Targeted EGFRC797S degradation agent and preparation and application thereof

Abstract

The invention provides a degradation agent targeting EGFRC797S as well as preparation and application thereof, and belongs to the technical field of medicines. The compound as shown in the formula I is prepared, the compound can degrade EGFR in a targeted mode, and expression of EGFR in H1975-M3 cells is remarkably reduced. The compound disclosed by the invention has obvious anti-proliferative activity on a non-small cell lung cancer cell line, particularly has good selectivity on an EGFRL858R / T790M / C797S mutant type H1975-M3 cell and an EGFRL858R / T790M mutant type NCI-H1975 cell, and is obvious in inhibition effect, and the compound disclosed by the invention is low in toxicity on normal cells. Meanwhile, the compound can obviously retard the cell cycle, promote cell apoptosis and inhibit cell invasion and migration. The compound provided by the invention can be used as an EGFRC797S degradation agent to be applied to preparation of non-small cell lung cancer cell resisting drugs. Formula I

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for targeting EGFR. C797S Novel EGFR PROTAC degrading agents and their preparation and application. Background Technology

[0002] Cancer is a major disease that seriously threatens human life and health, second only to cardiovascular and cerebrovascular diseases in its impact. Among 36 types of cancer, lung cancer has the highest incidence and mortality rates, making it the most deadly malignant tumor. Non-small cell lung cancer (NSCLC), the main histological subtype of lung cancer, accounts for approximately 85% of all lung cancer cases.

[0003] Mutations in the epidermal growth factor receptor (EGFR) gene are the most common oncogenic driver in non-small cell lung cancer (NSCLC). Over the past two decades, EGFR-targeting drugs have demonstrated significant efficacy in treating NSCLC, marking a major milestone in targeted therapy. First-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, approved in 2003 and 2004 respectively, showed significant efficacy in patients with advanced NSCLC harboring EGFR exon 19 deletion (19Del) or L858R mutations, significantly improving patient survival. However, after long-term use, 60% to 70% of patients developed the acquired resistance mutation T790M on EGFR exon 20, leading to treatment resistance. The third-generation EGFR-TKI osimertinib irreversibly alkylates the Cys797 site of EGFR, forming a covalent bond with its thiol group, thereby effectively inhibiting the EGFR T790M mutant kinase activity and suppressing aberrant activation of downstream signaling pathways. It has become a first-line treatment for patients with T790M-positive mutations. Osimertinib has strong blood-brain barrier penetration ability, can significantly delay the progression of central nervous system metastasis in patients with advanced EGFR mutation-positive non-small cell lung cancer, and prolong overall survival. Its efficacy and safety are significantly superior to first- and second-generation EGFR-TKI drugs.

[0004] However, most patients eventually develop resistance to osimertinib. The mechanisms of resistance are complex and diverse, primarily including intra-target resistance mechanisms, such as the C797S point mutation in EGFR (accounting for 20%), and extra-target resistance mechanisms, such as HER2 amplification, MET amplification, alterations in other tyrosine kinase receptors (AXL), oncogenic fusions (ALK, RET, FGFR), signaling pathway alterations, and histological changes. When the epidermal growth factor receptor (EGFR) signaling pathway is inhibited, tumors often activate other signaling pathways (such as anaplastic lymphoma kinase (ALK), HER2, and fibroblast growth factor receptor (FGFR)) to maintain survival and proliferation, thus leading to resistance.

[0005] In recent years, proteolytic targeting chimeras (PROTACs) have emerged as a novel therapeutic approach, demonstrating promising prospects for drug discovery and development. Recently, proteolytic targeting conjugates (PROTACs) derived from EGFR-TKIs have been developed for targeting drug-resistant EGFR in non-small cell lung cancer. PROTACs are heterobifunctional molecules, typically composed of a ligand that binds to the protein of interest, a chemical linker, and a ligand for an E3 ubiquitin ligase. PROTACs selectively degrade the target protein by inducing ubiquitination of the target protein through proteolytic enzymes. Therefore, designing and synthesizing EGFR-targeting degraders can overcome the resistance problem caused by EGFR-TKIs. This approach holds promise for providing more effective and durable treatment options for patients resistant to EGFR-TKIs, offering a new treatment strategy for this population. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a targeted EGFR... C797S Novel EGFR PROTAC degrading agents and their preparation and application.

[0007] This invention provides compounds of Formula I, or salts thereof, or stereoisomers thereof, or solvates thereof, or their crystal forms thereof: Formula I Wherein, R is selected from -CH2- or -C(O)-; n is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0008] Furthermore, the structure of the compound is shown in Formula II: Formula II Where n is selected from 2, 3, 4, 5, 6 or 7.

[0009] Furthermore, the structure of the compound is shown in Formula III: Formula III Where n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0010] Furthermore, the structure of the compound is selected from one of the following structures: .

[0011] The present invention also provides a method for preparing the above-mentioned compound, or its salt, or its stereoisomer, or its solvate, or its crystal form, the method comprising the following steps: (1) Compound 1 is reacted with thionyl chloride to obtain compound A; (2) Compound A and compound 2 are reacted to obtain compound B; (3) React compound B, compound C, nucleophile and organic base to obtain the compound described in formula I.

[0012] Furthermore, in step (1), the reaction temperature is 50~70℃ and the time is 1~3 hours; In step (2), the equivalent ratio of compound A and compound 2 is 1:0.1~0.3; In step (3), the equivalence ratio of compound B, compound C and nucleophile is 1:0.5~1.5:2~4; the nucleophile is potassium iodide; the organic base is N,N-diisopropylethylamine; the solvent for the reaction is an organic solvent, preferably N,N-dimethylformamide; the reaction temperature is 80~120℃ and the time is 1~5 hours.

[0013] The present invention also provides the use of the above-mentioned compound, or its salt, or its stereoisomer, or its solvate, or its crystal form, in the preparation of EGFR protein degrading agents.

[0014] Furthermore, the EGFR protein degrading agent is a drug for the prevention and / or treatment of non-small cell lung cancer.

[0015] Furthermore, the drug is targeted at individuals carrying EGFR. C797S Drugs for drug-resistant mutations in non-small cell lung cancer.

[0016] Furthermore, the EGFR C797S Drug resistance mutations include EGFR L858R / T790M / C797S Triple mutation or EGFR T790M / C797S Double mutation.

[0017] The present invention has achieved the following beneficial effects: Compound PB7 of this invention exhibits a significant inhibitory effect on the proliferation of non-small cell lung cancer cells, particularly on EGFR-mutant NCI-H1975 cells (L858R / T790M double mutation) and EGFR-mutant cells. C797S The mutated H1975-M3 (L858R / T790M / C797S triple mutation) exhibits significant selective inhibitory effects and low cytotoxicity to normal cells.

[0018] The compound PB7 of this invention significantly reduces the expression of EGFR protein in H1975-M3 cells and inhibits the phosphorylation of EGFR and its downstream signaling pathway proteins in a concentration-dependent manner; in addition, compound PB7 can arrest the cell cycle of H1975-M3 cells in the G0 / G1 phase, promote apoptosis, and inhibit cell invasion and migration.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 This is the proton NMR spectrum of compound PB7.

