Novel EGFR (epidermal growth factor receptor) degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a novel EGFR (epidermal growth factor receptor) degradation agent as well as a preparation method and application thereof. The EGFR degradation agent is PROTAC with GROs (such as AS1411) as an MDM2 recruitment element, target protein EGFR and E3 ubiquitin ligase MDM2 can be pulled close in vivo, so that the EGFR is labeled with ubiquitin, and then the EGFR is degraded through a ubiquitin-proteasome pathway. Experiments show that the EGFR degradation agent can effectively degrade EGFR and generate corresponding curative effects (such as inhibition of tumor cell proliferation and promotion of tumor cell apoptosis), and has very good application prospects and research values. In addition, the inventor also finds that AS1411 can specifically target and penetrate a hematoma barrier, and can be used for in-vivo imaging, diagnosis and detection of brain tumors.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and particularly to a novel EGFR degrader and its preparation method and application. Background Art

[0002] EGFR (Epidermal Growth Factor Receptor) is an important transmembrane receptor with tyrosine kinase activity, located on the cell membrane surface. It is activated by binding to epidermal growth factor EGF, transforming growth factor (TGF-α), etc., and is a constitutively expressed component in many normal epithelial tissues (such as skin and hair follicles). Overexpression of EGFR has been found in many human diseases, especially in various tumors and viral diseases. Activated EGFR is mainly related to the following signal transduction pathways: the mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) pathway, which initiates DNA replication and causes cell proliferation and differentiation; the PI3K-Akt pathway, which inhibits apoptosis; activation of downstream VEGF to promote the formation of microvascular networks; the EGFR-STAT3 pathway, which activates STAT3 and regulates the activity of multiple genes. The EGFR signal transduction pathway plays an important role in the proliferation, damage repair, invasion, and neovascularization of tumor cells. In recent years, drugs targeting EGFR have become a hot topic in tumor treatment. In addition to being closely related to the occurrence and development of tumors, EGFR also plays an important role in various viral diseases. After infection with various viruses, the expression level of EGFR in cells is upregulated. Viruses can use the EGFR-related signal pathway to invade target cells and successfully enter the host for proliferation; at the same time, they can regulate the host immune response through EGFR, triggering inflammation and immune antagonism. In addition, EGFR can regulate wound healing and fibrosis and is related to fibrosis diseases of multiple organs. Currently, studies have confirmed that pulmonary fibrosis after SARS-CoV and SARS-CoV-2 infections is related to the excessive response of the host to lung injury mediated by EGFR. Therefore, EGFR can also be used as a new target for antiviral therapy.

[0003] During the past few decades, researchers have developed four generations of EGFR inhibitors in total: the first-generation EGFR inhibitors are gefitinib and erlotinib based on the pyrimidine nucleus; the second-generation inhibitors are irreversible covalent inhibitors, including afatinib / dacomitinib; the third-generation EGFR inhibitors include osimertinib / omotorinib; the fourth-generation EGFR inhibitors focus on targeting allosteric sites, including EAI045 and JBJ-04-125-02. Despite the great therapeutic success, the clinical use of these EGFR inhibitors inevitably leads to acquired drug resistance, which poses new challenges to the targeted therapy of diseases. Moreover, inhibiting the EGFR protein requires maintaining the drug at a high concentration for a long time, and high-dose administration will cause the enrichment of the EGFR protein due to negative feedback, thus greatly weakening the inhibitory effect on it. Therefore, there is a need to further develop novel drugs that are more effective against the EGFR protein.

[0004] Targeted protein degradation is a brand-new and breakthrough drug development strategy that utilizes the inherent protein degradation pathways within cells to directly degrade pathogenic target proteins. This new type of drug form includes various types, such as PROTAC, molecular glue, LYTAC, ATAC, AbTAC, ATTEC, AUTAC, AUTOTAC, etc. PROTAC (PROteolysis Targeting Chimera) is a bifunctional molecule composed of a target protein ligand, a linker, and an E3 ubiquitin ligase recruitment element. After entering the cell, the target protein ligand in its structure specifically binds to the target protein, and the E3 ligase recruitment element at the other end binds to the E3 ligase, thus forming a ternary complex of target protein-PROTAC-E3. The E3 ubiquitin ligase mediates the ubiquitination of the target protein by ubiquitin-conjugating enzyme E2, and the target protein marked with polyubiquitination will be transported to the proteasome for degradation, thereby reducing the level of the target protein. In the above process, the target protein ligand does not need to occupy the binding site for a long time. Therefore, PROTAC can play a role in multiple cycles within the cell. Based on such a special mechanism of action of PROTAC, PROTAC drugs have important advantages for overcoming drug resistance and drug development for undruggable targets. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention provides a novel EGFR degrader and its preparation method and application. The EGFR degrader is a PROTAC using GROs (such as AS1411) as the MDM2 recruitment element, which can bring the target protein EGFR and the E3 ubiquitin ligase MDM2 closer in vivo, enabling EGFR to be tagged with ubiquitin and then degraded through the ubiquitin-proteasome pathway. Experiments show that the EGFR degrader can effectively degrade EGFR and produce corresponding therapeutic effects, such as inhibiting the proliferation of tumor (such as glioma) cells and promoting tumor cell apoptosis.

[0006] In the first aspect of the present invention, there is provided an EGFR degrader and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds, and the EGFR degrader has the following structure: Wherein, GRO is a guanine-rich oligonucleotide (G-rich oligonucleotides) residue capable of specifically binding to nucleolin (NCL), L is a linker structure part, EGFRB is an epidermal growth factor receptor EGFR recognition / binding part, and p is any suitable integer within 1-100.

[0007] Specifically, EGFRB can be the structure part of any suitable EGFR small molecule inhibitor or an EGFR antibody residue, and the EGFR inhibitor is, for example, the first to fourth generation EGFR-TKIs, such as quinazoline-based, pyridopyrimidine-based, pyrrolopyrimidine-based EGFR-TKIs, etc., especially 4-anilinoquinazoline-based EGFR-TKIs.

