An EGFR protein hydrolysis-targeted chimera and its preparation method, pharmaceutical composition and application

Abstract

The present invention belongs to the field of chemical medicine and discloses an EGFR proteolysis-targeted chimera, its preparation method, pharmaceutical composition, and application. The present invention provides an EGFR-degradable proteolysis-targeted chimera based on CP0371, a novel E3 ligase ligand with "molecular glue" functionality, and gefitinib, a target protein ligand. The EGFR proteolysis-targeted chimera prepared by the present invention can effectively induce EGFR degradation in cancer cell lines and has considerable bioavailability. It can be used to prepare EGFR inhibitors and can be used to form pharmaceutical compositions. It has a certain inhibitory effect on the proliferation of various tumor cells and is suitable for the development of cancer drugs for the treatment of colorectal cancer, lung cancer, esophageal cancer, glioblastoma, anal cancer, head and neck epithelial cancer, and other cancers.

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicines, and specifically relates to an EGFR protein hydrolysis-targeted chimera, a preparation method thereof, a pharmaceutical composition and applications thereof. Background Art

[0002] Proteolysis-targeting chimeras (PROTACs), which use bifunctional small molecules to induce target protein degradation, are one of the most exciting new drug modalities pioneered in recent years. PROTACs consist of a target protein ligand, an E3 ubiquitin ligase ligand, and an appropriate linker. They form a ternary complex called "target protein-PROTAC-E3 ligase" and degrade the target protein through the ubiquitin-proteasome system (UPS). Compared to traditional small molecule inhibitors, PROTACs offer unique advantages, including catalytic properties, reduced dosing and frequency, more potent and longer-lasting effects, increased selectivity, reduced potential toxicity, overcoming drug resistance, and expanding the target space. PROTAC design requires a known target protein ligand and an E3 ligase ligand as a protein decoy, and their development relies on the discovery and optimization of these ligands. The lack of available E3 ligase ligands and the low drugability of existing major E3 ligase ligands have limited PROTAC development.

[0003] Molecular glue degraders are small molecules that can induce interactions between E3 ubiquitin ligase substrate receptors and target proteins, leading to their degradation via ubiquitination. They possess dual-ligand structural characteristics with both E3 ubiquitin ligases and target proteins. This property of molecular glues binding to E3 ligases makes them an effective tool for discovering novel E3 ligase ligands.

[0004] The epidermal growth factor receptor (EGFR) is a transmembrane protein tyrosine kinase. As a receptor for members of the EGF family, it triggers the EGFR signaling pathway in human epithelial cells, thereby regulating cell proliferation, invasion, metastasis, apoptosis, and angiogenesis. Increased EGFR activity due to overexpression, mutation, or amplification of the EGFR gene leads to numerous human malignancies, including colorectal cancer, lung cancer, esophageal cancer, glioblastoma, anal cancer, and epithelial head and neck cancers. EGFR has been extensively studied in the biomedical community, and several EGFR tyrosine kinase inhibitors, such as gefitinib and afatinib, have been approved for marketing. However, clinical data indicate that current drug development still falls short of current medical needs, and some patients may develop drug resistance. Therefore, the development of EGFR-PROTACs that can overcome small molecule drug resistance and serve as E3 ligase ligands has become a critical issue. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide an EGFR proteolysis-targeted chimera, a preparation method, a pharmaceutical composition, and applications thereof, specifically employing the following technical solutions:

[0006] According to the first aspect of the present invention, there is provided a proteolytic targeting chimera of EGFR, the structural formula of which is shown in formula (I):

[0007] Formula (I);

[0008] Wherein, Linker is any chemically feasible connection structure.

[0009] Previously, the inventors accidentally discovered that the anti-tumor lead compound CP0371 with high membrane permeability and drugability, obtained by semi-synthetic modification of the natural triterpenoid oleanolic acid (OA), can act as a molecular glue to bind to DNA damage binding protein 1 (DDB1) and then mediate the degradation of phosphoglycerate dehydrogenase (PHGDH). Studies have shown that DDB1 is an important part of a variety of E3 ligases. The development of E3 ligase ligands that can bind to DDB1 may help expand the PROTAC target protein library and may be expected to target more undruggable proteins.

[0010] The present invention provides a proteolytically targeted chimera for EGFR degradation, based on CP0371, a novel E3 ligase ligand with "molecular glue" functionality, and gefitinib, a target protein ligand. The proteolytically targeted chimera produced by the present invention can effectively induce EGFR degradation in human colorectal cancer cell lines in a dose-dependent manner, exhibits considerable bioavailability, and demonstrates excellent in vivo anti-colon cancer efficacy.

[0011] Preferably, the linker is a saturated fatty chain, an unsaturated fatty chain, or a fatty acid chain. By selecting ideal linkers of different types and lengths, the goal of maintaining the binding of the two proteins while not interfering with their spatial binding can be achieved.

[0012] Preferably, the molecular structure of the EGFR proteolysis targeting chimera is any one of formula (II), formula (III), and formula (IV):

[0013] Formula (II);

[0014] Formula (III);

[0015] Formula (IV);

[0016] Wherein, in formula (II), n is any positive integer from 1 to 12; in formula (III), n is any positive integer from 0 to 8; in formula (IV), n is any positive integer from 1 to 9.

[0017] More preferably, the molecular structure of the EGFR proteolysis targeting chimera is represented by formula (V):

[0018] Formula (V).

[0019] The compound represented by formula (V) can effectively induce the degradation of EGFR in the human colorectal cancer cell line HCT-116 in a dose-dependent manner, has considerable bioavailability, and exhibits the most excellent in vivo anti-colon cancer effect, with a tumor inhibition rate of 72.3%.

[0020] According to the second aspect of the present invention, a method for preparing the above-mentioned EGFR protein hydrolysis targeted chimera is also provided. The route is simple, the raw materials used are cheap and readily available, and the overall reaction yield is high. The preparation route is route 1, route 2, or route 3:

[0021] Route 1: 1-Boc-4-(3-bromopropyl)piperazine and the gefitinib intermediate 4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ol are substituted with K2CO3 and DMF, followed by removal of Boc under TFA to obtain intermediate C. Oleanolic acid D reacts with N-Boc ethylenediamine under HATU to produce intermediate E. After removal of Boc, it reacts with 4-methoxyisocyanate to obtain urea intermediate F. F undergoes monoesterification with diacids of different lengths to produce G1-G5, which is then subjected to amide condensation with intermediate C to obtain the final products H1-H5.

[0022] ;

[0023] Route 2: 2-Azidoethanol and p-toluenesulfonyl chloride are reacted with triethylamine (TEA) to obtain intermediate K, which is then substituted with the gefitinib intermediate 4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-ol in the presence of K2CO3 and DMF to obtain intermediate L. Intermediate F is esterified with alkyne acids of different lengths to obtain M1-M4, which is then subjected to a click reaction with intermediate L to obtain the final products N1-N4.

[0024] ;

[0025] Route 3: Intermediate F undergoes substitution reaction with acetylene bromides of different lengths under the action of sodium hydride to obtain G1-G4, which then undergoes click reaction with intermediate L to obtain the final products P1-P4.

[0026] .