[0022] Figure 2 The EGFR protein degradation efficiency of the compounds of the present invention is as follows: (A) EGFR protein expression in H1975-M3 and A549 cells after treatment with PA2-PA7 1 µM for 24 h; (B) EGFR protein expression in H1975-M3 and A549 cells after treatment with PB2-PB10 1 µM for 24 h; (C) Quantitative analysis of the EGFR protein degradation efficiency of the compounds shown.

[0023] Figure 3 The compounds of this invention induce EGFR in a concentration-dependent manner. C797S Degradation: (A) Dose-response characteristics of the selected compounds in degrading EGFR in H1975-M3 cells after 24 h; (B) Graph showing the relationship between the concentration of the compound and the degradation efficiency of EGFR protein.

[0024] Figure 4The effects of the compounds of this invention on cell cycle and apoptosis in non-small cell lung cancer: (A) Effects of compounds PB7, osimertinib and brigatinib on the cell cycle of H1975-M3 cells; (B) Effects of compounds PB7, osimertinib and brigatinib on apoptosis of H1975-M3 cells; (C) Statistical analysis of cell cycle distribution and apoptosis of H1975-M3 cells after different drug treatment groups.

[0025] Figure 5 The effects of the compounds of this invention on the invasion and migration of non-small cell lung cancer cells are as follows: (A) Transwell migration experiment results after H1975-M3 cells were treated with different concentrations of the compound for 24 hours; (B) Transwell invasion experiment results after H1975-M3 cells were treated with different concentrations of the compound for 24 hours; (C) Quantitative results of Transwell migration and invasion experiments.

[0026] Figure 6 The compounds of this invention degrade EGFR C797S Mechanisms: (A) Effects of PB7 on EGFR-mediated signaling pathways in H1975-M3 cells; (B) Degradation activity of PB7 in H1975-M3 cells depends on the proteasome system. Detailed Implementation

[0027] Unless otherwise stated, the raw materials and equipment used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products. The invention will be further described below with reference to embodiments and accompanying drawings.

[0028] Compounds PA2~PA7 and PB2~PB10 were prepared according to the following synthetic route: Example 1: Preparation of compound PA2 3-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)propanamide (Compound PA2) (1) Compound 1 (3-bromopropionic acid, 1.0 equivalent) was dissolved in thionyl chloride (SOCl2) and heated and stirred at 60 °C for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry (LCMS) to confirm that the intermediate acyl chloride was completely formed. Subsequently, excess SOCl2 was removed by rotary evaporation under reduced pressure to obtain compound A in 95% yield; (2) No further purification was required. Pomalidomide (compound 2-1, 0.3 equivalents) was added directly to compound A to continue the reaction. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound B1, with a yield of 75%. (3) Under an inert atmosphere, compound B1 (1.0 equivalent), compound C (0.9 equivalent), potassium iodide (KI, 3.0 equivalent), and a catalytic amount of N,N-diisopropylethylamine (DIPEA) were dissolved in anhydrous N,N-dimethylformamide (DMF) and reacted at 100 °C for 3 hours with stirring. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was quenched with water and extracted multiple times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain product PA2, an orange-red solid, with a yield of 32%. 1 H NMR (400 MHz, DMSO- d 6) δ 11.21 (s, 1H), 11.15 (s, 1H), 10.35 (s, 1H), 8.52 (d, J = 8.4 Hz, 1H), 8.45 – 8.39 (m, 1H), 8.34 (d, J = 6.9 Hz, 2H), 8.16 (s, 1H), 7.84 (m 1H), 7.62 (d, J = 7.3 Hz, 1H),7.58 – 7.51 (m, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.12 – 7.06 (m, 1H), 6.80 (s,1H), 5.14 (m 1H), 3.92 (s, 3H), 3.66 – 3.59 (m, 2H), 3.15 (m 6H), 2.91 – 2.74(m, 6H), 2.51 (m, 2H), 1.79 (s, 3H), 1.76 (s, 3H). 13 C NMR (101 MHz, DMSO- d6) δ173.23, 170.28, 167.88, 167.14, 158.41, 156.38, 155.78, 155.26, 143.44,136.76, 136.54, 134.39, 132.29, 132.01, 131.23, 131.13, 127.08, 122.99,122.87, 121.94, 121.67, 121.26, 120.76, 118.89, 117.57, 105.88, 103.71,56.94, 55.40, 54.03, 52.69, 49.38, 42.33, 31.39, 19.03, 18.54, 18.33, 17.21,12.91. HRMS (ESI + m / z calcd for C 39 H 41 ClN 10 O9P + (M+H) + 859.2479, found 859.2459.

