Synthesis of purine derivative g4 targeting protein chimera based on protac technology and its application in antitumor activity

Abstract

This invention relates to purine derivative G4-targeting protein chimeras, and more particularly to a class of purine derivative G4-targeting protein chimeras synthesized using PROTAC technology and possessing antitumor activity. The general structural formula of the targeting chimera is as follows: where R is a linker, representing triethylene glycol, tetraethylene glycol, pentaethylene glycol, a four-carbon chain, a five-carbon chain, a six-carbon chain, a seven-carbon chain, an eight-carbon chain, a nine-carbon chain, azacyclobutane, tetrahydropyrrole, piperidine, or 1-phenylpiperazine. Spectroscopic and mass spectrometric data analysis show that the synthesized purine derivative G4-targeting protein chimeras are novel compounds. In vitro antitumor activity studies have demonstrated that the purine derivative G4-targeting protein chimeras provided by this invention exhibit strong antitumor activity against human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116. This is an excellent new antitumor compound with the potential to be developed into a new antitumor drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and in particular to the synthesis of a class of target protein chimeras (PROTACs) with antitumor activity and their application as antitumor drugs. Background Technology

[0002] Cancer is currently the leading cause of death and health threats to humanity, and its development has garnered significant attention from various sectors worldwide in recent years. With the aging population, changing lifestyles, and evolving environmental factors, cancer incidence and mortality rates are exhibiting increasingly complex trends, urgently driving medical researchers to relentlessly develop innovative anti-cancer drugs.

[0003] Purine derivatives hold great potential in antitumor activity. However, purine derivative compounds generally suffer from poor physicochemical properties, low bioavailability, and severe toxic side effects. Furthermore, current antitumor drugs generally have limited druggable targets and are prone to drug resistance, thus restricting antitumor drug development. In recent years, targeted protein degradation technology has received increasing attention. Compared to traditional tumor inhibitors, targeted protein degradation technology offers the following advantages: 1. It overcomes the limitations of undrugable protein targets, providing new potential treatments for some previously "incurable" diseases; 2. Due to its novel mechanism of action, the dosage is significantly reduced; 3. It can overcome drug resistance. Among these, protein degradation-targeted chimeras (PROTACs) have achieved significant breakthroughs and are currently the fastest-growing and most advantageous protein degradation technology pathway.

[0004] PROTAC is a hybrid bifunctional small molecule or polypeptide compound that functions via a linker. PROTAC molecules are synthesized by linking a protein-binding domain (PDB) to an E3 ligase ligand. The principle is to shorten the distance between the target protein and the ubiquitin ligase E3, thereby adding a ubiquitination tag to the target protein. Finally, the ubiquitin-proteasome system is used to degrade the target protein. After ubiquitination, the PROTAC molecule can detach from the target protein and E3 ligase and be recycled within the cell, achieving efficient degradation with a relatively low drug dose. This invention, based on the purine derivative G4, selects pomalidomide as the E3 ligase ligand, and uses glycol, carbon chain, and nitrogen heterocycle linkers to synthesize a series of novel antitumor molecules using PROTAC technology. Pharmacological activity tests on the obtained molecules show that these compounds have significant antitumor effects and can be developed as antitumor drugs.

[0005] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a class of target protein chimeras (PROTACs) with antitumor activity synthesized based on the purine derivative G4 and their application as antitumor drugs.

[0007] The technical solution adopted by this invention to solve its technical problem is: A class of target protein chimeras with antitumor activity, the general structural formula of which is as follows:

[0008] Where R stands for Linker, and can be triethylene glycol, tetraethylene glycol, pentaethylene glycol, four-carbon chain, five-carbon chain, six-carbon chain, seven-carbon chain, eight-carbon chain, nine-carbon chain, azacyclobutane, tetrahydropyrrole, piperidine, or 1-phenylpiperazine. Preferably, the purine derivative G4-targeting protein chimeric structure is as follows:

[0009]

[0010]

[0011]

[0012] The preparation method of the above-mentioned class of purine derivative G4-targeting protein chimeras with antitumor activity is as follows: Glycol chain synthesis route:

[0013]

[0014] Carbon chain synthesis route:

[0015] Synthetic route for nitrogen-containing heterocyclic chains:

[0016] The application of the above-mentioned class of purine derivative G4-targeting protein chimeras with antitumor activity in the preparation of antitumor drugs.

[0017] Furthermore, the antitumor drug is a drug for treating human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116.

[0018] The advantages and positive effects of this invention are as follows: This invention, through spectral and mass spectrometry data analysis, demonstrates that the synthesized purine derivative G4 targeting protein chimera is a novel compound. Furthermore, in vitro antitumor activity studies have proven that the purine derivative G4 targeting protein chimera provided by this invention exhibits strong antitumor activity against various tumor cells, including human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116. It is an excellent new antitumor compound with the potential to be developed into a novel antitumor drug. This invention presents a novel type of targeting protein chimera with antitumor activity, exhibiting significantly improved antitumor activity compared to the previously developed small-molecule drug G4, while requiring a reduced dosage. Attached image description: Figure 1 This is the 1H NMR spectrum of compound PROTAC 1 in deuterated CHCl3 in this invention; Figure 2 This is the 1H NMR spectrum of compound PROTAC 2 in deuterated CHCl3 in this invention; Figure 3 This is the 1H NMR spectrum of compound PROTAC 3 in deuterated CHCl3 in this invention; Figure 4 This is the 1H NMR spectrum of compound PROTAC 4 in deuterated CHCl3 in this invention. Figure 5 This is the 1H NMR spectrum of compound PROTAC 5 in deuterated CHCl3 in this invention; Figure 6 This is the 1H NMR spectrum of compound PROTAC 6 in deuterated CHCl3 in this invention; Figure 7 This is the 1H NMR spectrum of compound PROTAC 7 in deuterated CHCl3 in this invention; Figure 8 This is the 1H NMR spectrum of compound PROTAC 8 in deuterated CHCl3 in this invention; Figure 9 This is the 1H NMR spectrum of compound PROTAC 9 in deuterated CHCl3 in this invention; Figure 10 This is the 1H NMR spectrum of compound PROTAC 10 in deuterated CHCl3 in this invention; Figure 11 This is the 1H NMR spectrum of compound PROTAC 11 in deuterated CHCl3 in this invention; Figure 12 This is the 1H NMR spectrum of compound PROTAC 12 in deuterated CHCl3 in this invention; Figure 13This is the 1H NMR spectrum of compound PROTAC 13 in deuterated CHCl3 in this invention; Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. It should be noted that these embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0020] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0021] A class of target protein chimeras with antitumor activity, the general structural formula of which is as follows:

[0022] Where R stands for Linker, and can be triethylene glycol, tetraethylene glycol, pentaethylene glycol, four-carbon chain, five-carbon chain, six-carbon chain, seven-carbon chain, eight-carbon chain, nine-carbon chain, azacyclobutane, tetrahydropyrrole, piperidine, or 1-phenylpiperazine.

[0023] Preferably, the specific structural formula of the purine derivative G4 targeting protein chimeric complex is as follows:

[0024]

[0025]

[0026]

[0027] The preparation method of the above-mentioned class of purine derivative G4-targeting protein chimeras with antitumor activity is as follows: Glycol chain synthetic route:

[0028]

[0029] Carbon chain synthesis route:

[0030] Synthetic route for nitrogen-containing heterocyclic chains:

[0031] The application of the above-mentioned class of purine derivative G4-targeting protein chimeras with antitumor activity in the preparation of antitumor drugs.

[0032] Preferably, the antitumor drug is a drug for treating human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116.

[0033] The preferred implementation route for a class of purine derivative G4-targeting protein chimeras with antitumor activity is as follows: Reaction 1

[0034]

[0035] Preferably, the specific steps are as follows: Synthesis of compound PROTAC 1

[0036] In a 100 mL round-bottom flask, diethylene glycol (0.894 mL, 1 g, 9.42 mmol, 1.0 eq) was added and dissolved in 20 mL of tetrahydrofuran. Then, sodium hydride (1.44 mL, 1.05 g, 10.37 mmol, 1.1 eq) was slowly added under ice bath and magnetic stirring, and the mixture was stirred at room temperature for 30 min to ensure thorough mixing. Next, tert-butyl acrylate (1.62 g, 8.48 mmol, 0.9 eq) was slowly added dropwise to the reaction system, and the reaction was continued at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reactants had reacted completely, the reaction was quenched and extracted with dichloromethane (3 × 50 mL). The combined organic phases were washed successively with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300–400 mesh, elution system: dichloromethane / methanol = 100:1 → 50:1 gradient elution) to finally obtain intermediate 1a (1.64 g), with a yield of 86%.

[0037] Under nitrogen protection, compound 1a (100 mg, 0.33 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous tetrahydrofuran and placed in a 50 mL round-bottom flask. Triphenylphosphine (165 mg, 1.1 mmol, 1.1 eq) and carbon tetrabromide (0.30 mL, 1.8 mmol, 2.0 eq) were added sequentially with stirring at room temperature. The reaction mixture was stirred for 4 h, and the reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the mixture was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 min, and then extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300-400 mesh, elution system: dichloromethane / methanol = 70:1 → 50:1 gradient elution) to give intermediate 1b (163 mg, 99% yield).

[0038] In a 50 mL round-bottom flask, compound 1b (200 mg, 0.69 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. The reaction system was cooled in an ice bath, and trifluoroacetic acid (2-5 mL) was slowly added dropwise with stirring. After the addition was complete, the ice bath was removed, and the reaction system was allowed to rise to room temperature and the reaction was stirred for 2 h. The reaction was confirmed to be complete by thin-layer chromatography at 254 nm. The reaction solution was then slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. Subsequently, the mixture was extracted with dichloromethane (3 × 20 mL), and the combined organic phases were washed successively with saturated sodium bicarbonate solution (to neutralize excess trifluoroacetic acid) and deionized water. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to give intermediate 1c (198 mg, 100% yield). The purity of this product met the requirements for subsequent reactions and could be directly used for the next conversion.

[0039] Under argon protection, compound G4 (200 mg, 0.36 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Intermediate 1c (130.05 mg, 0.54 mmol, 1.5 eq), 1-hydroxybenzotriazole (HOBT, 33.38 mg, 0.72 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 74.45 mg, 0.72 mmol, 2.0 eq), and N-methylmorpholine (NMM, 42.41 mg, 0.72 mmol, 2.0 eq) were added sequentially. The reaction mixture was stirred in an ice bath for 10 min until completely dissolved, then transferred to room temperature and the reaction was continued for 12 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the product was extracted with ethyl acetate (3 × 20 mL) and water (30 mL). The combined organic phases were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 50:1 → 20:1 gradient elution) to give intermediate 1d (117 mg, yield 83.36%).

[0040] Under nitrogen protection, intermediate 1d (100 mg, 128.34 μmol, 1.0 eq) was dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Pomalidomide (52.6 mg, 192.51 μmol, 1.5 eq), potassium carbonate (20.20 mg, 192.51 μmol, 1.5 eq), and sodium iodide (2.39 mg, 19.25 μmol, 0.15 eq) were added sequentially. The reaction mixture was stirred at room temperature for 10 min until completely dissolved, then transferred to an 80°C oil bath and reacted for 6 h. The reaction progress was monitored by thin-layer chromatography at 365 nm. After the reaction was complete, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. The mixture was then extracted with dichloromethane (3 × 30 mL), and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 30:1 → 10:1 gradient elution) to obtain the target product PROTAC 1 (34 mg, yield 26.88%).