[0008] In particular, the EGFR degrader has the following structure:

[0009]

[0010] Wherein,

[0011] GRO is a guanine-rich oligonucleotide (G-rich oligonucleotides) residue capable of specifically binding to nucleolin (NCL);

[0012] L is a linker structure part, that is, a linker;

[0013] p is an integer from 1 to 100 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 40, 50, 60, 80, 100);

[0014] Ring A is an aliphatic ring or a heterocyclic ring;

[0015] Ring C is an aromatic ring or an aromatic heterocyclic ring;

[0016] B 1 、B 2 、B 3 is independently selected from: C or N;

[0017] Y 1 、Y 2 、Y 3 is independently selected from: a single bond, -O-, -S-, -C 0 -C 6 -alkylene-, -O-(C 0 -C 6 -alkylene)-, -S-(C 0 -C 6 -alkylene)-, -N-(C 0 -C 6 -alkyl), -C(O)-, -C(S)-, -C(O)-(C 0 -C 6 -alkylene)-, -C(S)-(C 0 -C 6 -alkylene)-, -C(O)-N(C 0 -C 6 -alkyl)-, -C(S)-N(C 0 -C 6 -alkyl)-, -SO 2 -(C 0 -C 6 -alkylene)-, -SO-(C 0 -C 6 -alkylene)-;

[0018] R 1 is one or more independent substituents on the benzene ring and is selected from: H, halogen, cyano, nitro, azide, C 1 -C 10 -alkyl, -(C 0 -C 6 -alkylene)-(C 3 -C 10 -cycloalkyl), -(C 0 -C 6 -alkylene)-(C 6 -C 10 -aryl), -(C 0 -C 6 -alkylene)-(4- to 10-membered heterocyclic group), C 1 -C 10 -haloalkyl, C 1 -C 10 -haloalkoxy, -N(C 0-10 -alkyl)(C 0-10 -alkyl), -N(C 0-10(alkyl)CO(C 0-10 (alkyl)、-N(C 0-10 (alkyl)CON(C 0-10 (alkyl)、-N(C 0-10 (alkyl)SO 2 (C 0-10 (alkyl)、-O(C 0-10 (alkyl)、-S(C 0-10 (alkyl)、-SO(C 0-10 (alkyl)、-SO 2 (C 0-10 (alkyl)、-SO 2 N(C 0-10 (alkyl)(C 0-10 (alkyl)、-COO(C 0-10 (alkyl)、-OCO(C 0-10 (alkyl)、-CON(C 0-10 (alkyl)(C 0-10 (alkyl)、-CO(C 0-10 (alkyl);

[0019] R 2 is one or more independent substituents on the benzene ring, and is selected from: H, halogen, cyano, nitro, azide, C 1 -C 10 (alkyl)、-(C 0 -C 6 (alkylene)-(C 3 -C 10 (cycloalkyl)、-(C 0 -C 6 (alkylene)-(C 6 -C 10 (aryl)、-(C 0 -C 6 (alkylene)-(4- to 10-membered heterocyclic group), C 1 -C 10 (haloalkyl), C 1 -C 10 (haloalkoxy), -N(C 0-10 (alkyl)(C 0-10 (alkyl)、-N(C 0-10 (alkyl)CO(C 0-10 (alkyl)、-N(C 0-10 (alkyl)CON(C 0-10 (alkyl)、-N(C 0-10 (alkyl)SO 2 (C 0-10 (alkyl)、-O(C 0-10 (alkyl)、-S(C 0-10 (alkyl)、-SO(C 0-10 (alkyl)、-SO 2(C 0-10 alkyl), -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl);

[0020] R 3 is one or more independent substituents on the C ring, selected from: H, halogen, cyano, nitro, azide, C 1 -C 10 alkyl, -(C 0 -C 6 alkylene)-(C 3 -C 10 cycloalkyl), -(C 0 -C 6 alkylene)-(C 6 -C 10 aryl), -(C 0 -C 6 alkylene)-(4- to 10-membered heterocyclic group), C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO 2 (C 0-10 alkyl), -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl).

[0021] In a preferred embodiment of the present invention, p is 1, that is, the general formula I is

[0022] Specifically, the GROs are guanine-rich oligonucleotides containing one or more GGT motifs and having G4 structural characteristics.

[0023] Preferably, the GROs have stable G4 structural characteristics, and the G4 structural characteristic signals can be detected by the methods described in the prior art (see, for example, Yu Yuan, Hu Fang, Xia Yuan, et al. Research progress on G-quadruplex detection methods and biology [J]. Chemistry of Life. 2021, 14(10): 2146-2155.; Gao Juan, Yuan Gu, Xu Ming. Detection, function and regulation of G-quadruplex structures [J]. Progress in Physiological Sciences. 2014, 45(5): 364-371.), for example, detected using probes (such as specific antibodies, specific fluorescent ligands, radiolabels, etc.), or detected by circular dichroism (CD), nuclear magnetic resonance (NMR), ultraviolet spectroscopy, molecular fluorescence spectroscopy, single molecule fluorescence resonance energy transfer (FRET), etc.

[0024] In some embodiments of the present invention, fluorescence probes (such as N-methyl mesoporphyrin IX (NMM), O-phenanthroline derivative) are used for detection, and the GROs have stable G4 structural characteristic signals (see, for example, Zhang Suge, Sun Hongxia, Tang Yalin. Research progress on DNA G-quadruplex recognition probes [J]. Chemistry Bulletin. 2016, 79(5): 387-394.).

[0025] In one embodiment of the present invention, when N-methyl mesoporphyrin IX (NMM) is used as a fluorescence probe for detection, stable G4 structural characteristics can be formed by AS1411 in the presence or absence of K + Specifically, the detection method may include the following steps: adding the GRO to be tested into a solution with or without K + heating at 90-99 °C for 5-10 minutes, incubating on ice, then adding NMM, incubating in the dark at room temperature, and detecting the fluorescence intensity (such as described in Example 2 of the present invention).

[0026] Specifically, the GROs include chemical modification on the GROs, nucleic acid unit replacement, or attachment of functional groups; these modifications or functional groups can be used to improve the stability of PROTAC, provide detection signals, or form compositions with other substances. Among them, the chemical modification is that at least one base is modified, and the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, isotopic labeling, phosphorothioate backbone modification, methoxy modification, and fluoro modification; the nucleic acid unit replacement is that at least one nucleic acid unit is replaced with LNA, UNA, or GNA; the functional groups include at least one of fluorescent groups, radioactive groups, therapeutic drugs, biotin, digoxin, nanoluminescent materials, nucleic acid substances, or enzyme markers.

[0027] Preferably, the GROs have 4 - 100 (such as 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 80, 100) nucleotides.

[0028] Preferably, the GROs are one of the DNA aptamer AS1411, AS1411 derivatives / analogues.

[0029] Among them, the AS1411 derivatives / analogues are GROs with a homology of more than 60% (such as more than 65%, 70%, 75%, 80%, 85%, 90%, 95%) to AS1411, and contain one or more GGT motifs, have G4 structural characteristics, and can specifically bind to NCL.

