dimethyl sulfoxide or acetonitrile) to obtain the compound shown in Formula XI.

[0069] Step 7: The compound shown in Formula XI is deprotected by an acid (such as trifluoroacetic acid or hydrogen chloride) to obtain the compound shown in Formula XII.

[0070] Step 8: The specific synthesis method is the same as in Step 6;

[0071] Step 9: The compound shown in Formula XII is reduced by a reducing agent (such as tetrahydroxydiboron, iron powder or zinc powder and a combination of acetic acid or hydrochloric acid) to obtain the compound shown in Formula XI;

[0072] Step 10: The specific synthesis method is the same as in Step 1;

[0073] Step 11: The specific synthesis method is the same as in step 5.

[0074] The design strategy of the ANXA3 protein degradation targeted degradation chimera provided by this invention is to select the potential anti-TNBC drug (R)-SL18 reported in the literature as a lead, based on the binding mode of (R)-SL18 and ANXA3, focus on the aromatic ring structure exposed to solvent in the molecule, and use the PROTAC strategy to modify its chemical structure, and finally obtain the ANXA3 protein degradation targeted degradation chimera molecule, which improves the anti-TNBC activity of (R)-SL18 and is conducive to achieving selective degradation of ANXA3.

[0075] Compared with existing technologies, the ANXA3 protein degradation-targeting chimeric structures and their pharmaceutically acceptable salts provided by this invention have not been reported by Scifinder. Furthermore, their in vitro antiproliferative activity and ANXA3 degradation activity in TNBC cell lines have not been reported, making them a novel class of ANXA3-targeting degradative agents. These compounds can bind to ANXA3 protein, selectively degrade ANXA3 protein, and inhibit the proliferation of breast cancer cells in vitro, thus exerting an anti-breast cancer effect. Detailed Implementation

[0076] The present invention will now be described in detail with reference to specific embodiments. However, these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0077] Example 1: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) The synthetic route for 3-yl)aminodithiocarbamate (Ia-1) is as follows:

[0078]

[0079]

[0080] Step 1, Preparation of Intermediate 3

[0081] Bromoacetamide (1, 3.8 g, 27.5 mmol) was dissolved in 50 mL of 1,2-dichloroethane, and oxaloyl chloride (5 mL) was added dropwise at 0 °C. The mixture was then heated to reflux for 5 h, and the solvent was removed by concentration under reduced pressure to obtain intermediate 2-bromoacetyl isocyanate, which was used directly in the next step without separation. 3-ethynylaniline (2.0 g, 17.1 mmol) was dissolved in dichloromethane, and the 2-bromoacetyl isocyanate obtained in the previous step was added dropwise at 0 °C. The mixture was then slowly heated to room temperature and stirred for 0.5 h. After filtration, the filter cake was washed successively with dichloromethane, icy diethyl ether, or methyl tert-butyl ether, and dried to give intermediate 3, a white solid of 4.33 g, with a yield of 90.2%. ESI-MS: m / z 303.0 [M+Na] + C 11 H9N2O2.

[0082] Step 2, Preparation of Intermediate 6

[0083] Compound 4 (lenalidomide, 518.52 mg, 2.0 mmol) was suspended in 20 mL of anhydrous tetrahydrofuran, and 3-bromopropionyl chloride (472.17 mg, 3.0 mmol) was added. The mixture was heated under reflux for 3 h, the solvent was removed under reduced pressure, and 30 mL of methyl tert-butyl ether was added and stirred. The mixture was filtered and dried to give compound 6 as a white solid, 624.3 mg, in 85.3% yield. ESI-MS: m / z 394.15 [M+H] + C 16 H 16 BrN3O4.

[0084] Step 3, Preparation of Intermediate 7

[0085] Compound 5a (394.2 mg, 1.0 mmol), sodium azide (390.0 mg, 6.0 mmol), and tetra-n-butylammonium iodide (74.0 mg, 0.2 mmol) were dissolved in 5 mL of N,N-dimethylformamide and reacted at 65 °C for 12 h. Most of the solvent was removed under reduced pressure, and the mixture was stirred with water for 1 h. The mixture was filtered, and the filter cake was collected, washed several times with water, and dried to give compound 7 as a white solid (352.5 mg), with a yield of 92.0%. ESI-MS: m / z 379.23 [M+Na] + C 16 H 16 N6O4

[0086] Step 4, Preparation of Intermediate 8

[0087] Copper sulfate pentahydrate (10.0 mg, 0.04 mmol) and benzoic acid (2.4 mg, 0.02 mmol) were thoroughly dissolved in 5.0 mL of a mixture of tetrahydrofuran and water (3:1, v / v). Sodium ascorbate (16.0 mg, 0.08 mmol) was then added, and the mixture was stirred at room temperature for 5 min. Intermediate 3 (67.4 mg, 0.24 mmol) and intermediate 7 (71.2 mg, 0.20 mmol) were then added, and the mixture was stirred overnight. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography to obtain intermediate 8 as a white solid, with a yield of 73.5%. HRMS (ESI) for C 27 H 25 BrN8O6[M+Na] + :calcd659.0973,found 659.0974.

