Compounds targeting degradation of nampt protein, and methods of synthesis and use thereof

Abstract

The application belongs to the technical field of drug chemical synthesis, discloses a compound for targeted degradation of NAMPT protein, and a structural general formula is as shown in formula (I). The application further discloses a synthesis method and application of the compound for targeted degradation of NAMPT protein, and solves the problem of a bottleneck existing in optimization of a connecting arm of the two ligands of NAMPT and PROTAC molecules.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemical synthesis technology, specifically relating to compounds that target and degrade NAMPT protein, as well as methods for synthesizing the aforementioned compounds that target and degrade NAMPT protein and their applications. Background Technology

[0002] Triple-negative breast cancer (TNBC) lacks expression of estrogen receptors, progesterone receptors, and HER2, making it insensitive to traditional endocrine and targeted therapies. Clinical treatment primarily relies on surgery and chemotherapy, but patients are prone to recurrence and metastasis, with a 5-year survival rate of only about 12.2%, necessitating new treatment strategies. Nicotinamide phosphoribosyltransferase (NAMPT) is an intracellular NAD+... + The activity of the key rate-limiting enzyme in the salvage synthesis pathway directly determines the NAD+ synthesis rate. + The synthesis rate of NAMPT is related to intracellular homeostasis. NAMPT is significantly highly expressed in TNBC tissues and is closely associated with poor patient prognosis and shorter survival. On the one hand, TNBC cells have a high metabolic rate and are highly reactive towards NAD+. + Demand surges, heavily reliant on NAMPT-mediated NAD + On the one hand, NAMPT is supplied to maintain proliferation and survival; on the other hand, NAMPT can also be secreted extracellularly to regulate NAD+. + Metabolic activity induces tumor-associated macrophages to polarize into the pro-cancer M2 type, suppresses anti-tumor immunity, and promotes angiogenesis and metastasis. Therefore, NAMPT is not only the metabolic core driving the autonomous growth of TNBC, but also a key factor in constructing an immunosuppressive microenvironment, making it a highly promising therapeutic target.

[0003] Protein degradation-targeting chimeras (PROTACs) are bifunctional small molecules that simultaneously bind to target proteins and E3 ubiquitin ligases, inducing the specific degradation of target proteins by the ubiquitin-proteasome system. Compared to traditional inhibitors, PROTACs offer advantages such as event-driven action, catalytic cycling, targeting of "drug-resistant" proteins, overcoming drug resistance, and high selectivity. Constructing a series of PROTAC molecules targeting NAMPT degradation can significantly improve the development efficiency and success rate of NAMPT degraders.

[0004] Currently, at the PROTAC molecular design level, existing general strategies face an empirical bottleneck in linker optimization: the linker length lacks precise guidance; if the linker is too short, the two ligands spatially repel each other, hindering effective binding to the target protein or E3 ligase, resulting in low efficiency of binary complex formation; if the linker is too long, degradation function is lost: although an excessively long flexible linker can avoid steric hindrance, it increases the molecular entropy, making it difficult for PROTAC to stably bring NAMPT and E3 ligase close to the effective ubiquitination distance (usually <10 Å), causing the target protein to fail to be successfully labeled and degraded.

[0005] The aforementioned contradiction of "too short for inactivation and too long for ineffectiveness" in the connecting arms makes the current PROTAC design highly dependent on a lot of trial and error, lacking a rational design strategy for specific target protein-ligand geometric relationships, which seriously restricts the development efficiency and success rate of NAMPT degraders.

[0006] While PROTAC technology holds great promise, its clinical translation faces multiple challenges. The primary obstacle is its large molecular weight, leading to significantly insufficient drug-like properties, manifested as low cell membrane permeability and oral bioavailability, poor water solubility, and complex and difficult-to-control pharmacokinetics. Secondly, the lack of tissue specificity makes it prone to unexpected off-target degradation in healthy tissues, and degradation efficiency is highly dependent on a few E3 ubiquitin ligases (such as CRBN and VHL), whose expression and activity differences limit universality and may lead to cytotoxicity. Simultaneously, the unique "hook effect" causes degradation activity to decrease rather than increase at high concentrations, and the prediction of ternary complex structures and structure-activity relationships is extremely complex, lacking universal design rules, making development highly dependent on experimental screening. Summary of the Invention

[0007] The first objective of this invention is to provide a compound that targets and degrades NAMPT protein. This compound has a simple structure and solves the bottleneck problem in the optimization of the linker arm of the existing NAMPT and PROTAC ligands.

[0008] The second objective of this invention is a method for synthesizing the above-mentioned compounds that target and degrade NAMPT protein.

[0009] A third objective of this invention is the use of the above-mentioned compounds that target and degrade NAMPT protein in the preparation of drugs for degrading NAMPT protein.

[0010] A fourth objective of this invention is the use of the above-mentioned compounds that target and degrade NAMPT protein in the preparation of drugs for the treatment of tumors.

[0011] The first technical solution adopted in this invention is: a compound that targets and degrades NAMPT protein, with the general structural formula shown in formula (Ⅰ): .

[0012] The first technical solution adopted in this invention is also characterized by: In formula (Ⅰ) Each is independently selected from one of the following structures:

[0013] in, Indicates the connection site.

[0014] The CRBN ligands in formula (Ⅰ) are each independently selected from one of the following structures:

[0015] in, Indicates the connection site.

[0016] The above compounds have structural formulas of any one of formulas A1–A20: .

[0017] The second technical solution adopted in this invention is: a method for synthesizing the above-mentioned compound that targets and degrades NAMPT protein, comprising: The Sonogashira coupling reaction was carried out using methyl 3-bromo-4-fluorobenzoate and trimethylsilylacetylene, and the reaction product was deprotected to give compound OT. Compound OT was added to thalidomide containing linker arms of different lengths. HATU and DIPEA were added to the system to carry out the reaction. After the reaction was completed, the reaction solution was diluted with water, extracted, washed, dried, filtered, concentrated and purified to obtain the target compound as shown in formula (I).

[0018] The second technical solution adopted in this invention is further characterized by: The above synthesis method includes the following steps: (1) Synthesis of compound OT Methyl 3-bromo-4-fluorobenzoate was mixed with trimethylsilylacetylene and reacted in the presence of triphenylphosphine palladium and cuprous iodide to give intermediate 2a. 2a is deprotected by tetrabutylammonium fluoride (TBAF) to obtain intermediate 2b; Intermediate 2b was mixed with 1-tert-butyloxycarbonylpiperazine, and the two reacted to give intermediate 2c. Intermediate 2c was mixed with 4-bromofluorobenzene and reacted in the presence of triphenylphosphine palladium and cuprous iodide to obtain intermediate 2d. Intermediate 2d is hydrolyzed by lithium hydroxide to give intermediate 2e; Intermediate 2e was mixed with 3-(1H-pyrazol-4-yl)propane-1-amine hydrochloride and reacted in the presence of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU) and N,N-diisopropylethylamine (DIPEA) to give intermediate 2f. Intermediate 2f was deprotected by hydrochloric acid to give compound OT; (2) Synthesis of thalidomide with linker arms of different lengths 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione reacts with 1-tert-butoxycarbonylpiperazine to give intermediate 3a; 3a is deprotected under acidic conditions and then reacted with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3b; 3b is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3c; 3c is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3d; 3a is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3e; 3e is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3f; 3a is deprotected under acidic conditions and then reacted with 1'-(tert-butyloxycarbonyl)-1,4'-bipiperidine-4-carboxylic acid to give intermediate 3j; 3b was deprotected under acidic conditions and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3g; 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione reacts with 1-BOC-4-piperidinylpiperazine to give intermediate 3h; The protecting group was removed under acidic conditions for 3 hours, and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3i; 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione reacts with 1-tert-butyloxycarbonylpiperazine to give intermediate 4a; 4a is deprotected under acidic conditions and then reacted with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4b; 4b is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4c; 4c is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4d; 4a was deprotected under acidic conditions and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 4b; The aforementioned intermediates 3a-3g, 3i-3j, and 4a-4e are thalidomide with connecting arms of different lengths; (3) Synthesis of the final product Compound OT was reacted with thalidomide containing different structural linking arms, namely intermediates 3a-3g, 3i-3j, and 4a-4e, to obtain the target compound as shown in formula (I).

[0019] The third technical solution adopted in this invention is: the application of the above-mentioned compound that targets and degrades NAMPT protein in the preparation of drugs for degrading NAMPT protein.

[0020] The fourth technical solution adopted in this invention is: the application of the above-mentioned compound that targets and degrades NAMPT protein in the preparation of drugs for tumor treatment.

[0021] The fourth technical solution adopted in this invention is further characterized by: Application of compounds that target and degrade NAMPT protein as active ingredients in the preparation of tumor therapeutic drugs.

[0022] The dosage form of the drug is oral preparation, mucosal delivery preparation or injection.