[0027] According to the third aspect of the present invention, there is also provided the use of the above-mentioned EGFR proteolysis-targeting chimera or a pharmaceutically acceptable salt thereof in the preparation of an EGFR inhibitor.

[0028] Preferably, EGFR inhibitors can be used to prepare drugs for treating related cancers with EGFR abnormalities. Related cancers include: gynecological cancers, such as ovarian cancer, cervical cancer, vaginal cancer, pudendal cancer, uterine / endometrial cancer, gestational trophoblastic tumor, fallopian tube cancer, uterine sarcoma; endocrine cancers, such as adrenal cortical cancer, pituitary cancer, pancreatic cancer, thyroid cancer, parathyroid cancer, thymic cancer, multiple endocrine neoplasia; bone cancers, such as osteosarcoma, Ewing's sarcoma, chondrosarcoma, etc.; lung cancers, such as small cell lung cancer and non-small cell lung cancer; brain and CN S tumors, such as neuroblastoma, acoustic neuroma, glioma and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, advanced colorectal adenocarcinoma; gastrointestinal cancers, such as liver cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumors, gallbladder cancer, stomach cancer, esophageal cancer, small intestine cancer; genitourinary cancers, such as penile cancer, testicular cancer, prostate cancer; head and neck tumors, such as nasal cancer, paranasal sinus cancer, nasopharyngeal cancer, oral cancer, lip cancer, salivary gland cancer, laryngeal cancer, lower limb cancer, Pharyngeal cancer, pharyngeal cancer; blood cancers, such as acute myeloid leukemia, acute lymphocytic leukemia, childhood leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia; bone marrow cancer blood diseases, such as myelodysplastic syndrome, myeloproliferative disorders, Fanconi anemia, aplastic anemia, essential macroglobulinemia; lymphoma, such as Hodgkin's disease, non-Hodgkin's disease The invention also includes the following cancers: retinoblastoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, AIDS-related lymphoma; eye cancers, including retinoblastoma and uveal melanoma; skin cancers, such as melanoma, non-melanoma skin cancer, and Merkel cell carcinoma; soft tissue sarcomas, such as Kaposi's sarcoma, childhood soft tissue sarcoma, and adult soft tissue sarcoma; urinary system cancers, such as renal cell carcinoma, Wilms' tumor, bladder cancer, urethral cancer, and metastatic cell carcinoma. More preferably, the related cancers include colorectal cancer, lung cancer, esophageal cancer, glioblastoma, anal cancer, and head and neck epithelial cancer.

[0029] According to the fourth aspect of the present invention, a pharmaceutical composition is also provided, which comprises the above-mentioned EGFR proteolysis-targeting chimera or a pharmaceutically acceptable salt thereof as a main active ingredient.

[0030] Preferably, the pharmaceutical composition comprises an excipient, a solvent, and a pharmaceutically acceptable carrier. The excipient includes at least one of gum arabic, syrup, lanolin, and starch. The excipient is stable, has no incompatibility with the main drug, does not produce side effects, does not affect efficacy, is resistant to deformation, cracking, mildew, or insect damage at room temperature, is harmless to the human body, has no physiological effects, does not chemically or physically interact with the main drug, and does not affect the determination of the main drug content. The solvent includes water, glycerin, or ethanol.

[0031] The beneficial effects of the present invention are as follows: the present invention obtains a novel EGFR protein hydrolysis targeting chimera, the preparation process is simple and easy, and a variety of EGFR protein hydrolysis targeting chimera compounds are obtained through different pathways, which can be used to prepare EGFR inhibitors and can be composed into pharmaceutical compositions, which have a certain inhibitory effect on the proliferation of various tumor cells and are suitable for the development of cancer drugs for the treatment of colorectal cancer, lung cancer, esophageal cancer, glioblastoma, anal cancer, head and neck epithelial cancer, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Shown is a screening diagram for the effect of compound N2 on degradation of EGFR protein in HCT-116 cell line;

[0033] Figure 2 The graph shows the concentration-dependent degradation of EGFR protein by compound N2 in the HCT-116 cell line. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments and drawings to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, scheme and effect of the present invention. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict.

[0035] Example 1

[0036] An EGFR proteolysis-targeting chimera (denoted as compound H1), compound G1, compound F, and compound C, whose structures are shown below:

[0037] Compound H1, Compound C, Compound G1, Compound F,

[0038] The specific preparation method is:

[0039] 1) Preparation of compound G1: Compound F (648 mg, 1.00 mmol) was dissolved in dichloromethane (10 mL) under an ice bath. N,N'-dicyclohexylcarbodiimide (413 mg, 2.00 mmol), 4-dimethylaminopyridine (61.3 mg, 0.500 mmol), and pimelic acid (192 mg, 1.20 mmol) were added, respectively. The ice bath was removed after 5 minutes. After stirring at room temperature overnight, the liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), extracted with ethyl acetate (3 × 20 mL), and the organic phase was dried over anhydrous sodium sulfate. After concentration, the mixture was purified by silica gel flash column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound G1.

[0040] Compound G1 was tested and the test results were as follows: HRMS (ESI) calculated for C 47 H 70 N3O7 - [MH] - : 788.5219, found. 788.5217.

[0041] 2) Preparation of compound H1: G1 (158 mg, 0.200 mmol) and compound C (89.2 mg, 0.0.200 mmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (152 mg, 0.400 mmol) and diisopropylethylamine (64.6 mg, 0.500 mmol) were added. The mixture was reacted at room temperature overnight. After completion of the reaction, the reaction solution was washed with 1N HCl solution (10 mL), saturated sodium bicarbonate solution (10 mL), and saturated NaCl solution (10 mL), respectively, and extracted with ethyl acetate (3×10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1 to 2:1) to obtain compound H1 (white solid, 177 mg, 73%).

[0042] Compound H1 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s,1H), 8.29 (s, 1H), 7.65 – 7.62 (m, 2H), 7.56 (s, 1H), 7.49 (dd, J = 7.5, 2.1Hz, 1H), 7.34 – 7.28 (m, 2H), 7.26 (d,J = 7.5 Hz, 1H), 7.21 (s, 1H), 6.90 –6.78 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H),4.71 (s, 1H), 4.13 (d, J = 0.8 Hz, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 3.52 (s,2H), 3.47 (s, 2H), 3.40 (d, J = 1.0 Hz, 2H), 3.38 – 3.26 (m, 2H), 2.65 (d, J =0.9 Hz, 2H), 2.49 (d, J = 0.7 Hz, 4H), 2.33 (s, 2H), 2.31 – 2.20 (m, 3H), 2.13(dd, J = 18.0, 0.9 Hz, 1H), 2.03 – 1.87 (m, 8H), 1.75 (d, J = 13.0 Hz, 1H), 1.70(d, J = 12.9 Hz, 1H), 1.65 (dd, J = 13.0, 12.5 Hz, 2H), 1.59 – 1.56 (m, 4H), 1.54(d, J = 5.7 Hz, 1H), 1.51 (d, J = 5.7 Hz, 1H), 1.46 – 1.39 (m, 3H), 1.38 – 1.34(m, 4H), 1.33 (d, J = 7.3 Hz, 1H), 1.30 (d, J = 9.7 Hz, 1H), 1.24 (s, 1H), 1.07 –1.01 (m, 7H), 1.00 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.86 (d, J = 3.1 Hz,6H). 13C NMR (100 MHz, CDCl3) δ 177.2, 174.3, 173.8, 159.3, 159.2, 157.0,156.1, 153.0, 151.6, 148.1, 146.4, 145.1, 144.9, 134.1, 127.3, 124.3, 123.0,120.6, 118.6, 117.6, 116.5, 112.0, 108.6, 103.2, 83.5, 70.1, 61.7, 56.7,55.9, 55.1, 54.0, 53.2, 49.9, 48.2, 47.8, 46.9, 43.2, 42.1, 41.7, 40.5, 40.3,38.9, 38.2, 38.0, 37.9, 37.8, 37.7, 36.9, 35.4, 32.9, 32.5, 31.0, 29.9, 29.4,29.2, 29.1, 27.2, 25.7, 25.3, 23.4, 22.6, 20.0, 17.6. HRMS (ESI): m / z calcdfor C 69 H 95 ClFN8O8 + [M+H] + : 1217.6940; found 1217.6945.