[0029] Example 2: Preparation of compound PA3 4-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)butanamide (Compound PA3) By replacing 3-bromopropionic acid with 4-bromobutyric acid in step (1) of Example 1, and keeping all other steps the same, compound PA3, an orange-red solid, is obtained with a yield of 33%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.75 (s, 1H),9.39 (s, 1H), 8.76 – 8.72 (m, 2H), 8.35 (m, 1H), 8.06 (s, 1H), 7.64 (t, J = 8.0Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.43 – 7.35 (m, 2H), 7.24 (d, J= 7.8 Hz, 1H),7.11 – 7.04 (m, 1H), 6.49 (s, 1H), 4.89 (m, 1H), 3.85 (s, 3H), 3.35 (m, 4H),3.00 (m, 2H), 2.66 (m, 4H), 2.50 (m, 4H), 1.78 (s, 3H), 1.75 (s, 3H), 1.55(m, 4H). 13 C NMR (101 MHz, Chloroform- d ) δ 171.99, 171.06, 169.16, 168.16,166.73, 156.92, 156.07, 154.75, 152.09, 143.25 (d, J = 2.5 Hz), 141.97, 137.83,136.63, 136.46, 132.76, 131.14, 129.69 (d, J = 10.7 Hz), 125.35, 124.27, 123.02(d, J = 12.0 Hz), 122.63 (d, J = 7.2 Hz), 120.47 (d, J = 95.6 Hz), 118.44, 116.20,115.35, 107.40, 102.29, 70.61, 57.20, 56.22, 52.97, 51.75, 49.35, 35.67,31.45, 26.50, 22.68, 21.81, 18.88, 18.17. HRMS (ESI + ) m / z calcd forC 40 H 43 ClN 10 O9P + (M+H) + 873.2635, found 873.2618. Example 3: Preparation of compound PA4 5-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)pentanamide (Compound PA4) By replacing 3-bromopropionic acid with 5-bromopentanoic acid in step (1) of Example 1, and keeping all other steps the same, compound PA4, an orange-red solid, is obtained with a yield of 35%. 1H NMR (400 MHz, Chloroform- d ) δ 10.73 (d, J = 2.7Hz, 1H), 9.36 (d, J = 2.4 Hz, 1H), 8.73 (m, 2H), 8.34 (m, 1H), 8.05 (d, J = 2.7Hz, 1H), 7.63 (m, 1H), 7.48 – 7.41 (m, 2H), 7.40 – 7.35 (m, 1H), 7.21 – 7.17(m, 1H), 7.06 (m, 1H), 6.52 (d, J = 2.6 Hz, 1H), 4.92 – 4.84 (m, 1H), 3.84 (s,3H), 3.34 (m, 2H), 3.02 (p, J = 2.6 Hz, 4H), 2.75 – 2.67 (m, 2H), 2.61 (t, J =4.1 Hz, 4H), 2.44 (m, 4H), 1.77 (s, 3H), 1.73 (s, 3H), 1.62 – 1.52 (m, 4H). 13 CNMR (101 MHz, Chloroform- d ) δ 172.04, 171.50, 169.19, 168.46, 166.74, 156.93,156.05, 154.65, 152.21, 143.18, 142.23 (d, J = 2.4 Hz), 137.73, 136.52 (d, J =4.1 Hz), 136.40, 132.74, 131.11, 129.69 (d, J = 10.9 Hz), 125.25, 124.13,123.02 (d, J = 12.1 Hz), 122.63 (d, J = 7.2 Hz), 120.47 (d, J= 95.8 Hz), 118.42,116.39, 115.36, 107.31, 107.29, 102.22, 70.59, 57.77, 56.16, 53.04, 51.91,49.33, 37.56, 31.46, 26.48, 25.83, 23.05, 22.68, 18.82, 18.11. HRMS (ESI + m / z calcd for C 41 H 45 ClN 10 O9P + (M+H) + 887.2792, found 887.2781. Example 4: Preparation of compound PA5 6-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)hexanamide (Compound PA5) By replacing 3-bromopropionic acid with 6-bromohexanoic acid in step (1) of Example 1, and keeping all other steps the same, compound PA5, an orange-red solid, is obtained with a yield of 35%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.82 (s, 1H), 9.43 (d, J = 7.4 Hz, 1H), 8.84 – 8.80 (m, 2H), 8.42 (m, 1H), 8.14 (s, 1H), 7.71(m, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.50 – 7.44 (m, 2H), 7.31 (m, 1H), 7.15 (m,1H), 6.62 (s, 1H), 4.99 – 4.93 (m, 1H), 3.94 (s, 3H), 3.12 (t, J = 4.8 Hz, 4H),2.89 (m, 2H), 2.78 (m, 2H), 2.71 (t, J = 4.8 Hz, 4H), 2.50 (m, 4H), 1.85 (s,3H), 1.82 (s, 3H), 1.81 – 1.76 (m, 2H), 1.66 – 1.61 (m, 2H), 1.46 – 1.41 (m,2H). 13C NMR (101 MHz, Chloroform- d ) δ 172.18, 171.27, 169.22, 168.31, 166.73,160.80, 156.93, 156.08, 154.72, 152.14, 143.22 (d, J = 2.5 Hz), 142.11, 137.79,136.69, 136.45, 132.76, 131.12, 129.69 (d, J = 10.9 Hz), 125.27, 124.29, 123.04(d, J = 12.2 Hz), 122.66 (d, J = 6.9 Hz), 120.50 (d, J = 95.9 Hz), 118.45, 115.81 (d, J = 93.0 Hz), 107.38, 102.25, 58.10, 56.18, 53.05, 51.88, 49.33, 45.59,39.93, 37.76, 31.44, 26.90, 26.02, 25.03, 22.71, 18.85, 18.14. HRMS (ESI + m / z calcd for C 42 H 47 ClN 10 O9P + (M+H) + 901.2948, found 901.2941. Example 5: Preparation of compound PA6 7-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)heptanamide (Compound PA6) By replacing 3-bromopropionic acid with 7-bromoheptanoic acid in step (1) of Example 1, and keeping all other steps the same, compound PA6, an orange-red solid, is obtained with a yield of 32%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.82 (s, 1H),9.44 (s, 1H), 8.84 – 8.79 (m, 2H), 8.42 (m, 1H), 8.14 (s, 1H), 7.71 (m, 1H),7.54 (d,J = 7.3 Hz, 1H), 7.50 – 7.44 (m, 2H), 7.32 (m, 1H), 7.14 (m, 1H), 6.63(s, 1H), 5.00 – 4.93 (m, 1H), 3.95 (s, 3H), 3.41 (m, 2H), 3.17 (t, J = 4.8 Hz,4H), 2.78 (d, J = 8.1 Hz, 4H), 2.54 (m, 2H), 2.49 – 2.45 (m, 2H), 1.85 (s, 3H),1.82 (s, 3H), 1.76 (q, J = 7.1 Hz, 2H), 1.61 (m, J = 10.7, 6.4 Hz, 4H), 1.45 –1.38 (m, 4H). 13 C NMR (101 MHz, CDCl3) δ 172.27, 171.36, 169.24, 168.42,166.74, 160.80, 156.91, 156.07, 154.70, 152.14, 143.21 (d, J = 2.5 Hz), 141.89,137.78, 136.80, 136.44, 132.75, 131.12, 129.70 (d, J = 11.0 Hz), 125.29,124.47, 123.04 (d, J = 12.1 Hz), 122.65 (d, J = 7.1 Hz), 120.51 (d, J = 95.8 Hz),118.44, 115.78 (d, J = 83.3 Hz), 107.41, 102.38, 70.60, 58.22, 56.24, 52.95,51.63, 49.32, 37.80, 31.42, 28.86, 27.09, 26.48, 25.85, 25.12, 22.74, 18.85,18.14. HRMS (ESI + ) m / z calcd for C 43 H 49 ClN 10 O9P + (M+H) +915.3105, found 915.3099. Example 6: Preparation of compound PA7 8-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1,3-dioxoisoindolin-4-yl)octanamide (Compound PA7) By replacing 3-bromopropionic acid with 8-bromooctanoic acid in step (1) of Example 1, and keeping all other steps the same, compound PA7, an orange-red solid, is obtained with a yield of 33%. 1 H NMR (400 MHz, Chloroform- d ) δ 10.73 (s, 1H),9.39 (s, 1H), 8.73 (d, J = 8.3 Hz, 2H), 8.35 (m, 1H), 8.06 (s, 1H), 7.67 – 7.61(m, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.39 (d, J = 4.7 Hz, 2H), 7.24 (m, 1H), 7.06(m, 1H), 6.57 (s, 1H), 4.90 – 4.83 (m, 1H), 3.87 (s, 3H), 3.05 (m, 4H), 2.71– 2.59 (m, 6H), 2.44 – 2.32 (m, 6H), 1.78 (s, 3H), 1.74 (s, 3H), 1.68 (m,2H), 1.51 – 1.43 (m, 2H), 1.33 (m, 4H), 1.26 – 1.21 (m, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 172.44, 171.57, 169.31, 168.62, 166.76, 160.75, 156.95,156.08, 154.75, 152.10, 143.22 (d, J = 2.6 Hz), 142.20, 137.77, 136.75, 136.43,132.77 (d, J = 2.2 Hz), 131.16, 129.68 (d, J = 10.8 Hz), 125.27, 124.24, 123.04(d, J= 12.1 Hz), 122.68 (d, J = 7.0 Hz), 120.49 (d, J = 95.9 Hz), 118.45, 115.80 (d, J = 86.2 Hz), 107.35, 102.34, 70.61, 58.53, 56.19, 53.06, 51.78, 49.33,37.73, 31.45, 29.15, 28.80, 27.57, 26.49, 26.37, 25.34, 22.84, 18.85, 18.14.HRMS (ESI + m / z calcd for C 44 H 51 ClN 10 O9P + (M+H) + 929.3261, found 929.3258. Example 7: Preparation of compound PB2 3-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)propanamide (Compound PB2) By replacing pomalidomide in step (2) of Example 1 with lenalidomide (compound 2-2), and keeping the other steps the same, compound PB2, an orange-red solid, is obtained with a yield of 30%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.96 (s,1H), 10.03 (s, 1H), 7.85 – 7.82 (m, 1H), 7.51 – 7.43 (m, 3H), 6.47 (m, 1H), 6.24 (m, 1H), 5.75 (m, 1H), 5.08 (m, 2H), 4.35 (m, 2H), 3.55 (m, 2H), 3.11 –3.02 (m, 2H), 2.92 – 2.79 (m, 2H), 2.59 – 2.51 (m, 2H), 2.45 (s, 3H), 2.31(m, 2H), 1.96 (m, 2H), 1.71 (d, J = 13.5 Hz, 1H), 1.22 (m, 2H), 1.20 (s, 3H), 1.19 (s, 3H), 1.17 – 1.13 (m, 2H).13 C NMR (101 MHz, DMSO- d 6) δ 173.37, 171.52,168.26, 163.73, 134.41, 133.93, 133.21, 131.64, 129.21, 128.11, 125.84,119.89, 54.01, 52.02, 47.10, 46.76, 42.29, 31.66, 23.07, 19.24, 18.52, 17.19,12.89. HRMS (ESI + m / z calcd for C 39 H 43 ClN 10 O8P + (M+H) + 845.2686, found 845.2663. Example 8: Preparation of compound PB3 4-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)butanamide (Compound PB3) By replacing 3-bromopropionic acid in step (1) of Example 1 with 4-bromobutyric acid, and pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB3, an orange-red solid, can be obtained with a yield of 30%. 1 H NMR (400MHz, Chloroform- d ) δ 10.82 (s, 1H), 9.33 (s, 1H), 8.85 (s, 1H), 8.43 (m, 1H), 8.13 (s, 1H), 7.67 (m, 2H), 7.46 (m, 3H), 7.34 – 7.28 (m, 1H), 7.14 (m, 1H),6.52 (s, 1H), 5.08 (m, 1H), 4.43 (s, 2H), 3.90 (s, 3H), 3.07 (m, 4H), 2.73(m, 4H), 2.58 (m, 4H), 2.27 – 2.13 (m, 2H), 1.97 (m, 4H), 1.85 (s, 3H), 1.81 (s, 3H). 13 C NMR (101 MHz, Chloroform- d) δ 171.61, 171.53, 170.08, 168.98,156.87, 156.07, 154.76, 152.01, 143.26, 141.69, 136.82, 134.68, 133.12,132.84, 132.78, 129.72 (d, J = 10.7 Hz), 129.03, 126.27, 124.60, 123.04 (d, J =12.2 Hz), 122.62 (d, J = 7.1 Hz), 120.95, 120.00, 115.96, 107.52, 102.36,57.60, 56.25, 53.07, 52.04, 51.86, 47.12, 35.34, 31.50, 29.71, 23.31, 22.08,18.88, 18.17. HRMS (ESI + m / z calcd for C 40 H 45 ClN 10 O8P + (M+H) + 859.2842, found 859.2828. Example 9: Preparation of compound PB4 5-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)pentanamide (Compound PB4) By replacing 3-bromopropionic acid in step (1) of Example 1 with 5-bromopentanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB4, an orange-red solid, can be obtained with a yield of 31%. 1 H NMR (400MHz, Chloroform- d ) δ 10.78 (s, 1H), 8.96 (s, 1H), 8.82 (s, 1H), 8.43 (m, 1H), 8.10 (s, 1H), 7.77 (m, 1H), 7.60 (d, J= 7.4 Hz, 1H), 7.49 – 7.38 (m, 3H), 7.30(m, 1H), 7.12 (m, 1H), 6.57 (s, 1H), 4.40 (s, 2H), 3.91 (s, 3H), 3.08 (m,4H), 2.69 (m, 4H), 2.54 – 2.43 (m, 4H), 2.31 – 2.01 (m, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.72 (m, 2H), 1.64 – 1.57 (m, 2H), 1.51 (t, J = 7.4 Hz, 1H), 1.45(d, J = 6.6 Hz, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 172.13, 170.50, 169.18,156.90, 156.02, 154.66, 152.29, 143.18, 142.09, 136.31, 134.25, 133.27,132.80, 132.56, 129.81, 129.71, 129.02, 126.32, 124.28, 123.12, 122.56,120.89, 119.94, 116.20, 107.38, 102.35, 57.68, 56.31, 54.11, 52.95, 52.05,51.57, 42.41, 36.36, 31.51, 29.69, 25.56, 23.36, 23.12, 18.85, 18.13. HRMS(ESI + m / z calcd for C 41 H 47 ClN 10 O8P + (M+H) + 873.2999, found 873.2985. Example 10: Preparation of compound PB5 6-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)hexanamide (Compound PB5) By replacing 3-bromopropionic acid in step (1) of Example 1 with 6-bromohexanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB5, an orange-red solid, can be obtained with a yield of 31%. 1 H NMR (400MHz, DMSO- d 6) δ 11.22 (s, 1H), 11.04 (s, 1H), 9.86 (d, J = 4.2 Hz, 1H), 8.41(m, 2H), 8.34 (s, 1H), 8.17 (s, 1H), 7.84 (m, 1H), 7.59 – 7.47 (m, 3H), 7.32(m, 1H), 7.20 – 7.09 (m, 1H), 6.84 – 6.80 (m, 1H), 4.46 – 4.32 (m, 2H), 3.93(s, 3H), 3.18 (m, 4H), 2.96 – 2.87 (m, 2H), 2.62 (m, 2H), 2.50 (m, 4H), 2.40(m, 2H), 2.04 (m, 2H), 1.79 (s, 3H), 1.76 (s, 3H), 1.64 (m, 4H), 1.37 (m, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ 173.38, 171.84, 171.72, 171.55, 169.41,168.35, 158.44, 155.79, 155.30, 144.09, 143.44, 134.55, 134.26, 134.15,133.12, 132.70, 132.28, 131.17 (d, J = 10.9 Hz), 129.22 (d, J = 20.5 Hz), 126.05,125.73, 121.71, 118.19 (d, J = 259.6 Hz), 110.90, 105.89, 103.82, 56.94, 55.37,52.02, 51.98, 49.07, 47.03, 46.04, 36.13, 31.70, 31.67, 26.70, 25.34, 23.22,23.12, 19.00, 18.30. HRMS (ESI + m / z calcd for C42 H 49 ClN 10 O8P + (M+H) + 887.3155, found 887.3142. Example 11: Preparation of compound PB6 7-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- (2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)heptanamide (Compound PB6) By replacing 3-bromopropionic acid in step (1) of Example 1 with 7-bromoheptanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB6, an orange-red solid, can be obtained with a yield of 30%. 1 H NMR (400MHz, Chloroform- d ) δ 10.77 (s, 1H), 8.90 (s, 1H), 8.83 (s, 1H), 8.43 (m, 1H),8.11 (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.44 (m, 3H),7.29 – 7.25 (m, 1H), 7.12 (m, 1H), 6.59 (s, 1H), 5.04 (m, 1H), 4.37 (s, 2H),3.92 (s, 3H), 3.14 – 3.01 (m, 4H), 2.68 (s, 6H), 2.43 (m, 4H), 2.23 – 2.02(m, 2H), 1.83 (s, 3H), 1.80 (s, 3H), 1.69 (m, 2H), 1.51 (m, 2H), 1.36 – 1.26(m, 4H). 13 C NMR (101 MHz, CDCl3) δ 172.35, 172.09, 170.51, 169.18, 156.92,156.01, 154.68, 152.27, 143.14, 142.20, 136.37, 134.30, 133.30, 132.81,132.55, 129.73 (d, J = 10.7 Hz), 129.00, 126.33, 124.21, 123.07 (d, J= 11.8 Hz), 122.62 (d, J = 6.9 Hz), 120.90, 119.95, 116.24, 107.32, 102.31, 58.17, 56.26,52.95, 51.91, 51.68, 46.91, 36.61, 31.49, 29.68, 28.95, 27.06, 26.02, 25.52,23.19, 18.81, 18.10. HRMS (ESI + m / z calcd for C 43 H 51 ClN 10 O8P + (M+H) + 901.3312, found 901.3303. Example 12: Preparation of compound PB7 8-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)octanamide (Compound PB7) By replacing 3-bromopropionic acid in step (1) of Example 1 with 8-bromooctanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB7, an orange-red solid, can be obtained with a yield of 33%. 1 H NMR (400MHz, Chloroform- d ) δ 10.74 (s, 1H), 8.84 (m, 2H), 8.42 (m, 1H), 8.11 (d, J =1.9 Hz, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.46 (d, J= 6.2Hz, 2H), 7.41 (m, 1H), 7.33 – 7.29 (m, 1H), 7.13 (m, 1H), 6.60 (s, 1H), 5.07(m, 1H), 4.47 – 4.30 (m, 2H), 3.93 (s, 3H), 3.10 (m, 4H), 2.72 (s, 4H), 2.45(m, 4H), 2.27 – 2.04 (m, 2H), 1.84 (s, 3H), 1.80 (s, 3H), 1.68 (m, 2H), 1.53– 1.46 (m, 2H), 1.43 (m, 2H), 1.34 – 1.24 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ172.38, 172.21, 170.57, 169.19, 156.92, 156.02, 154.68, 152.27, 143.10,142.15, 136.39, 134.44, 133.25, 132.81, 132.55, 129.74 (d, J = 10.7 Hz),128.96, 126.35, 124.25, 123.10 (d, J = 11.9 Hz), 122.65 (d, J = 7.3 Hz), 120.94,119.98, 116.22, 107.31, 102.37, 58.37, 56.31, 54.01, 52.96, 51.84, 51.55,36.74, 31.51, 29.13, 28.93, 27.23, 25.93, 25.60, 23.27, 18.79, 18.08, 12.38.HRMS (ESI + m / z calcd for C 44 H 53 ClN 10 O8P + (M+H) + 915.3468, found 915.3462. The proton NMR spectrum of compound PB7 is as follows. Figure 1 As shown.