[0041] 1 H NMR (400 MHz, Chloroform-d) δ=1.45-1.75 (m, 13H), 1.86-1.96 (tt,J= 4.7, 6.7 Hz, 2H), 1.96-2.09 (m, 6H), 2.29-2.36 (m, 2H), 2.43-2.50 (m, 5H), 2.54-2.61 (t, J =6.7 Hz, 2H), 2.71-2.79 (t, J =7.1 Hz, 2H), 3.33-3.41 (m, 4H), 3.53-3.60 (m, 4H), 3.63-3.67 (s, 4H), 3.67-3.74 (t, J =6.8 Hz, 2H), 4.35-4.43(t, J =7.1 Hz, 2H), 4.63-4.67 (s, 2H), 5.07-5.18 (m, 2H), 6.95-7.02 (dd, J =1.5,7.9 Hz, 1H), 7.26-7.36 (m, 2H), 7.42-7.50 (m, 2H), 7.50-7.55 (d, J =7.9 Hz, 1H), 7.70-7.79 (dd, J =1.5, 7.7 Hz, 1H). For example... Figure 1 Synthesis of compound PROTAC 2

[0042] In a 100 mL round-bottom flask, triethylene glycol (0.894 mL, 1 g, 9.42 mmol, 1.0 eq) was added and dissolved in 20 mL of tetrahydrofuran. Then, sodium hydride (1.44 mL, 1.05 g, 10.37 mmol, 1.1 eq) was slowly added under ice bath and magnetic stirring, and the mixture was stirred at room temperature for 30 min to ensure thorough mixing. Next, tert-butyl acrylate (1.62 g, 8.48 mmol, 0.9 eq) was slowly added dropwise to the reaction system, and the reaction was continued at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reactants had reacted completely, the reaction was quenched and extracted with dichloromethane (3 × 50 mL). The combined organic phases were washed successively with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300–400 mesh, elution system: dichloromethane / methanol = 100:1 → 50:1 gradient elution) to finally obtain intermediate 2a (1.64 g), with a yield of 86%.

[0043] Under nitrogen protection, compound 2a (100 mg, 0.33 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous tetrahydrofuran and placed in a 50 mL round-bottom flask. Triphenylphosphine (165 mg, 1.1 mmol, 1.1 eq) and carbon tetrabromide (0.30 mL, 1.8 mmol, 2.0 eq) were added sequentially with stirring at room temperature. The reaction mixture was stirred for 4 h, and the reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the mixture was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 min, and then extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300-400 mesh, elution system: dichloromethane / methanol = 70:1 → 50:1 gradient elution) to give intermediate 2b (163 mg, 99% yield).

[0044] In a 50 mL round-bottom flask, compound 2b (200 mg, 0.69 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. The reaction system was cooled in an ice bath, and trifluoroacetic acid (2-5 mL) was slowly added dropwise with stirring. After the addition was complete, the ice bath was removed, and the reaction system was allowed to rise to room temperature and the reaction was stirred for 2 h. The reaction was confirmed to be complete by thin-layer chromatography at 254 nm. The reaction solution was then slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. Subsequently, the mixture was extracted with dichloromethane (3 × 20 mL), and the combined organic phases were washed successively with saturated sodium bicarbonate solution (to neutralize excess trifluoroacetic acid) and deionized water. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to give intermediate 2c (198 mg, 100% yield). The purity of this product met the requirements for subsequent reactions and could be directly used for the next conversion.

[0045] Under argon protection, compound G4 (200 mg, 0.36 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Intermediate 2c (130.05 mg, 0.54 mmol, 1.5 eq), 1-hydroxybenzotriazole (HOBT, 33.38 mg, 0.72 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 74.45 mg, 0.72 mmol, 2.0 eq), and N-methylmorpholine (NMM, 42.41 mg, 0.72 mmol, 2.0 eq) were added sequentially. The reaction mixture was stirred in an ice bath for 10 min until completely dissolved, then transferred to room temperature and the reaction was continued for 12 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 20 mL) and water (30 mL). The combined organic phases were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 50:1 → 20:1 gradient elution) to give intermediate 2d (117 mg, yield 81%).

[0046] Under nitrogen protection, intermediate 2d (100 mg, 128.34 μmol, 1.0 eq) was dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Pomalidomide (52.6 mg, 192.51 μmol, 1.5 eq), potassium carbonate (20.20 mg, 192.51 μmol, 1.5 eq), and sodium iodide (2.39 mg, 19.25 μmol, 0.15 eq) were added sequentially. The reaction mixture was stirred at room temperature for 10 min until completely dissolved, then transferred to an 80°C oil bath for 6 h. The reaction progress was monitored by thin-layer chromatography at 365 nm. After the reaction was complete, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. The mixture was then extracted with dichloromethane (3 × 30 mL), and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 30:1 → 10:1 gradient elution) to obtain the target product PROTAC 2 (34 mg, yield 22%).

[0047] 1H NMR (DMSO-d6, 400 MHz) δ=0.97-1.02 (s, 4H), 2.42-2.64 (m, 16H),2.68-2.73 (d, J =1.9 Hz, 8H), 2.82-2.86 (s, 4H), 2.92-3.09 (m, 17H), 3.24-3.46(m, 18H), 3.48-3.53 (s, 4H), 3.62-3.73 (m, 6H), 4.28-4.33 (d, J =1.5 Hz, 7H),4.33-4.44 (m, 4H), 4.54-4.61 (m, 19H), 4.61-4.65 (m, 4H), 5.28-5.36 (dq, J =2.2, 4.3 Hz, 4H), 5.56-5.60 (s, 3H), 6.00-6.10 (m, 2H), 7.85-7.95 (dd, J =1.5, 7.9 Hz, 1H), 8.19-8.32 (m, 5H), 8.35-8.48 (m, 3H), 8.62-8.74 (m, 2H). For example... Figure 2 Synthesis of compound PROTAC 3

[0048] In a 100 mL round-bottom flask, tetraethylene glycol (0.894 mL, 1 g, 9.42 mmol, 1.0 eq) was added and dissolved in 20 mL of tetrahydrofuran. Then, sodium hydride (1.44 mL, 1.05 g, 10.37 mmol, 1.1 eq) was slowly added under ice bath and magnetic stirring, and the mixture was stirred at room temperature for 30 min to ensure thorough mixing. Next, tert-butyl acrylate (1.62 g, 8.48 mmol, 0.9 eq) was slowly added dropwise to the reaction system, and the reaction was continued at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reactants had reacted completely, the reaction was quenched and extracted with dichloromethane (3 × 50 mL). The combined organic phases were washed successively with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300–400 mesh, elution system: dichloromethane / methanol = 100:1 → 50:1 gradient elution) to finally obtain intermediate 3a (1.64 g), with a yield of 86%.

