Bivalent ligand molecules targeting egfr and uses thereof

Abstract

This invention discloses a bivalent ligand molecule targeting EGFR and its applications, belonging to the field of drug development technology. Its general structural formula is: [Formula omitted for brevity], where L is a linking group, and M1 and M2 are EGFR protein ligands. This invention forms a bivalent EGFR ligand molecule by covalently linking two EGFR ligands through a linking group. This bivalent ligand molecule can induce additional protein-protein interactions between EGFR monomers, which greatly enhances the binding strength and stability of the drug to EGFR, thereby overcoming the drug resistance problem of traditional EGFR inhibitors and providing a new treatment strategy for cancer patients carrying EGFR mutations and other patients with other diseases.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, specifically to a divalent ligand molecule targeting EGFR and its applications. Background Technology

[0002] EGFR, as the core transmembrane tyrosine kinase receptor of the ErbB / HER family, regulates key physiological processes such as cell proliferation, differentiation, and survival. Exon 19 deletion (Del746-750) and exon 21 L858R point mutations are major oncogenic mutations, and EGFR is overexpressed in most solid tumors, including non-small cell lung cancer, breast cancer, colorectal cancer, and head and neck squamous cell carcinoma, making it an important target for targeted cancer therapy. However, almost all patients receiving EGFR-TKIs treatment inevitably develop acquired resistance after long-term use, becoming a core bottleneck limiting the efficacy of targeted therapy. The main resistance mechanism is EGFR-dependent resistance, primarily due to EGFR gene mutations, including secondary point mutations such as T790M and C797S, and gene amplification, which weaken the drug's binding ability to the target or enhance EGFR signaling pathway activation. For different types of EGFR resistance, various treatment strategies exist to overcome EGFR resistance. Based on existing resistance mechanisms, potential treatment strategies include the development of fourth-generation EGFR tyrosine kinase inhibitors, combination therapies targeting multiple pathways, and protein-degrading agents. However, developing novel drug molecules to overcome EGFR resistance remains important. Meanwhile, chimeric molecules have become a hot topic in the development of new drugs against EGFR resistance due to their advantages such as overcoming undrugable targets and directly degrading target proteins.

[0003] Multivalent ligand drugs are a novel class of drugs containing multiple action fragments. Their core advantage lies in their ability to simultaneously interact with multiple domains or monomers of the target protein through different structural fragments within the molecule. This multivalent interaction generates binding forces far exceeding those of traditional monovalent drugs (affinity can be increased by several orders of magnitude) and may induce entirely new protein-protein interactions, thereby achieving richer biological effects. These novel interaction modes between multivalent ligand molecules and target proteins give them significant advantages over monovalent drugs in drug development targeting specific targets, achieving pharmacological effects that monovalent drugs cannot achieve in areas such as multifunctional drug regulation, target selectivity, target affinity, novel pharmacological effects, and overcoming drug resistance. For protein dimer / oligomery drug targets, the "multivalent ligand molecule within oligomer" strategy remains the mainstream approach, while the emerging "multivalent ligand molecule between oligomers" strategy exhibits more unique advantages: it is no longer limited to inhibiting the function of a single oligomer, but rather induces interactions between multiple oligomers to produce new polymerized forms, thereby achieving an effect transition from "inhibition" to "isolation" or "degradation".

[0004] In summary, in order to address the resistance problem of traditional EGFR inhibitors (including first-generation reversible inhibitors and second / third-generation irreversible inhibitors) and provide new treatment strategies for cancer patients carrying EGFR mutations, a new class of multivalent ligands targeting EGFR urgently needs to be developed. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a bivalent ligand molecule targeting EGFR and its application. This type of EGFR bivalent ligand molecule can induce additional protein-protein interactions in EGFR monomers, which can greatly enhance the binding strength and stability of drugs with EGFR. As a result, it can show therapeutic effects on non-small cell lung cancer carrying EGFR L858, T790, C797, 19del and other site mutations, overcome the drug resistance problem of traditional EGFR inhibitors (including first-generation reversible inhibitors and second / third-generation irreversible inhibitors), and provide a new treatment strategy for tumor patients carrying EGFR mutations.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a divalent ligand molecule targeting EGFR, the general structural formula of which is shown in formula (I): Formula (I): ; In equation (I), L is ; L1 is , , -(CH2) n -、-(CH2CH2O) n CH2CH2-,-CO(CH2CH2O) n CH2CH2CO- or a single bond; X1 and X2 are independently -(CH2). n -、-(CH2CH2O) n CH2CH2-、-CO(CH2) n -、-(CH2) n CO-, -CO-CH=CH-(CH2) n -、-(CH2) n -CH=CH-CO- or a single bond; n is any integer between 0 and 10; n1, n2, n3, and n4 are independent integers between 1 and 4; X3 and X4 are independent or ; M1 and M2 are independent EGFR protein ligands that may be structurally identical or different.