[0030] Example 13: Preparation of compound PB8 9-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)nonanamide (Compound PB8) By replacing 3-bromopropionic acid in step (1) of Example 1 with 9-bromononanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB8, an orange-red solid, can be obtained with a yield of 32%. 1 H NMR (400MHz, Chloroform- d ) δ 10.77 (s, 1H), 8.84 (d, J = 4.7 Hz, 2H), 8.43 (m, 1H), 8.12 (s, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.47 (m, 2H), 7.41 (m, 1H), 7.31 (m, 1H), 7.13 (m, 1H), 6.62 (s, 1H), 5.09 (m, 1H), 4.50 –4.32 (m, 2H), 3.93 (s, 3H), 3.15 (m, 4H), 2.80 – 2.74 (m, 4H), 2.54 – 2.40(m, 4H), 2.29 – 2.05 (m, 2H), 1.84 (s, 3H), 1.81 (s, 3H), 1.70 (t, J = 7.3 Hz,2H), 1.53 (m, 2H), 1.46 (m, 2H), 1.28 (m, 8H). 13 C NMR (101 MHz, CDCl3) δ172.47, 172.12, 170.50, 169.18, 156.91, 156.02, 154.68, 152.24, 143.14,141.98, 136.54, 134.40, 133.29, 132.80, 132.54, 129.75 (d, J = 11.2 Hz),128.94, 126.38, 124.41, 123.09 (d, J = 12.4 Hz), 122.63 (d, J= 6.9 Hz), 120.94,119.99, 116.15, 107.36, 102.46, 58.11, 56.33, 54.24, 52.87, 51.83, 51.43,42.51, 36.78, 31.58, 29.19, 29.06, 29.01, 27.07, 25.85, 25.61, 23.28, 18.82,18.11, 12.29. HRMS (ESI + m / z calcd for C 45 H 55 ClN 10 O8P + (M+H) + 929.3625, found 929.3620. Example 14: Preparation of compound PB9 10-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)decanamide (Compound PB9) By replacing 3-bromopropionic acid in step (1) of Example 1 with 10-bromodecanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB9, an orange-red solid, can be obtained with a yield of 33%. 1 H NMR (400MHz, Chloroform- d ) δ 10.73 (d, J = 11.4 Hz, 1H), 9.07 – 8.94 (m, 1H), 8.83 (d, J = 8.5 Hz, 1H), 8.44 – 8.37 (m, 1H), 8.13 – 8.08 (m, 1H), 7.76 (d, J = 7.9 Hz,1H), 7.63 – 7.56 (m, 1H), 7.48 (m, 2H), 7.39 (d, J = 7.4 Hz, 1H), 7.28 (d, J=2.9 Hz, 1H), 7.18 – 7.09 (m, 1H), 6.64 – 6.60 (m, 1H), 5.10 (m, 1H), 4.51 –4.34 (m, 2H), 3.94 (s, 3H), 3.21 – 3.13 (m, 4H), 2.84 (m, 4H), 2.73 (m, 2H), 2.56 (m, 2H), 2.47 (m, 2H), 2.27 (s, 1H), 2.10 (m, 1H), 1.84 (s, 3H), 1.81(s, 3H), 1.68 (m, 2H), 1.52 (m, 2H), 1.45 (m, 4H), 1.27 – 1.24 (m, 6H). 13 C NMR(101 MHz, CDCl3) δ 172.60, 172.26, 170.54, 169.22, 156.89, 155.99, 154.61,152.31, 143.04, 141.88, 136.45 (d, J = 8.2 Hz), 134.44, 133.33, 132.78, 132.50,129.77 (d, J = 10.7 Hz), 128.85, 126.44, 124.41, 123.13 (d, J = 12.1 Hz), 122.63(d, J = 6.9 Hz), 120.96, 120.00, 116.17, 107.28, 102.56, 58.11, 56.42, 54.49,52.80, 51.84, 51.19, 42.75, 36.77, 31.62, 29.21, 29.12, 29.07, 27.10, 25.65,25.54, 23.28, 18.79, 18.08, 17.99, 12.32. HRMS (ESI + ) m / z calcd forC 46 H 57 ClN 10 O8P + (M+H) + 943.3781, found 943.3762. Example 15: Preparation of compound PB10 11-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin- 2-yl)amino)-5-methoxy-2-nitrophenyl)piperazin-1-yl)-N-(2-(2,6-dioxopiperidin- 3-yl)-1-oxoisoindolin-4-yl)undecanamide(Compound PB10) By replacing 3-bromopropionic acid in step (1) of Example 1 with 11-bromoundecanoic acid, and replacing pomalidomide in step (2) with lenalidomide, while keeping the other steps the same, compound PB10, an orange-red solid, can be obtained with a yield of 30%. 1 H NMR (400MHz, Chloroform- d ) δ 10.77 (d, J = 2.7 Hz, 1H), 8.85 (m, 2H), 8.42 (m, 1H),8.12 (m, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.62 (m, 1H), 7.49 (m, 2H), 7.40 (m,1H), 7.32 (d, J = 7.7 Hz, 1H), 7.16 – 7.11 (m, 1H), 6.64 (s, 1H), 5.12 – 5.04(m, 1H), 4.51 – 4.36 (m, 2H), 3.94 (s, 3H), 3.20 (m, 4H), 2.84 (m, 4H), 2.62– 2.55 (m, 2H), 2.46 (m, 2H), 2.32 – 2.23 (m, 1H), 2.16 – 2.05 (m, 1H), 1.84(s, 3H), 1.81 (s, 3H), 1.70 (m, 2H), 1.60 (m, 2H), 1.48 (m, 4H), 1.27 (m, 10H). 13 C NMR (101 MHz, CDCl3) δ 172.50, 172.08, 170.43, 169.21, 156.89,156.03, 154.66, 152.23, 143.10, 141.78, 136.67, 134.37, 133.29, 132.79,132.54, 129.76 (d, J = 11.0 Hz), 128.91, 126.35, 124.58, 123.11 (d, J = 12.5 Hz), 122.65 (d, J= 6.9 Hz), 120.97, 120.02, 116.06, 107.38, 102.60, 58.17, 56.38,54.41, 52.85, 51.86, 51.23, 47.12, 42.65, 36.82, 31.60, 29.67, 29.26, 29.19,29.10, 27.13, 25.63, 23.24, 18.82, 18.10, 18.01, 12.26. HRMS (ESI + ) m / z calcdfor C 47 H 59 ClN 10 O8P + (M+H) + 957.3938, found 957.3921. The following experimental examples demonstrate the beneficial effects of the present invention.