[0030] Specifically, the AS1411 derivatives / analogues are one of GRO29A, GRO15A, AT11, AT11-L0, AT21, AT27, GRO29-2, GRO29-3, GRO29-5, GRO29-13, GRO14C, GRO25A, GRO28A, GRO28B, GRO29-6, GRO32A, GRO32B, GRO56A, GRO A, GRO B, GRO C, GRO D, GRO E, GRO F, GRO G, GRO H, GRO I, GRO J, GROK, GRO L, GRO M, and their nucleotide sequences are shown as SEQ ID NO: 8 - 38 respectively.

[0031] Specifically, the sequence of AS1411 is as shown in SEQ ID NO: 1: 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

[0032] In some preferred embodiments of the present invention, the GROs are AS1411.

[0033] In some other embodiments of the present invention, the GROs are selected from: GRO29A, GRO15A, AT11.

[0034] Specifically, L can be any suitable linking moiety that links the GRO to an epidermal growth factor receptor EGFR recognition / binding moiety (such as the structural moiety of an EGFR-TKI).

[0035] Specifically, L can be linked to any base, sugar, or phosphate backbone at the 3'-end, 5'-end, or in the middle of the GRO.

[0036] In some embodiments of the present invention, L is linked to the 3'-end of the GRO.

[0037] In some embodiments of the present invention, L is linked to the 5'-end of the GRO.

[0038] In some embodiments of the present invention, the GRO moiety in General Formula I has the following structure:

[0039]

[0040] Specifically, L has the following structure: Wherein,

[0041] L 1 is a divalent group linked to the GRO, and it can be selected from: a single bond, -O-(C 0 -C 6 alkylene)-, -S-(C 0 -C 6 alkylene)-, -N(R L1 )-(C 0 -C 6 alkylene)-, -N(R L2 )C(O)-(C 0 -C 6 alkylene)-, -OP(O)(OR L1 )O-(C 0 -C 6 alkylene)-, -C(O)-(C 0 -C 6 alkylene)-, -C(S)-(C 0 -C 6 alkylene)-, -CON(R L1 )-(C 0 -C 6 alkylene)-;

[0042] L 3 is a divalent group linked to the A ring, and it can be selected from: a single bond, -O-(C0 -C 6 -alkylene)-, -S-(C 0 -C 6 -alkylene)-, -C(O)-(C 0 -C 6 -alkylene)-, -C(S)-(C 0 -C 6 -alkylene)-, -N(R L3 )-(C 0 -C 6 -alkylene)-, -CON(R L3 )-(C 0 -C 6 -alkylene)-, -N(R L3 )CO-(C 0 -C 6 -alkylene)-, -SO 2 -(C 0 -C 6 -alkylene)-, -SO-(C 0 -C 6 -alkylene)-, 4- to 10-membered heteroalkylene;

[0043] L 2 is a single bond or a divalent saturated or unsaturated straight-chain or branched-chain C1-C50 hydrocarbon chain (e.g., a C1-C20 alkyl chain), and 0 to 6 methylene units in the hydrocarbon chain (e.g., alkyl chain) are independently substituted by: -CY-, -O-, -S-, -S-S-, -C(O)-, -C(S)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R L2 )-, -N(R L2 )C(O)-, -N(R L2 )C(O)O-, -N(R L2 )C(O)N(R L2 )-, -N(R L2 )-, -S(O) 2 -,-S(O) 2 N(R L2 )-, -N(R L2 )S(O) 2 -,-S(O)-, -S(O)N(R L2 )-, -N(R L2 )S(O)-, -P(O)(OR L2 )O-, -P(O)-, -P(O)N(R L2 )-, -P(O)(N(R L2 ) 2 )-, -OP(O)(OR L2 )2 N(R L2 )-, -P(O)(OR L2 ) 2 N(R L2 )-, -N(R L2 )P(O)(OR L2 )O-, -N(R L2 )P(O)-, -Si(R L2 ) 2 -, -C(=N-CN)-,

[0044] An amino acid residue, a nucleotide residue, an oligonucleotide residue, an oligopeptide residue, wherein, m2 is selected from an integer between 1 and 10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and each -CY- is independently an optionally substituted divalent ring selected from the following: arylene, cycloalkylene, heterocyclylene; the H in the hydrocarbon chain may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, -OR L0 , -C(O)R L0 , -C(S)R L0 , -C(O)OR L0 , -C(S)SR L0 , -OC(O)R L0 , -OC(S)R L0 , -OC(S)SR L0 , -C(O)N(R L0 ) 2 , -OC(O)N(R L0 ) 2 , -N(R L0 )C(O)OR L0 , -N(R L0 )SO 2 R L0 , -SO 2 N(R L0 ) 2 , -OSO 2 N(R L0 ) 2 , -N(R L0 )C(O)R L0 , -N(R L0 ) 2 , -SR L0 , -SOR L0 , -SO 2 R L0 , -OSO 2 R L0 , C 1 , -C10 Alkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, C 1 -C 10 Haloalkyl, C 1 -C 10 Haloalkoxy, -(C 0 -C 6 Alkylene)-(C 3 -C 10 Cycloalkyl), -(C 0 -C 6 Alkylene)-(C 6 -C 10 Aryl), -(C 0 -C 6 Alkylene)-(4- to 10-membered heterocyclic group);

[0045] R L0 、R L1 、R L2 and R L3 are independently selected from: H, C 1 -C 10 Alkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, -(C 0 -C 6 Alkylene)-(C 3 -C 10 Cycloalkyl), -(C 0 -C 6 Alkylene)-(C 6 -C 10 Aryl), -(C 0 -C 6 wherein the H in the Alkyl, C 1 -C 10 Alkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, C 0 -C 6 Alkylene, C 3 -C 10 Cycloalkyl, C 6 -C 10 Aryl, 4- to 10-membered heterocyclic group may optionally be substituted by one or more groups selected from: halogen, cyano, nitro, azide, hydroxy, amino, mercapto, carboxy, C 1 -C 10 Alkyl, C2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -(C 0 -C 6 alkylene)-(C 3 -C 10 cycloalkyl), -(C 0 -C 6 alkylene)-(C 6 -C 10 aryl), -(C 0 -C 6 alkylene)-(4- to 10-membered heterocyclic group).

[0046] Specifically, each -CY- is independently selected from the following optionally substituted divalent rings: phenylene, bicyclic arylene, tricyclic arylene, monocyclic cycloalkylene, bicyclic cycloalkylene, tricyclic cycloalkylene, monocyclic heteroarylene, bicyclic heteroarylene, tricyclic heteroarylene, monocyclic heterocycloalkylene, bicyclic heterocycloalkylene, tricyclic heterocycloalkylene.