[0007] The beneficial effects of this invention are as follows: This invention forms a divalent EGFR ligand molecule by covalently linking two EGFR ligands through a linking group. This divalent ligand molecule can induce additional protein-protein interactions between EGFR monomers. Through this interaction, the binding strength and stability of the drug to EGFR can be greatly enhanced, thereby overcoming the drug resistance problem of traditional EGFR inhibitors and providing a new treatment strategy for tumor patients carrying EGFR mutations.

[0008] Furthermore, L1 is , , -(CH2) n -、-(CH2CH2O) n CH2CH2- or a single bond; n is any integer between 0 and 10; n1, n2, n3, and n4 are independent integers between 1 and 4; X3 and X4 are .

[0009] Furthermore, the EGFR protein ligand is: or ; Where W is either -O- or -NH-.

[0010] Furthermore, the EGFR protein ligand is: , or .

[0011] Furthermore, the divalent ligand molecule targeting EGFR is: , , , , , , , , , , , , , , , , , , , , , , , or .

[0012] A second aspect of the present invention provides the use of the above-described EGFR-targeting divalent ligand molecule, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, in the preparation of a medicament.

[0013] Furthermore, the drugs include anti-tumor drugs.

[0014] Furthermore, anti-tumor drugs include treatments for any of the following cancers: non-small cell lung cancer, colorectal cancer, head and neck squamous cell carcinoma, pancreatic cancer, esophageal cancer, breast cancer, gastric cancer, and ovarian cancer.

[0015] Furthermore, anti-tumor drugs are therapeutic agents for small cell lung cancer carrying EGFR mutations.

[0016] Furthermore, the anti-tumor drugs are for the treatment of non-small cell lung cancer carrying mutations at sites such as EGFR L858, T790, C797, and 19del.

[0017] A third aspect of the present invention provides an antitumor drug comprising the above-described EGFR-targeting divalent ligand molecule, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0018] Furthermore, this includes pharmaceutically acceptable excipients.

[0019] The present invention has the following beneficial effects: The present invention provides a divalent ligand molecule targeting EGFR, or its stereoisomer, or a pharmaceutically acceptable salt thereof, or its solvate, which can induce additional protein-protein interactions with EGFR monomers, thereby exerting a therapeutic effect on EGFR-dependent tumors or other diseases. These compounds hold promise as components of pharmaceutical formulations for treating EGFR-resistant tumors or other diseases. Attached Figure Description

[0020] Figure 1 The graph shows the phosphorylation levels of EGFR and key downstream signaling pathway proteins in H1975 cells after treatment with the EGFR-targeting divalent ligand molecule prepared in Example 17 at different concentrations and treatment times. In the graph, A shows the Western blot results of H1975 cells after treatment with 1 μmol / L EGFR-targeting divalent ligand molecule for 6, 12, 24, 48 and 72 h, and B shows the Western blot results of H1975 cells after treatment with 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μmol / L EGFR-targeting divalent ligand molecule for 24 h. Detailed Implementation

[0021] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] The structural formulas of the EGFR-targeting divalent ligand molecules obtained in the embodiments of the present invention are shown in Table 1: Table 1 Structural Formulas of Embodiments of the Invention

[0023] The following specific embodiments illustrate the EGFR-targeting divalent ligand molecule, its preparation method, and its application provided by the present invention.

[0024] Example 1: A method for synthesizing a divalent ligand molecule targeting EGFR, the synthetic reaction formula is as follows: ; Specifically, the following steps are included: Intermediate 1 (100 mg, 0.31 mmol, 1 eq) was dissolved in DMF (10 mL) solution, and anhydrous potassium carbonate (216 mg, 1.564 mmol, 5 eq) was added to the solution. After stirring at room temperature for 40 min, 1,4-dibromobutane (27 mg, 0.125 mmol, 0.4 eq) was added to the reaction solution. The mixture was stirred for 3 h under nitrogen protection at 50 °C. The reaction progress was monitored by TLC. After the starting material was completely converted, 20 mL of water was added, and a large amount of solid was observed to precipitate. The mixture was filtered, and the filter cake was collected and dried under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography (DCM:MeOH = 15:1, volume ratio). After concentration under reduced pressure, 70.5 mg of white solid was obtained (yield: 65%).