[0023] The beneficial effects of this invention are: 1. In this invention, an analogue of compound OT-82 was used as a NAMPT protein ligand. A linker combining flexible and rigid chains was used to link the E3 ubiquitin ligase ligands 5-F and 4-F thalidomide to prepare 20 PROTAC molecules. 2. The PROTACs molecules designed in this invention have the ability to degrade NAMPT protein, and the PROTACs molecules can degrade NAMPT protein in a concentration-dependent manner in human breast cancer cells; these PROTACs molecules have shown good anti-tumor effects and have broad application prospects in the diagnosis and treatment of tumors. Attached Figure Description

[0024] Figure 1 This is a predicted diagram of the binding of the compound OT-82 of this invention to the NAMPT protein (PDB: 2GVJ); Figure 2 This is a synthetic route diagram of the OT compound of the present invention; Figure 3a Synthetic route diagram of thalidomide containing different structural linking arms 3a-3g and 3i-3j as intermediates of the present invention; Figure 3b Synthetic route diagram for thalidomide containing different structural linking arms 4a-4e as intermediates of the present invention; Figure 4 This is a synthetic route diagram for the target PROTACs molecules of this invention; Figure 5 The degradation results of NAMPT protein by the PROTACs molecules of this invention; Figure 6 To understand the mechanism of NAMPT protein degradation induced by compound A13 after using the lysosomal degradation pathway blocker MG132; Figure 7To understand the mechanism by which compound A13 induces NAMPT protein degradation after using CRBN ligands thalidomide and OT-82, respectively. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] This invention provides compounds for targeted degradation of NAMPT protein, their synthesis methods, and applications. Using analogues of compound OT-82 as NAMPT protein ligands, and introducing saturated cycloalkanes with different structures as linking arms to E3 ubiquitin ligase ligands 5-fluoro and 4-fluorothalidomide, 20 PROTAC molecules, i.e., the target compounds, were prepared. The PROTAC molecules designed in this invention have the ability to degrade NAMPT protein, such as… Figure 1 As shown, PROTACs molecules can degrade NAMPT protein in a concentration-dependent manner; these PROTACs molecules exhibit good anti-tumor effects and have broad application prospects in the diagnosis and treatment of tumors. Their general structural formula is shown in formula (I): .

[0027] I. Design Concept of the Invention OT-82 possesses a classic NAMPT binding mode with strong target specificity. Its rigid molecular skeleton facilitates linker modification, and its in vivo activity and safety have been validated, demonstrating a promising foundation for drug development. This invention selects OT-82 as a target for a novel series of PROTACs and, based on the small molecular weight, compact structure, moderate lipophilicity, and high oral bioavailability of the CRBN ligand thalidomide, designs and synthesizes a series of PROTAC molecules that target and degrade NAMPT. Molecular docking studies show that the pyridine ring of OT-82 is exposed in the solvent, indicating that this site is suitable as a linker connection site without affecting target binding. To achieve linker coupling, the pyridine ring is replaced with a piperazine group (named compound OT), which provides a universal linker arm for structural derivatization.

[0028] II. Specific Synthetic Methods of Compounds Targeting NAMPT Protein Degradation like Figure 2As shown, methyl 3-bromo-4-fluorobenzoate, a commercially available raw material, was subjected to a Sonogashira coupling reaction with trimethylsilylacetylene to give intermediate 2a. Intermediate 2a was deprotected by tetrabutylammonium fluoride (TBAF) to give intermediate 2b. Intermediate 2b then underwent a substitution reaction with 1-tert-butoxycarbonylpiperazine to give intermediate 2c. Subsequently, intermediate 2c underwent a Sonogashira coupling reaction with 4-bromofluorobenzene to give intermediate 2d. Intermediate 2d was hydrolyzed by lithium hydroxide to give intermediate 2e. Intermediate 2e then underwent a dehydration condensation reaction with 3-(1H-pyrazol-4-yl)propane-1-amine hydrochloride to give intermediate 2f. Intermediate 2f was deprotected by hydrochloric acid to give compound OT.

[0029] Using the commercially available starting material 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione, a nucleophilic substitution reaction was first carried out with 1-tert-butyloxycarbonylpiperazine to yield intermediate 3a. Intermediate 3a underwent deprotection under acidic conditions, followed by reaction with... N -Boc-piperidine-4-carboxylic acid undergoes a condensation reaction to give intermediate 3b. Intermediates 3c and 3d are prepared by similar one-step and two-step reactions, respectively. Intermediate 3a, after deprotection under acidic conditions, undergoes a reductive amination reaction with tert-butyl 4-formylpiperidine-1-carboxylic acid to give intermediate 3e. Intermediate 3e, after acidic deprotection, undergoes a condensation reaction with N-tert-butoxycarbonylpiperidine-4-carboxylic acid to give intermediate 3f. Intermediate 3j is prepared by reacting 1′-(tert-butoxycarbonyl)-[1,4′-bipiperidine]-4-carboxylic acid with intermediate 3a, using a method similar to that used for intermediate 3b. Intermediate 3b, after deprotection under acidic conditions, undergoes a reductive amination reaction with tert-butyl 4-formylpiperidine-1-carboxylic acid to give compound 3g. 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindol-1,3-dione undergoes a nucleophilic substitution reaction with tert-butyl 4-(piperidin-4-yl)piperazine-1-carboxylic acid to give intermediate 3h. Intermediate 3h is then subjected to an acidic deprotection reaction, followed by... N -tert-butyloxycarbonylpiperidine-4-carboxylic acid undergoes a condensation reaction to give intermediate 3i. Intermediates 4a-4e are prepared from 2-(2,6-dioxopiperidine-3-yl)-4-fluoroisoindoline-1,3-dione using methods similar to those used for intermediates 3a-3e, such as... Figure 3a , Figure 3b As shown.

[0030] The general synthesis method for all PROTACs is as follows: Figure 4As shown, compound OT was added to thalidomide containing linker arms of different lengths, namely intermediates 3a-3g, 3i-3j, and 4a-4e, for reaction. HATU and DIPEA were added to the system for further reaction. After the reaction was completed, the reaction solution was diluted with water, and the target compounds A1-A20 were obtained by extraction, washing, drying, filtration, concentration, and purification.

[0031] The synthesis process is illustrated using compound A1 as an example. It begins with an acid-mediated deprotection reaction of intermediate 3a, followed by a dehydration condensation reaction with 3-tert-butoxy-3-oxopropionic acid. Subsequently, the tert-butyl protecting group is removed via acid-mediated deprotection to yield the key carboxylic acid intermediate 3a1. Intermediate 3a1 then undergoes a second condensation reaction with OT to obtain the target compound A1. All other target compounds were synthesized using a similar method to A1.

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Synthesis of intermediate 2c K₂CO₃ (1.17 g, 8.5 mmol) was added to a DMF solution of methyl 4-fluoro-3-((trimethylsilyl)ethynyl)benzoate (600 mg, 3.4 mmol) and 1-Boc-piperazine (940 mg, 5 mmol). After stirring at 100 °C for 6 hours, the reaction mixture was extracted with DCM (30 mL × 3) and washed with brine. The organic phase was dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by rapid column chromatography on silica gel to give product 2b (1.06 g, 92% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 7.92 (d, J = 2.1 Hz, 1H), 7.87 (dd, J = 8.7, 2.3 Hz, 1H), 7.07 (d, J =8.7 Hz, 1H), 4.55 (s, 1H), 3.82 (s, 3H), 3.47 (t, J = 5.0 Hz, 4H), 3.23 (t, J =5.0 Hz, 5H), 1.42 (s, 9H). Example 2: Synthesis of intermediate 2d PdCl₂(PPh₃)₂ (1 g, 0.5 mmol), CuI (552 mg, 1 mmol), 4-bromofluorobenzene (603 mg, 1.2 mmol), and 2b (1 g, 1 mmol) were added to a Schlenk tube under argon protection, followed by the addition of MeCN (100 mL) and Et₃N (1.26 mL, 3 mmol). The resulting solution was stirred overnight at 70 °C. The reaction progress was monitored by TLC. After the reaction was complete, the mixture was diluted with EtOAc (30 mL), filtered through a diatomaceous earth mat, and washed with EtOAc (10 mL). The filtrate was concentrated under vacuum and purified by rapid silica gel column chromatography to give intermediate 2c (865 mg, 68% yield).

[0034] Example 3: Synthesis of intermediate 2e Compound 2d (600 mg, 1.4 mmol) and lithium hydroxide (LiOH, 6 mmol) were dissolved in a mixed solvent of THF / MeOH / H2O (21 mL, volume ratio 4:2:1). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the solution was concentrated, neutralized to weakly acidic with 1 M HCl aqueous solution, and then evaporated to dryness under reduced pressure to give the crude product (523 mg, yield 90%).