[0043] Example 2

[0044] An EGFR proteolysis-targeting chimera (denoted as compound H2) has the following structure:

[0045] The structure of compound H2 is as follows:

[0046] Compound H2.

[0047] The specific preparation method is: Compound H2 is obtained according to the synthesis steps of Compound H1 described in Example 1, except that pimelic acid is replaced with suberic acid compared to Example 1.

[0048] Compound H2 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s,1H), 8.29 (s, 1H), 7.68 – 7.59 (m, 2H), 7.56 (s, 1H), 7.49 (dd, J= 7.5, 2.1Hz, 1H), 7.33 – 7.28 (m, 2H), 7.26 (d, J = 7.5 Hz, 1H), 7.21 (s, 1H), 6.90 –6.79 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H),4.71 (s, 1H), 4.13 (d, J = 0.9 Hz, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 3.52 (s,2H), 3.47 (s, 2H), 3.40 (d, J = 1.0 Hz, 2H), 3.37 – 3.26 (m, 2H), 2.65 (d, J =0.8 Hz, 2H), 2.49 (d, J = 0.7 Hz, 4H), 2.33 (s, 2H), 2.31 – 2.21 (m, 3H), 2.13(dd, J = 18.0, 0.9 Hz, 1H), 2.05 – 1.87 (m, 8H), 1.75 (d, J = 13.0 Hz, 1H), 1.70(d, J = 12.9 Hz, 1H), 1.65 (dd, J = 13.0, 12.5 Hz, 2H), 1.58 (s, 2H), 1.56 – 1.48(m, 4H), 1.45 – 1.38 (m, 3H), 1.38 – 1.27 (m, 8H), 1.24 (s, 1H), 1.08 – 1.01(m, 7H), 1.00 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.86 (d, J = 3.1 Hz, 6H). 13CNMR (100 MHz, CDCl3) δ 177.5, 174.6, 173.7, 159.2, 159.2, 156.6, 156.2,153.9, 151.2, 148.0, 146.6, 145.6, 144.7, 134.5, 127.3, 124.3, 123.5, 120.8,118.0, 117.2, 116.9, 111.9, 108.1, 103.8, 83.4, 70.3, 62.3, 56.5, 55.6, 55.3,53.7, 53.7, 49.9, 48.4, 47.4, 46.8, 43.6, 42.8, 41.4, 40.1, 40.0, 38.9, 38.7,38.2, 37.2, 37.0, 36.8, 35.6, 32.5, 32.3, 31.0, 29.4, 29.9, 29.4, 29.1, 29.0,27.5, 26.2, 25.8, 23.5, 22.5, 21.3, 19.5, 17.9. HRMS (ESI): m / z calcd forC 70 H 97 ClFN8O8 + [M+H] + : 1231.7096; found 1231.7095.

[0049] Example 3

[0050] An EGFR proteolysis-targeting chimera (denoted as compound H3) has the following structure:

[0051] Compound H3.

[0052] The specific preparation method is: Compound H3 is obtained according to the synthesis steps of Compound H1 described in Example 1. Compared with Example 1, only pimelic acid is replaced by azelaic acid.

[0053] Compound H3 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (d, J =38.7 Hz, 2H), 7.96 (s, 2H), 7.60 (d, J = 39.3 Hz, 2H), 7.21 (s, 2H), 7.12 (d, J=19.7 Hz, 2H), 5.75 (s, 1H), 5.36 (s, 1H), 4.46 (s, 1H), 4.17 (s, 2H), 3.92(s, 4H), 3.79 (d, J = 18.3 Hz, 6H), 3.66 (d, J = 18.6 Hz, 4H), 2.85 (d, J = 24.3Hz, 20H), 2.32 – 2.23 (m, 6H), 1.84 (s, 3H), 1.58 (s, 14H), 1.45 (d, J = 14.8Hz, 14H), 1.12 (s, 6H), 0.70 (s, 3H). HRMS (ESI) calculated for C 71 H 99 ClFN8O8 + [M+H] + : 1245.7253, found. 1245.7255.

[0054] Example 4

[0055] An EGFR proteolysis-targeting chimera (denoted as compound H4) has the following structure:

[0056] Compound H4.

[0057] The specific preparation method is: Compound H4 is obtained according to the synthesis steps of Compound H1 described in Example 1. Compared with Example 1, only pimelic acid is replaced by sebacic acid.

[0058] Compound H4 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.64 (s,1H), 7.86 (dd, J = 6.5, 2.7 Hz, 1H), 7.79 (s, 1H), 7.59 – 7.55 (m, 1H), 7.25 –7.09 (m, 5H), 6.88 – 6.78 (m, 3H), 6.52 (t, J = 5.4 Hz, 1H), 5.58 (d, J = 5.7 Hz,1H), 5.36 (t, J = 3.6 Hz, 1H), 4.47 (t, J= 7.9 Hz, 1H), 4.23 – 4.16 (m, 2H),3.99 (s, 3H), 3.77 (s, 3H), 3.65 – 3.59 (d, J = 6.8 Hz, 3H), 3.52 – 3.46 (m,2H), 3.44 – 3.19 (m, 4H), 3.15 (dd, J = 12.1, 5.9 Hz, 1H), 2.60 – 2.52 (m, 4H),2.45 (dt, J = 14.2, 4.9 Hz, 5H), 2.29 (td, J = 7.6, 2.3 Hz, 6H), 2.13 – 1.92 (m,7H), 1.81 – 1.67 (m, 8H), 1.36 – 1.24 (m, 17H), 1.13 (s, 3H), 0.91 – 0.86 (m,13H), 0.71 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 181.3, 178.0, 175.4, 163.2,158.6, 153.9, 146.2, 144.4, 141.7, 138.8, 134.0, 125.5, 124.3, 121.2, 117.1,116.4, 114.6, 108.1, 103.9, 102.2, 81.1, 65.6, 63.0, 59.6, 56.4, 55.7, 55.3,55.0, 54.0, 51.4, 47.6, 46.9, 46.5, 45.7, 42.4, 41.9, 40.8, 40.2, 39.5, 38.9,37.9, 37.0, 36.0, 35.0, 34.2, 33.4, 33.1, 32.3, 30.8, 29.8, 29.8, 29.5, 29.2,27.8, 26.7, 25.9, 25.2, 23.7, 23.3, 22.2, 18.7, 18.4, 16.9, 15.5, 12.8, 10.5.HRMS (ESI) calculated for C 72 H 101 ClFN8O8 + [M+H] + : 1259.7409, found. 1259.7404.