[0031] Experimental Example 1: MTT assay for cell growth inhibition rate 1. Experimental Methods BEAS-2B cells are a normal lung bronchial epithelial cell line, cultured in DMEM high-glucose medium (supplemented with 10% fetal bovine serum); H1975-M3 (EGFR T790M / L858R / C797S ) cells, NCI-H1975 (EGFR T790M / L858R The cells were non-small cell lung cancer cell lines, cultured in RPMI 1640 medium (supplemented with 10% fetal bovine serum); A549 WT Non-small cell lung cancer cell lines were cultured in DMEM high-glucose medium (supplemented with 10% fetal bovine serum). When cells reached the logarithmic growth phase, they were seeded at a density of 5000 cells / well in 96-well cell culture plates and incubated at 37 °C for 12 h in a 5% CO2 incubator. The test compound was added to three replicates at different concentrations (50, 16.67, 5.56, 1.85, 0.62, 0.2, 0.068, 0.023 μM), with 0.4% DMSO as a control. After incubation at 37 °C for 72 h in a 5% CO2 incubator, 10 μL of MTT staining solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 h. The culture medium was then aspirated, and 100 μL of DMSO was added to each well, shaken until all crystals were dissolved, and the cells were analyzed at 490 nm using VICTOR® Nivo. TM A multi-mode plate reader was used to measure the absorbance (OD) of each well in a 96-well plate during the experiment. Cell lethality (%) was calculated as follows: [1 - (OD)] 490 Dosing port - OD 490 Background hole) / (OD)490 Reference Hole - OD 490 [Background wells] × 100%, and the half-maximum inhibition concentration (IC50) was calculated using SPSS 26.0. 50 Value (means ± SD, n=3) (each of the above parallel experiments was independently repeated three times).