[0047] In some embodiments of the present invention, each -CY- is independently selected from the following:

[0048]

[0049]

[0050] wherein, R L4 、R L5 are independently selected from: H, OH, halogen, C 1-8 alkyl, O(C 1-8 alkyl), S(C 1-8 alkyl), NH(C 1-8 alkyl), N(C 1-8 alkyl) 2 、C 3-11 cycloalkyl, C 3-11 heterocycloalkyl, O(C 1-8 cycloalkyl), S(C 1-8 cycloalkyl), NH(C 1-8 cycloalkyl), N(C 1-8 cycloalkyl)(C 1-8 alkyl), OH, NH 2 、SH, SO 2 (C1-8 alkyl), P(=O)(OC 1-8 alkyl)(C 1-8 alkyl), P(=O)(OC 1-8 alkyl) 2 , C 1-8 alkynyl, CH=CH(C 1-8 alkyl), C(C 1-8 alkyl)=CH(C 1-8 alkyl), C(C 1-8 alkyl)=C(C 1-8 alkyl) 2 , Si(OH) 3 , Si(C 1-8 alkyl) 3 , Si(OH)(C 1-8 alkyl) 2 , C(=O)(C 1-8 alkyl), CO 2 H, CN, CF 3 , CHF 2 , CH 2 F, NO 2 , SF 5 , SO 2 NH(C 1-8 alkyl), SO 2 N(C 1-8 alkyl) 2 , S(=O)N(C 1-8 alkyl) 2 , C(=O)NH(C 1-8 alkyl), C(=O)N(C 1-8 alkyl) 2 , N(C 1-8 alkyl)C(=O)NH(C 1-8 alkyl), N(C 1-8 alkyl)C(=O)N(C 1-8 alkyl) 2 , NHC(=O)NH(C 1-8 alkyl), NHC(=O)N(C 1-8 alkyl) 2 , NHC(=O)NH 2 , N(C 1-8 alkyl)SO 2 NH(C 1-8 alkyl), N(C 1-8 alkyl)SO 2 N(C 1-8 alkyl) 2 , NHSO 2 NH(C 1-8 alkyl), NHSO2 N(C 1-8 alkyl) 2 or NHSO 2 NH 2 ; or, R L4 、R L5 together with the atom to which it is attached form a cycloalkylidene or heterocycloalkylidene group.

[0051] More specifically, R L4 、R L5 are independently selected from: -CH 3 , -OH,

[0052] or, R L4 、R L5 together with the atom to which it is attached form a three- to six-membered cycloalkylidene group (such as ) or a four- to six-membered heterocycloalkylidene group (such as ).

[0053] In some embodiments of the present invention, R L1 is H.

[0054] In some embodiments of the present invention, R L3 is H.

[0055] In some embodiments of the present invention, R L4 is H.

[0056] In some embodiments of the present invention, R L4 is OH.

[0057] In some embodiments of the present invention, R L5 is H.

[0058] In some embodiments of the present invention, R L5 is OH.

[0059] In one embodiment of the present invention, L 2 adopts the following scheme (1): L 2 is a C1-C20 straight-chain alkylene group, and 0-6 methylene units in the alkylene group are independently substituted by the following groups: -O-, -S-, -S-S-, -C(O)-, -C(O)O-, -OC(O)-, -N(R L2 )-, -C(O)N(R L2 )-, -N(R L2 )C(O)-,

[0060] Among them, each R L2 is independently selected from: H, C 1 -C 6 alkyl, and each R L4 is independently selected from: H, OH, C 1 -C 6 alkoxy.

[0061] More specifically, L 2 can be selected from: C1-C20 straight-chain alkylene, -(CH 2 CH 2 O) m2 -CH 2 -、-(CH 2 CH 2 O) m2 -CH 2 CH 2 -、-CH 2 -(CH 2 CH 2 O) m2 -CH 2 -、-CH 2 CH 2 -(CH 2 CH 2 O) m2 -CH 2 -、-CH 2 CH 2 -(CH 2 CH 2 O) m2 -CH 2 CH 2 -、-(C 1 -C 10 alkylene)-O-(C 1 -C 10 alkylene)-、-(C 1 -C 10 alkylene)-NH-(C 1 -C 10 alkylene)-、-(C 1 -C 10 alkylene)-C(O)NH-(C 1 -C 10 alkylene)-、-(C 1 -C 10 alkylene)-NHC(O)-(C 1 -C 10 alkylene)-、-(C 1 -C 6 alkylene)-O-(C 1 -C 6(Alkylene)-C(O)NH-(C 1 -C 6 (Alkylene)-、-(C 1 -C 6 (Alkylene)-O-(C 1 -C 6 (Alkylene)-NHC(O)-(C 1 -C 6 (Alkylene)-、

[0062]

[0063]

[0064] wherein, m2 is selected from integers between 1 and 10, g is 0 or 1, h is selected from integers between 0 and 10 (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), i is selected from integers between 0 and 10 (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and G is any suitable trivalent group.

[0065] In some specific embodiments of the present invention, L 2 is selected from:

[0066]

[0067] In some embodiments of the present invention, L 2 is selected from C1-C20 linear alkylene, -(C 0 -C 6 (Alkylene)-(CH 2 CH 2 O) m2 (Alkylene)-、-(C 1 -C 6 (Alkylene)-、-(C 1 -C 10 (Alkylene)-O-(C 1 -C 10 (Alkylene)-、-(C 1 -C 10 (Alkylene)-N(C 1 -C 6 (Alkyl)-(C 1 -C 10 (Alkylene)-、-(C 1 -C 10 (Alkylene)-C(O)N(C 1 -C 6 (Alkyl)-(C 1 -C 10 (Alkylene)-、-(C1 -C 10 -alkylene)-N(C 1 -C 6 -alkyl)C(O)-(C 1 -C 10 -alkylene)-, -(C 1 -C 6 -alkylene)-O-(C 1 -C 6 -alkylene)-C(O)NH-(C 1 -C 6 -alkylene)-, -(C 1 -C 6 -alkylene)-O-(C 1 -C 6 -alkylene)-NHC(O)-(C 1 -C 6 -alkylene)-, wherein one methylene unit in the alkylene is independently replaced by a group capable of connecting to a solid support (e.g., G is any suitable trivalent group).

[0068] In some embodiments of the present invention, is wherein the J ring is a 4- to 10-membered saturated heterocyclic ring.

[0069] Specifically, the J ring is a 4- to 6-membered saturated heterocyclic ring, optionally further containing other heteroatoms, such as More specifically, can be, for example,

[0070] In some embodiments of the present invention, G is a sugar residue, such as can be

[0071] In some embodiments of the present invention, L 2 is Especially (the x end is connected to L 3 , and the y end is connected to L 1 ), for example

[0072] In another embodiment of the present invention, L 2 adopts the following scheme (2): L 2 is a C1-C20 straight-chain alkylene, wherein 1 to 3 methylene units are independently replaced by the following groups: -CY-, Optionally, L 2also contains a group selected from the following: -O-, -C(O)-, -N(R L2 )-, -C(O)N(R L2 )-, -N(R L2 )C(O)-,

[0073]

[0074] wherein each R L2 is independently selected from: H, C 1 -C 6 alkyl, and each R L4 is independently selected from: H, OH, C 1 -C 6 alkoxy.