[0059] Example 5

[0060] An EGFR proteolysis-targeting chimera (denoted as compound H5) has the following structure:

[0061] Compound H5.

[0062] The specific preparation method is: Compound H5 is obtained according to the synthesis steps of Compound H1 described in Example 1. Compared with Example 1, only pimelic acid is replaced by undecanedioic acid.

[0063] Compound H5 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.59 (s,1H), 7.93 (d, J = 4.0 Hz, 1H), 7.74 – 7.50 (m, 2H), 7.37 (s, 1H), 7.22 (d, J =8.8 Hz, 2H), 7.10 (t, J = 8.7 Hz, 1H), 6.78 (s, 2H), 6.65 (d, J = 4.9 Hz, 1H), 5.91 (s, 1H), 5.36 (s, 1H), 4.47 (t, J = 8.0 Hz, 1H), 4.18 (d, J = 6.7 Hz, 2H), 3.95 (s, 3H), 3.76 (s, 3H), 3.68 (d, J = 15.0 Hz, 2H), 3.56 (t, J = 4.9 Hz, 2H),3.45 – 3.22 (m, 3H), 3.16 (t, J = 7.2 Hz, 1H), 2.68 (t, J = 7.1 Hz, 2H), 2.57 (d, J = 14.5 Hz, 4H), 2.35 – 2.23 (m, 4H), 2.11 (p, J= 8.1, 6.5 Hz, 2H), 2.01 –1.92 (m, 1H), 1.88 – 1.80 (m, 2H), 1.77 – 1.66 (m, 2H), 1.63 – 1.52 (m, 11H), 1.43 (s, 4H), 1.33 – 1.28 (m, 17H), 1.15 – 1.12 (m, 5H), 1.02 – 0.93 (m, 3H), 0.91 – 0.83 (m, 15H). 13 C NMR (100 MHz, CDCl3) δ 179.9, 173.9, 171.9, 157.4,155.3, 153.5, 148.9, 144.3, 135.8, 131.8, 125.6, 124.4, 123.2, 123.0, 122.2,116.6, 116.4, 114.4, 107.0, 102.7, 80.7, 67.5, 56.3, 55.6, 55.3, 55.0, 53.4,52.9, 47.5, 46.7, 46.4, 45.0, 42.0, 41.8, 40.9, 39.4, 38.1, 37.8, 36.9, 35.0,34.2, 33.3, 33.1, 32.9, 32.0, 30.8, 30.4, 29.8, 29.8, 29.5, 29.5, 29.4, 29.3,29.2, 28.2, 27.4, 25.9, 25.4, 25.3, 23.8, 23.6, 22.8, 18.3, 17.0, 16.9, 15.5,14.2. HRMS (ESI) calculated for C 73 H 103 ClFN8O8 + [M+H] + : 1273.7566, found.1273.7569.

[0064] Example 6

[0065] An EGFR proteolysis-targeting chimera (denoted as compound N1), compound M1, compound F, and compound L, whose structures are shown below:

[0066] Compound N1, Compound F,

[0067] Compound M1, Compound L.

[0068] The specific preparation method is:

[0069] 1) Preparation of compound M1: Compound F (648 mg, 1.00 mmol) was dissolved in dichloromethane (10 mL) under an ice bath, and N,N'-dicyclohexylcarbodiimide (413 mg, 2.00 mmol), 4-dimethylaminopyridine (61.3 mg, 0.500 mmol), and propiolic acid (84.1 mg, 1.20 mmol) were added respectively. The ice bath was removed after 5 minutes, and the mixture was stirred at room temperature overnight. The liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), and extracted with ethyl acetate (3 × 20 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel flash column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound M1.

[0070] Compound M1 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 7.24 –7.20 (m, 2H), 7.11 (s, 1H), 6.82 (d, J = 8.7 Hz, 2H), 6.61 (t, J = 4.9 Hz, 1H),5.85 (t, J = 5.3 Hz, 1H), 5.37 (t, J = 3.6 Hz, 1H), 4.65 (dt, J = 29.3, 8.4 Hz,1H), 3.77 (s, 3H), 3.42 (ddd, J = 19.7, 15.3, 7.9 Hz, 2H), 3.31 (q, J = 6.4, 5.8Hz, 1H), 3.17 (dt, J = 12.3, 5.4 Hz, 1H), 2.61 – 2.55 (m, 1H), 2.04 – 1.84 (m,4H), 1.79 (s, 2H), 1.73 – 1.57 (m, 8H), 1.52 – 1.42 (m, 3H), 1.37 – 1.30 (m,2H), 1.25 (s, 2H), 1.14 (d, J = 5.6 Hz, 3H), 1.01 (d, J= 14.2 Hz, 2H), 0.93 –0.89 (m, 6H), 0.88 (s, 6H), 0.85 (s, 3H), 0.73 (d, J = 5.4 Hz, 3H). 13 C NMR (100MHz, CDCl3) δ 179.7, 157.2, 156.1, 152.8, 144.2, 131.9, 122.9, 122.7, 114.3,83.6, 75.1, 74.2, 55.5, 55.2, 47.4, 46.6, 46.3, 41.9, 41.7, 39.3, 36.9, 36.8,34.1, 32.9, 32.8, 31.9, 30.7, 29.7, 29.6, 29.4, 27.9, 27.3, 25.8, 23.5, 22.7,18.1, 16.6, 15.4, 14.0. HRMS (ESI) calculated for C 43 H 62 N3O5 + [M+H] + : 700.4684,found. 700.4687.

[0071] 2) Preparation of compound N1: M1 (140 mg, 0.200 mmol) and compound L (77.8 mg, 0.0.200 mmol) were dissolved in a tBuOH / H2O (6.4 mL, 1:1) mixed solvent, and CuSO4•5H2O (25.0 mg, 0.100 mmol) and sodium L-ascorbate (35.6 mg, 0.180 mmol) were added. The mixture was reacted at room temperature overnight. After completion of the reaction, CuSO4•5H2O was filtered out, and the filtrate was concentrated in vacuo and isolated by silica gel flash column chromatography (dichloromethane:methanol = 8:1) to obtain compound N1 (white solid, 74.1 mg, 34%).