[0088] Preparation of intermediate 9

[0089] Following the synthetic method described in ACS Medicinal Chemistry Letters, 2015, 6(5):523-527, intermediate 9 was obtained as a brown oily substance. ESI-MS: m / z 266.0 [M+H] + C 16 H 15 N3O.

[0090] Step 5, Preparation of target compound Ia-1

[0091] Intermediate 8 (38.2 mg, 0.144 mmol) was dissolved in 1.0 mL of anhydrous N,N-dimethylformamide, and anhydrous potassium phosphate (30.6 mg, 0.144 mmol) was added. After stirring at room temperature for 15 min, carbon disulfide (52 μL, 5.0 mmol) was added, and the mixture was stirred for 1 h. Then, intermediate 8 (76.32 mg, 0.12 mmol) was added, and the mixture was stirred overnight at room temperature. Most of the solvent was removed under reduced pressure, and the mixture was separated by HPLC to obtain the target compound Ia-1 as a white solid of 23.3 mg, with a yield of 21.6%. 1 H NMR (400MHz, DMSO) δ11.50(d,J=6.5Hz,1H),11.00(s,1H),10.96(s,1H),10.42(s,1H),9.80(s,1H),8.61(s,1H),8.01(s,1H) ,7.81(d,J=7.5Hz,1H),7.71(m,2H),7.60(d,J=7.5Hz,2H),7.55–7.51(m,2H),7.51–7.47(m,4H),7.44–7.36(m,1H),7.36–7.2 9(m,2H),5.99(d,J=6.3Hz,1H),5.14(dd,J=13.3,5.0Hz,1H),4.44(t,J=6.7Hz,2H),4.36(q,J=17.4Hz,2H),4.28(s,2H),3.39 HRMS(ESI)for C 44 H 39 N 11 O7S2[M+H] + :calcd 898.2548,found898.2536.

[0092] Example 2: 2-(3-(3-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-2-oxoethoxy)ethyl)carbamoyl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) The preparation route of 3-yl)aminodithiocarbamate (Ic-1) is as follows:

[0093] Step 1, Preparation of Intermediate 10:

[0094] Compound 4 (259.15 mg, 1.0 mmol) was suspended in 10 mL of anhydrous acetonitrile. 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (264.0 mg, 1.20 mmol) and N-methylimidazolium (318.5 μL, 4.00 mmol) were added in portions with stirring at room temperature. After the addition was complete, the mixture was reacted overnight at room temperature. The solvent was removed under reduced pressure, and the solution was purified by column chromatography to give 392.3 mg of a white solid, in 85.3% yield. ESI-MS: m / z 497.30 [M+Na] + C 22 H 28 N4O7.

[0095] Step 2, Preparation of intermediate 11:

[0096] Intermediate 10 (100.0 mg, 0.2 mmol) was dissolved in 2 mL of methanol, and 2 mL of a 4N dioxane solution of hydrogen chloride was added. The mixture was stirred at room temperature for 30 min, and the solvent and excess hydrogen chloride were removed under reduced pressure to obtain the hydrochloride salt of intermediate 11, which was used directly in the next reaction. ESI-MS: m / z 361.20 [M+H] + C 17 H 20 N4O5.

[0097] Step 3, Preparation of intermediate 12:

[0098] The hydrochloride salt of intermediate 11 (80.1 mg, 0.2 mmol) was redissolved in 1.0 mL of anhydrous N,N-dimethylformamide. Then, m-nitrobenzoic acid (36.0 mg, 0.21 mmol) and diisopropylethylamine (110 μL, 0.63 mmol) were added. After complete dissolution, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 91.5 mg, 0.24 mmol) was added in portions at room temperature. After the addition was complete, the mixture was stirred at room temperature for 2 h. The reaction was quenched by adding 5 mL of saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (5 mL × 6). The organic phases were combined and washed successively with 10% citric acid solution, water, and saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The mixture was purified by column chromatography to obtain intermediate 12 as a white solid (53.8 mg). The overall yield of the two steps was 52.8%. ESI-MS: m / z 502.19 [M+Na] + C 24 H 25 N5O6.

[0099] Step 4, Preparation of intermediate 13:

[0100] Intermediate 12 (110.1 mg, 0.21 mmol) and 4,4'-bipyridine (1.60 mg, 0.011 mmol) were dissolved in 1 mL of anhydrous N,N-dimethylformamide. After stirring at room temperature for 5 min, tetrahydroxydiboron (66.0 mg, 0.74 mmol) was added in portions, and the mixture was stirred at room temperature for 30 min. Most of the solvent was removed under reduced pressure, and the mixture was purified by column chromatography to give intermediate 13 as a white solid, 90.3 mg, with a yield of 89.7%. ESI-MS: m / z 510.25 [M+Na] + C 24 H 23 N5O8

[0101] Step 5, Preparation of intermediate 14:

[0102] For the specific method, refer to step 3 of Example 1, and replace intermediate 2 with intermediate 13.