[0049] Under nitrogen protection, compound 3a (100 mg, 0.33 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous tetrahydrofuran and placed in a 50 mL round-bottom flask. Triphenylphosphine (165 mg, 1.1 mmol, 1.1 eq) and carbon tetrabromide (0.30 mL, 1.8 mmol, 2.0 eq) were added sequentially with stirring at room temperature. The reaction mixture was stirred for 4 h, and the reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the mixture was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 min, and then extracted with dichloromethane (3 × 30 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (300-400 mesh, elution system: dichloromethane / methanol = 70:1 → 50:1 gradient elution) to give intermediate 3b (163 mg, 99% yield).

[0050] In a 50 mL round-bottom flask, compound 3b (200 mg, 0.69 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. The reaction system was cooled in an ice bath, and trifluoroacetic acid (2-5 mL) was slowly added dropwise with stirring. After the addition was complete, the ice bath was removed, and the reaction system was allowed to rise to room temperature and the reaction was stirred for 2 h. The reaction was confirmed to be complete by thin-layer chromatography at 254 nm. The reaction solution was then slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. Subsequently, the mixture was extracted with dichloromethane (3 × 20 mL), and the combined organic phases were washed successively with saturated sodium bicarbonate solution (to neutralize excess trifluoroacetic acid) and deionized water. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to give intermediate 3c (198 mg, 100% yield). The purity of this product met the requirements for subsequent reactions and could be directly used for the next conversion.

[0051] Under argon protection, compound G4 (200 mg, 0.36 mmol, 1.0 eq) was dissolved in 15 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Intermediate 2c (130.05 mg, 0.54 mmol, 1.5 eq), 1-hydroxybenzotriazole (HOBT, 33.38 mg, 0.72 mmol, 2.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 74.45 mg, 0.72 mmol, 2.0 eq), and N-methylmorpholine (NMM, 42.41 mg, 0.72 mmol, 2.0 eq) were added sequentially. The reaction mixture was stirred in an ice bath for 10 min until completely dissolved, then transferred to room temperature and the reaction was continued for 12 h. The reaction progress was monitored by thin-layer chromatography at 254 nm. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 20 mL) and water (30 mL). The combined organic phases were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 50:1 → 20:1 gradient elution) to give intermediate 3d (117 mg, yield 84%).

[0052] Under nitrogen protection, intermediate 3d (100 mg, 128.34 μmol, 1.0 eq) was dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) in a 50 mL round-bottom flask. Pomalidomide (52.6 mg, 192.51 μmol, 1.5 eq), potassium carbonate (20.20 mg, 192.51 μmol, 1.5 eq), and sodium iodide (2.39 mg, 19.25 μmol, 0.15 eq) were added sequentially. The reaction mixture was stirred at room temperature for 10 min until completely dissolved, then transferred to an 80°C oil bath for 6 h. The reaction progress was monitored by thin-layer chromatography at 365 nm. After the reaction was complete, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, and stirred for 5 min. The mixture was then extracted with dichloromethane (3 × 30 mL), and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (300-400 mesh, dichloromethane / methanol = 30:1 → 10:1 gradient elution) to obtain the target product PROTAC 3 (34 mg, yield 29%).

[0053] 1 H NMR (400 MHz, Chloroform-d) δ=0.84-0.90 (t, J=6.7 Hz, 2H), 1.10-1.17 (d, J = 11.8 Hz, 2H), 1.21-1.24 (d, J =7.0 Hz, 2H), 1.24-1.27 (d, J =1.8 Hz, 6H), 1.28-1.29 (d, J =2.3 Hz, 2H), 1.65-1.69 (s, 2H), 1.69-1.74 (s, 4H), 1.74-1.78 (s, 2H), 1.85-2.12 (m, 8H), 2.15-2.25 (d, J =8.3 Hz, 2H), 2.57-2.64 (m,2H), 2.64-2.76 (m, 3H), 2.97-3.07 (s, 2H), 3.43-3.50 (t, J =6.3 Hz, 1H), 3.57-3.63 (m, 5H), 3.63-3.68 (dd, J =1.4, 6.5 Hz, 8H), 3.70-3.78 (m, 3H), 3.78-3.84(t, J =6.3 Hz, 2H), 4.17-4.32 (d, J =17.9 Hz, 2H), 4.66-4.80 (td, J =5.0, 8.8, 10.3Hz, 5H), 5.98-6.10 (s, 1H), 7.06-7.11 (d, J =8.6 Hz, 1H), 7.15-7.21 (dd, J =1.9,9.9 Hz, 1H), 7.27-7.41 (m, 2H), 7.46-7.55 (d, J =15.4 Hz, 1H). For example... Figure 3 Synthesis of compound PROTAC 4

[0054] At room temperature, in a 50 mL round-bottom flask, tert-butyl 5-bromopentanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC was used to confirm complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 4a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0055] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 4a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 4b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 4b, with a yield of 86%.

[0056] Under nitrogen protection, 100 mg (139.05 μmol) of compound 4b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. After the reaction was complete, as monitored by thin-layer chromatography at 365 nm, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 4, with a yield of 26.8%.

[0057] 1 H NMR (DMSO-d6, 400 MHz) δ=0.72-1.44 (8H, m), 1.45-1.69 (6H, m), 1.69-2.12 (7H, m), 2.15-2.37 (2H, m), 2.38-2.50 (2H, s), 2.52-2.78 (3H, m),2.79-3.07 (3H, d, J =21.4 Hz), 3.11-3.50 (6H, d, J =33.4 Hz), 3.50-3.70 (2H, dt, J =5.5, 40.5 Hz), 4.00-4.14 (1H, t, J =6.0 Hz), 4.44-4.74 (4H, m), 6.46-6.57 (1H,s), 6.90-7.07 (1H, dd, J =7.8, 10.4 Hz), 7.13-7.66 (3H, m), 7.75-7.84 (1H, s), 7.91-8.10 (1H, m). For example... Figure 4 Synthesis of compound PROTAC 5

[0058] At room temperature, in a 50 mL round-bottom flask, tert-butyl 6-bromohexanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC was used to confirm complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 5a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0059] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 5a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 5b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 5b, with a yield of 86%.