[0035] Example 4: Synthesis of intermediate 2f 2e (500 mg, 1.2 mmol) and 3-(1 H -pyrazol-4-yl)propyl-1-amine (180 mg, 1.4 mmol) dissolved in N , N Dimethylformamide (DMF, 5 mL) was added to the solution. Triethylamine (520 μL, 3.6 mmol) and HBTU (684 mg, 1.8 mmol) were added, and the reaction mixture was stirred at room temperature for 4 h. After the reaction was complete, the reaction mixture was extracted with ethyl acetate (30 mL × 3) and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give the target product 2d (514 mg, 82% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 12.51 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.01 (d, J= 2.2 Hz, 1H), 7.83 (dd, J =8.5, 2.2 Hz, 1H), 7.62 (dd, J = 8.6, 5.6 Hz, 2H), 7.43 (s, 2H), 7.31 (t, J =8.8 Hz, 2H), 7.07 (d, J = 8.6 Hz, 1H), 3.53 (t, J = 4.8 Hz, 4H), 3.27 (q, J =6.6 Hz, 2H), 3.20 (s, 4H), 2.46 (t, J = 7.5 Hz, 2H), 1.76 (p, J = 7.3 Hz, 2H), 1.43 (s, 9H). Example 5: OT synthesis Compound 2f (400 mg, 0.75 mmol) was dissolved in dichloromethane (DCM, 3 mL), and trifluoroacetic acid (TFA, 0.6 mL) was added. The reaction mixture was stirred at room temperature for 4 h. The solvent was removed under reduced pressure to obtain the crude product, which was used directly in the next reaction without further purification.

[0036] Example 6: Synthesis of intermediate 3a To a DMF solution of 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (1 g, 3.6 mmol), 1-tert-butyloxycarbonylpiperazine (1 g, 4.3 mmol) and DIPEA (793 μL, 4.3 mmol) were added, and the mixture was stirred at 100 °C for 4 h. After the reaction was complete, the reaction mixture was extracted with ethyl acetate (30 mL × 3) and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by rapid silica gel column chromatography to give the target product 3a (1.33 g, 83% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 11.07 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 2.3 Hz, 1H), 7.26 (dd, J = 8.6, 2.4 Hz, 1H), 5.08 (dd,J = 12.8, 5.5 Hz, 1H), 3.62 (dd, J = 6.8, 4.1 Hz, 2H), 3.59 -3.52 (m, 4H), 3.48 (dd, J = 6.7 Hz, 4H), 2.89 (ddd, J = 16.9, 13.8, 5.5 Hz,1H), 2.59 (m, J = 18.1 Hz, 1H), 2.53 (dd, J = 13.4, 4.4 Hz, 1H), 2.06 - 1.99(m, 1H), 1.42 (s, 9H). Example 7: Synthesis of intermediate 3b Step 1: Add TFA (0.6 mL) to a DCM (10 mL) solution of compound 3a (1 g, 2.6 mmol), and stir the resulting mixture at room temperature for 4 h. After the reaction is complete, remove the solvent under reduced pressure to obtain the crude product, which does not require purification and can be used directly in the next step of the reaction.

[0037] Step 2: Add DIPEA (526 μL, 3 mmol) to a DMF (5 mL) solution of the above crude product (500 mg, 1.5 mmol), and stir at room temperature for 10 min; then add... N -tert-butyloxycarbonylpiperidine-4-carboxylic acid (527 mg, 2.3 mmol) and O-(7-azabenzotriazol-1-yl)- N , N , N ', N '-Tetramethylurea hexafluorophosphate (HATU, 874 mg, 2.3 mmol) was added, and the resulting mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with DCM (30 mL × 3) and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give the target product 3b (506 mg, 61% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 11.07 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.25 (dd, J= 8.6, 2.3 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 3.94 (d, J = 13.0 Hz, 2H), 3.66 (dt, J =56.2, 5.3 Hz, 4H), 3.49 (dt, J = 33.2, 5.3 Hz, 4H), 2.93 - 2.74 (m, 4H), 2.59(dt, J = 18.3, 3.4 Hz, 1H), 2.53 (dd, J = 13.4, 4.5 Hz, 1H), 2.06 - 2.00 (m,1H), 1.63 (d, J = 14.2 Hz, 2H), 1.43 (d, J = 16.3 Hz, 2H), 1.40 (s, 9H). Example 8: Synthesis of intermediate 3a1 Step 1: Add TFA (0.3 mL) to a DCM (10 mL) solution of compound 3a (100 mg, 0.26 mmol), and stir the resulting mixture at room temperature for 4 h. After the reaction is complete, remove the solvent under reduced pressure to obtain the crude product, which does not require purification and can be used directly in the next reaction.

[0038] Step 2: The crude product (80 mg, 0.23 mmol) was dissolved in DMF (5 mL), and N,N-diisopropylethylamine (DIPEA, 85 μL, 0.46 mmol) was added. The mixture was stirred at room temperature for 10 min. Then, 3-tert-butoxy-3-oxopropionic acid (55 mg, 0.35 mmol) and HATU (132 mg, 0.35 mmol) were added, and the resulting mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was extracted with DCM (30 mL × 3), washed with saturated brine, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give product 3a1 (74 mg, 65% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 11.07 (s, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 2.3 Hz, 1H), 7.26 (dd, J= 8.6, 2.4 Hz, 1H), 5.08 (dd, J = 12.8, 5.5 Hz, 1H), 3.62 (dd, J =6.8, 4.1 Hz, 2H), 3.59 - 3.52 (m, 4H), 3.48 (dd, J = 6.7 Hz, 4H), 2.89 (ddd, J = 16.9, 13.8, 5.5 Hz, 1H), 2.59 (m, J = 18.1 Hz, 1H), 2.53 (dd, J = 13.4, 4.4Hz, 1H), 2.06 - 1.99 (m, 1H), 1.42 (s, 9H). Example 9: Synthesis of intermediate 3j Step 1: Add TFA (0.6 mL) to a DCM (10 mL) solution of compound 3a (1 g, 2.6 mmol), and stir the resulting mixture at room temperature for 4 h. After the reaction is complete, remove the solvent under reduced pressure to obtain the crude product, which does not require purification and can be used directly in the next step of the reaction.

[0039] Step 2: To a DMF (5 mL) solution of the above crude product (200 mg, 0.6 mmol), DIPEA (210 μL, 1.2 mmol) was added, and the mixture was stirred at room temperature for 10 min. Subsequently, 1'-(tert-butyloxycarbonyl)-[1,4'-bipiperidine]-4-carboxylic acid (280 mg, 0.9 mmol) and HATU (342 mg, 0.9 mmol) were added, and the resulting mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with DCM (30 mL × 3) and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the residue was purified by rapid silica gel column chromatography to obtain the target product 3j (280 mg, 73% yield). 1 HNMR (600 MHz, DMSO- d 6) δ 11.07 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.38 (d, J =2.3 Hz, 1H), 7.27 (dd, J = 8.7, 2.3 Hz, 1H), 5.08 (dd, J= 12.8, 5.4 Hz, 1H), 4.07 (s, 2H), 3.73 - 3.60 (m, 4H), 3.51 (dt, J = 40.4, 5.3 Hz, 6H), 3.37 (s,2H), 3.03 (s, 2H), 2.93 - 2.84 (m, 1H), 2.75 (s, 2H), 2.59 (dt, J = 20.0, 4.6Hz, 1H), 2.53 (dd, J = 13.3, 4.5 Hz, 1H), 2.05 - 2.00 (m, 1H), 1.97 (d, J =11.3 Hz, 2H), 1.93 - 1.79 (m, 4H), 1.50 (m, J = 12.7 Hz, 2H), 1.41 (s, 9H). Example 10: Synthesis of intermediate 4a To a DMF (5 mL) solution of 2-(2,6-dioxadiidine-3-yl)-4-fluoroisoindoline-1,3-dione (1 g, 3.6 mmol), 1-tert-butyloxycarbonylpiperazine (1 g, 4.3 mmol) and DIPEA (793 μL, 4.3 mmol) were added, and the mixture was stirred at 100 °C for 4 h. After the reaction was complete, the reaction solution was extracted with DCM (30 mL × 3) and washed with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give the target product 4a (1.4 g, 85% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 11.10 (s, 1H), 7.72 (dd, J = 8.4, 7.1 Hz, 1H), 7.39 (d, J = 7.1 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 5.11 (dd, J = 12.9, 5.4 Hz, 1H), 3.51 (t, J = 4.9 Hz, 4H), 3.25 (t, J = 5.0Hz, 4H), 2.88 (ddd, J= 17.0, 13.8, 5.5 Hz, 1H), 2.60 (dt, J = 17.3, 3.4 Hz,1H), 2.57 - 2.52 (m, 1H), 2.06 - 2.00 (m, 1H), 1.43 (s, 9H). Example 11: General method for synthesizing target PROTACs Compound OT (0.1 mmol, 1.0 eq) and thalidomide with linker arms of varying lengths (0.1 mmol, 1.0 eq) were dissolved in DMF (2 mL). HATU (0.12 mmol, 1.2 eq) and DIPEA (0.2 mmol, 2 eq) were added to the system. After addition, the reaction was allowed to proceed at room temperature for 12 h. The reaction was then monitored by TLC. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase using dichloromethane (3 × 30 mL). The aqueous and organic phases were monitored separately by TLC to ensure that all product had entered the organic phase and that no product remained in the aqueous phase. The organic phases were then combined, washed with saturated brine to absorb excess water, dried thoroughly with anhydrous sodium sulfate, and filtered to remove the anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure using a rotary evaporator and then purified by silica gel column chromatography.