[0103] Step 6, Preparation of target compound Ic-1:

[0104] For the specific method, refer to step 5 of Example 1, and replace intermediate 8 with intermediate 14. 1 H NMR (400MHz, DMSO) δ11.53(d,J=6.8Hz,1H),11.01(s,2H),10.44(s,1H),9.76(s,1H),8.61(s,1H),7.93(s,1H),7.75–7. 66(m,4H),7.61–7.56(m,3H),7.57–7.52(m,2H),7.53–7.45(m,3H),7.41–7.35(m,1H),7.35–7.29(m,2H),5.98(d,J=6.7H z,1H),5.12(dd,J=13.3,5.0Hz,1H),4.38(t,J=12.0Hz,2H),4.27(s,2H),4.16(s,2H),3.68(t,J=5.5Hz,2H),3.52(dd,J= 10.1,4.6Hz,2H),3.39(s,3H),2.96–2.84(m,1H),2.64–2.53(m,1H),2.44–2.21(m,1H),2.03–1.95(m,1H); HRMS(ESI)for C 44 H 41 N9O9S2[M+H] + :calcd 904.2541, found 904.2552.

[0105] Example 3: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-2)

[0106]

[0107] The preparation method is the same as in Example 1, except that compound 5 in step 2 is replaced with 4-bromobutyryl chloride. 1 H NMR (400MHz, DMSO) δ11.53(d,J=6.7Hz,1H),11.03(s,1H),10.99(s,1H),10.45(s,1H),9.84(s,1H),8.66(s,1H),8.04(s,1H),7.87-7.80(m,J=7. 2Hz,1H),7.78-7.72(m,1H),7.72-7.67(m,1H),7.64-7.61(m,1H),7.61- 7.59(m,1H),7.59-7.55(m,2H),7.55-7.51(m,2H),7.51-7.47(m,4H),7. 44-7.36(m,1H),7.36-7.29(m,2H),6.00(d,J=6.7Hz,1H),5.15(dd,J=13 .3,5.1Hz,1H),4.50(t,J=6.7Hz,2H),4.38(q,J=17.6Hz,2H),4.29(s,2H ),3.40(s,3H),2.98-2.87(m,1H),2.68-2.57(m,,1H),2.45-2.39(m,2H) ,2.39-2.29(m,1H),2.27-2.17(m,2H),2.08-1.97(m,1H); HRMS(ESI)for C 45 H 41 N 11 O7S2[M+Na] + :calcd 934.2524, found 934.2529.

[0108] Example 4: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-3)

[0109]

[0110] The preparation method is the same as in Example 1, except that compound 5 in step 2 is replaced with 5-bromopentanoyl chloride. 1 H NMR(400MHz,DMSO)δ11.53(d,J=6.7Hz,1H),11.03(s,1H),10.99(s,1H),10 .45(s,1H),9.84(s,1H),8.66(s,1H),8.04(s,1H),7.87-7.80(m,J=7.2Hz, 1H),7.78-7.72(m,1H),7.72-7.67(m,1H),7.64-7.61(m,1H),7.61-7.59(m ,1H),7.59-7.55(m,2H),7.55-7.51(m,2H),7.51-7.47(m,4H),7.44-7.36(m ,1H),7.36-7.29(m,2H),5.99(d,J=6.3Hz,1H),5.14(dd,J=13.3,5.0Hz,1H ),4.44(t,J=6.7Hz,2H),4.36(q,J=17.4Hz,2H),4.28(s,2H),3.39(s,3H), 2.95-2.86(m,1H),2.63-2.56(m,1H),2.43(t,J=7.2Hz,2H),2.40-2.31(m, 1H),2.06-1.99(m,1H),1.98-1.90(m,2H),1.66-1.59(m,2H); HRMS(ESI)for C 46 H 43 N 11 O7S2[M+H] + :calcd 926.2861,found926.2832.

[0111] Example 5: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-6-oxohexyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-4)

[0112]

[0113] The preparation method is the same as in Example 1, except that compound 5 in step 2 is replaced with 6-bromohexanoyl chloride. 1 H NMR (400MHz, DMSO) δ11.51(d,J=6.7Hz,1H),11.02(s,1H),10.98(s,1H),10.43(s,1H),9.76(s,1H),8.61(s,1H),8.00(s,1H),7 .84-7.79(m,1H),7.77-7.69(m,2H),7.64-7.60(m,2H),7.59-7.54(m,2H),7.53-7.47(m,5H),7.43-7.38(m,1H),7.38-7.31(m, 2H),5.99(d,J=6.7Hz,1H),5.14(dd,J=13.2,5.1Hz,1H),4.45-4.34(m,4H),4.29(s,2H),3.41(s,3H),2.98-2.87(m,1H),2.64( t,J=19.2Hz,1H),2.43-2.30(m,3H),2.08-1.99(m,1H),1.95-1.86(m,2H),1.67-1.55(m,2H),1.35-1.27(m,2H); HRMS(ESI)for C 47 H 45 N 11 O7S2[M+H] + :calcd 940.3018, found 940.3020.