[0060] Under nitrogen protection, 100 mg (139.05 μmol) of compound 5b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. After the reaction was completed, as monitored by thin-layer chromatography at 365 nm, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 5, with a yield of 22%.

[0061] 1 H NMR (Chloroform-d, 400 MHz) δ=1.08-1.21 (2H, m), 1.22-1.54 (9H,m), 1.55-1.82 (9H, m), 1.82-2.03 (8H, m), 2.11-2.48 (6H, m), 2.56-2.79 (4H,m), 2.93-3.02 (2H, m), 3.37-3.43 (2H, t, J =6.7 Hz), 4.06-4.27 (2H, t, J =5.8Hz), 4.68-4.78 (4H, m), 6.20-6.37 (1H, s), 7.06-7.12 (1H, d, J =8.2 Hz), 7.15-7.22 (1H, d, J =9.9 Hz), 7.23-7.33 (2H, d, J =13.1 Hz), 7.45-7.51 (1H, s). For example... Figure 5 Synthesis of compound PROTAC 6

[0062] At room temperature, in a 50 mL round-bottom flask, tert-butyl 7-bromoheptanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC analysis confirmed complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated by vacuum distillation to obtain 1.0 g of intermediate 6a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0063] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 6a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 6b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 6b, with a yield of 86%.

[0064] Under nitrogen protection, 100 mg (139.05 μmol) of compound 6b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. After the reaction was completed, as monitored by thin-layer chromatography at 365 nm, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 6, with a yield of 22%.

[0065] 1 H NMR (400 MHz, Chloroform-d) δ=1.07-1.20 (dd, J =3.7, 12.5 Hz, 3H),1.29-1.37 (d, J =8.0 Hz, 4H), 1.40-1.47 (t, J =7.6 Hz, 1H), 1.50-1.64 (dt, J =7.3,15.2 Hz, 4H), 1.64-1.73 (s, 6H), 1.73-1.78 (dd, J =4.6, 7.0 Hz, 2H), 1.84-1.89(m, 2H), 1.89-1.96 (m, 3H), 1.96-2.00 (d, J =6.3 Hz, 2H), 2.00-2.04 (s, 2H), 2.16-2.23 (d, J =7.8 Hz, 2H), 2.29-2.37 (m, 2H), 2.59-2.66 (d, J =6.3 Hz, 2H),2.66-2.76 (m, 3H), 2.96-3.04 (d, J =11.2 Hz, 2H), 3.76-3.82 (t, J =7.4 Hz, 1H), 4.18-4.25 (d, J =5.7 Hz, 2H), 4.51-4.57 (t,J =6.6 Hz, 1H), 4.67-4.80 (t, J =6.7Hz, 5H), 6.11-6.22 (s, 1H), 7.06-7.11 (d, J =8.0 Hz, 1H), 7.16-7.20 (m, 1H),7.27-7.32 (t, J =7.9 Hz, 1H), 7.36-7.43 (m, 1H), 7.46-7.50 (d, J =5.2 Hz, 1H), 7.50-7.58 (dd, J =7.6, 18.7 Hz, 1H), 7.98-8.04 (m, 1H). For example... Figure 6 Synthesis of compound PROTAC 7

[0066] At room temperature, in a 50 mL round-bottom flask, tert-butyl 8-bromooctanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC was used to confirm complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated by vacuum distillation to obtain 1.0 g of intermediate 7a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0067] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 7a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 7b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 7b, with a yield of 86%.

[0068] Under nitrogen protection, 100 mg (139.05 μmol) of compound 7b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. The reaction was monitored by thin-layer chromatography at 365 nm until completion. The reaction solution was then slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 7, with a yield of 28%.

[0069] 1 H NMR (Chloroform-d, 400 MHz) δ=0.97-1.27 (6H, m), 1.27-1.55 (14H,d, J =7.1 Hz), 1.60-1.88 (4H, d, J =49.4 Hz), 1.89-2.08 (6H, d, J=6.8 Hz), 2.19-2.55 (8H, s), 2.69-2.93 (2H, s), 3.05-3.23 (2H, s), 3.31-3.51 (4H, s), 3.99-4.16 (2H, d, J =7.0 Hz), 5.32-5.55 (3H, s), 7.01-7.21 (2H, d, J =8.8 Hz), 7.44-7.54 (4H, d, J =19.1 Hz), 7.61-7.73 (1H, d, J =15.6 Hz), 7.82-7.96 (1H, d, J =15.6Hz). Figure 7 Synthesis of compound PROTAC 8

[0070] At room temperature, in a 50 mL round-bottom flask, tert-butyl 9-bromononanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction system. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC was used to confirm complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 8a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0071] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 8a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 8b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 8b, with a yield of 86%.

[0072] Under nitrogen protection, 100 mg (139.05 μmol) of compound 8b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. After the reaction was completed, as monitored by thin-layer chromatography at 365 nm, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 8, with a yield of 29%.

[0073] 1 H NMR (DMSO-d6, 400 MHz) δ=1.19-1.29 (8H, s), 1.35-1.89 (18H, m), 1.92-2.09 (6H, m), 2.51-2.69 (8H, m), 2.84-2.89 (2H, m), 3.68-3.76 (3H, s),4.47-4.71 (5H, m), 4.97-5.10 (2H, dd, J=5.3, 12.9 Hz), 6.44-6.57 (2H, s), 6.96-7.04 (3H, t, J =8.3 Hz), 7.15-7.25 (1H, d, J =8.4 Hz), 7.30-7.37 (1H, d, J =10.5 Hz), 7.44-7.53 (2H, m), 7.82-7.90 (1H, s). For example... Figure 8 Synthesis of compound PROTAC 9

[0074] At room temperature, in a 50 mL round-bottom flask, tert-butyl 10-bromodecanoate (500 mg, 2.11 mmol, 1.0 eq) was dissolved in 10.0 mL of dichloromethane. Under ice bath conditions, 2–5 mL of trifluoroacetic acid (TFA) solution was slowly added dropwise to the reaction mixture. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 2 h. TLC was used to confirm complete reaction. The reaction mixture was then slowly poured into an ice-water mixture and stirred for 5 minutes. The mixture was then extracted three times with 20 mL of dichloromethane. The combined organic phases were neutralized with excess TFA using saturated sodium bicarbonate solution and washed successively with deionized water. The organic phase was dried over anhydrous sodium sulfate, and the mixture was concentrated by vacuum distillation to obtain 1.0 g of intermediate 9a, with a yield of 99%. No further purification was required, and the next reaction could proceed.