[0040] A1: Yellow powder, 67 mg, 57%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.51 (s, 1H), 11.09 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6, 2.3 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.67 - 7.52 (m, 2H), 7.44 (s, 2H), 7.37(d, J = 2.3 Hz, 1H), 7.35 - 7.21 (m, 3H), 7.08 (d, J = 8.7 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 3.70 (m, 4H), 3.62 (dd, J = 14.6, 7.9 Hz, 6H), 3.55 (d, J= 5.4 Hz, 2H), 3.49 (dd, J = 7.4, 4.2 Hz, 2H), 3.44 (t, J = 7.0 Hz, 1H), 3.25 (dd, J = 11.2, 5.7 Hz, 4H), 2.89 (ddd, J = 17.3, 13.9, 5.5 Hz, 1H), 2.64 - 2.52(m, 2H), 2.47 (t, J = 7.5 Hz, 2H), 2.03 (tt, J = 8.3, 3.2 Hz, 1H), 1.77 (q, J =7.3 Hz, 2H), 1.06 (t, J = 7.0 Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ : 173.25,170.53, 167.99, 167.44, 166.45, 166.35, 165.39, 163.77, 161.31, 155.83,155.30, 134.34, 133.96, 133.88, 133.04, 129.56, 128.12, 125.40, 119.66,118.99, 118.27, 118.22, 116.75, 116.53, 108.47, 94.18, 87.93, 56.50, 51.05,50.75, 49.27, 47.20, 46.92, 46.24, 45.20, 41.90, 41.12, 40.69, 31.46, 31.05,22.66, 21.58, 19.03. HRMS-ESI m / z : calcd for C 45 H 44 FN9O7[M + H] + : 842.3420; found 842.3444. Purity: 99.08%. A2: Yellow powder, 58 mg, 53%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.50 (s, 1H), 11.08 (s, 1H), 8.44 (t,J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.5,2.3 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.67 - 7.60 (m, 2H), 7.44 (s, 2H), 7.37- 7.27 (m, 3H), 7.25 (dd, J = 8.7, 2.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 5.08(dd, J = 12.9, 5.4 Hz, 1H), 3.83 - 3.63 (m, 6H), 3.62 (d, J = 5.4 Hz, 4H), 3.52- 3.41 (m, 3H), 3.32 - 3.24 (m, 4H), 3.20 (t, J = 4.9 Hz, 2H), 2.96 - 2.82 (m,1H), 2.64 - 2.55 (m, 4H), 2.46 (d, J = 7.5 Hz, 2H), 2.09 - 1.97 (m, 1H), 1.81- 1.72 (m, 2H), 1.06 (t, J = 7.0 Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.25,170.78, 170.65, 170.53, 168.00, 167.45, 165.39, 155.88, 155.32, 134.35,133.98, 133.89, 133.03, 129.54, 128.10, 125.41, 119.66, 119.41, 119.38,118.90, 118.17, 116.75, 116.53, 114.80, 108.33, 94.20, 87.95, 56.50, 51.18,50.78, 49.27, 47.12, 46.99, 45.41, 44.47, 41.82, 40.99 , 40.69, 40.48, 39.68,39.47, 31.46, 31.05, 28.00, 27.93, 22.66, 21.59, 19.03. HRMS-ESI m / z : calcd for 46 H 46 FN9O7[M + H] + : 856.3577; found 856.3593. Purity: 96.66%. A3: yellow powder, 61 mg, 52%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.44 (s, 1H), 11.08(s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 8.5,2.3 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.68 - 7.57 (m, 2H), 7.43 (s, 2H), 7.38- 7.24 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H), 4.37(d, J = 12.7 Hz, 1H), 3.82 (d, J = 13.2 Hz, 1H), 3.67 (d, J= 37.4 Hz, 9H), 3.55- 3.44 (m, 4H), 3.30 - 3.17 (m, 6H), 3.10 (t, J = 12.3 Hz, 1H), 2.99 - 2.84(m, 2H), 2.69 (t, J = 12.0 Hz, 1H), 2.65 - 2.53 (m, 2H), 2.47 (s, 2H), 2.02(d, J = 11.9 Hz, 1H), 1.76 (t, J = 7.3 Hz, 2H), 1.66 (s, 3H), 1.31 - 1.23 (m, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ : 173.46, 173.16, 170.73, 168.18, 167.63, 166.66, 166.00, 165.56, 161.49, 156.03, 155.57, 134.49, 134.15, 134.07, 133.20, 129.75, 128.29, 125.60, 119.26, 118.43, 116.95, 116.73, 114.99, 108.69, 94.37, 88.10, 51.22, 50.94, 49.45, 48.39, 47.80, 47.18, 46.40, 44.61,42.06, 41.24, 37.58, 31.64, 31.24, 29.66, 29.26, 22.83, 21.77. HRMS-ESI m / z :calcd for C 51 H 53 FN 10 O8[M+H] + : 953.4105; found 953.4135. Purity: 97.52%. A4: Yellow powder, 64 mg, 55%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.48 (s, 1H), 11.08 (s, 1H), 8.43 (t, J = 5.6 Hz, 1H), 8.02 (d, J= 2.2 Hz, 1H), 7.84 (dd, J = 8.6,2.2 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.67 - 7.60 (m, 2H), 7.44 (s, 2H), 7.36(d, J = 2.2 Hz, 1H), 7.33 - 7.24 (m, 3H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H), 4.37 (d, J = 12.7 Hz, 1H), 3.95 (d, J = 13.2 Hz, 1H), 3.80- 3.57 (m, 8H), 3.58 - 3.45 (m, 4H), 3.43 - 3.30 (m, 1H), 3.28 - 3.17 (m,4H), 3.11 (t, J = 12.1 Hz, 1H), 3.00 - 2.84 (m, 2H), 2.73 - 2.53 (m, 7H), 2.47(t, J = 7.5 Hz, 2H), 2.04 (ddd, J = 12.4, 6.7, 2.7 Hz, 1H), 1.78 (q, J = 7.3 Hz,2H), 1.69 (t, J = 16.7 Hz, 2H), 1.61 - 1.49 (m, 1H), 1.41 - 1.31 (m, 1H), 1.28- 1.21 (m, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ: 172.17, 171.95, 169.68, 169.45, 169.02, 166.92, 166.37, 164.34, 162.70, 160.24, 154.82, 154.31, 133.25, 132.90, 132.82, 131.96, 128.47, 127.02, 124.32, 118.62, 118.01, 117.27, 117.11, 115.68, 115.45, 113.73, 107.41, 93.12, 86.90, 50.12, 49.71, 48.22, 46.55, 45.93, 44.35, 43.59, 43.38, 40.74, 40.01, 36.47, 30.39, 29.99, 28.12, 27.49, 26.93, 26.81, 21.59, 20.52. HRMS-ESI m / z : calcd for C 52 H 55 FN 10 O8[M+H] + :967.4261; found 967.4279. Purity: 96.07%. A5: yellow powder, 60 mg, 58%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.51 (s, 1H), 11.10 (s, 1H), 8.44 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6, 2.2 Hz, 1H), 7.73 (dd, J = 8.4, 7.1 Hz, 1H), 7.68 - 7.55 (m, 2H), 7.50 - 7.22 (m, 6H), 7.07 (d, J = 8.6 Hz, 1H), 5.12 (dd, J = 12.8, 5.4 Hz, 1H), 4.44 - 4.31 (m, 3H), 4.02 (d, J= 13.0 Hz, 1H), 3.80 - 3.60 (m, 10H), 3.50 - 3.39 (m, 2H), 3.31 - 3.21 (m, 6H), 3.16 - 3.05 (m, 2H), 3.01 - 2.86 (m, 3H), 2.73 - 2.52(m, 4H), 2.47(t, J = 7.5 Hz, 2H), 2.09 - 1.99 (m, 1H), 1.81 - 1.72 (m, 2H), 1.70 - 1.51 (m, 5H), 1.45 - 1.31 (m, 2H), 1.28 - 1.23 (m, 1H), 1.05 (t, J =7.0 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6) δ : 173.26, 172.88, 172.47, 170.42, 167.47, 166.85, 166.47, 165.78, 165.40, 163.77, 161.31, 155.84, 149.86, 136.42, 134.10, 133.96, 133.87, 133.03, 129.55, 128.11, 124.33, 119.66, 119.37, 118.22, 117.40, 116.75, 116.53, 115.75, 114.81, 94.18, 87.93, 56.50,54.03, 51.71, 51.03, 50.71, 49.32, 46.22, 45.56, 45.34, 44.66, 41.88, 41.56,41.11, 37.60, 37.42, 31.43, 31.05, 29.69, 28.74, 22.53, 21.58, 19.03. HRMS-ESI m / z : calcd for C 57 H 62 FN 11 O9[M+H] + : 1064.4789; found 1064.4834. Purity:95.56%. A6: Yellow powder, 65 mg, 54%. 1 H NMR (400 MHz, DMSO- d 6)δ : 12.49 (s, 1H), 11.10(s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6,2.3 Hz, 1H), 7.73 (dd, J = 8.4, 7.1 Hz, 1H), 7.68 - 7.58 (m, 2H), 7.47 - 7.23(m, 6H), 7.07 (d, J = 8.7 Hz, 1H), 5.12 (dd, J = 12.8, 5.4 Hz, 1H), 4.43 - 4.31(m, 4H), 4.01 (s, 2H), 3.80 - 3.60 (m, 10H), 3.47 - 3.41 (m, 2H), 3.30 - 3.20(m, 6H), 3.10 (d, J = 11.1 Hz, 3H), 2.96 - 2.86 (m, 3H), 2.70 - 2.58 (m, 4H),2.47 (t, J = 7.5 Hz, 2H), 2.08 - 2.00 (m, 1H), 1.81 - 1.73 (m, 2H), 1.71 -1.48 (m, 8H), 1.41 - 1.30 (m, 3H), 1.23 (s, 2H), 1.06 (t, J = 7.0 Hz, 3H). 13 CNMR (100 MHz, DMSO- d 6) δ: 173.26, 172.88, 172.45, 170.42, 167.47, 166.85, 166.47, 165.78, 165.40, 163.77, 161.31, 155.84, 149.86, 136.43, 134.10, 133.96, 133.88, 133.03, 129.55, 128.11, 124.34, 119.65, 119.40, 118.22, 117.40, 116.75, 116.53, 115.75, 114.81, 94.18, 87.93, 56.50, 51.71, 51.03, 50.74, 49.32, 46.21, 45.56, 45.35, 44.66, 41.88, 41.55, 41.11, 37.59, 37.43, 31.43, 31.05, 29.69, 28.73, 22.53, 21.58, 19.03. HRMS-ESI m / z : calcd for C 63 H 71 FN 12 O 10 [M+H] + : 1175.5473; found 1175.5545. Purity: 97.04%. A7: yellow powder, 66 mg, 61%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.46 (s, 1H), 11.08 (s, 1H), 8.45 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.84 (dd, J = 8.6, 2.3 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 8.6, 5.5 Hz, 2H), 7.43 (s, 2H), 7.39 - 7.27 (m, 3H), 7.25 (dd, J = 8.6, 2.4 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J= 12.9, 5.4 Hz, 1H), 4.42 - 4.29 (m, 2H), 3.77 (d, J = 12.8Hz, 1H), 3.73 - 3.55 (m, 10H), 3.54 - 3.41 (m, 6H), 3.25 (d, J = 21.7 Hz, 5H),3.02 - 2.82 (m, 4H), 2.73 - 2.54 (m, 4H), 2.46 (d, J = 7.4 Hz, 2H), 2.24 -1.94 (m, 4H), 1.81 - 1.60 (m, 8H), 1.40 - 1.23 (m, 2H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ : 173.25, 170.52, 167.99, 167.44, 166.50, 165.70, 165.39, 163.77, 161.31, 155.83, 155.36, 134.32, 133.96, 133.87, 133.05, 129.56, 128.11, 125.40, 119.65, 119.40, 119.37, 119.04, 118.30, 118.21, 116.75, 116.53, 114.80, 108.44, 94.17, 87.94, 56.50, 51.02, 50.76,49.27, 47.60, 46.99, 46.21, 44.44, 41.86, 41.63, 41.06, 31.46, 31.05, 22.65,21.58, 19.03. m / z : calcd for C 57 H 64 FN 11 O8[M+H] + : 1050.4996; found1050.4990. Purity: 96.45%. A8: Yellow powder, 62 mg, 54%. 