[0114] Example 6: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-7-oxoheptyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-5)

[0115]

[0116] The preparation method is the same as in Example 1, except that compound 5 in step 2 is replaced with 7-bromoheptyl chloride. 1 H NMR (400MHz, DMSO) δ δ 11.53 (d, J = 6.7 Hz, 1H), 11.03 (s, 1H), 10.99 (s, 1H), 10.44 (s, 1H), 9.78 (s, 1H), 8.62 (s, 1H), 8.02 (s, 1H), 7.84-7.79(m,1H),7.77-7.69(m,2H),7.64-7.60(m,2H),7.59-7.54(m,2H),7.53-7.47(m,5H),7.43-7.38(m,1H),7.38-7.31(m ,2H),6.00(d,J=6.7Hz,1H),5.16(dd,J=13.2,5.1Hz,1H),4.45-4.34(m,4H),4.29(s,2H),3.41(s,3H),2.98-2.87(m,1H),2.64 (t,J=19.2Hz,1H),2.43-2.30(m,3H),2.08-1.99(m,1H),1.95-1.86(m,2H),1.67-1.55(m,2H),1.43-1.29(m,4H); HRMS(ESI)for C 48 H 47 N 11 O7S2[M+H] + :calcd 976.2994, found 976.3007.

[0117] Example 7: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-8-oxooctyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-6)

[0118]

[0119] The preparation method is the same as in Example 1, except that compound 5 in step 2 is replaced with 8-bromooctanoyl chloride. 1H NMR (400MHz, DMSO) δ11.53(d,J=6.7Hz,1H),11.05(s,1H),10.99(s,1H),10.44(s,1H),9.77(s,1H),8.62(s,1H),8.03(s,1H),7.86-7.79 (m,1H),7.78-7.73(m,1H),7.72-7.67(m,1H),7.65-7.62(m,1H),7.62-7.60(m,1H),7.59-7.55(m,2H),7.53-7.47(m,5H),7.43-7.37(m, 1H),7.37-7.30(m,2H),6.00(d,J=6.7Hz,1H),5.16(dd,J=13.2,5.1Hz,1H),4.45-4.34(m,4H),4.29(s,2H),3.41(s,3H),2.99-2.87(m,1 H),2.64(t,J=18.9Hz,1H),2.43-2.28(m,3H),2.09-1.99(m,1H),1.94-1.82(m,2H),1.67-1.55(m,2H),1.48-1.27(m,6H); HRMS(ESI)for C 49 H 49 N 11 O7S2[M+H] + :calcd 968.3331,found 968.3335.

[0120] Example 8: 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)amino)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-7)

[0121]

[0122] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are 3-(5-amino-1-oxoisoindolinyl)piperidine-2,6-dione and 4-bromobutyryl chloride. 1H NMR (400MHz, DMSO) δ11.52(d,J=6.6Hz,1H),10.98(s,1H),10.43(s,1H),10.28(s,1H),8.66-8.61(m,1H),8.01(s,1H),7 .96(s,1H),7.76-7.71(m,1H),7.71-7.67(m,1H),7.66-7.62(m,1H),7.62-7.52(m,5H),7.52-7.44(m,3H),7.42-7.36(m ,1H),7.36-7.28(m,2H),5.98(d,J=6.6Hz,1H),5.08(dd,J=13.4,5.1Hz,1H),4.48(t,J=6.9Hz,2H),4.43-4.22(m,3H),3 .39(s,3H),2.98-2.86(m,1H),2.69-2.54(m,1H),2.45-2.33(m,3H),2.25-2.16(m,2H),2.04-1.93(m,1H); HRMS(ESI)for C 45 H 42 N 11 O7S2[M+Na] + :calcd 934.2524,found934.2532.

[0123] Example 9:

[0124] 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-8)

[0125]

[0126] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are 3-(5-amino-1-oxoisoindolinyl)piperidine-2,6-dione and 5-bromobutyryl chloride. 1H NMR (400MHz, DMSO) δ11.54(d,J=6.8Hz,1H),10.99(s,2H),10.42(s,1H),10.21(s,1H),8.60(s,1H),7.98(s,2H),7.94(s,1 H),7.73-7.65(m,2H),7.64-7.59(m,1H),7.59-7.57(m,1H),7.57-7.54(m,2H),7.54-7.50(m,2H),7.48-7.43(m,3H),7.38- 7.27(m,3H),5.95(d,J=6.7Hz,1H),5.06(dd,J=13.2,4.8Hz,1H),4.44-4.19(m,6H),3.36(s,3H),2.94-2.79(m,1H),2.63- 2.52(m,1H),2.40-2.26(m,3H),2.01-1.92(m,1H),1.92-1.81(m,2H),1.73-1.54(m,2H),1.35-1.23(m,2H); HRMS(ESI)forC 46 H 43 N 11 O7S2[M+H] + :calcd 926.2861, found 926.2890.