[0075] In some embodiments of the present invention, -CY- is selected from:

[0076]

[0077]

[0078] In some specific embodiments of the present invention, L 2 is selected from:

[0079] In another embodiment of the present invention, L 2 adopts the following scheme (3): L 2 is an oligonucleotide residue, such as a DNA oligonucleotide residue, an RNA oligonucleotide residue, a DNA / RNA hybrid oligonucleotide residue, which can be single-stranded or double-stranded. In this case, L 1 can be a single bond or O, that is, GRO is connected to L ) through a phosphoester bond (that is, 2 ).

[0080] Specifically, the oligonucleotide is composed of A and / or T.

[0081] In some embodiments of the present invention, L 2 is a single-stranded oligonucleotide residue, which contains 3-30 (for example, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30) A or T, for example, a single-stranded oligonucleotide residue composed of 6 A.

[0082] In some embodiments of the present invention, L 2 is a double-stranded oligonucleotide residue comprising 3 - 30 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30) A-T base pairs, for example, a double-stranded oligonucleotide residue consisting of 6 A-T base pairs.

[0083] In some embodiments of the present invention, L 2 is a single-stranded DNA oligonucleotide residue consisting of 6 A's.

[0084] In some embodiments of the present invention, L 2 is a DNA double-stranded oligonucleotide consisting of 6 A-T base pairs.

[0085] In some embodiments of the present invention, L 2 is a DNA double-stranded oligonucleotide consisting of 10 A-T base pairs.

[0086] In some embodiments of the present invention, L 2 is a DNA / RNA hybrid double-stranded oligonucleotide, wherein the DNA single strand consists of 6 T's and the RNA single strand consists of 6 A's.

[0087] In another embodiment of the present invention, L 2 adopts the following scheme (4): L 2 is an oligopeptide residue consisting of 2 - 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid residues.

[0088] In some embodiments of the present invention, L 3 is a single bond.

[0089] In some embodiments of the present invention, L 3 is -N(H)-.

[0090] In some embodiments of the present invention, L 3 is -C(O)-.

[0091] In some embodiments of the present invention, L 3 is -CON(R L3 )-, R L3 is selected from: H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, for example,

[0092] Specifically, the epidermal growth factor receptor EGFR recognition / binding moiety is the structural moiety of a small molecule inhibitor of EGFR, and the small molecule inhibitor can be gefitinib, afatinib, osimertinib, lapatinib, erlotinib, dacomitinib, poziotinib, neratinib, alflutinib (AST 2818), almonertinib (HS10296), BBT-176, BI-4020, CH7233163, gilitertinib, JND-3229, lazertinib, neratinib (EGF816), PCC-0208027, rezivertinib (BPI-7711), TQB3804, zorifertinib (AZ-3759), or DZD9008.

[0093] In some embodiments of the present invention, the epidermal growth factor receptor EGFR recognition / binding moiety is the structural moiety of a 4-anilinoquinazoline-based EGFR-TKI (such as the quinazoline derivative shown in CN1182421A).

[0094] Specifically, ring A is a 4- to 6-membered cycloalkyl or a 4- to 6-membered nitrogen-containing heterocycloalkyl, for example, In some embodiments of the present invention, ring A is

[0095] In some embodiments of the present invention, is

[0096] Specifically, R 1 is selected from: H, C 1 -C 6 alkyl, halogen, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, -O(C 1-6 alkyl); in some embodiments of the present invention, R 1 is -O(C 1-3 alkyl), such as -OCH 3 .

[0097] In some embodiments of the present invention, is

[0098] Specifically, R 2 is selected from: H, C 1 -C6 Alkyl, halogen, C 1 -C 6 Halogenated alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Halogenated alkoxy, -O(C 1-6 alkyl); In some embodiments of the present invention, R 2 is H.

[0099] Specifically, B 1 , B 2 , B 3 are all C, such as Or, B 1 , B 2 , B 3 At least one of them is N, such as In some embodiments of the present invention, B 1 is N, B 2 is C, B 3 is N, such as

[0100] In some embodiments of the present invention, is

[0101] Specifically, R 3 is selected from: H, C 1 -C 6 alkyl, halogen, C 1 -C 6 halogenated alkyl, C 1 -C 6 alkoxy, C 1 -C 6 halogenated alkoxy, -O(C 1-6 alkyl); In some embodiments of the present invention, R 3 is halogen, such as F, Cl, Br, I.

[0102] Specifically, the C ring is a benzene ring or a 5-6 membered nitrogen-containing aromatic heterocycle, for example

[0103] In some embodiments of the present invention, the C ring is

[0104] In some embodiments of the present invention, is Wherein, it has R 3 ' as defined above for R 3 .

[0105] Specifically, Y 1 is selected from: a single bond, -C 0 -C 6 alkylene-, -O-(C 0 -C 6 alkylene)-; in some embodiments of the present invention, Y 1 is -C 0 -C 3 alkylene-, such as

[0106] Specifically, Y 2 is selected from: a single bond, -O-, -S-, -O-(C 0 -C 6 alkylene)-; in some embodiments of the present invention, Y 2 is -O-.

[0107] Specifically, Y 3 is selected from: -N-(C 0 -C 6 alkyl), -C(O)-N(C 0 -C 6 alkyl)-, -C(S)-N(C 0 -C 6 alkyl)-; in some embodiments of the present invention, Y 3 is -N-(C 0 -C 3 alkyl), such as -NH-.

[0108] In some embodiments of the present invention, the moiety is

[0109]

[0110] In some embodiments of the present invention, the EGFR degrader has the following structure:

[0111]

[0112] In some embodiments of the present invention, the EGFR degrader has the following structure:

[0113]

[0114] Specifically, in Formulas II and III, GRO, L 1 and L 2 are as described above.

[0115] Specifically, in Formulas II and III, L 1 is attached to any base, sugar or phosphate backbone at the 3'-end, 5'-end or in the middle of GRO.

[0116] In some embodiments of the present invention, L 1 is a single bond or O, that is, GRO is connected to L through a phosphate ester bond (that is, ). 2 connected.

[0117] In some embodiments of the present invention, L 2 adopts the above-mentioned scheme (1) and / or (2), especially scheme (1), for example

[0118] More specifically, the EGFR degrader has the following structure:

[0119]

[0120] where h is an integer between 1 and 10, and i is an integer between 0 and 10.