1 H NMR (400 MHz, DMSO- d 6) δ: 12.52 (s, 1H), 11.09(s, 1H), 8.45 (t, J = 5.7 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.6,2.3 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.67 - 7.57 (m, 2H), 7.44 (s, 2H), 7.37- 7.23 (m, 4H), 7.07 (d, J = 8.7 Hz, 1H), 5.09 (dd, J = 12.9, 5.4 Hz, 1H), 4.43- 4.33 (m, 3H), 4.03 (d, J = 13.1 Hz, 1H), 3.82 - 3.61 (m, 9H), 3.53 - 3.43(m, 5H), 3.30 - 3.21 (m, 5H), 3.09 (s, 2H), 3.00 - 2.83 (m, 3H), 2.72 - 2.54(m, 4H), 2.47 (t, J = 7.4 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.81 - 1.72 (m, 2H),1.43 - 1.29 (m, 4H), 1.36 (m, 2H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz,DMSO- d 6) δ: 173.25, 172.97, 172.47, 170.52, 167.99, 167.44, 166.47, 165.78,165.39, 161.31, 155.84, 155.37, 134.31, 133.96, 133.88, 133.03, 129.55,128.11, 125.40, 119.65, 119.08, 118.33, 118.22, 116.75, 116.53, 114.81,108.49, 94.18, 87.93, 56.50, 51.03, 50.74, 49.28, 47.62, 46.99, 46.22, 45.56,44.45, 41.88, 41.10, 37.56, 37.42, 31.46, 31.05, 29.66, 28.71, 22.65, 21.58,19.03. HRMS-ESI m / z : calcd for C 57 H 62 FN 11 O9[M+H] + : 1064.4789; found 1064.4803.Purity: 95.34%. A9: yellow powder, 63 mg, 53%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.48 (s, 1H), 11.08(s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.83 (dd, J = 8.5,2.2 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.68 - 7.57 (m, 2H), 7.44 (s, 2H), 7.38- 7.23 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H), 4.44- 4.32 (m, 3H), 3.98 (dd, J= 39.2, 13.1 Hz, 2H), 3.74 - 3.60 (m, 7H), 3.54 -3.39 (m, 6H), 3.28 (t, J = 6.4 Hz, 3H), 3.21 (d, J = 4.8 Hz, 2H), 3.11 (d, J =13.0 Hz, 2H), 2.97 - 2.87 (m, 2H), 2.66 - 2.56 (m, 6H), 2.46 (d, J = 7.5 Hz, 2H), 2.03 (dd, J = 10.5, 5.4 Hz, 1H), 1.76 (t, J = 7.3 Hz, 2H), 1.69 - 1.51 (m,4H), 1.35 - 1.23 (m, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ :173.25, 172.97, 172.51, 170.74, 170.53, 170.05, 167.99, 167.44, 165.40, 161.30, 155.90, 155.38, 134.32, 133.98, 133.89, 133.03, 129.54, 128.08, 125.41, 119.41, 119.08, 118.34, 118.18, 116.75, 116.53, 114.80, 108.49, 94.19, 87.96, 56.50, 51.20, 50.78, 49.28, 47.61, 47.01, 45.43, 44.68, 41.80,41.09, 37.56, 31.46, 31.05, 29.48, 28.74, 27.88, 22.65, 21.59, 19.03. HRMS-ESI m / z : calcd for C 58 H 64 FN 11 O9[M+H] + : 1078.4945; found 1078.4977. Purity:95.97%. A10: Yellow powder, 60 mg, 55%.1 H NMR (400 MHz, DMSO- d 6) δ : 12.50 (s, 1H),11.09 (s, 1H), 8.44 (t, J = 5.7 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.83 (dd, J =8.5, 2.2 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.68 - 7.51 (m, 2H), 7.40 (d, J =26.4 Hz, 2H), 7.37 - 7.23 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J =12.8, 5.4 Hz, 1H), 4.44 - 4.32 (m, 4H), 4.02 (s, 2H), 3.74 - 3.42 (m, 15H),3.25 (dd, J = 13.7, 6.7 Hz, 4H), 3.11 (d, J = 11.8 Hz, 3H), 2.99 - 2.84 (m,4H), 2.68 - 2.55 (m, 4H), 2.47 (t, J = 7.4 Hz, 2H), 2.08 - 1.96 (m, 1H), 1.79- 1.73 (m, 2H), 1.71 - 1.47 (m, 8H), 1.43 - 1.21 (m, 3H), 1.06 (t, J = 7.0 Hz,3H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.25, 172.96, 172.44, 170.53, 167.99, 167.44, 166.47, 165.78, 165.39, 155.84, 155.37, 134.32, 133.96, 133.88, 133.03, 129.55, 128.11, 125.41, 119.66, 119.08, 118.34, 118.23, 116.75, 116.53, 114.81, 108.49, 94.18, 87.93, 56.50, 51.03, 50.74, 49.28, 47.62,47.00, 46.22, 45.59, 44.65, 44.44, 41.87, 41.11, 40.69, 37.58, 37.43, 31.46,31.05, 29.67, 28.70, 22.65, 21.58, 19.03. HRMS-ESI m / z : calcd for C 63 H 71 FN 12 O 10 [M+H] + : 1175.5473; found 1175.5485. Purity: 97.82%. A11: Yellow powder, 58 mg, 51%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.50 (s, 1H), 11.06 (s, 1H), 8.44 (t, J = 5.7 Hz, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.83 (dd, J =8.5, 2.2 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.67 - 7.58 (m, 2H), 7.53 - 7.37(m, 2H), 7.37 - 7.24 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J= 12.8, 5.4Hz, 1H), 4.42 - 4.32 (m, 4H), 4.08 - 3.88 (m, 3H), 3.74 - 3.60 (m, 7H), 3.53- 3.41 (m, 6H), 3.30 - 3.17 (m, 6H), 3.11 (d, J = 12.8 Hz, 2H), 2.99 - 2.82(m, 4H), 2.63 - 2.55 (m, 6H), 2.46 (d, J = 7.6 Hz, 2H), 2.05 -1.99 (m, 1H),1.80 - 1.73 (m, 2H), 1.70 -1.61 (m, 4H), 1.55 -1.48 (m, 2H), 1.37 -1.31 (m,2H), 1.28 -1.18 (m, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ :173.26, 172.48, 170.75, 170.53, 170.05, 167.99, 167.44, 165.40, 161.31,155.90, 155.38, 134.32, 133.99, 133.90, 129.55, 128.08, 125.41, 119.66,119.42, 119.08, 118.35, 118.20, 116.76, 116.54, 114.80, 108.49, 94.19, 87.95,61.68, 56.50, 54.38, 51.19, 50.80, 50.76, 50.48, 49.28, 47.66, 47.00, 46.13,44.67, 44.47, 41.10, 40.86, 37.56, 31.46, 31.05, 29.49, 28.71, 27.88, 22.65,21.58, 19.03. HRMS-ESI m / z : calcd for C 64 H 73 FN 12 O 10 [M+H] + : 1189.5629; found1189.5657. Purity: 98.56%. A12: yellow powder, 55 mg, 49%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.49 (s, 1H), 11.08 (s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.5, 2.2 Hz, 1H), 7.70 - 7.57 (m, 3H), 7.43 (s, 2H), 7.36 - 7.27 (m, 3H), 7.25 (dd, J = 8.8, 2.3 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.35 (t, J = 5.1 Hz, 2H), 4.08 (d, J = 12.7 Hz, 2H), 3.79 (d, J = 12.9 Hz, 1H), 3.73 - 3.58 (m, 6H), 3.52 - 3.41 (m, 5H), 3.29 - 3.21 (m, 5H), 3.06 (t, J = 11.6 Hz, 1H), 2.96 (t, J = 12.1 Hz, 2H), 2.92 - 2.78 (m, 2H), 2.69 - 2.53 (m, 4H), 2.47 (t, J = 7.6 Hz, 2H), 2.07 - 1.96 (m, 1H), 1.89 - 1.71 (m, 4H), 1.64 - 1.56 (m, 2H), 1.47 (d, J = 11.0 Hz, 2H), 1.38 - 1.22 (m, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.26, 172.48, 170.56, 168.07, 167.42, 166.46, 165.78, 165.39, 161.31, 155.83, 155.21, 134.50, 133.96, 133.87, 133.03, 129.56, 128.11, 125.46, 119.65, 119.36, 118.19, 118.15, 116.75, 116.52, 114.80, 108.29, 94.17, 87.93, 56.50, 51.04, HRMS-ESI m / z : calcd for C 56 H 62 FN 11 O8[M+H] + :1036.4840; found 1036.4899. Purity: 95.88%. A13: Yellow powder, 66 mg, 68%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.50 (s, 1H), 11.08 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.83 (dd, J =8.5, 2.2 Hz, 1H), 7.71 - 7.56 (m, 3H), 7.43 (s, 2H), 7.35 - 7.21 (m, 4H),7.07 (d, J = 8.6 Hz, 1H), 5.07 (dd, J = 12.9, 5.4 Hz, 1H), 4.35 (t, J = 5.1 Hz, 2H), 4.08 (d, J = 12.8 Hz, 2H), 3.92 (d, J= 13.4 Hz, 1H), 3.79 - 3.62 (m, 4H), 3.53 - 3.39 (m, 6H), 3.31 - 3.17 (m, 6H), 3.08 (t, J = 12.4 Hz, 1H), 3.02 -2.84 (m, 4H), 2.64 - 2.55 (m, 6H), 2.48 - 2.37 (m, 2H), 2.07 - 1.96 (m, 1H),1.89 - 1.79 (m, 2H), 1.81 - 1.69 (m, 2H), 1.62 (s, 2H), 1.52 - 1.44 (m, 2H), 1.42 - 1.20 (m, 3H), 1.06 (t, J = 7.0 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6) δ :173.25, 172.51, 170.73, 170.56, 170.05, 167.42, 165.39, 155.89, 155.21, 133.97, 133.89, 133.02, 129.53, 128.08, 125.46, 119.65, 118.18, 118.14, 116.75, 116.52, 114.80, 108.29, 94.19, 87.95, 61.03, 56.50, 51.19, 50.78, 49.76, 49.22, 49.04, 47.09, HRMS-ESI m / z:calcd for C 57 H 64 FN 11 O8[M + H] + : 1050.4996; found 1050.5065. Purity: 95.58%. A14: Yellow powder, 56 mg, 53%. 1 H NMR (400 MHz, DMSO- d 6) δ: 12.45 (s, 1H),11.09 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J =8.6, 2.3 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.66 - 7.57 (m, 2H), 7.43 (s, 2H),7.37 - 7.22 (m, 4H), 7.07 (d, J = 8.7 Hz, 1H), 5.08 (dd, J = 12.8, 5.4 Hz, 1H),4.51 - 4.31 (m, 2H), 3.83 (d, J = 12.9 Hz, 1H), 3.72 - 3.58 (m, 8H), 3.54 -3.41 (m, 5H), 3.29 - 3.23 (m, 4H), 3.02 - 2.82 (m, 4H), 2.69 - 2.53 (m, 4H),2.46 (t, J = 7.5 Hz, 2H), 2.28 (s, 2H), 2.08 - 1.97 (m, 1H), 1.83 - 1.70 (m,4H), 1.69 - 1.53 (m, 4H), 1.45 (d, J = 11.9 Hz, 1H), 1.35 - 1.25 (m, 1H), 1.06(t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.33, 173.25, 170.52, 167.99, 167.44, 166.46, 165.73, 165.38, 155.82, 155.37, 134.32, 133.96, 133.87, 133.04, 129.56, 128.12, 125.40, 119.65, 119.04, 118.31, 118.21, 116.75, 116.53, 114.81, 108.45, 94.18, 87.93, 56.50, 51.05, 50.76, 49.28,48.81, 48.57, 47.61, 45.55, 41.88, 41.07, 31.46, 31.05, 29.01, 28.48, 27.69,22.65, 21.58, 19.03. m / z : calcd for C 56 H 62 FN 11 O8[M+H] + : 1036.4840; found1036.4907. Purity: 95.32%. A15: Yellow powder, 50 mg, 53%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.44 (s, 1H),11.08 (s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J =8.5, 2.2 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.68 - 7.59 (m, 2H), 7.43 (s, 2H), 7.36 - 7.23 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H),4.47 - 4.34 (m, 2H), 3.98 (d, J= 13.2 Hz, 1H), 3.71 - 3.60 (m, 7H), 3.54 -3.42 (m, 6H), 3.29 - 2.25 (m, 2H), 3.21 (d, J = 5.5 Hz, 2H), 3.04 - 2.83 (m,4H), 2.57 (d, J = 4.3 Hz, 5H), 2.48 (d, J = 7.5 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.87 - 1.55 (m, 8H), 1.50 - 1.40 (m, 1H), 1.30 - 1.19 (m, 4H), 1.06 (t, J =7.0 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ : 173.25, 170.72, 170.52, 170.08, 167.99, 167.44, 165.39, 161.31, 155.89, 155.37, 134.32, 133.98, 133.89, 133.04, 129.55, 125.41, 119.65, 119.42, 119.05, 118.33, 118.18, 116.75, 116.53, 114.80, 108.46, 94.19, 87.96, 56.49, 51.19, 50.78, 49.28, 48.71,48.67, 45.42, 44.41, 41.81, 41.07, 38.89, 31.46, 31.05, 29.48, 28.03, 27.87,22.65, 21.58, 19.03. m / z : calcd for C 57 H 64 FN 11 O8[M+H] + : 1050.4996; found1050.5044. Purity: 96.24%. A16: Yellow powder, 62 mg, 55%. 