[0032] 2. Experimental Results The results in Table 1 show that the compounds of this invention exhibit significant selective inhibitory effects on non-small cell lung cancer cell lines (NCI-H1975 and H1975-M3) carrying EGFR mutations. Among them, compound PB7 showed the most prominent anti-proliferative activity against H1975-M3 cells (EGFR triple mutation), with an IC50 value of [missing value]. 50 The value was as low as 0.044 ± 0.004 µM, significantly superior to other PB series compounds and the positive control drug brigatinib (IC50). 50 =1.95 ± 0.97 µM) and osimertinib (IC50) 50 =4.55 ±0.47 µM). Meanwhile, PB7 showed an IC50 value of 4.55 ± 0.47 µM against NCI-H1975 cells (EGFR double mutant). 50 The concentration was 0.33 ± 0.04 µM, also exhibiting excellent inhibitory activity. More importantly, PB7 showed low toxicity to normal bronchial epithelial cells BEAS-2B (IC50). 50 =17.80 ± 0.83 µM), with a selectivity index approximately 400 times higher than that of H1975-M3 cells, indicating a good therapeutic window.

[0033] Table 1. IC50 of the compounds against NSCLC cell proliferation 50 value.

[0034] As shown in Table 1, the antiproliferative activity of the PB series compounds exhibits a clear structure-activity relationship. The activity significantly increases with increasing linker chain length from PB6 (n=6) to PB7 (n=7); however, the activity decreases when the linker chain is further extended to PB8 (n=8) and PB9 (n=9). PB7 exhibits the best antitumor activity among the PB series, with its inhibitory activity against H1975-M3 cells being approximately 35-fold higher than PB6, approximately 19-fold higher than PB8, and approximately 84-fold higher than PB9. This result suggests that linker chain length has a crucial influence on compound activity, with the optimal balance achieved at n=7.

[0035] Experimental Example 2: EGFR protein degradation efficiency of the compounds of this invention 1. Experimental Methods (1) Extraction of total cellular protein: Cells from various cell lines in the logarithmic growth phase were extracted using a method involving 3 × 10⁻⁶ cells / cells. 5 Cells were evenly seeded into 6-well plates at a concentration of [number] cells / well. After cell adhesion, 1 µM of each compound was added to each well, and the cells were treated for 24 h. The supernatant was discarded, and the cells were washed once with pre-chilled PBS and the supernatant was discarded. 100 μL of RIPA lysis buffer (Beyotime Biotechnology) containing 2% protease inhibitor and 2% phosphatase inhibitor was added to each well, and the cells were lysed on ice for 30 min. After lysis, the lysis buffer was collected, centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatant was collected and stored at -20 °C. Protein quantification was performed on the resulting protein solution according to the BCA quantitative kit (Beyotime, China) instructions. 5× protein loading buffer was added, and the cells were boiled at 95 °C for 15 min.