[0072] Compound N1 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.62 (s,1H), 8.42 (s, 1H), 8.38 (s, 1H), 7.93 (dd, J = 6.7, 2.6 Hz, 1H), 7.64 (t, J= 5.5Hz, 1H), 7.39 (s, 1H), 7.25 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.10 (t, J =8.8 Hz, 1H), 6.79 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 6.5 Hz, 1H), 5.90 (s, 1H),5.34 (d, J = 3.5 Hz, 1H), 4.83 (d, J = 4.9 Hz, 2H), 4.69 (t, J = 8.1 Hz, 1H), 4.44(t, J = 5.0 Hz, 2H), 3.95 (s, 3H), 3.75 (s, 3H), 3.50 – 3.22 (m, 4H), 3.17 (dd, J = 11.6, 5.9 Hz, 1H), 2.58 – 2.52 (m, 1H), 1.95 – 1.87 (m, 6H), 1.84 – 1.78(m, 3H), 1.67 (t, J = 13.3 Hz, 5H), 1.59 – 1.47 (m, 8H), 1.25 (s, 3H), 1.11 (s,3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H), 0.67 (s, 3H). 13CNMR (100 MHz, CDCl3) δ 179.7, 179.5, 160.5, 157.2, 156.6, 156.2, 154.8,154.1, 151.3, 151.0, 147.5, 144.2, 140.6, 137.3, 132.4, 129.0, 124.2, 123.1,121.9, 120.8, 116.5, 116.3, 114.4, 103.5, 82.3, 66.5, 60.4, 56.2, 55.5, 55.1,50.1, 47.4, 46.6, 46.3, 41.9, 40.9, 39.9, 39.3, 37.9, 36.8, 34.1, 32.9, 32.2,31.9, 30.7, 29.7, 29.2, 28.1, 27.3, 25.8, 25.1, 23.5, 18.1, 16.8, 15.4,14.1. 19 F NMR (376 MHz, CDCl3) δ -62.7. HRMS (ESI): m / z calcd for C 60 H 76 ClFN9O7 + [M+H] + : 1088.5535; found 1088.5533.

[0073] Example 7

[0074] An EGFR proteolysis-targeting chimera (denoted as compound N2) has the following structure:

[0075] Compound N2.

[0076] The specific preparation method is: Compound N2 is obtained according to the synthesis steps of Compound N1 described in Example 6. Compared with Example 6, only propiolic acid is replaced by butynic acid.

[0077] Compound N2 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.60 (s,1H), 8.29 (d, J = 27.8 Hz, 1H), 7.94 (dd, J = 6.6, 2.6 Hz, 1H), 7.87 (s, 1H),7.64 (dt, J = 7.3, 3.4 Hz, 1H), 7.22 (d,J = 2.8 Hz, 2H), 7.20 (s, 1H), 7.11 (t, J = 8.8 Hz, 1H), 7.02 (s, 1H), 6.83 – 6.78 (m, 2H), 6.56 (d, J = 6.3 Hz, 1H),5.68 (s, 1H), 5.35 (d, J = 3.8 Hz, 1H), 4.78 (t, J = 5.0 Hz, 2H), 4.48 (t, J = 5.0Hz, 2H), 4.34 (dd, J = 11.6, 4.5 Hz, 1H), 3.98 (s, 3H), 3.80 (s, 2H), 3.76 (s,3H), 3.40 (dd, J = 19.0, 11.4 Hz, 2H), 3.28 (t, J = 9.4 Hz, 1H), 3.15 (dd, J =11.8, 6.0 Hz, 1H), 2.56 (d, J = 12.9 Hz, 1H), 2.09 – 1.91 (m, 5H), 1.82 – 1.78(m, 2H), 1.71 (t, J = 13.6 Hz, 2H), 1.60 – 1.54 (m, 3H), 1.47 (d, J = 9.3 Hz,3H), 1.25 (s, 6H), 1.12 (s, 3H), 0.97 (d, J = 13.5 Hz, 2H), 0.88 (d, J = 2.5 Hz,3H), 0.85 (s, 3H), 0.80 (s, 3H), 0.75 (s, 3H), 0.73 (s, 3H), 0.67 (s, 3H). 13CNMR (100 MHz, CDCl3) δ 181.1, 179.7, 174.6, 172.9, 170.0, 168.4, 157.2,156.7, 156.4, 155.5, 153.8, 147.8, 144.2, 141.2, 131.3, 124.6, 123.3, 122.9,122.4, 122.3, 116.2, 114.4, 105.5, 82.3, 69.4, 61.2, 56.2, 55.5, 55.0, 50.1,47.4, 46.6, 46.3, 42.4, 41.9, 40.7, 40.0, 39.9, 39.3, 37.8, 37.7, 36.7, 32.9,32.1, 32.0, 30.7, 29.7, 29.4, 28.0, 27.3, 25.8, 23.5, 22.7, 18.1, 16.6, 15.3,14.1. 19 F NMR (376 MHz, CDCl3) δ -62.7. HRMS (ESI): m / z calcd for C 61 H 78 ClFN9O7 + [M+H] + : 1102.5691; found 1102.5694.

[0078] Example 8

[0079] An EGFR proteolysis-targeting chimera (denoted as compound N3) has the following structure:

[0080] Compound N3.

[0081] The specific preparation method is: Compound N3 is obtained according to the synthesis steps of Compound N1 described in Example 6. Compared with Example 6, only propiolic acid is replaced by pentynoic acid.

[0082] Compound N3 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.64 (s,1H), 8.38 (s, 1H), 8.00 (dd, J = 6.7, 2.6 Hz, 1H), 7.68 (s, 1H), 7.64 (d, J= 8.4Hz, 1H), 7.24 – 7.21 (m, 2H), 7.16 – 7.06 (m, 2H), 6.84 – 6.79 (m, 2H), 6.58(t, J = 5.1 Hz, 1H), 5.70 (s, 1H), 5.33 (d, J = 3.6 Hz, 1H), 4.75 (t, J = 4.7 Hz,2H), 4.45 (t, J = 4.8 Hz, 2H), 4.32 (dd, J = 11.7, 4.4 Hz, 1H), 4.00 (s, 3H),3.77 (s, 3H), 3.44 – 3.33 (m, 2H), 3.29 (d, J = 5.4 Hz, 1H), 3.15 (dd, J = 12.5,5.1 Hz, 1H), 3.04 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H), 2.60 – 2.53 (m,1H), 1.96 (t, J = 13.1 Hz, 2H), 1.71 (t, J = 12.9 Hz, 3H), 1.57 (d, J = 12.1 Hz,4H), 1.47 – 1.42 (m, 2H), 1.39 (d, J = 10.6 Hz, 2H), 1.34 (d, J = 3.3 Hz, 2H),1.31 (d, J = 4.3 Hz, 2H), 1.28 (s, 1H), 1.15 (d, J = 4.3 Hz, 2H), 1.10 (s, 3H),0.95 (d, J = 14.2 Hz, 2H), 0.88 (d, J = 2.1 Hz, 5H), 0.85 (s, 3H), 0.76 (s, 3H),0.74 (s, 3H), 0.64 (s, 3H), 0.62 (s, 3H). HRMS (ESI): m / z calcd forC 62 H 80 ClFN9O7+ [M+H] + : 1116.5848; found 1116.5844.

[0083] Example 9

[0084] An EGFR proteolysis-targeting chimera (denoted as compound N4) has the following structure:

[0085] Compound N4.

[0086] The specific preparation method is: Compound N4 is obtained according to the synthesis steps of Compound N1 described in Example 6. Compared with Example 6, only propiolic acid is replaced by hexynoic acid.