[0075] Under nitrogen protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 9a (120.31 mg, 0.54 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. After stirring in an ice bath for 10 minutes to ensure complete dissolution, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was extracted three times with 20 mL of ethyl acetate and 30 mL of water. The combined organic phases were washed sequentially with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 9b was purified by silica gel column chromatography (300-400 mesh, eluent ratio of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 9b, with a yield of 86%.

[0076] Under nitrogen protection, 100 mg (139.05 μmol) of compound 9b was dissolved in 5 mL of DMF in a 50 mL round-bottom flask. After complete dissolution, pomalidomide (56.99 mg, 208.58 μmol), potassium carbonate (21.20 mg, 208.58 μmol), and a catalytic amount of sodium iodide (2.39 mg, 20.86 μmol) were added sequentially. After magnetic stirring at room temperature for 10 minutes, the reaction mixture was transferred to a 90°C oil bath and reacted for 6 hours. After the reaction was complete, as monitored by thin-layer chromatography at 365 nm, the reaction solution was slowly poured into an ice-water mixture to quench the reaction, stirred for 5 minutes, and extracted three times with 30 mL of dichloromethane. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate, then concentrated under reduced pressure. The product was purified by silica gel column chromatography (300-400 mesh, gradient elution of dichloromethane:methanol = 50:1 to 20:1) to obtain 34 mg PROTAC 9, with a yield of 30%.

[0077] 1 H NMR (400 MHz, Chloroform-d) δ=0.98-1.13 (d, J =12.4 Hz, 1H), 1.16-1.49 (d, J =41.3 Hz, 20H), 1.52-1.72 (qd, J =7.5, 10.2, 14.5 Hz, 6H), 1.72-1.98(qt, J =11.8, 25.1 Hz, 8H), 2.14-2.25 (m, 1H), 2.25-2.37 (t, J =7.6 Hz, 4H), 2.41-2.55 (t, J =12.0 Hz, 1H), 2.65-2.78 (t, J =12.7 Hz, 1H), 2.88-3.01 (s, 1H), 3.31-3.49 (t, J =6.8 Hz, 3H), 3.54-3.65 (d, J =11.4 Hz, 1H), 3.85-3.95 (s, 1H), 4.61-4.81 (dd, J =9.1, 21.8 Hz, 3H), 6.98-7.15 (m, 3H), 7.16-7.24 (d, J =10.1 Hz,1H), 7.47-7.58 (s, 1H). Synthesis of compound PROTAC 10

[0078] Under argon protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 10a (44.54 mg, 0.43 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. The reaction mixture was stirred in an ice bath for 10 minutes until completely dissolved, and then transferred to room temperature for 20 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was subjected to three liquid-liquid extractions with 20 mL of ethyl acetate and 30 mL of water. The combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 10b was purified by silica gel column chromatography (300-400 mesh, elution system of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 10b, with a separation yield of 71%.

[0079] At room temperature, in a 50 mL round-bottom flask, intermediate 10b (100 mg) was dissolved in 10 mL of 4 M HCl in 1,4-dioxane solution. The reaction was stirred for 2 h at room temperature. After the reaction was complete, the reaction system was slowly poured into water to quench the reaction. The mixture was extracted with ethyl acetate, and the combined organic phases were washed successively with saturated sodium bicarbonate solution and deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 10c, with a yield of 98%. No further purification was required, and the next reaction could proceed.

[0080] At room temperature, intermediate 10c was dissolved in 10 ml of DMSO, and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and DIEA were added sequentially, stirring for 5 min until fully dissolved. The mixture was then reacted in an oil bath at 100 °C for 6 h. After the reaction was complete as detected by TLC, the reaction solution was slowly poured into an ice-water mixture and stirred for 5 min. The mixture was then extracted three times with 20 ml of dichloromethane, and the organic phases were combined. The reaction system was then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. Purification was performed by silica gel column chromatography (dichloromethane:methanol = 50:1-20:1 gradient elution) to obtain 34 mg of PROTAC 10, with a yield of 46%.

[0081] 1H NMR (DMSO-d6, 400 MHz) δ=1.22-1.44 (9H, s), 1.49-1.75 (5H, s), 1.77-2.13 (9H, m), 2.14-2.33 (3H, d, J =11.4 Hz), 2.78-2.97 (3H, t, J =13.0 Hz),3.46-3.62 (4H, s), 3.76-4.09 (4H, d, J =48.8 Hz), 4.52-4.81 (4H, m), 4.96-5.14(2H, dd, J =5.0, 12.7 Hz), 6.92-7.08 (1H, t, J =7.2 Hz), 7.13-7.42 (2H, dd, J =8.8,58.0 Hz), 7.41-7.56 (3H, m), 7.71-7.87 (1H, s). Synthesis of compound PROTAC 11

[0082] Under argon protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 11a (44.54 mg, 0.43 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. The reaction mixture was stirred in an ice bath for 10 minutes until completely dissolved, and then transferred to room temperature for 20 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was subjected to three liquid-liquid extractions with 20 mL of ethyl acetate and 30 mL of water. The combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 11b was purified by silica gel column chromatography (300-400 mesh, elution system of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 11b, with a separation yield of 65%.