[0121] In one embodiment of the present invention, the EGFR degrader has the following structure:

[0122]

[0123] In other embodiments of the present invention, the EGFR degrader has the following structure:

[0124]

[0125]

[0126]

[0127] In the second aspect of the present invention, there is provided a method for preparing the EGFR degrader described in the first aspect, which includes the step of coupling a small molecule ligand moiety with GROs through L.

[0128] In some embodiments of the present invention, the preparation method includes the following steps: first prepare connect it to a solid-phase carrier, and then synthesize oligonucleotides through solid-phase synthesis to obtain the target product;

[0129] Specifically, the solid-phase synthesis adopts the phosphoramidite method, which includes four steps: deprotection, coupling, capping, and oxidation, as described in Kosuri S, Church GM. Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 2014 May; 11(5):499-507. Figure 2 shown.

[0130] Specifically, in solid-phase synthesis, L' contains (such as ).

[0131] In some embodiments of the present invention, R F is -ODMTr.

[0132] In some embodiments of the present invention, the solid-phase carrier is controlled-pore glass beads (CPG), and the pore size of the CPG is determined according to the length of the synthesized oligonucleotide.

[0133] In some embodiments of the present invention, the preparation method includes the following steps: React and link with GROs; or,

[0134] First prepare GROL'R F , and then link with a small molecule ligand; or,

[0135] Separate prepare GRO-L”RF' and and then couple the two;

[0136] wherein, L', L”, L”' are optionally suitable linking groups, and R F , R F ', R F ” are optionally suitable reactive groups.

[0137] In the third aspect of the present invention, a pharmaceutical composition comprises the EGFR degrader described in the first aspect or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, and one or more pharmaceutically acceptable excipients.

[0138] Specifically, the pharmaceutically acceptable excipients can be selected from one or more of the following: fillers, binders, lubricants, disintegrants, antioxidants, buffers, bacteriostatic agents, suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc.

[0139] Specifically, the pharmaceutical composition can be administered by any suitable route, such as enteral administration (such as oral, sublingual, rectal administration) or parenteral administration (such as intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory administration, etc.) routes.

[0140] Specifically, the pharmaceutical composition can be prepared into pharmaceutical preparations in the following forms: injections, syrups, elixirs, suspensions, powders, granules, tablets, capsules, lozenges, creams, ointments, lotions, gels, emulsions, etc.

[0141] When preparing an injection, any commonly used carrier in the art can be used, such as: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyethoxylated isostearyl alcohol, and fatty acid esters of polyvinyl sorbitan, etc. In addition, commonly used solubilizers and buffers can also be added.

[0142] Specifically, the pharmaceutical composition is preferably in unit dosage form. In this form, the preparation is further divided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form can be a capsule, a tablet or any dosage form; in addition, the unit dosage form can also be a packaged preparation, such as tablets, capsules and powders packaged in vials or ampoules, etc.

[0143] Specifically, in the pharmaceutical composition, the EGFR degrader can be used alone or in combination with other types of active ingredients, such as other EGFR (or EGFR mutants) inhibitors, such as afatinib, osimertinib, lapatinib, erlotinib, dacomitinib, poziotinib, neratinib, alflutinib (AST 2818), almonertinib (HS10296), BBT-176, BI-4020, CH7233163, gilitertinib, JND-3229, lazertinib, EGF816, PCC-0208027, rezivertinib (BPI-7711), TQB3804, zorifertinib (AZ-3759) or DZD9008; EGFR antibodies, such as cetuximab, panitumumab, necitumumab, HLX07, JMT101; or bispecific EGFR and MET antibodies (e.g., amivantamab (JNJ-61186372, JNJ-372)).

[0144] For tumor treatment, the EGFR degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof according to the present invention, or the pharmaceutical composition disclosed herein, can be administered in combination with other anti-cancer agents that are not EGFR inhibitors. For example, it can be administered in combination with the following: MEK, including mutant MEK inhibitors (trametinib, cobimtetinib, binimetinib, selumetinib, refametinib); c-MET, including mutant c-Met inhibitors (savolitinib, cabozantinib, foretinib, glumetinib, tepotinib) and MET antibodies (emibetuzumab, telisotuzumab vedotin, ABBV 339); mitotic kinase inhibitors (CDK4 / 6 inhibitors, such as palbociclib, ribociclib, abemacicilb, GIT38); anti-angiogenic agents, for example, bevacizumab, nintedanib; apoptosis inducers, such as Bcl-2 inhibitors, for example, venetoclax, obatoclax, navitoclax, palcitoclax (APG-1252), and Mcl-1 inhibitors, for example, AZD-5991, AMG-176, S-64315; mTOR inhibitors, for example, rapamycin, temsirolimus, everolimus, ridoforolimus; RET inhibitors, such as pralsetinib and selpercatinib, and PI3K inhibitors dactolisib (BEZ235), pictilisib (GDC-0941), LY294002, idelalisib (CAL-101); JAK inhibitors (for example, AZD4205, itacitinib), Aurora A inhibitors (for example, alisertib); BCR / ABL and / or Src family tyrosine kinase inhibitors (for example, dasatinib); VEGF inhibitors (for example, MP0250;Ramucirumab; multi-kinase protein inhibitors (e.g., anlotinib, midostaurin); PARP inhibitors (e.g., niraparib); platinum therapies (e.g., cisplatin (CDDP), carboplatin (CBDCA) or nedaplatin (CDGP)); PD-L1 inhibitors (e.g., durvalumab (MEDI 4736)); HER2 / neu receptor inhibitors (e.g., trastuzumab); anti-HER2 or anti-HER3 antibody-drug conjugates (e.g., patritumab deruxtecan (U3-1402), trastuzumab emtansine); or immunogene therapy (e.g., oncoprex).;

[0145] Specifically, the amount of the active ingredient in the unit dose preparation can be changed or adjusted between 0.1 mg and 1000 mg (e.g., 0.1, 1, 5, 10, 20, 40, 50, 100, 200, 400, 500, 1000 mg), depending on the specific application and potency of the active ingredient. If necessary, the composition may further contain other suitable therapeutic agents.

[0146] In the fourth aspect of the present invention, a delivery system for an EGFR degrader is provided, which comprises the above-mentioned EGFR degrader and a carrier, and can deliver the EGFR degrader to target cells or target tissues without NCL expression on the cell surface, etc.