[0087] Compound N4 was tested, and the test results are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s,1H), 8.29 (s, 1H), 7.66 (s, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 6.0 Hz,2H), 7.49 (dd, J = 7.5, 2.1 Hz, 1H), 7.33 – 7.28 (m, 2H), 7.27 – 7.19 (m, 2H), 6.96 – 6.76 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s,1H), 4.71 (s, 1H), 4.48 (d, J = 3.2 Hz, 2H), 4.39 (d, J = 0.7 Hz, 2H), 3.89 (s,3H), 3.80 (s, 3H), 3.40 (d, J = 1.1 Hz, 2H), 3.38 – 3.27 (m, 2H), 2.88 – 2.68(m, 2H), 2.51 – 2.35 (m, 2H), 2.28 (t, J = 1.0 Hz, 1H), 2.13 (dd, J= 18.0, 0.9Hz, 1H), 2.03 – 1.94 (m, 4H), 1.93 – 1.89 (m, 2H), 1.88 – 1.75 (m, 2H), 1.72(d, J = 5.1 Hz, 1H), 1.70 – 1.60 (m, 2H), 1.53 (dd, J = 13.0, 5.7 Hz, 2H), 1.46 –1.39 (m, 3H), 1.39 – 1.27 (m, 4H), 1.24 (s, 1H), 1.07 – 1.01 (m, 7H), 1.00(s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.86 (d, J = 3.1 Hz, 6H). 13 C NMR (100 MHz, CDCl3) δ 177.7, 174.2, 159.1, 159.2, 156.6, 156.1, 153.0, 151.5, 148.9,146.5, 145.4, 144.5, 142.6, 134.7, 128.0, 127.4, 124.3, 123.0, 120.0, 118.0,117.2, 116.9, 111.9, 108.5, 103.8, 83.5, 74.3, 66.3, 57.3, 56.5, 55.6, 55.3,49.9, 48.4, 47.4, 43.6, 43.2, 42.8, 41.4, 40.1, 40.0, 38.4, 38.1, 38.0, 37.9,37.6, 35.7, 32.5, 32.3, 31.0, 29.7, 29.5, 28.8, 25.2, 25.2, 23.5, 22.5, 19.5,17.9. HRMS (ESI): m / z calcd for C 63 H 82 ClFN9O7 + [M+H] + : 1130.6004; found1130.6008.

[0088] Example 10

[0089] An EGFR proteolysis-targeting chimera (denoted as compound P1), compound O1, compound F, and compound L, whose structures are shown below:

[0090] Compound P1, Compound O1,

[0091] Compound F, Compound L.

[0092] The specific preparation method is:

[0093] 1) Preparation of compound O1: The preparation method is as follows: Compound F (648 mg, 1.00 mmol) was dissolved in tetrahydrofuran (10 mL) under an ice bath, and sodium hydride (40 mg, 1.00 mmol) was slowly added. After 10 minutes, 3-bromopropyne (143 mg, 1.20 mmol) was slowly added. The ice bath was removed after 10 minutes, and the mixture was stirred at room temperature overnight. The liquid in the bottle was concentrated using a rotary evaporator, then washed with water (20 mL), and extracted with ethyl acetate (3 × 20 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel flash column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain compound O1.

[0094] Compound O1 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.50 (s,1H), 7.55 (dd, J = 9.0, 2.7 Hz, 2H), 6.85 – 6.81 (m, 2H), 6.41 (d, J = 6.3 Hz,1H), 5.39 (d, J = 3.5 Hz, 1H), 4.27 – 4.17 (m, 2H), 3.77 (d, J = 2.7 Hz, 3H),3.47 (q, J = 11.1 Hz, 3H), 3.32 (d, J = 8.1 Hz, 1H), 3.21 (d, J = 9.1 Hz, 1H), 2.54(d, J = 12.8 Hz, 1H), 2.27 (d, J = 3.1 Hz, 1H), 2.05 – 1.88 (m, 4H), 1.79 – 1.65(m, 4H), 1.60 – 1.44 (m, 8H), 1.38 (dd, J= 20.7, 12.0 Hz, 4H), 1.28 – 1.23 (m,2H), 1.19 (s, 1H), 1.16 (d, J = 2.7 Hz, 3H), 0.98 (d, J = 2.7 Hz, 3H), 0.89 (s, 6H), 0.87 (d, J = 2.6 Hz, 3H), 0.77 (d, J = 2.7 Hz, 3H), 0.75 (d, J = 2.6 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 180.3, 155.4, 155.3, 144.6, 133.2, 123.1, 121.3,113.9, 80.2, 78.9, 72.1, 55.5, 55.1, 47.5, 46.7, 46.2, 45.5, 42.0, 39.5,39.4, 38.7, 38.5, 36.9, 36.7, 34.1, 32.9, 32.5, 32.3, 30.7, 28.1, 27.3, 27.1,25.7, 23.8, 23.5, 18.2, 17.0, 15.6, 15.4. HRMS (ESI): m / z calcd for C 43 H 64 N3O4 + [M+H] + : 686.4891; found 686.4888.

[0095] 2) Preparation of compound P1: Dissolve O1 (137 mg, 0.200 mmol) and compound L (77.8 mg, 0.0.200 mmol) in t CuSO4•5H2O (25.0 mg, 0.100 mmol) and sodium L-ascorbate (35.6 mg, 0.180 mmol) were added to a BuOH / H2O (6.4 mL, 1:1) mixed solvent and reacted at room temperature overnight. After the reaction was completed, CuSO4•5H2O was filtered out and the filtrate was concentrated in vacuo and separated by silica gel flash column chromatography (dichloromethane: methanol = 8:1) to obtain compound P1 (white solid, 98.9 mg, 44%).

[0096] Compound P1 was tested, and the test results were as follows: 1H NMR (400 MHz, CDCl3) δ 8.82 (d, J =22.5 Hz, 2H), 8.57 (d, J = 1.7 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.81 (dt, J =6.7, 2.2 Hz, 1H), 7.58 – 7.55 (m, 1H), 7.52 (s, 1H), 7.36 – 7.32 (m, 2H),7.14 (d, J = 1.7 Hz, 1H), 7.07 – 7.03 (m, 1H), 6.68 – 6.64 (m, 2H), 6.50 (d, J =5.2 Hz, 1H), 5.35 (s, 1H), 4.67 (t, J = 5.2 Hz, 2H), 4.59 (d, J = 15.8 Hz, 1H),4.49 (d, J = 15.8 Hz, 1H), 4.34 (t, J = 5.4 Hz, 2H), 3.89 (d, J = 1.7 Hz, 3H), 3.68(d, J = 1.6 Hz, 3H), 3.47 – 3.40 (m, 2H), 3.18 (dd, J = 11.0, 4.9 Hz, 2H), 2.44(dd, J = 12.9, 4.2 Hz, 1H), 2.27 (s, 2H), 1.97 – 1.82 (m, 4H), 1.68 (t, J = 13.3Hz, 2H), 1.54 (dt, J = 29.7, 7.0 Hz, 8H), 1.40 – 1.22 (m, 8H), 1.10 (s, 3H),0.96 – 0.95 (m, 3H), 0.85 (s, 6H), 0.78 (s, 3H), 0.75 (d, J = 1.7 Hz, 3H), 0.66(s, 3H). 13C NMR (100 MHz, CDCl3) δ 180.5, 156.8, 156.5, 155.6, 155.5, 154.2,148.3, 147.2, 145.7, 144.1, 135.8, 132.6, 124.4, 124.2, 123.4, 122.1, 116.3,116.1, 113.8, 109.2, 108.2, 106.8, 78.9, 68.9, 56.1, 55.4, 55.1, 49.7, 47.5,46.7, 46.6, 46.2, 43.7, 41.9, 41.8, 39.6, 39.3, 38.7, 38.5, 36.9, 34.0, 32.9,32.5, 32.2, 30.6, 28.1, 27.2, 27.1, 25.7, 23.8, 23.5, 23.4, 18.2, 16.9, 15.6,15.4. 19 F NMR (376 MHz, CDCl3) δ -62.7. HRMS (ESI): m / z calcd for C 60 H 78 ClFN9O6 + [M+H] + : 1074.5742; found 1074.5743.