[0025] Example 2-10: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 1, except that the linking group of the divalent ligand molecule is different.

[0026] Example 11: A method for synthesizing a divalent ligand molecule targeting EGFR, the synthetic reaction formula is as follows:

[0027] Specifically, the following steps are included: S1. Intermediate 1 (2 g, 6.25 mmol, 1 eq) was dissolved in DMF (50 mL) solution, and anhydrous potassium carbonate (4.3 g, 31.28 mmol, 5 eq) was added to the solution. After stirring at room temperature for 40 min, tert-butyl bromoacetate (1.2 g, 6.25 mmol, 1 eq) was added to the reaction solution. The mixture was stirred for 3 h under nitrogen protection at 50 °C. The reaction progress was monitored by TLC. After the starting material was completely converted, 20 mL of water was added. A large amount of solid was observed to precipitate. The mixture was filtered, and the filter cake was collected and dried under reduced pressure to obtain the crude product. The crude product was purified by chromatography (DCM:MeOH = 15:1, volume ratio) and concentrated under reduced pressure to obtain intermediate 2.

[0028] S2. Dissolve intermediate 2 (2.3 g, 5.31 mmol) in dioxane hydrochloride solution (20 mL), stir at room temperature for 3 h, monitor the reaction progress by TLC, and after the starting material is completely converted, dry the reaction solution under reduced pressure to obtain intermediate 3.

[0029] S3. Intermediate 3 (2 g, 5.31 mmol, 1 eq) was dissolved in DMF (50 mL) solution, and then NMM (1.6 g, 19.93 mmol, 3 eq), HOBT (1.08 g, 7.97 mmol, 1.5 eq), and EDCI (1.53 g, 7.97 mmol, 1.5 eq) were added. The mixture was stirred at room temperature for 20 min, and then N-Boc piperazine (1 g, 5.31 mmol, 1 eq) was added. The mixture was stirred at room temperature for another 1 h. The reaction progress was monitored by TLC. After the reaction was complete, a small amount of water was added, and the aqueous layer was extracted three times with 50 mL EA. The organic layer was washed twice with saturated saline solution, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 4.

[0030] S4. Dissolve intermediate 4 (2.37 g, 4.35 mmol) in dioxane hydrochloride solution (20 mL), stir at room temperature for 3 h, monitor the reaction progress by TLC, and after the starting material is completely converted, dry the reaction solution under reduced pressure to obtain intermediate 5.

[0031] S5. Intermediate 3 (100 mg, 0.26 mmol, 1 eq) was dissolved in DMF (10 mL), and then NMM (80 mg, 0.79 mmol, 3 eq), HOBT (53 mg, 0.39 mmol, 1.5 eq), and EDCI (75 mg, 0.39 mmol, 1.5 eq) were added. The mixture was stirred at room temperature for 20 min, and then intermediate 5 (116 mg, 0.26 mmol, 1 eq) was added. The mixture was stirred at room temperature for another 1 h. The reaction progress was monitored by TLC. After the starting material was completely converted, a small amount of water was added, and the aqueous layer was extracted three times with 20 mL of EA. The organic layer was washed twice with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product. The product was purified by thin-layer chromatography (DCM:MeOH = 10:1, v / v) and concentrated under reduced pressure to obtain 136 mg of white solid (yield: 65%) as described in Example 11.

[0032] Examples 12-14: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 11, except that the linking group of the divalent ligand molecule is different.

[0033] Example 15: A method for synthesizing a divalent ligand molecule targeting EGFR, the synthetic reaction formula is as follows:

[0034] Specifically, the following steps are included: S1. Intermediate 1 (500 mg, 1.57 mmol, 1 eq) was dissolved in DMF (10 mL) solution, and anhydrous potassium carbonate (1.08 g, 7.84 mmol, 5 eq) was added to the solution. After stirring at room temperature for 40 min, 1,2-dibromoethane (876 mg, 4.71 mmol, 3 eq) was added to the reaction solution. The mixture was stirred for 3 h under nitrogen protection at 50 °C. The reaction progress was monitored by TLC. After the starting material was completely converted, 20 mL of water was added. A large amount of solid was observed to precipitate. The mixture was filtered, and the filter cake was collected and dried under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography (DCM:MeOH = 15:1, volume ratio) and concentrated under reduced pressure to obtain intermediate 6.