1 H NMR (400 MHz, DMSO- d 6) δ: 12.49 (s, 1H),11.08 (s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J =8.6, 2.3 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.66 - 7.58 (m, 2H), 7.43 (s, 2H),7.35 - 7.23 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J = 12.9, 5.4 Hz, 1H),4.42 - 4.32 (m, 3H), 3.99 (d, J = 13.0 Hz, 1H), 3.80 (d, J = 13.0 Hz, 1H), 3.72- 3.56 (m, 6H), 3.49 - 3.39 (m, 6H), 3.31 - 3.20 (m, 6H), 3.07 (s, 2H), 2.95- 2.85 (m, 2H), 2.73 - 2.59 (m, 2H), 2.47 (t, J = 8.0 Hz, 2H), 2.19 (d, J = 7.1Hz, 2H), 2.06 - 1.98 (m, 1H), 1.85 - 1.70 (m, 5H), 1.66 - 1.54 (m, 3H), 1.42- 1.32 (m, 1H), 1.30 - 1.22 (m, 2H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz,DMSO- d 6) δ: 173.25, 172.36, 170.53, 168.03, 167.44, 166.47, 165.76, 165.39, 161.31, 155.83, 155.70, 134.32, 133.96, 133.87, 133.03, 129.55, 128.11, 125.35, 119.65, 119.40, 118.82, 118.23, 116.75, 116.52, 114.81, 108.35, 94.17, 87.93, 56.50, 53.16, 51.04, 50.74, 49.26, 47.39, 46.23, 45.57, 45.22,41.88, 41.64, 41.12, 37.51, 33.26, 31.88, 31.46, 31.05, 29.48, 22.66, 21.58,19.03. HRMS-ESI m / z : calcd for C 57 H 64 FN 11 O8[M+H] + : 1050.4996; found 1050.5012.Purity: 98.58%. A17: Yellow powder, 60 mg, 58%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.48 (s, 1H), 11.08 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.1 Hz, 1H), 7.84 (dd, J =8.6, 2.3 Hz, 1H), 7.72 - 7.59 (m, 3H), 7.43 (s, 2H), 7.35 - 7.18 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.08 (dd, J= 12.9, 5.4 Hz, 1H), 4.44 - 4.30 (m, 3H), 4.03 - 3.89 (m, 2H), 3.78 - 3.62 (m, 4H), 3.52 - 3.38 (m, 6H), 3.32 - 3.15(m, 6H), 3.15 - 2.97 (m, 2H), 2.95 - 2.84 (m, 2H), 2.64 - 2.53 (m, 7H), 2.47(t, J = 8.0 Hz, 2H), 2.19 (d, J = 6.8 Hz, 2H), 2.08 - 1.97 (m, 1H), 1.87 - 1.70(m, 5H), 1.62 (s, 3H), 1.39 - 1.24 (m, 2H), 1.06 (t, J = 7.0 Hz, 3H). 13 C NMR (100 MHz, DMSO-) d 6) δ : 173.25, 172.39, 170.74, 170.54, 170.04, 168.03, 167.45, 165.40, 163.77, 161.31, 155.90, 155.72, 134.33, 133.98, 133.89, 133.03, 129.54, 128.09, 125.36, 119.65, 119.41, 118.80, 118.23, 118.18, 116.75, 116.53, 114.80, 108.34, 94.19, 87.96, 56.50, 53.18, 51.19, 50.78, 49.25,47.40, 45.42, 45.24, 44.70, 41.81, 41.11, 37.65, 33.29, 31.46, 31.05, 28.00,27.88, 22.65, 21.59, 19.03. HRMS-ESI m / z : calcd for C 58 H 66 FN 11 O8[M+H] + :1064.5153; found 1064.5220. Purity: 98.35%. A18: Yellow powder, 57 mg, 54%. 1H NMR (400 MHz, DMSO- d 6) δ : 12.51 (s, 1H),11.10 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J =8.5, 2.3 Hz, 1H), 7.73 (dd, J = 8.4, 7.1 Hz, 1H), 7.67 - 7.57 (m, 2H), 7.45(s, 2H), 7.41 - 7.28 (m, 4H), 7.08 (d, J = 8.6 Hz, 1H), 5.12 (dd, J = 12.9, 5.4Hz, 1H), 3.85 - 3.55 (m, 10H), 3.45 (dd, J = 7.4, 3.4 Hz, 1H), 3.31 - 3.22 (m,7H), 2.95 - 2.82 (m, 1H), 2.72 - 2.52 (m, 2H), 2.47 (t, J = 7.4 Hz, 2H), 2.10- 1.98 (m, 1H), 1.78 (q, J = 7.3 Hz, 2H), 1.31 - 1.23 (m, 1H), 1.06 (t, J = 7.0Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.25, 170.43, 167.49, 166.85, 166.44, 166.38, 165.41, 163.78, 161.31, 155.84, 149.85, 136.41, 134.09, 133.97, 133.88, 133.04, 129.56, 128.11, 124.33, 119.40, 118.23, 117.37, 116.75, 116.53, 115.69, 114.81, 94.19, 87.93, 56.50, 51.24, 51.04, 50.75, 50.58, 49.32, 46.21, 46.12, 41.89, 41.65, 31.43, 31.05, 22.52, 21.60, 19.02. MS-ESI m / z : calcd for C 45 H 44 FN9O7[M+H] + : 842.3420; found 842.3421. Purity: 95.25%. A19: yellow powder, 56 mg, 53%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.50 (s, 1H), 11.09 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.84 (dd, J =8.5, 2.3 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.63 (dd, J = 8.6, 5.6 Hz, 2H), 7.43 (s, 2H), 7.38 - 7.28 (m, 4H), 7.07 (d, J = 8.6 Hz, 1H), 5.10 (dd, J =12.8, 5.4 Hz, 1H), 4.63 - 4.09 (m, 2H), 3.79 (d, J = 13.0 Hz, 1H), 3.73 - 3.52 (m, 6H), 3.48 - 3.42 (m, 1H), 3.32 - 3.20 (m, 9H), 3.00 (t,J = 12.3 Hz, 1H),2.94 - 2.83 (m, 1H), 2.63 - 2.54 (m, 4H), 2.47 (t, J = 7.4 Hz, 2H), 2.19 (d, J = 7.4 Hz, 2H), 2.07 - 1.99 (m, 1H), 1.89 - 1.68 (m, 5H), 1.25 (d, J = 10.8 Hz, 1H), 1.06 (t, J = 7.0 Hz, 2H), 0.94 (d, J = 12.0 Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ : 173.25, 170.45, 167.52, 166.76, 166.50, 165.70, 165.39, 163.77, 161.31, 155.83, 150.18, 136.33, 134.15, 133.96, 133.87, 133.04, 129.56, 128.11, 124.17, 119.65, 119.40, 118.21, 116.99, 116.75, 116.53, 115.28, 114.80, 94.17, 87.95, 64.11, 56.50, 53.51,51.02,50.76,49.28,46.23,46.14,41.87,41.70,33.08,31.43,31.24,31.05,30.61,22.54,21.58,19.03. m / z :calcd for C 51 H 55 FN 10 O7[M+H] + : 939.4312; found 939.4396. Purity: 97.14%. A20: Yellow powder, 60 mg, 51%. 1 H NMR (400 MHz, DMSO- d 6) δ : 12.44 (s, 1H), 11.08 (s, 1H), 8.43 (t, J= 5.6 Hz, 1H), 8.02 (d, J = 2.2 Hz, 1H), 7.84 (dd, J =8.6, 2.3 Hz, 1H), 7.71 (dd, J = 8.4, 7.2 Hz, 1H), 7.63 (ddd, J = 8.4, 5.3, 2.4Hz, 2H), 7.43 (s, 2H), 7.40 - 7.24 (m, 4H), 7.07 (d, J = 8.7 Hz, 1H), 5.09(dd, J = 12.8, 5.4 Hz, 1H), 4.35 (d, J = 12.9 Hz, 1H), 3.91 (d, J = 13.4 Hz,1H), 3.68 (d, J = 17.9 Hz, 4H), 3.27 (d, J = 8.3 Hz, 4H), 3.20 (s, 2H), 3.10 -2.79 (m, 3H), 2.64 - 2.53 (m, 8H), 2.47 (t, J = 7.4 Hz, 2H), 2.23 (s, 2H),2.02 (dd, J = 12.8, 6.5 Hz, 1H), 1.89 - 1.67 (m, 5H), 1.34 - 1.20 (m, 6H),1.13 - 1.02 (m, 1H), 0.93 - 0.84 (m, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ: 173.32,170.79, 170.51, 170.03, 167.56, 166.81, 165.42, 163.80, 161.34, 155.94,136.41, 134.17, 134.02, 133.06, 129.58, 128.11, 124.24, 119.69, 119.45,118.23, 117.07, 116.80, 116.58, 115.39, 114.84, 94.24, 87.99, 53.48, 51.23,50.81, 49.31, 45.44, 45.19, 41.84, 41.69, 41.44, 31.47, 31.10, 29.53, 28.04,22.57, 21.62, 14.47. HRMS-ESI m / z : calcd for C 52 H 57 FN 10 O7[M+H] + : 953.4468; found953.4104. Purity: 95.23%. IV. Pharmacological activity testing of PROTACs molecules The obtained target compound degraded NAMPT protein in human breast cancer cells 4T1 and MDA-MB-231. The Western blotting method included: after cell plating and treatment with the appropriate drug, cells were collected and gently washed three times with pre-chilled PBS to remove residual culture medium and serum components. Then, pre-chilled cell lysis buffer was added to the cell-containing culture dish, and the cells were lysed on ice for 30 min. Cell lysates were scraped from the cell walls using a cell scraper and transferred to centrifuge tubes for centrifugation (4 ℃, 12000 rpm, 15 min), collecting the protein supernatant. The total protein concentration of the sample was determined and calculated using the BCA protein quantification kit, following the kit's instructions. The sample protein concentration was leveled using protein loading buffer and denatured in a 100 ℃ water bath for 10 min. The denatured protein sample and pre-stained protein marker were loaded separately into a Bio-Rad vertical electrophoresis apparatus, and polyacrylamide gel electrophoresis was performed under the set electrophoresis conditions. After electrophoresis, the transfer sandwich was assembled in the order of "gel-PVDF membrane-filter paper," and the assembled transfer device was placed in the Bio-Rad transfer kit for transfer. After transfer, the PVDF membrane was removed and placed in 5% skim milk blocking buffer for 1 hour at room temperature. The PVDF membrane was then transferred to a diluted primary antibody solution and fully immersed, and incubated overnight at 4°C. The PVDF membrane was removed and repeatedly washed with TBST buffer for 8 minutes each time, for a total of 3 washes, to remove unbound primary antibody. The PVDF membrane was then placed in a diluted secondary antibody solution and incubated on a shaker at room temperature for 2 hours, followed by 3 washes with TBST buffer for 8 minutes each time, to thoroughly remove non-specific binding. The washed PVDF membrane was then immersed in a mixture containing ultrasensitive ECL chemiluminescent reagent, ensuring that the luminescent solution uniformly covered the entire membrane surface. Subsequently, it was scanned and developed in an imaging system, and the grayscale values ​​of the bands were quantitatively analyzed using ImageJ software.