[0097] Example 11

[0098] An EGFR proteolysis-targeting chimera (denoted as compound P2) has the following structure:

[0099] Compound P2.

[0100] The specific preparation method is: Compound P2 is obtained according to the synthesis steps of Compound P1 described in Example 10. Compared with Example 10, only 3-bromopropyne is replaced by 4-bromo-n-butyne.

[0101] Compound P2 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s,1H), 8.29 (s, 1H), 7.66 (s, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.58 (s, 1H), 7.56(s, 1H), 7.49 (dd, J= 7.5, 2.1 Hz, 1H), 7.33 – 7.28 (m, 2H), 7.26 (d, J = 7.5Hz, 1H), 7.22 (s, 1H), 6.93 – 6.77 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.18 (d, J = 0.7 Hz, 1H), 5.49 (s, 1H), 4.48 (d, J = 3.2 Hz, 2H), 4.39 (d, J = 0.7 Hz, 2H),3.89 (s, 3H), 3.80 (s, 3H), 3.69 – 3.50 (m, 2H), 3.44 – 3.27 (m, 5H), 2.96 –2.69 (m, 2H), 2.28 (t, J = 1.0 Hz, 1H), 2.13 (dd, J = 18.0, 0.9 Hz, 1H), 2.04 –1.96 (m, 2H), 1.95 – 1.84 (m, 4H), 1.75 (d, J = 13.0 Hz, 1H), 1.63 (dd, J = 13.0,2.5 Hz, 3H), 1.53 (dd, J = 13.0, 5.9 Hz, 3H), 1.42 (dd, J = 13.1, 1.2 Hz, 2H),1.38 (s, 1H), 1.37 – 1.27 (m, 3H), 1.20 (s, 1H), 1.08 – 0.99 (m, 7H), 0.96(d, J = 2.8 Hz, 6H), 0.91 (s, 3H), 0.86 (d, J = 2.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 177.7, 159.4, 159.2, 156.6, 156.1, 153.0, 151.5, 148.9, 146.5, 145.4, 144.5, 141.5, 134.7, 127.4, 126.2, 124.3, 123.0, 120.0, 118.0, 117.2,116.9, 111.9, 108.6, 103.8, 87.9, 74.3, 71.1, 62.2, 57.5, 56.5, 55.6, 54.5,49.8, 48.6, 47.4, 43.6, 42.8, 41.5, 40.1, 39.1, 39.0, 38.7, 38.2, 38.0, 37.9,34.5, 32.5, 32.5, 30.9, 29.9, 29.7, 27.6, 25.8, 23.2, 22.4, 19.5, 17.2. HRMS(ESI): m / z calcd for C 61 H 80 ClFN9O6 + [M+H] + : 1088.5899; found 1088.5903.

[0102] Example 12

[0103] An EGFR proteolysis-targeting chimera (denoted as compound P3) has the following structure:

[0104] Compound P3.

[0105] The specific preparation method is: Compound P3 is obtained according to the synthesis steps of Compound P1 described in Example 10. Compared with Example 10, only 3-bromopropyne is replaced by 5-bromo-n-pentyne.

[0106] Compound P3 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.91 (s,1H), 8.66 (s, 1H), 8.57 (s, 1H), 7.81 (dd, J = 6.7, 2.6 Hz, 1H), 7.67 (s, 1H),7.58 – 7.55 (m, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H), 7.19 (s, 1H), 7.04(t,J = 8.8 Hz, 1H), 6.67 (d, J = 8.6 Hz, 2H), 6.62 (s, 1H), 5.34 (d, J = 3.7 Hz,1H), 4.69 (t, J = 5.0 Hz, 2H), 4.40 (t, J = 5.2 Hz, 2H), 3.93 (s, 3H), 3.66 (s,3H), 3.46 (s, 1H), 3.32 (h, J = 7.6 Hz, 6H), 3.17 (d, J = 4.9 Hz, 1H), 2.64 (t, J =6.5 Hz, 2H), 2.52 (dd, J = 12.8, 4.3 Hz, 1H), 1.92 – 1.83 (m, 4H), 1.75 – 1.40(m, 16H), 1.32 – 1.21 (m, 4H), 1.11 (s, 3H), 0.95 (s, 3H), 0.87 (s, 3H), 0.85(s, 3H), 0.81 (s, 3H), 0.74 (s, 3H), 0.68 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ179.9, 156.7, 155.7, 155.2, 155.1, 153.3, 147.5, 147.2, 144.3, 135.8, 133.2,124.1, 123.1, 122.9, 122.0, 121.9, 121.7, 120.6, 120.4, 116.4, 116.2, 113.8,107.7, 104.5, 78.9, 68.2, 56.1, 55.4, 55.1, 49.6, 47.5, 46.6, 46.4, 46.2,45.4, 41.9, 41.1, 39.5, 39.3, 38.7, 38.4, 36.9, 34.0, 32.9, 32.6, 32.3, 30.7,28.1, 27.3, 27.1, 25.8, 23.7, 23.5, 23.4, 18.2, 16.9, 15.6, 15.2. 19F NMR (376MHz, CDCl3) δ -62.7. HRMS (ESI): m / z calcd for C 62 H 82 ClFN9O6 + [M+H] + : 1102.6055;found 1102.6053.

[0107] Example 13

[0108] An EGFR proteolysis-targeting chimera (denoted as compound P4) has the following structure:

[0109] Compound P4.

[0110] The specific preparation method is: Compound P4 is obtained according to the synthesis steps of Compound P1 described in Example 10. Compared with Example 10, only 3-bromopropyne is replaced by 6-bromo-1-hexyne.