[0127] Example 10:

[0128] 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)amino)-7-oxoheptyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-9)

[0129]

[0130] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are 3-(5-amino-1-oxoisoindolinyl)piperidine-2,6-dione and 7-bromoheptanoyl chloride. 1H NMR (400MHz, DMSO) δ11.54(d,J=6.8Hz,1H),10.99(s,2H),10.42(s,1H),10.21(s,1H),8.60(s,1H),7.98(s,2H),7.94(s,1H),7. 73-7.65(m,2H),7.64-7.59(m,1H),7.59-7.57(m,1H),7.57-7.54(m,2H),7.54-7.50(m,2H),7.48-7.43(m,3H),7.38-7.27(m,3H) ,5.95(d,J=6.7Hz,1H),5.06(dd,J=13.2,4.8Hz,1H),4.39(s,1H),4.38-4.33(m,1H),4.24(s,1H),3.36(s,3H),2.94-2.79(m,1H) ,2.63-2.52(m,1H),2.40-2.26(m,3H),2.01-1.92(m,1H),1.92-1.81(m,1H),1.73-1.54(m,2H),1.43-1.29(m,4H); HRMS(ESI)for C 48 H 47 N 11 O7S2[M+Na] + :calcd 976.2994,found 976.3001.

[0131] Example 11:

[0132] 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)amino)-8-oxooctyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-10)

[0133]

[0134] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are 3-(5-amino-1-oxoisoindolinyl)piperidine-2,6-dione and 8-bromooctanoyl chloride. 1H NMR (400MHz, DMSO) δ11.54(d,J=6.8Hz,1H),10.99(s,2H),10.42(s,1H),10.21(s,1H),8.60(s,1H),7.98(s,2H),7.94(s,1H),7.73- 7.65(m,2H),7.64-7.59(m,1H),7.59-7.57(m,1H),7.57-7.54(m,2H),7.54-7.50(m,2H),7.48-7.43(m,3H),7.38-7.27(m,3H),5.98 (d,J=6.7Hz,1H),5.14(dd,J=13.2,5.1Hz,1H),4.39(d,J=7.8Hz,2H),4.35(d,J=9.2Hz,2H),4.27(s,2H),3.39(s,3H),2.98-2.83(m ,1H),2.62-2.52(m,1H),2.37-2.32(m,3H),1.99(s,1H),1.93-1.80(m,2H),1.64-1.53(m,2H),1.37-1.28(br.m,6H); HRMS(ESI)for C 49 H 49 N 11 O7S2[M+Na] + :calcd 1004.3307, found 1004.3302.

[0135] Example 12:

[0136] 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-4-oxobutyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-11)

[0137]

[0138] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are pomalidomide and 4-bromobutyryl chloride. 1HNMR(400MHz,DMSO)δ11.53(d,J=6.8Hz,1H),11.16(s,1H),10.99(s,1H),10.43(s,1H),9.80(s,1H),8.64(s,1H) ,8.38(d,J=8.4Hz,1H),8.00(s,1H),7.84-7.78(m,1H),7.76-7.67(m,2H),7.63-7.58(m,3H),7.57-7.52(m,2H),7 .52-7.46(m,3H),7.41-7.29(m,3H),5.98(d,J=6.7Hz,1H),5.14(dd,J=12.8,5.3Hz,1H),4.50(t,J=6.7Hz,2H),4. 28(s,2H),3.39(s,3H),2.95-2.82(m,1H),2.63-2.54(m,4H),2.28-2.18(m,2H),2.09-2.01(m,1H); HRMS(ESI)for C 45 H 39 N 11 O8S2[M+H] + :calcd 926.2497, found 926.2524.

[0139] Example 13:

[0140] 2-(3-(3-(1-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-6-oxohexyl)-1H-1,2,3-triazol-4-yl)phenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ia-12)

[0141]

[0142] The preparation method is the same as in Example 1, wherein the reaction raw materials in step 2 are pomalidomide and 6-bromohexanoyl chloride. 1HNMR(400MHz,DMSO)δ11.54(d,J=6.7Hz,1H),11.17(s,1H),11.00(s,1H),10.43(s,1H),9.71(s,1H),8.61(s,1H),8.46-8.44 (d,J=8.4Hz,1H),8.01(s,1H),7.83-7.79(m,1H),7.76-7.69(m,2H),7.61-7.54(m,5H),7.41-7.32(m,3H),6.00(d,J=6.7Hz,1 H),5.15(dd,J=12.7,5.4Hz,1H),4.52(t,J=6.8Hz,2H),4.40-4.44(s,2H),3.41(s,3H),2.99-2.82(m,1H),2.64-2.59(m,1H), 2.49(m,2H),2.25(p,J=6.9Hz,2H),2.13-2.02(m,1H),1.97-1.89(m,2H),1.72-1.66(m,2H),1.39-1.32(m,2H); HRMS(ESI)for C 47 H 41 N 11 O8S2[M+H] + :calcd954.2810,found954.2837.