[0041] Western blot results (see details) Figure 5 The 20 PROTAC molecules designed and synthesized in this invention can all degrade NAMPT protein in 4-T1 and MDA-MB-231 cells. Among them, A13 showed the best protein degradation ability in DCs of MDA-MB-231 cells. 50 The value was 9.31 ± 1.65 nM, and the DC50 value in 4T1 cells was 12.54 ± 3.36 nM.

[0042] Next, using A13 as an example, the mechanism of competitive inhibition was used to confirm that A13 induces the degradation of NAMPT protein through the ubiquitin-proteasome pathway (see details). Figure 6 , Figure 7First, A13 significantly degraded NAMPT protein. Next, 4-T1 and MDA-MB-231 cells were pretreated with the proteasome inhibitor MG132, followed by treatment with compound A13. After treatment, the degradation activity of NAMPT by A13 was significantly reduced, suggesting that the degradation of the target protein occurs via the proteasome pathway. Then, 4-T1 and MDA-MB-231 cells were pretreated with the CRBN ligand thalidomide and the NAMPT protein ligand OT-82, respectively, before the addition of compound A13. After treatment, the degradation activity of NAMPT by A13 was also significantly reduced, suggesting that the degradation of the target protein occurs through the formation of a NAMPT-PROTACs-CRBN ternary complex.