[0147] Specifically, the above-mentioned carrier can be any carrier suitable for delivering nucleic acid drugs into target tissues or target cells, etc., such as those disclosed in the prior art (see, for example, Wang Junfeng, Tan Manman, Wang Ying, etc. Research progress on modification and delivery of nucleic acid drugs [J]. Journal of Zhejiang University (Medical Sciences), 2023, 52(04): 417-428. which is incorporated herein by reference), for example, viral vectors (such as lentivirus, adenovirus, adeno-associated virus vectors), non-viral vectors (such as lipid nanoparticles (LNP), polymer nanocarriers, inorganic nanocarriers, protein carriers, exosomes, etc.).

[0148] In some embodiments of the present invention, the carrier is a lipid nanoparticle (LNP).

[0149] In the fifth aspect of the present invention, there is provided the use of the EGFR degrader described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, in the preparation of a drug for preventing and / or treating EGFR-related diseases.

[0150] Specifically, the diseases are those that can benefit from the prevention and / or treatment by degrading EGFR, such as, but not limited to, tumors, autoimmune diseases, inflammatory diseases, diseases related to pathogen infections, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, fibrotic diseases, etc.

[0151] Specifically, the tumors include but are not limited to: lung cancer, malignant melanoma, brain tumors, tumors of the digestive organs, uterine cancer, fallopian tube cancer, papillary thyroid cancer, testicular cancer, maxillary cancer, nasopharyngeal cancer, throat cancer, tongue cancer, oral cancer, adenoid cystic carcinoma, various sarcomas, retinoblastoma, ameloblastoma, osteosarcoma, hematological malignancies, nervous system tumors, glioma, glioblastoma multiforme, neuroglioma, skin cancer, skin appendage cancer and skin metastatic cancer, medulloblastoma, blastoma, liposarcoma, neuroendocrine tumor, synovial sarcoma, gastrinoma, carcinoid tumor, mesothelioma, islet cell carcinoma, schwannoma, meningioma, melanoma, acoustic neuroma, adenocarcinoma, lymphoid malignancies, epithelial squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, squamous cell carcinoma of the lung, hepatocellular carcinoma, gastric cancer, gastric adenocarcinoma, intestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, thyroid cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, cholangiocarcinoma, prostate cancer, salivary gland adenocarcinoma, kidney cancer, vulvar cancer, anal cancer, penile cancer, esophageal cancer, cardia cancer, biliary tract tumors and head and neck cancers. More specifically, the tumors are selected from: non-small cell lung cancer, breast cancer, cervical cancer, gastric cancer, bladder cancer, glioma.

[0152] Specifically, the hematological malignancies include: leukemia, lymphoma, multiple myeloma (MM).

[0153] Specifically, leukemia can be chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL), chronic myelogenous leukemia (CML) (e.g., B-cell CML, T-cell CML), acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), acute monocytic leukemia, etc.

[0154] Specifically, the lymphoma can be Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström macroglobulinemia)), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma, and T-cell NHL, such as precursor T lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sézary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma, NK / T-cell lymphoma, especially diffuse large B-cell lymphoma (DLBCL).

[0155] In some embodiments of the present invention, the tumor is glioma.

[0156] Specifically, the autoimmune diseases include but are not limited to: organ-specific autoimmune diseases, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune diseases, ulcerative colitis, etc.

[0157] Specifically, the inflammatory diseases include but are not limited to: osteoarthritis, acute gout, multiple sclerosis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), neuroinflammation, asthma, chronic obstructive airway disease, pneumonia, myositis, eczema, dermatitis, acne, cellulitis, occlusive diseases, thrombosis, alopecia, nephritis, vasculitis, retinitis, uveitis, scleritis, sclerosing cholangitis, hypophysitis, thyroiditis, septic shock, systemic inflammatory response syndrome (SIRS), toxic shock syndrome, acute lung injury, ARDS (adult respiratory distress syndrome), acute renal failure, burns, pancreatitis (e.g., acute pancreatitis), postoperative syndrome, sarcoidosis, Jarisch-Herxheimer reaction, encephalitis, myelitis, meningitis, and malaria, etc.

[0158] Specifically, the neurodegenerative diseases include, but are not limited to: Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.

[0159] Specifically, the pathogen can be a microorganism, a parasite (protozoa, worms, etc.) or other vectors.

[0160] Specifically, the microorganism can be selected from one or more of the following: virus, chlamydia, rickettsia, mycoplasma, bacterium, spirochete, fungus, etc.

[0161] In one embodiment of the present invention, the pathogen is a virus, such as, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Epstein - Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, vaccinia virus), Papovaviridae (such as papillomavirus (HPV)), Parvoviridae (such as B19 virus), Hepadnaviridae (such as hepatitis B virus), Polyomaviridae (such as polyomavirus), Reoviridae (such as reovirus, rotavirus), Picornaviridae (such as enterovirus, foot - and - mouth disease virus), Caliciviridae (such as Norwalk virus, hepatitis E virus), Togaviridae (such as rubella virus), Arenaviridae (such as lymphocytic choriomeningitis virus), Retroviridae (HIV - 1, HIV - 2, HTLV - 1), Flaviviridae (such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus type 1 (HPIV), HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV - 229E, HCoV - OC43, HCoV - NL63, HCoV - HKU1, SARS - CoV, MERS - CoV, SARS - CoV - 2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus).

[0162] Specifically, the diseases related to pathogen infection include, but are not limited to: influenza, SARS, COVID - 19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease, etc.

[0163] Specifically, the cardiovascular diseases include, but are not limited to: coronary heart disease, peripheral artery disease, atherosclerosis, ischemic heart disease, ischemic cardiomyopathy, myocardial infarction, heart failure, angina pectoris, myocarditis, hypercholesterolemia, hypertension, ischemia-reperfusion injury, cerebral ischemia (stroke), embolism (such as pulmonary embolism, renal embolism, hepatic embolism, gastrointestinal embolism or peripheral limb embolism), or myocardial ischemia, etc.

[0164] Specifically, the metabolic diseases include, but are not limited to: diabetes (such as type I diabetes, type II diabetes or gestational diabetes), obesity, fatty liver (NASH or others), cachexia, hypercholesterolemia, gout, etc.

[0165] Specifically, the fibrotic diseases include, but are not limited to: myocardial fibrosis, pulmonary fibrosis, renal fibrosis, postoperative stenosis, keloid formation, liver cirrhosis, biliary cirrhosis, scleroderma, etc.