[0036] (2) SDS-PAGE protein electrophoresis: Prepare a 10% separating gel and pour it between two glass plates, avoiding air bubbles. Pour the separating gel to 1 cm below the bottom edge of the comb and gently add double-distilled water for water sealing. After pouring the separating gel, let it stand at room temperature for 30 min until the separating gel has fully polymerized. Slowly pour off the upper layer of double-distilled water and absorb any remaining double-distilled water with filter paper. Prepare a stacking gel and quickly pour it to the top of the glass plate. Insert the comb to prevent air bubbles and let it stand at room temperature for 30 min before use. Load the aliquoted total cell protein sample. Add 4 μl of pre-stained protein marker to both wells of the protein sample. Then turn on the electrophoresis apparatus and separate the desired bands by electrophoresis. The electrophoresis can then be stopped.

[0037] (3) Transfer: Immerse an appropriately sized PVDF membrane in methanol for about 30 s, then transfer it to the transfer buffer to prepare a "sandwich" of sponge pad-filter paper-separating gel-PVDF membrane-filter paper-sponge pad, and place it in the transfer tank. Pour in transfer buffer, cover with ice, and transfer at a constant current of 300 mA for 90 min. After the transfer is complete, remove the PVDF membrane and mark the front and back sides and the position of the standard molecular weight reference protein.

[0038] (4) Blocking, primary antibody incubation, and secondary antibody incubation: Place the successfully transferred membrane in rapid blocking buffer and block at room temperature for about 10 min. Dilute EGFR protein and its downstream protein with primary antibody dilution buffer and incubate overnight at 4 °C. Use β-actin antibody as an internal control. Wash the membrane 3 times with 1×TBST for 5 min each time, then place it in secondary antibody dilution buffer and incubate at room temperature for 1 h. Finally, wash the membrane 3 times with 1×TBST for 15 min each time.

[0039] (5) ECL development: ECL chemiluminescent solutions A and B are mixed in a 1:1 ratio and kept well. The mixed ECL reagent is added to the PVDF membrane, and chemiluminescence is obtained to form bands.

[0040] (6) Take pictures using a gel imaging system.

[0041] 2. Experimental Results See results Figure 2 AC. Western blot analysis showed that the PB series compounds (especially PB7) efficiently degraded the EGFR triple mutant protein in H1975-M3 cells, while having almost no effect on EGFR protein levels in A549 cells (EGFR wild-type). The PA series compounds showed relatively weak degradation effects on EGFR. This result indicates that the compounds of the present invention have selective degradation effects on EGFR mutants, with PB7 exhibiting the most significant degradation efficiency.

[0042] Experimental Example 3: The compound of the present invention induces EGFR in a concentration-dependent manner. C797S degradation 1. Experimental Methods H1975-M3 cells in logarithmic growth phase were harvested at 3 × 10⁻⁶. 5 Cells were evenly seeded into 6-well plates at a concentration of cells / well. After cell adhesion, compounds PB6, PB7, PB8, and PB9 (concentrations of 1000, 500, 250, 125, 63, 32, and 16 nM, respectively) were added and treated for 24 h. Subsequent protein extraction and immunoblotting experiments were the same as in Experiment 2.

[0043] 2. Experimental Results See results Figure 3 AB. This invention, through systematic structure-property relationship studies, discovered that in PB series PROTAC molecules, the linker chain length (n) has a significant impact on EGFR. C797S Degradation activity has a decisive influence. When n=7 (compound PB7), the degradation activity reaches its optimum, and its DC... 50 The value was 55.67±9.26 nM, which can effectively reduce EGFR. L858R / T790M / C797S Triple mutant protein levels. DC levels of the remaining compounds. 50 The activity levels were PB6 (189.77±39.04 nM), PB8 (250.56±30.64 nM), and PB9 (370.03±4.55 nM), respectively. In particular, PB7 (n=7) exhibited unique optimal activity among the PB series, showing the best activity against EGFR. C797S The degradation activity of PB7 was 3.4 times and 4.5 times higher than that of its neighboring PB6 and PB8, respectively. This unexpected technical effect highlights the potential of PB7 as a target for EGFR. C797SSignificant advantages of degradation agents.

[0044] Experimental Example 4: Effects of the Compounds of the Present Invention on Cell Cycle and Apoptosis in Non-Small Cell Lung Cancer 1. Experimental Methods (1) Cell cycle detection: H1975-M3 cells in good growth condition and in the logarithmic growth phase were collected, digested with trypsin, and then the culture medium was pipetted into a cell suspension. 5 × 10⁶ cells were seeded per well of a six-well plate. 5 Cells were cultured at 37°C in an incubator with 5% CO2 and saturated humidity. After cell adhesion, different concentrations of drugs were added for 24 h. After 24 h of culture, cells were collected by centrifugation at 1000 rpm for 5 min in a pre-chilled centrifuge at 4°C. Cells were washed once with PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was removed. 500 µL of pre-chilled 70% ethanol was added to the cells and the cells were fixed overnight at 4°C. After centrifugation, the fixative was washed off with PBS before staining. 500 µL of prepared propidium iodide staining solution (500 µL staining buffer, 25 µL propidium iodide staining solution, 10 µL RNase A solution) was added to the cell pellet, and the cells were incubated at 37°C in the dark for 30 min. Cell cycle distribution was analyzed using an Agilent NovoCyte flow cytometer.

[0045] (2) Apoptosis detection: Cells in good growth condition and in the logarithmic growth phase were routinely digested with trypsin and then the culture medium was pipetted to form a cell suspension. 3 × 10⁶ cells were seeded per well of a six-well plate. 5 Cells were cultured at 37 °C in an incubator with 5% CO2 and saturated humidity. After cell adhesion, different concentrations of drugs were added for 48 h. After 48 h of culture, cells were collected and centrifuged again at 2000 rpm for 5 min in a pre-cooled centrifuge at 4 °C. After centrifugation, the supernatant was carefully aspirated, and the cells were resuspended in 500 μL of 1× Binding Buffer. 5 μL of Annexin V-FITC (BioSharp) was added to the sample and mixed well, followed by 5 μL of propidium iodide staining solution. After staining at room temperature in the dark for 15 min, the cells were analyzed using an Agilent NovoCyte flow cytometer.

[0046] 2. Experimental Results See results Figure 4 AC. Compound PB7 significantly induced cell cycle arrest in H1975-M3 cells at the G0 / G1 phase and, in a concentration-dependent manner, induced apoptosis. This result is related to PB7's efficient degradation of EGFR. C797S The mutant protein exhibited consistent activity, further confirming its mechanism of exerting anti-tumor effects by targeting and degrading EGFR.