[0035] S2. Intermediate 6 (100 mg, 0.24 mmol, 1 eq) was dissolved in DMF (10 mL) solution, and anhydrous potassium carbonate (1.6 g, 1.18 mmol, 5 eq) was added to the solution. After stirring at room temperature for 40 min, piperazine (8.26 mg, 0.096 mmol, 0.4 eq) was added to the reaction solution. The mixture was stirred for 3 h under nitrogen protection at 50 °C. The reaction progress was monitored by TLC. After the starting material was completely converted, 20 mL of water was added. A large amount of solid was observed to precipitate. The mixture was filtered, and the filter cake was collected and dried under reduced pressure to obtain the crude product. The crude product was purified by thin-layer chromatography (DCM:MeOH = 15:1, volume ratio). After concentration under reduced pressure, 56 mg of white solid (yield: 75%) of Example 15 was obtained.

[0036] Examples 16-18: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 15, except that the linking group of the divalent ligand molecule is different.

[0037] Example 19: A method for synthesizing a divalent ligand molecule targeting EGFR, the synthetic reaction formula is as follows:

[0038] Specifically, the following steps are included: Intermediate 7 (100 mg, 0.21 mmol, 1 eq) was dissolved in a mixed solution of MeOH (1 mL) and DCM (4 mL), and then 2,2'-oxybis(ethylamine) (11 mg, 0.11 mmol, 0.5 eq) was added. One drop of acetic acid was added, and the mixture was stirred at room temperature for 3 h under nitrogen protection. TLC monitoring showed the formation of an imine product. Sodium cyanoborohydride was then added, and the mixture was stirred at room temperature for another 3 h under nitrogen protection. The reaction progress was monitored by TLC. After the starting material was completely converted, the reaction solution was evaporated to dryness to obtain a yellow solid. This solid was purified by thin-layer chromatography (DCM:MeOH = 10:1) and concentrated under reduced pressure to obtain 84 mg of the yellow solid of Example 19 (yield: 75%).

[0039] Examples 20-21: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 19, except that the linking group of the divalent ligand molecule is different.

[0040] Example 22: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 1, except that the linking group of the divalent ligand molecule is different.

[0041] Example 23: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 19, except that the linking group of the divalent ligand molecule is different.

[0042] Example 24: A method for synthesizing a divalent ligand molecule targeting EGFR, the synthetic reaction formula is as follows:

[0043] Specifically, the following steps are included: Intermediate 8 (100 mg, 0.31 mmol, 1 eq) was dissolved in DMF (5 mL) solution, and then HATU (236 mg, 0.62 mmol, 2 eq), DIEA (80 mg, 0.62 mmol, 2 eq), and (E)-4-bromo-2-butenoic acid (77 mg, 0.47 mmol, 1.5 eq) were added. The mixture was stirred at room temperature for 3 h, and the reaction progress was monitored by TLC. After the reaction was complete, 20 mL of water was added and the aqueous layer was extracted three times with 50 mL of EA. The organic layer was washed twice with saturated saline solution and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and purified by thin-layer chromatography (DCM:MeOH = 10:1, volume ratio). After concentration under reduced pressure, 120 mg of intermediate 9 yellow solid was obtained. Intermediate 9 (120 mg, 0.26 mmol, 1 eq) was dissolved in DMF (5 mL) solution, and piperazine (11 mg, 0.13 mmol, 0.5 eq) and potassium carbonate (180 mg, 1.31 mmol, 5 eq) were added. The mixture was stirred at room temperature for 3 h, and the reaction progress was monitored by TLC. After the reaction was complete, 20 mL of water was added and the aqueous layer was extracted three times with 50 mL of EA. The organic layer was washed twice with saturated saline solution and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and purified by thin-layer chromatography (DCM:MeOH = 10:1, volume ratio). After concentration under reduced pressure, 82 mg of the yellow solid of Example 24 (yield: 74%) was obtained.

[0044] Example 25: A divalent ligand molecule targeting EGFR is prepared using the same method as in Example 24, except that the linking group of the divalent ligand molecule is different.