[0143] Example 14:

[0144] 2-(3-(3-(((1-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)phenyl)ureido)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza -3-yl)aminodithiocarbamate (Ib-1)

[0145]

[0146] The preparation method is the same as in Example 1, wherein the reaction raw material in step 1 is 3-amino-N-(prop-2-yn-1-yl)benzamide. 1H NMR (400MHz, DMSO) δ11.52(d,J=6.6Hz,1H),11.04(s,1H),11.01(s,1H),10.45(s,1H),9.98(s,1H),9.04(s,1H),7.93-7.98 (m,2H),7.77-7.68(m,4H),7.62(d,J=12.1Hz,1H),7.61-7.54(m,3H),7.53-7.44(m,4H),7.42-7.36(m,1H),7.35-7.29(m,2H ),5.97(d,J=6.6Hz,1H),5.15(dd,J=13.4,5.1Hz,1H),4.64(t,J=6.7Hz,2H),4.47(d,J=5.7Hz,2H),4.28(d,J=15.7Hz,3H),3 .39(s,3H),3.00(t,J=6.7Hz,2H),2.97-2.86(m,1H),2.65-2.56(t,1H),2.42-2.27(m,1H),2.09-1.98(m,1H); HRMS(ESI)for C 46 H 42 N 12 O8S2[M+H] + :calcd 955.2763, found 955.2771.

[0147] Example 15:

[0148] 2-(3-(4-((1-(4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)4-oxobutyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-nitrophenyl)ureo)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza -3-yl)aminodithiocarbamate (Ib-2)

[0149]

[0150] The preparation method is the same as in Example 1, wherein the reaction raw material in step 1 is 2-nitro-5-(prop-2-yn-1-oxy)aniline, and the bromoacyl chloride in step 2 is 4-bromobutyryl chloride. 1H NMR(400MHz,DMSO)δ11.57(s,1H),11.14(s,1H),11.04(s,1H),9.86(s,1H),8.40-8.18(m,2H ),7.88-7.66(m,4H),7.66-7.55(m,2H),7.54-7.42(m,5H),7.40-7.29(m,2H),5.99(s,1H),5. 16(d,J=13.9Hz,1H),4.47(s,2H),4.42-4.30(m,2H),4.27(s,2H),3.41(s,3H),2.93(t,J=15. 3Hz,1H),2.71-2.57(m,1H),2.45-2.29(m,3H),2.18(s,2H),2.11-1.95(m,1H); HRMS(ESI)for C 46 H 42 N 12 O 10 S2[M+H] + :calcd986.2586,found 987.2667.

[0151] Example 16:

[0152] 2-(3-(3-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-6-oxohexyl)carbamoyl)phenyl)ureido)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ic-2)

[0153]

[0154] The preparation method is the same as in Example 2, wherein the reaction raw material in step 1 is 6-((tert-butoxycarbonyl)amino)hexanoic acid. 1H NMR (400MHz, DMSO) δ11.53(d,J=6.8Hz,1H),11.01(s,2H),10.44(s,1H),9.76(s,1H),8.61(s,1H),7.93(s,1H),7.75-7 .66(m,4H),7.61-7.56(m,3H),7.57-7.52(m,2H),7.53-7.45(m,3H),7.41-7.35(m,1H),7.35-7.29(m,2H),5.97(d,J=6 .7Hz,1H),5.12(dd,J=13.3,5.0Hz,1H),4.36(q,J=17.5Hz,2H),4.26(s,2H),3.39(s,3H),2.98-2.83(m,1H),2.65-2.5 4(m,1H),2.43-2.29(m,3H),2.05-1.97(m,1H),1.73-1.60(m,2H),1.59-1.49(m,2H),1.41-1.30(m,2H); HRMS(ESI)for C 46 H 45 N9O8S2[M+H] + :calcd 938.2725,found938.2732.

[0155] Example 17:

[0156] 2-(3-(3-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-6-oxohexyl)carbamoyl)phenyl)ureido)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ic-3)

[0157]

[0158] The preparation method is the same as in Example 2, wherein the reaction raw material in step 1 is 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid. 1H NMR (400MHz, DMSO) δ11.53(d,J=6.8Hz,1H),11.01(s,2H),10.44(s,1H),9.76(s,1H),8.61(s,1H),7.93(s,1H),7 .75-7.66(m,4H),7.61-7.56(m,3H),7.57-7.52(m,2H),7.53-7.45(m,3H),7.41-7.35(m,1H),7.35-7.29(m,2H),5 .98(d,J=6.7Hz,1H),5.12(dd,J=13.3,5.0Hz,1H),,4.36(q,J=17.5Hz,2H),4.26(m,2H),4.16(m,2H),3.68(t,J=5 .5Hz,2H),3.39(s,3H),2.98-2.83(m,1H),2.65-2.54(m,1H),2.43-2.29(m,3H),2.05-1.97(m,1H); HRMS(ESI)for C 45 H 43 N9O9S2[M+H] + :calcd 918.2626, found 918.2625.