[0111] Compound P4 was tested, and the test results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.54 (s,1H), 8.29 (s, 1H), 7.66 (s, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 6.0 Hz,2H), 7.49 (dd, J = 7.5, 2.1 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.26 (d, J = 7.5 Hz,1H), 7.22 (s, 1H), 6.93 – 6.79 (m, 2H), 6.52 (d, J = 0.7 Hz, 1H), 6.18 (d, J =0.7 Hz, 1H), 5.49 (s, 1H), 4.48 (d, J = 3.2 Hz, 2H), 4.39 (d, J = 0.7 Hz, 2H),3.89 (s, 3H), 3.80 (s, 3H), 3.60 – 3.44 (m, 2H), 3.40 (d, J = 1.1 Hz, 2H), 3.38– 3.28 (m, 3H), 2.77 (s, 2H), 2.28 (t, J= 1.0 Hz, 1H), 2.13 (dd, J = 18.0, 0.9Hz, 1H), 2.00 (d, J = 13.0 Hz, 1H), 1.98 – 1.93 (m, 2H), 1.93 – 1.83 (m, 3H),1.75 (d, J = 13.0 Hz, 1H), 1.68 (d, J = 3.2 Hz, 2H), 1.66 – 1.49 (m, 8H), 1.42(dd, J = 13.1, 1.2 Hz, 2H), 1.39 – 1.27 (m, 4H), 1.20 (s, 1H), 1.08 – 0.99 (m,7H), 0.96 (d, J = 2.8 Hz, 6H), 0.91 (s, 3H), 0.85 (s, 6H). 13 C NMR (100 MHz,CDCl3) δ 177.7, 159.1, 159.2, 156.6, 156.1, 153.0, 151.5, 148.9, 146.5,145.4, 144.8, 143.0, 134.7, 128.3, 127.3, 124.3, 123.0, 120.0, 118.0, 117.2,116.9, 111.9, 108.6, 103.1, 87.9, 74.3, 70.0, 57.3, 56.5, 55.8, 54.3, 49.9,48.2, 47.4, 43.6, 42.8, 41.5, 40.1, 39.3, 39.0, 38.1, 38.1, 38.0, 38.0, 37.6,32.8, 32.5, 31.4, 30.9, 30.6, 30.5, 29.8, 29.7, 27.2, 25.2, 23.2, 22.7, 19.5,17.5. HRMS (ESI): m / z calcd for C 63 H 84 ClFN9O6 + [M+H] + : 1116.6212; found1116.6213.

[0112] Example 14

[0113] In this example, the anti-tumor cell proliferation experiment screening was carried out on the PROTAC molecules (the proteolysis-targeting chimeras prepared in Examples 1-13) based on the pentacyclic triterpene molecular glue CP0371 as the E3 ligase ligand and gefitinib as the target protein ligand. MTT experiments were carried out in three different malignant tumor cell lines (human colorectal cancer cells HCT-116, HCT-15, and human lung cancer cells HCC-827).

[0114] To more intuitively compare the differences in compound activities, IC 50 was divided into four categories: 1 μM < IC 50 < 3 μM (A), 3 μM < IC50 < 10 μM (B), 10 μM < IC50 (C). The specific test results are shown in Table 1 below:

[0115] Table 1

[0116]

[0117] As can be seen from the results in Table 1, compounds H3, H4, H5, N1, N2, N3, P1, and P3 all showed good anti-proliferative activities in several different cell lines, and these compounds generally performed well in colorectal cancer cells, especially in the HCT-116 cell line. Therefore, these compounds will be selected for further evaluation in the HCT-116 cell line in the subsequent evaluation.

[0118] Example 15

[0119] In this example, the ability of the selected compounds H3, H, 5, N1, N2, N3, P1, and P3 in Example 14 to degrade EGFR was evaluated by Western blotting in the human colorectal cancer cell line HCT-116.

[0120] The results are as Figure 1 shown. Compounds H3, N1, N2, N3, and P1 all had obvious effects on degrading EGFR, and among them, compound N2 had the best effect. Compound N2 was selected for Western blotting analysis of its effect on degrading EGFR in a concentration gradient. As Figure 2 shown, with the increase in the concentration of compound N2, the degradation amount of EGFR in HCT-116 cells increased significantly, indicating that the degradation of EGFR by compound N2 was concentration-dependent.

[0121] Example 16

[0122] In this example, an evaluation experiment on the bioavailability of compound N2 was carried out.

[0123] The low bioavailability of PROTAC molecules usually limits their in vivo efficacy. One of the key factors limiting the bioavailability of PROTAC molecules is the poor solubility of conventional E3 ligase ligands. The novel E3 ligase ligand CP0371 selected in this invention has the characteristics of good solubility and is expected to exert higher in vivo bioavailability.

[0124] The specific method is as follows: after fasting overnight, three rats were given the drug by gavage (20 mg / kg) and three rats were given the drug by tail vein (5 mg / kg). Then, blood was collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively, and placed in a pre-heparinized sodium tube. The blood and sodium heparin were thoroughly mixed by flicking several times, and then centrifuged (4 ℃, 3000 rpm, 10 min) to obtain plasma. 50 μL of plasma sample was taken, 50 μL of diluent (50% methanol / water) and 250 μL of methanol precipitant were added, and the sample was vortexed and centrifuged (4 ℃, 12000 rpm, 10 min). The supernatant was passed through a membrane, sealed, and sent for LC-MS / MS detection.

[0125] Parameters were analyzed using WinNonlin software, and the results showed that compound N2 had good oral bioavailability of 44.6%, which was a significant improvement compared to other PROTAC molecules.

[0126] Example 17

[0127] In this example, the in vivo efficacy of compound N2 was evaluated, and HCT-116 cells were used to establish a xenograft model.

[0128] The specific method is: HCT-116 cells were inoculated subcutaneously in the armpits of nude mice, and the tumors grew to 70-110 mm 3 The mice were randomly divided into a control group (6 mice) and a Compound N2-treated group (50 mpk, 6 mice) for 18 days. There was no significant weight fluctuation or abnormality in either the treated or control groups, indicating the safety of Compound N2. Tumor weight and volume in the treated group were significantly lower than those in the control group, with a tumor inhibition rate ((1 - tumor weight in the treated group / tumor weight in the control group) * 100%) of 72.3%. This demonstrates that Compound N2 has strong in vivo efficacy.

[0129] Although the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but rather should be construed as providing a broad possible interpretation of these claims in view of the prior art by reference to the appended claims, thereby effectively encompassing the intended scope of the invention. In addition, the invention has been described above in terms of embodiments foreseen by the inventors for the purpose of providing a useful description, and those insubstantial modifications of the invention that are not currently foreseen may still represent equivalent modifications of the invention.

Claims

1. An EGFR proteolysis targeting chimera, characterized in that The molecular structure of the EGFR proteolysis targeting chimera is any one of formula (II), formula (III), and formula (IV): Formula (II); Formula (III); Formula (IV); Wherein, in formula (II), n is any positive integer of 5, 7-9; in formula (III), m is any integer of 0-2; in formula (IV), o is any positive integer of 1-3.

2. The EGFR proteolysis targeting chimera according to claim 1, characterized in that The molecular structure of the EGFR proteolysis targeting chimera is shown in formula (V): Formula (V).

3. A method for preparing the EGFR protein hydrolysis targeting chimera according to claim 1, characterized in that: Its preparation route is route 1, route 2 or route 3: Route 1: ； Route 2: ； Route 3: 。 4. Use of the EGFR proteolysis targeting chimera or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 in the preparation of an EGFR inhibitor, characterized in that: The EGFR inhibitor can be used to prepare drugs for treating related cancers with EGFR abnormalities; the related cancers are colorectal cancer and lung cancer.

5. A pharmaceutical composition, characterized in that The invention comprises the EGFR proteolysis targeting chimera or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition comprises an excipient, a solvent, and a pharmaceutically acceptable carrier.

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