[0045] The structural characterization data of the EGFR-targeting divalent ligand molecules obtained in Examples 1-25 are shown in Table 2: Table 2 Structural characterization data of embodiments of the present invention

[0046] Experimental Example 1: Assay on Tumor Cell Proliferation Inhibition Activity I. Experimental Methods: This experiment primarily used the MTT assay to detect the inhibitory activity of each embodiment on the proliferation of two non-small cell lung cancer cell lines (H1975 and HCC827), specifically including the following steps: First, logarithmically grown cells were collected, digested with trypsin, resuspended in culture medium, counted, and seeded at an appropriate density (100 µL per well) into 96-well plates. The plates were then incubated at 37°C with 5% CO2. After cell adhesion, 100 µL of a divalent EGFR-targeting ligand molecule at different concentrations was added, with three replicates for each concentration, and a blank control was included. After a specific treatment time, 20 µL of 5 mg / mL MTT solution was added, and the plates were incubated at 37°C with 5% CO2 for 3 h. The supernatant was discarded, and 150 µL of DMSO was added to each well to dissolve the purple crystals. Finally, the absorbance (A) was measured at 570 nm using a microplate reader (A value is proportional to the number of viable cells). The fitted drug growth inhibition curve was calculated using Graphpad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ) value, cell growth inhibition rate = (control group A) 570 -Experimental Group A 570 ) / Control Group A 570 ×100%.

[0047] II. Analysis of Experimental Results The inhibitory activity (IC50) of the EGFR-targeting bivalent ligand molecules obtained in Examples 1-25 against tumor cell lines 50 As shown in Table 3, "-" indicates IC 50 >10 μmol / L; "+" indicates 10 μmol / L >IC 50 >1 μmol / L; "++" indicates 1 μmol / L >IC 50 >0.1 μmol / L; "+++" indicates IC50 50 <0.1 μmol / L).

[0048] Table 3 Results of tumor cell proliferation inhibition activity assay

[0049] Experimental Example 2: Assay on the inhibitory activity of gefitinib against the proliferation of different tumor cells This embodiment tests the inhibitory activity of some embodiments on the proliferation of tumor cells from other tissues. The experiment is mainly conducted by the MTT assay. The specific experimental method is the same as in Experiment 1, except that the cell lines selected are different.

[0050] Examples 9 / 15 / 16 / 17 / 18: Inhibitory activity (IC50) against different tumor cell lines 50 As shown in Table 4, "-" indicates IC 50 >10 μmol / L; "+" indicates 10 μmol / L >IC 50 >1 μmol / L; "++" indicates 1 μmol / L >IC 50 >0.1 μmol / L; "+++" indicates IC50 50 <0.1 μmol / L).

[0051] Table 4 Results of the assay on the inhibitory activity of the embodiments on the proliferation of tumor cells from different tissue sources

[0052] Experimental Example 3: Western Blot Analysis of Proteins I. Experimental Methods This experimental example characterizes the effects of the divalent EGFR ligand molecule obtained in Example 17 on the phosphorylation levels of EGFR and its key downstream signaling pathway proteins in H1975 cells at different concentrations and treatment times. The specific steps include: (1) Cell culture and drug treatment: The HCC827 and NCI-H1975 cells used in this experiment were both obtained from the National Collection of Authenticated Cell Cultures (NCACC) of the Chinese Academy of Sciences. Cells were proliferated or cultured in RPMI-1640 medium (BasaIMedia, Shanghai, China), supplemented with penicillin and streptomycin antibiotics (1 wt%, P1400, Solarbio) and fetal bovine serum (10 wt%, GeminiBio, West Sacramento, CA, USA), and cultured in a 5% CO2, 37°C cell culture incubator.

[0053] (2) Protein extraction and quantification: All cells used in this experiment were adherent cells. After the drug was applied for a specific time, the culture medium was discarded, and the cells were washed twice with PBS buffer to remove residual serum protein from affecting the assay. The cells were then digested with trypsin, collected, and washed twice more with PBS buffer. After centrifugation, residual liquid was removed as much as possible. An appropriate amount of RIPA lysis buffer was added, and the cells were lysed on ice for 30 min. The cells were then sonicated (5 s / time, 1 s interval, for a total of 5 times). After the cells were fully lysed, the cells were centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected, and the protein concentration was quantified using a BCA protein assay kit. The volume was adjusted to ensure that the protein concentration of the samples was the same. 5× protein loading buffer was added, and the cells were boiled at 100 °C for 10 min to denature the protein. The cells were then frozen and stored at -20 °C.