[0159] Example 18:

[0160] 2-(3-(3-((6-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)amino)-8-oxooctyl)carbamoyl)phenyl)ureido)-2-oxoethyl(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diaza) -3-yl)aminodithiocarbamate (Ic-4)

[0161]

[0162] The preparation method is the same as in Example 2, wherein the reaction raw material in step 1 is 8-((tert-butoxycarbonyl)amino)octanoic acid. 1H NMR (400MHz, DMSO) δ11.53(d,J=6.8Hz,1H),11.01(s,2H),10.45(s,1H),9.76(s,1H),8.61(s,1H),7.92(s,1H),7.75-7 .66(m,4H),7.61-7.56(m,3H),7.58-7.53(m,2H),7.53-7.45(m,3H),7.42-7.36(m,1H),7.35-7.29(m,2H),5.97(d,J=6 .7Hz,1H),5.12(dd,J=13.3,5.0Hz,1H),4.36(q,J=17.5Hz,2H),4.26(s,2H),3.39(s,3H),2.98-2.83(m,1H),2.66-2.5 8(m,1H),2.43-2.29(m,3H),2.05-1.97(m,1H),1.73-1.60(m,2H),1.59-1.49(m,2H),1.41-1.30(m,2H); HRMS(ESI)for C 48 H 49 N9O8S2[M+H] + :calcd 944.3148,found944.3150.

[0163] Example 19: Antiproliferative activity of the compounds of the present invention against TNBC cell lines MDA-MB-231 and MDA-MB-468

[0164] The effect of the target compound on the proliferation of MDA-MB-231 and MDA-MB-468 cells was determined using the CCK-8 assay, and its IC50 was calculated. 50 The value was used to evaluate the antitumor proliferative activity of the compound in vitro.

[0165] Specific method: When the MDA-MB-231 or MDA-MB-468 cell lines reach a density greater than 80%, discard the culture medium, wash the cells 1-2 times with 2 mL PBS, add 0.1% trypsin, digest at 37°C for 1 min, add 2 mL DMEM complete culture medium to stop digestion, centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend the cells in 1 mL DMEM complete culture medium, measure and adjust the cell density, and seed the cell suspension into 96-well plates (100 μL / well) to achieve a density of 7000 MDA-MB-231 / well and 10000 MDA-MB-468 / well. Set up a drug treatment group, a negative control group, and a blank control group. The drug treatment group and the negative control group were seeded with the same cell density, while the blank control group was not seeded with cells.

[0166] Prepare a 1000× dimethyl sulfoxide solution (analyte stock solution) of the test compound. Dilute the test compound to 1000 times its final concentration with culture medium, i.e., add 1 μL of the test compound stock solution to 99 μL of culture medium to obtain the working solution.

[0167] After incubating the 96-well plates at 37°C with 5% CO2 for approximately 24 hours, the cells were seeded and allowed to adhere. The culture medium was discarded, and 100 μL of the working solution of the test compound (final concentrations of 50 μM, 25 μM, 10 μM, 5 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.1 μM) was added to each well. Three replicates were set up for each concentration. 100 μL of complete culture medium containing 0.1% DMSO was added to the negative control and blank control groups. The cells were cultured for another 72 hours before CCK-8 assay.

[0168] CCK-8 stock solution was diluted 10-fold with complete culture medium to obtain CCK-8 working solution. After cell treatment for 72 hours, the culture medium in the 96-well plate was discarded, and 100 μL of CCK-8 working solution was added to each well. After incubation in a cell culture incubator for 2-4 hours, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 The inhibition rate (I%) was calculated according to the formula, and then the IC50 of the target compound against the TNBC cell line was calculated using GraphPad Prism 8. 50 value.

[0169] The formula for calculating the inhibition rate I% is as follows:

[0170]

[0171] Table 1. Evaluation results of the in vitro anti-TNBC proliferation activity of the target compounds.

[0172]

[0173] Note: (R)-SL18 is a positive control. The above experiment was repeated three times, and the mean and standard deviation of the three experiments were calculated.

[0174] The experimental results showed that, compared with the positive compound (R)-SL18, all 18 target compounds exhibited better or comparable levels of anti-proliferative activity against the TNBC cell lines MDA-MB-231 and MDA-MB-468. Among them, the compound with the best activity was Ib-1, whose anti-TNBC activity was 1.2 to 1.3 times that of the positive control (R)-SL18.