[0047] Experimental Example 5: Effects of the Compounds of the Present Invention on the Invasion and Migration of Non-Small Cell Lung Cancer Cells 1. Experimental Methods (1) Transwell migration assay: H1975-M3 cells in logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed once with PBS. Then, serum-free RPMI 1640 medium was added for starvation treatment for 4 h. Afterwards, the cells were digested with trypsin and resuspended in serum-free RPMI 1640 medium to adjust the cell concentration to 4 × 10⁻⁶ cells / mL. 5 Cells were cultured at a density of 1 cell / mL. 200 μL of the medium was added to the upper chamber of a Transwell chamber, and 700 μL of RPMI 1640 medium containing 20% ​​fetal bovine serum was added to the lower chamber. Different concentrations of the drug were then added to treat the cells. After incubation for 24 h, the medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by staining with 0.1% crystal violet at room temperature for 15-30 min. The cells were gently washed three times with PBS, and any unmigrated cells on the upper layer were gently wiped away with a cotton swab. The cells were then photographed and observed using a research-grade inverted fluorescence microscope (Fluorescence Microscope).

[0048] (2) Transwell invasion assay: H1975-M3 cells in logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed once with PBS. Then, serum-free RPMI 1640 medium was added for starvation treatment for 4 h. Simultaneously, Matrigel (BDBiosciences) was rapidly diluted 1:7 on ice with the corresponding serum-free RPMI 1640 medium. 100 μL of the diluted Matrigel was pre-spread onto the upper chamber of the transwell chamber. After incubating at 37 ℃ for 30 min, 100 μL of warm serum-free medium was added to the upper chamber to rehydrate the basement membrane for 1 h. After removing the medium from the upper chamber, cells with a density of 10-1 were harvested. 6 200 μL of H1975 cells / mL were added to the upper chamber, and 700 μL of RPMI 1640 medium containing 20% ​​FBS was added to the lower chamber. Simultaneously, different concentrations of drugs were added to treat the cells. During this period, some invasive H1975 cells degraded matrix proteins in Matrigel, passed through the pores of the polycarbonate membrane, and eventually adhered to the underside of the polycarbonate membrane. After 24 h of incubation, the medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by staining with 0.1% crystal violet at room temperature for 15–30 min. The cells were gently washed three times with PBS, and the unmigrated cells in the upper layer were gently wiped away with a cotton swab. The cells were then observed using a research-grade inverted fluorescence microscope (Fluorescencemicroscope).

[0049] 2. Experimental Results Figure 5 AC represents the effect of the selected compounds on the migration and invasion of lung cancer H1975-M3 cells. The figure shows that the compound PB7 of this invention, as demonstrated by Transwell migration and invasion assays, can inhibit the migration of lung cancer H1975-M3 cells in a concentration-dependent manner with statistical significance.

[0050] Experimental Example 6: Degradation of EGFR by the Compound of the Present Invention C797S Mechanism 1. Experimental Methods H1975-M3 cells in logarithmic growth phase were harvested at 3 × 10⁻⁶. 5 Cells were evenly seeded into 6-well plates at a concentration of [number] cells / well. After cell adhesion, 10 μM of proteasome inhibitor MG132 or 10 μM of E1 activator inhibitor MLN4924 were added to each well, and the treatment was carried out for 4 h. Then, 0.25 μM of compound PB7 was added to one well. In half of the wells, only 10 μM of proteasome inhibitor MG132 or 10 μM of E1 activator inhibitor MLN4924 were added, and in one well, only 0.25 μM of compound PB7 was added. Protein extraction and Western blotting experiments were performed as in Experiment 2.

[0051] 2. Experimental Results See results Figure 6 AB. The results showed that PB7 (0.25 μM)-induced EGFR degradation could be completely blocked by the proteasome inhibitor MG132 or the E1 activator inhibitor MLN4924. Specifically, PB7 alone significantly reduced EGFR protein levels; however, pretreatment with MG132 or MLN4924 for 4 hours, followed by PB7 treatment for 24 hours, did not reduce EGFR protein levels. This result indicates that PB7-mediated EGFR degradation depends on the ubiquitin-proteasome pathway.

[0052] In summary, this invention provides a method for targeting EGFR C797S The degradation agents and uses of the compounds of this invention. The antiproliferative activity of the compounds in NSCLC tumor cells is consistent with their efficiency in degrading EGFR, and degradation effect is the main mechanism of action. Among them, the choice of E3 ubiquitin ligase ligand and the length of the linker chain have a key influence on the degradation efficiency. When lenalidomide is used as the E3 ligand and a seven-carbon chain is used as the linker, the resulting compound PB7 has the best degradation activity, and its half-maximal concentration (DC) for EGFR degradation is [not specified]. 50 The concentration reached 65 nM. Compound PB7 of this invention can effectively reduce the levels of downstream proteins and their phosphorylation in the EGFR pathway. Compound PB7 of this invention simultaneously targets VHL and EGFR, promoting the degradation of EGFR via the ubiquitin-proteasome pathway, thereby inhibiting its downstream signaling. Compound PB7 of this invention can act as an EGFR...C797S Degrading agents have broad application prospects in the preparation of drugs for treating non-small cell lung cancer.

Claims

1. The compound of formula I, or its salt, its stereoisomer, its solvate, or its crystal form: Formula I in, R is selected from -CH2- or -C(O)-; n is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10.

2. The compound, or its salt, or its stereoisomer, or its solvate, or its crystal form according to claim 1, characterized in that: The structure of the compound is shown in Formula II: Formula II Where n is selected from 2, 3, 4, 5, 6 or 7.

3. The compound, or its salt, or its stereoisomer, or its solvate, or its crystal form according to claim 1, characterized in that: The structure of the compound is shown in Formula III: Formula III Where n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

4. The compound, or its salt, or its stereoisomer, or its solvate, or its crystal form according to claim 1, characterized in that: The structure of the compound is selected from one of the following structures: 。 5. A method for preparing the compound, salt thereof, stereoisomer thereof, solvate thereof, or crystal form thereof according to any one of claims 1 to 4, characterized in that: The method includes the following steps: (1) Compound 1 is reacted with thionyl chloride to obtain compound A; (2) Compound A and compound 2 are reacted to obtain compound B; (3) React compound B, compound C, nucleophile and organic base to obtain the compound described in formula I.

6. The method according to claim 5, characterized in that: In step (1), the reaction temperature is 50~70℃ and the time is 1~3 hours; In step (2), the equivalent ratio of compound A and compound 2 is 1:0.1~0.3; In step (3), the equivalence ratio of compound B, compound C and nucleophile is 1:0.5~1.5:2~4; the nucleophile is potassium iodide; the organic base is N,N-diisopropylethylamine; the solvent for the reaction is an organic solvent; the reaction temperature is 80~120℃ and the time is 1~5 hours.

7. Use of the compound, salt thereof, stereoisomer thereof, solvate thereof, or crystal form thereof according to any one of claims 1 to 4 in the preparation of an EGFR protein degrading agent.

8. The use according to claim 7, characterized in that: The EGFR protein degrading agent is a drug for the prevention and / or treatment of non-small cell lung cancer.

9. The use according to claim 8, characterized in that: The drug is for patients with EGFR. C797S Drugs for drug-resistant mutations in non-small cell lung cancer.

10. The use according to claim 9, characterized in that: The EGFR C797S Drug resistance mutations include EGFR L858R / T790M / C797S Triple mutation or EGFR T790M / C797S Double mutation.

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