[0083] At room temperature, in a 50 mL round-bottom flask, intermediate 11b (100 mg) was dissolved in 10 mL of 4 M HCl in 1,4-dioxane solution. The reaction was stirred for 2 h at room temperature. After the reaction was confirmed to be complete by thin-layer chromatography at 254 nm, the reaction system was slowly poured into water to quench the reaction. The mixture was extracted with ethyl acetate, and the combined organic phases were washed successively with saturated sodium bicarbonate solution and deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 11c, with a yield of 98%. No further purification was required, and the next reaction could proceed.

[0084] At room temperature, intermediate 11c was dissolved in 10 ml of DMSO, and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and DIEA were added sequentially, stirring for 5 min until fully dissolved. The mixture was then reacted in an oil bath at 100 °C for 6 h. After the reaction was complete as detected by TLC, the reaction solution was slowly poured into an ice-water mixture and stirred for 5 min. The mixture was then extracted three times with 20 ml of dichloromethane, and the organic phases were combined. The reaction system was then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. Purification was performed by silica gel column chromatography (dichloromethane:methanol = 50:1-20:1 gradient elution) to obtain 34 mg of PROTAC 11, with a yield of 26%.

[0085] 1 H NMR (DMSO-d6, 400 MHz) δ=1.03-1.47 (11H, m), 1.53-1.86 (9H, m), 1.88-2.13 (8H, m), 2.57-2.71 (2H, m), 2.90-3.08 (1H, s), 3.15-3.52 (8H, m),4.45-4.73 (4H, m), 4.96-5.19 (2H, dd, J =5.2, 12.8 Hz), 6.95-7.07 (1H, t, J =7.6Hz), 7.16-7.38 (2H, dd, J =9.1, 57.6 Hz), 7.43-7.53 (3H, m), 7.767.86 (1H, s). Synthesis of compound PROTAC 12

[0086] Under argon protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 12a (44.54 mg, 0.43 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. The reaction mixture was stirred in an ice bath for 10 minutes until completely dissolved, and then transferred to room temperature for 20 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was subjected to three liquid-liquid extractions with 20 mL of ethyl acetate and 30 mL of water. The combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 12b was purified by silica gel column chromatography (300-400 mesh, elution system of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 12b, with a separation yield of 62%.

[0087] At room temperature, in a 50 mL round-bottom flask, intermediate 12b (100 mg) was dissolved in 10 mL of 4 M HCl in 1,4-dioxane solution. The reaction was stirred for 2 h at room temperature. After the reaction was complete, the reaction system was slowly poured into water to quench the reaction by thin-layer chromatography at 254 nm. The mixture was extracted with ethyl acetate, and the combined organic phases were washed successively with saturated sodium bicarbonate solution and deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 12c, with a yield of 98%. No further purification was required, and the next reaction could proceed.

[0088] At room temperature, intermediate 12c was dissolved in 10 ml of DMSO, and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and DIEA were added sequentially, stirring for 5 min until fully dissolved. The mixture was then reacted in an oil bath at 100 °C for 6 h. After the reaction was complete as detected by TLC, the reaction solution was slowly poured into an ice-water mixture and stirred for 5 min. The mixture was then extracted three times with 20 ml of dichloromethane, and the organic phases were combined. The reaction system was then washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. Purification was performed by silica gel column chromatography (dichloromethane:methanol = 50:1-20:1 gradient elution) to obtain 34 mg of PROTAC 12, with a yield of 28%.

[0089] 1 H NMR (Chloroform-d, 400 MHz) δ=1.53-2.09 (23H, m), 2.11-2.31 (2H,d,J =7.9 Hz), 2.36-2.55 (3H, m), 2.58-2.79 (4H, dd, J =15.9, 28.0 Hz), 2.79-3.04(4H, m), 3.91-4.08 (2H, s), 4.16-4.29 (2H, t, J =5.7 Hz), 4.61-4.78 (2H, d, J =7.5 Hz), 7.06-7.35 (6H, m), 7.47 -7.54 (1H, s). Synthesis of compound PROTAC 13

[0090] Under argon protection, 200 mg (0.36 mmol) of compound G4 was dissolved in 15 mL of anhydrous N,N-dimethylformamide in a 50 mL round-bottom flask. Then, intermediate 13a (44.54 mg, 0.43 mmol), HOBT (33.38 mg, 0.72 mmol), EDCI (74.45 mg, 0.72 mmol), and NMM (42.41 mg, 0.72 mmol) were added sequentially. The reaction mixture was stirred in an ice bath for 10 minutes until completely dissolved, then transferred to room temperature and reacted for 20 hours. The reaction progress was monitored by thin-layer chromatography. After termination, the mixture was subjected to three liquid-liquid extractions with 20 mL of ethyl acetate and 30 mL of water. The combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the intermediate 13b was purified by silica gel column chromatography (300-400 mesh, elution system of dichloromethane:methanol = 70:1 to 50:1) to obtain 156 mg of intermediate 13b, with a separation yield of 60%.

[0091] At room temperature, in a 50 mL round-bottom flask, intermediate 13b (100 mg) was dissolved in 10 mL of 4 M HCl in 1,4-dioxane solution. The reaction was stirred for 2 h at room temperature. After the reaction was confirmed to be complete by thin-layer chromatography at 254 nm, the reaction system was slowly poured into water to quench the reaction. The mixture was extracted with ethyl acetate, and the combined organic phases were washed successively with saturated sodium bicarbonate solution and deionized water. The organic phase was dried over anhydrous sodium sulfate, and the system was concentrated by vacuum distillation to obtain 1.0 g of intermediate 13c, with a yield of 98%. No further purification was required, and the next reaction could proceed.

[0092] At room temperature, intermediate 13c was dissolved in 10 ml of DMSO, and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and DIEA were added sequentially, stirring for 5 min until fully dissolved. The mixture was then reacted in an oil bath at 100 °C for 6 h. After the reaction was complete as detected by TLC, the reaction solution was slowly poured into an ice-water mixture and stirred for 5 min. The mixture was then extracted three times with 20 ml of dichloromethane. The organic phases were combined, and the reaction system was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated by vacuum distillation. The solution was purified by silica gel column chromatography (dichloromethane:methanol = 50:1-20:1 gradient elution) to obtain 34 mg of PROTAC 13, with a yield of 30%.