[0054] (3) Western blot analysis of proteins: Prepare the required concentration of SDS-PAGE gel, place the gel in the electrophoresis tank, and pour in the electrophoresis buffer. Add appropriate amounts of protein sample and pre-stained marker to the gel wells sequentially, and begin electrophoresis. Electrophoresis is first performed at 80 V for approximately 30 minutes until the sample enters the separating gel. Then, adjust the voltage to 120 V and electrophoresis for approximately 60 minutes to separate the sample. After electrophoresis, remove the gel and place it in the transfer buffer. Place a PVDF membrane of similar size to the separating gel in the transfer buffer, and also soak two appropriately sized filter papers in the transfer buffer. Arrange the gel, filter paper, and PVDF membrane in the following order: (black side) sponge → filter paper → gel → PVDF membrane → filter paper → sponge (white side) to form a transfer "sandwich." Remove any air bubbles between the layers and quickly insert the gel into the transfer clamp. Adjust the current to a suitable level (250 mA) and begin transfer. Set the transfer time according to the protein molecular weight. After transfer, the PVDF membrane was removed and placed in TBS / T buffer for 10 min, then transferred to blocking buffer (TBS / T solution containing 5 wt% skim milk powder) and blocked on a horizontal shaker at room temperature for 1.5 h. After blocking, the membrane was washed three times in TBS / T buffer for 5 min each time. Primary antibody was diluted to an appropriate concentration using primary antibody dilution buffer and incubated overnight at 4°C. After primary antibody incubation, the PVDF membrane was washed three times with TBS / T buffer for 5 min each time, followed by incubation with a suitable concentration of secondary antibody on a shaker at room temperature for 1 h. After incubation, the PVDF membrane was eluted three times with TBS / T buffer for 20-30 min each time, then rinsed in TBS / T buffer for 10 min each time. Finally, developing solution was evenly added to the PVDF membrane and exposed on an exposure unit. The resulting Western blot images were quantitatively analyzed using ImageJ software.

[0055] II. Analysis of Experimental Results The characterization results of the effects of the divalent EGFR ligand molecules obtained in Example 17 on the phosphorylation levels of EGFR and its key downstream signaling pathway proteins in H1975 cells at different concentrations and treatment times are as follows: Figure 1 As shown in the figure. The results show that the bivalent ligand molecule of EGFR can downregulate the phosphorylation level of EGFR protein in a time- and concentration-dependent manner, further inhibiting the transduction of downstream signaling pathways, downregulating the phosphorylation levels of key target proteins of downstream signaling pathways, AKT and STAT5, thereby inhibiting the survival and proliferation of tumor cells, proving that it can overcome the problem of EGFR drug resistance.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A divalent ligand molecule targeting EGFR, characterized in that, Its general structural formula is shown in equation (I): Equation (I): ; In equation (I), L is ; L1 is , , -(CH2) n -、-(CH2CH2O) n CH2CH2-、-CO(CH2CH2O) n CH2CH2CO- or a single bond; X1 and X2 are independently -(CH2). n -、-(CH2CH2O) n CH2CH2-、-CO(CH2) n -、-(CH2) n CO-, -CO-CH=CH-(CH2) n -、-(CH2) n -CH=CH-CO- or a single bond; n is any integer between 0 and 10; n1, n2, n3, and n4 are independent integers between 1 and 4; X3 and X4 are independent or ; M1 and M2 are independent EGFR protein ligands that may be structurally identical or different.

2. The EGFR-targeting divalent ligand molecule according to claim 1, characterized in that, The EGFR protein ligand is: or ; Where W is either -O- or -NH-.

3. The EGFR-targeting divalent ligand molecule according to claim 1 or 2, characterized in that, The EGFR protein ligand is: , or .

4. The EGFR-targeting divalent ligand molecule according to claim 1, characterized in that, The divalent ligand molecule targeting EGFR is: , , , , , , , , , , , , , , , , , , , , , , , or .

5. The use of the EGFR-targeting divalent ligand molecule, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, as described in any one of claims 1-4, in the preparation of a medicament.

6. The application according to claim 5, characterized in that, The drugs mentioned include anti-tumor drugs.

7. The application according to claim 6, characterized in that, The antitumor drugs include treatments for any one of non-small cell lung cancer, colorectal cancer, head and neck squamous cell carcinoma, pancreatic cancer, esophageal cancer, breast cancer, gastric cancer, and ovarian cancer.

8. The application according to claim 6, characterized in that, The antitumor drug is a treatment for small cell lung cancer carrying EGFR mutations.

9. An antitumor drug, characterized in that, Includes the EGFR-targeting divalent ligand molecule, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, as described in any one of claims 1-4.

10. The antitumor drug according to claim 9, characterized in that, This includes pharmaceutically acceptable excipients.

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