[0175] Example 20: Evaluation of the effect on ANXA3 protein expression levels

[0176] Take MDA-MB-231 cells in logarithmic growth phase, add an appropriate amount of trypsin to digest the cells, collect the cells, centrifuge, and discard the supernatant. Resuspend the cells in serum-containing culture medium, count them, and adjust the cell density. Seed the cell suspension into 6-well plates (1.5 × 10⁻⁶). 4 Cells were cultured in 100 cells / dish at 37°C under a 5% carbon dioxide atmosphere for 24 hours. After cell attachment, the culture medium was aspirated, and the test compound solution (final concentration 5 μM) dissolved in the culture medium was added to the cells as the experimental group. A culture medium containing 0.1% DMSO was added to the cells as the blank control group.

[0177] ANXA3 protein expression was detected using Western blotting. After incubating cells for 24 h, the culture medium was discarded, and an appropriate amount of RIAP protein lysis buffer (containing 1% PMSF) was added. Cells were incubated on ice for 5 min, and the lysis buffer was transferred to Eppendorf tubes. The cells were vortexed every 10 min for a total of three cycles. The cells were then centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected. Total protein concentration was quantified using the BCA method. 5× loading buffer was added to the extracted total protein solution, and the mixture was boiled for 10 min. An SDS-PAGE gel was prepared, and the loading volume was adjusted according to the total protein concentration to ensure 50 μg of total protein per lane. Electrophoresis was performed at a constant voltage of 110 V for approximately 70 min, depending on the protein marker position. A PVDF membrane was activated by soaking in methanol for 1 min. A transfer system was prepared using the following sequence: sponge pad, three layers of filter paper, gel, PVDF membrane, three layers of filter paper, sponge pad. Transfer was performed at a constant voltage of 110 V for 90 min. After transfer, the PVDF membrane was blocked with Western blotting buffer at room temperature for 1 hour. The primary antibody was diluted with the appropriate ratio using primary antibody dilution buffer and incubated on the PVDF membrane at 4°C for 8 to 12 hours. After incubation, the membrane was washed three times with TBST for 10 minutes each time. The secondary antibody was diluted with the appropriate ratio using secondary antibody dilution buffer and incubated on the PVDF membrane at room temperature for 1 hour. After incubation, the membrane was washed three times with TBST for 10 minutes each time. ECL developing solution A and B were mixed in a 1:1 ratio and evenly dropped onto the PVDF membrane. The membrane was then quickly transferred to a chemiluminescence imaging system for development. The experimental results were analyzed using ImageJ software.

[0178] Table 2. Screening results of the in vitro ANXA3 degradation activity of the target compounds.

[0179]

[0180] Note: deg.% indicates the degradation rate of ANXA3 or ANXA6 protein; degradation rate is graded as follows (Grade A: above 40%; Grade B: 10-40%; Grade C: 0-10%; Grade D: no degradation).

[0181] Experimental results showed that all 18 target compounds exhibited varying degrees of ANXA3 degradation activity. Compared with the positive compound (R)-SL18, their degradation activity for ANXA6 was significantly weaker than that for ANXA3, demonstrating better selectivity for ANXA3. Among them, compound Ib-1 showed the best ANXA3 / ANXA6 degradation selectivity.

[0182] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An ANXA3 protein-targeting degradation chimera, or a pharmaceutically acceptable salt thereof, characterized in that, The ANXA3 protein-targeting degradation chimera is selected from one of the following structures: Ia-2, Ia-4, Ia-12, Ib-1, and Ic-3. (Ia-2), (Ia-4), (Ia-12), (Ib-1), (Ic-3); The ANXA3 protein-targeting degradation chimera selectively degrades the ANXA3 protein, and the degradation effect of the ANXA3 protein-targeting degradation chimera on ANXA6 is weaker than that on ANXA3.

2. Use of the ANXA3 protein-targeting degradation chimera of claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating breast cancer.

3. The use according to claim 2, characterized in that, Use of ANXA3 protein-targeting degradation chimeras, or pharmaceutically acceptable salts thereof, in the preparation of selective ANXA3 degraders.

4. The use according to claim 2 or 3, characterized in that, Breast cancer is selected from one of the molecular subtypes of triple-negative breast cancer, Luminal A, Luminal B, or Her2+ breast cancer.

5. An antitumor pharmaceutical composition that exerts its antitumor effect by acting as an ANXA3 degrading agent, characterized in that, The antitumor drug composition uses the ANXA3 protein-targeting degradation chimera of claim 1, or a pharmaceutically acceptable salt thereof, as the active ingredient.

6. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is in the form of tablets.

7. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is in the form of capsules.

8. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is in the form of pills.

9. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is an injectable formulation.

10. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, characterized in that, The antitumor drug composition is a sustained-release formulation.

11. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is a spray.

12. The antitumor pharmaceutical composition according to claim 5, which exerts its antitumor effect by acting as an ANXA3 degrading agent, is characterized in that, The antitumor drug composition is a nano-delivery system.

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