[0093] 1 H NMR (DMSO-d6, 400 MHz) δ=1.52-1.75 (6H, m), 1.75-2.13 (8H, m),2.24-2.47 (2H, d, J =35.6 Hz), 2.52-2.68 (4H, m), 2.86-2.98 (2H, d, J =10.6 Hz), 3.20-3.39 (6H, d, J =5.2 Hz), 3.39-3.57 (6H, dd, J =5.5, 14.8 Hz), 4.50-4.71 (2H,m), 4.98-5.09 (2H, dd, J =5.4, 12.8 Hz), 6.90-7.06 (3H, m), 7.14-7.26 (1H, d, J =8.2 Hz), 7.26-7.41 (1H, d, J =10.6 Hz), 7.42-7.53 (2H, q, J =7.4 Hz), 7.73-7.85(2H, m), 7.92-8.05 (1H, s). The above-mentioned application of a type of purine derivative G4-targeting protein chimera with antitumor activity in the preparation of antitumor drugs.

[0094] Preferably, the antitumor drug is a drug for treating human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116.

[0095] Assay of the antitumor activity of the purine derivative G4-targeting protein chimera in this invention Solution preparation: Preparation of PRMI 1640 and DMEM low-glucose culture medium: Purchase Gibco PRMI 1640 medium and DMEM low-glucose medium, 500 mL per bottle, add 10% fetal bovine serum and 1% penicillin-streptomycin solution, that is, add 50 mL of fetal bovine serum and 5 mL of penicillin-streptomycin per bottle of medium. The medium is prepared in a clean bench and then stored in a refrigerator at 4°C.

[0096] Preparation of PBS buffer: In a 1000 mL Erlenmeyer flask, weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, and 0.2 g of potassium dihydrogen phosphate. Add 800 mL of purified water, stir thoroughly to dissolve, and bring the volume to 1000 mL. After autoclaving, store in a refrigerator at 4°C.

[0097] Preparation of MTT solution: Weigh 0.5 g of MTT dry powder, dissolve it in 100 mL of PBS buffer, filter it through a 0.22 μM filter membrane for sterilization, and store it at -12℃.

[0098] Specific steps for determining antitumor activity: The tumor cells used in the antitumor activity assay of this invention were: human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT116. Activity assays were performed using human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT116.

[0099] MDA-MB-231 and HepG2 cells were cultured in PRMI 1640 cell culture medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum. MGC803 and HCT116 cells were cultured in DMEM low-glucose medium with identical preparation conditions. The culture conditions were a 37°C incubator containing 5% CO2. Specific steps: (1) After counting the cells using a hemocytometer, dilute them to 5 x 10⁻⁶ with culture medium. 4 cells / mL; (2) Add 100 μL of cell suspension to each well of a 96-well plate and incubate at 37°C. ◦ MDA-MB-231, MGC803, HepG2, and HCT116 cells were cultured in a C incubator for 24 h.

[0100] (3) Dissolve the required test compound in dimethyl sulfoxide at concentrations of 0.01, 0.1, 1, 10, and 100 µM, and add 0.5 μL / well according to the concentration. Incubate at 37°C for 48 h. (4) Add MTT at a concentration of 5 mg / mL and incubate at 37°C for 4 h; (5) MDA-MB-231, MGC803, HepG2, and HCT116 cells were lysed with DMSO. The OD values ​​of MDA-MB-231, MGC803, HepG2, and HCT116 cells at 492 and 630 nm were measured using an ELISA reader. (6) Process the data and calculate the survival rate based on the OD value.

[0101] The results are shown in Figure 1. Table 1. Results of antitumor activity assays for purine derivative G4-targeting protein chimeras.

[0102] Note: Human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT116. The chemotherapy drug camptothecin (CPT) was used as a positive control drug.

[0103] Experimental results showed that, in antigen activity tests on human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human hepatocellular carcinoma cells HepG2, and human colon cancer cells HCT116, the 13 target protein chimeras based on compound G4 exhibited excellent activity. In comparison, compound PROTAC 6 showed better activity in human triple-negative breast cancer cells MDA-MB-231. Therefore, compounds PROTAC 1, PROTAC 2, PROTAC 3, PROTAC 4, PROTAC 5, PROTAC 6, PROTAC 7, PROTAC 8, PROTAC 9, PROTAC 10, PROTAC 11, PROTAC 12, and PROTAC 13 all have the potential to become drugs for treating tumors. A comprehensive analysis of the effects of these 13 compounds revealed that PROTAC... Six pairs of human triple-negative breast cancer cells MDA-MB-231, human liver cancer cells HepG2, and human colon cancer cells HCT116 all showed good activity, making them the compounds with the best activity in this invention.

[0104] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments and drawings.

Claims

1. A type of purine derivative G4-targeting protein chimera synthesized using PROTAC technology and possessing antitumor activity, characterized in that, The structure is as follows: Where R stands for Linker, and can be triethylene glycol, tetraethylene glycol, pentaethylene glycol, four-carbon chain, five-carbon chain, six-carbon chain, seven-carbon chain, eight-carbon chain, nine-carbon chain, azacyclobutane, tetrahydropyrrole, piperidine, or 1-phenylpiperazine.

2. The purine derivative G4-targeting protein chimera with antitumor activity as described in claim 1, characterized in that: The specific structural formula of the chimera is as follows:

3. A method for preparing a type of azaflavonoid-targeting protein chimera with antitumor activity as described in claim 1 or 2, characterized in that: The synthesis route is as follows: Glycol chain synthesis route: Carbon chain synthesis route: Synthetic route for nitrogen-containing heterocyclic chains:

4. A type of purine derivative G4-targeting protein chimera synthesized according to PROTAC as described in claim 1 or 2, which has the application in the preparation of antitumor drugs, characterized in that: The antitumor drugs mentioned are those used to treat human triple-negative breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells MGC803, human liver cancer cells HepG2, and human colon cancer cells HCT-116.

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