Claims

1. A compound that targets and degrades NAMPT protein, characterized in that, The general structural formula is shown in equation (Ⅰ): 。 2. The compound for targeted degradation of NAMPT protein according to claim 1, characterized in that, In the formula (Ⅰ) Each is independently selected from one of the following structures: in, Indicates the connection site.

3. The compound for targeted degradation of NAMPT protein according to claim 1, characterized in that, The CRBN ligands in formula (Ⅰ) are each independently selected from one of the following structures: in, Indicates the connection site.

4. The compound for targeted degradation of NAMPT protein according to claim 1, characterized in that, The compound has a structural formula of any one of formulas A1–A20: 。 5. The method for synthesizing the compound for targeted degradation of NAMPT protein according to claim 1 or 2, characterized in that, The synthesis method includes: The Sonogashira coupling reaction was carried out using methyl 3-bromo-4-fluorobenzoate and trimethylsilylacetylene, and the reaction product was deprotected to give compound OT. Compound OT was added to thalidomide containing linker arms of different lengths. HATU and DIPEA were added to the system to carry out the reaction. After the reaction was completed, the reaction solution was diluted with water, extracted, washed, dried, filtered, concentrated and purified to obtain the target compound as shown in formula (I).

6. The method for synthesizing the compound for targeted degradation of NAMPT protein according to claim 5, characterized in that, The synthesis method includes the following steps: (1) Synthesis of compound OT Methyl 3-bromo-4-fluorobenzoate was mixed with trimethylsilylacetylene and reacted in the presence of triphenylphosphine palladium and cuprous iodide to give intermediate 2a. 2a is deprotected by tetrabutylammonium fluoride to obtain intermediate 2b; Intermediate 2b was mixed with 1-tert-butyloxycarbonylpiperazine, and the two reacted to give intermediate 2c. Intermediate 2c was mixed with 4-bromofluorobenzene and reacted in the presence of triphenylphosphine palladium and cuprous iodide to obtain intermediate 2d. Intermediate 2d is hydrolyzed by lithium hydroxide to give intermediate 2e; Intermediate 2e was mixed with 3-(1H-pyrazol-4-yl)propane-1-amine hydrochloride and reacted with N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and N,N-diisopropylethylamine to give intermediate 2f. Intermediate 2f was deprotected by hydrochloric acid to give compound OT; (2) Synthesis of thalidomide with linker arms of different lengths 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione reacts with 1-tert-butoxycarbonylpiperazine to give intermediate 3a; 3a is deprotected under acidic conditions and then reacted with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3b; 3b is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3c; 3c is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3d; 3a is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3e; 3e is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 3f; 3a is deprotected under acidic conditions and then reacted with 1'-(tert-butyloxycarbonyl)-1,4'-bipiperidine-4-carboxylic acid to give intermediate 3j; 3b was deprotected under acidic conditions and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3g; 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione reacts with 1-BOC-4-piperidinylpiperazine to give intermediate 3h; The protecting group was removed under acidic conditions for 3 hours, and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 3i; 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione reacts with 1-tert-butyloxycarbonylpiperazine to give intermediate 4a; 4a is deprotected under acidic conditions and then reacted with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4b; 4b is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4c; 4c is deprotected under acidic conditions and then reacts with 1-tert-butoxycarbonyl-4-piperidinecarboxylic acid to give intermediate 4d; 4a was deprotected under acidic conditions and then reacted with 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde to give intermediate 4b; The aforementioned intermediates 3a-3g, 3i-3j, and 4a-4e are thalidomide with connecting arms of different lengths. (3) Synthesis of the final product Compound OT was reacted with thalidomide containing different structural linking arms, namely intermediates 3a-3g, 3i-3j, and 4a-4e, to obtain the target compound as shown in formula (I).

7. The use of the compound that targets and degrades NAMPT protein according to any one of claims 1-4 in the preparation of a drug for degrading NAMPT protein.

8. The use of the compound that targets and degrades NAMPT protein according to any one of claims 1-4 in the preparation of a medicament for the treatment of tumors.

9. The use of the compound according to claim 8 that targets and degrades NAMPT protein in the preparation of a drug for tumor treatment, characterized in that, Application of compounds that target and degrade NAMPT protein as active ingredients in the preparation of tumor therapeutic drugs.

10. The use of the compound according to claim 9, which targets and degrades NAMPT protein, in the preparation of a drug for tumor treatment, characterized in that, The dosage form of the drug is oral preparation, mucosal delivery preparation or injection.

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