[0166] In particular, the diseases are selected from: head and neck cancer (such as head and neck squamous cell carcinoma), colon cancer, rectal cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung cancer (such as non-small cell lung cancer), lung adenocarcinoma (such as lung invasive adenocarcinoma), esophageal cancer (such as esophageal squamous cell carcinoma), gastric cardia cancer, gastric cancer, gastric adenocarcinoma, intestinal cancer (such as colorectal cancer), breast cancer (such as triple-negative breast cancer, extramammary Paget's disease), cervical cancer (such as cervical squamous cell carcinoma), ovarian cancer (such as epithelial ovarian cancer, ovarian serous carcinoma), uterine cancer (such as endometrial cancer), fallopian tube cancer, prostate cancer, bladder cancer, kidney cancer, thyroid cancer (such as papillary thyroid carcinoma), glioma (such as ependymoma, astrocytoma), medulloblastoma, glioblastoma multiforme, osteosarcoma, nasopharyngeal cancer, pharyngeal and laryngeal cancer (such as laryngeal cancer, hypopharyngeal cancer), retinoblastoma, tongue cancer (such as tongue squamous cell carcinoma), oral cancer (such as oral squamous cell carcinoma, oropharyngeal squamous cell carcinoma, oral epidermoid carcinoma, oral leukoplakia, oral floor cancer, buccal mucosa cancer), salivary gland cancer (such as mucoepidermoid carcinoma), adenoid cystic carcinoma, ameloblastoma, skin cancer (cutaneous squamous cell carcinoma, malignant melanoma), pulmonary fibrosis (such as idiopathic pulmonary interstitial fibrosis), papillomavirus, seborrheic keratosis, psoriasis.

[0167] In the sixth aspect of the present invention, there is provided a method for treating EGFR-related diseases, which comprises the step of administering to a subject in need thereof the EGFR degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof described in the first aspect of the present invention, or the pharmaceutical composition described in the third aspect of the present invention, or the delivery system described in the fourth aspect of the present invention.

[0168] Specifically, the diseases are as described in the fifth aspect of the present invention.

[0169] Specifically, the subject is a mammal, particularly a human.

[0170] Specifically, the administration can be carried out by any suitable route of administration, such as enteral administration (e.g., oral, sublingual, rectal administration) or parenteral administration (e.g., intravenous, intramuscular, intranasal, intraocular, intracerebral, intravaginal, intraperitoneal, transdermal, subcutaneous, intradermal, respiratory administration, etc.).

[0171] In the seventh aspect of the present invention, there is provided the use of GROs in the preparation of (in vivo) imaging, diagnostic, and detection reagents for brain tumors.

[0172] Specifically, the GROs are as described in the first aspect of the present invention, particularly AS1411.

[0173] Specifically, the reagent further comprises a label, such as a fluorophore, a colorimetric label, gold nanoparticles, quantum dots, biotin, and other tag molecules that can be used for detection (such as alkynyl groups for Raman diffraction imaging, cycloolefins for click reactions, initiating groups for polymer labeling), and can also be selected from polypeptide / protein molecules, LNA / PNA, unnatural amino acids and their analogs (such as peptidomimetics), unnatural nucleic acids and their analogs (nucleomimetics), and nanostructures (including inorganic nanoparticles, NV-center, aggregation / assembly-induced emission molecules, rare earth ion ligand molecules, polyoxometalates, etc.). In some embodiments of the present invention, the label is a fluorophore, such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, FAM, TexasRed, ROX, TET, VIC, JOE, HEX, LC RED640, LC RED705, etc. In some embodiments of the present invention, the reagent is a GROs labeled with a fluorophore, such as AS1411 labeled with Cy5.

[0174] Specifically, the brain tumor can be a primary brain tumor or a brain metastasis.

[0175] In the eighth aspect of the present invention, there is provided a brain tumor imaging agent, which can be used for in vivo imaging and targeting of brain tumors, and for auxiliary diagnosis, treatment, etc., and which comprises GROs labeled with a label for imaging.

[0176] Specifically, the GROs are as described in the first aspect of the present invention, particularly AS1411.

[0177] Specifically, the label is as described in the seventh aspect of the present invention.

[0178] In the ninth aspect of the present invention, there is provided a method for (in vivo) imaging, diagnosing, and detecting brain tumors, which comprises the step of using GROs.

[0179] More specifically, the method includes the step of using GROs labeled with markers for imaging (as described in the eighth aspect of the present invention).

[0180] The present invention provides a PROTAC for degrading EGFR prepared with GROs as a novel recruitment element of MDM2, and its preparation method and application. The inventors found through research a new function of GROs in recruiting MDM2, and prepared a PROTAC targeting EGFR using the same, which can effectively degrade EGFR and produce corresponding therapeutic effects (such as inhibiting the proliferation of tumor cells and promoting apoptosis of tumor cells), and has very good application prospects and research value. In addition, it is generally considered that macromolecules are difficult to be used for in vivo imaging, targeting and other treatments of brain tumors, while the inventors found that AS1411 can specifically target and penetrate brain tumors in vivo and can be used for in vivo imaging, diagnosis and detection of brain tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] Figure 1 The figure shows the result of NCL binding to MDM2.

[0182] Figure 2 The figure shows the result of AS1411 being a GRO with stable G4 structural characteristics.

[0183] Figure 3 The figure shows the result of the weak G4 structural characteristics of iSN04.

[0184] Figure 4 A shows the result of CRO being unable to capture NCL and MDM2; Figure 4 B shows the result of AS1411 concentration-dependent capture of NCL and MDM2; Figure 4 C shows the result of high-concentration iSN04 being able to capture NCL, but iSN04 being unable to capture MDM2.

[0185] Figure 5 A shows the result of CRO being unable to bind to NCL and recruit MDM2; Figure 5 B shows the result of AS1411 recruiting a large amount of MDM2 depending on its interaction with NCL; Figure 5 C shows that iSN04 can only bind to NCL, but cannot recruit MDM2 depending on its interaction with NCL.

[0186] Figure 6 A shows the result of AS1411 not affecting the interaction between NCL and MDM2; Figure 6 B shows the result of iSN04 blocking the binding between NCL and MDM2.

[0187] Figure 7The figure shows the results that AS1411 can specifically recognize glioma cells in vitro.

[0188] Figure 8 The figure shows the results that AS1411 can enter glioma cells in vitro.

[0189] Figure 9 The figure shows the results that AS1411 can penetrate and enter the interior of glioma cell spheres.

[0190] Figure 10 The figure shows the results that AS1411 can specifically target glioma in vivo without entering normal brain tissue.

[0191] Figure 11 The figure shows the results that AS1411 can specifically accumulate in glioma in vivo.

[0192] Figure 12 The figure shows the results that there is a large amount of angiogenesis in glioma and NCL is highly expressed in glioma and vascular endothelial cells.

[0193] Figure 13 The figure shows the results that AS1411 can penetrate the blood-tumor barrier.

[0194] Figure 14 The figure shows the results that AS1411 recruits MDM2 and silences NCL to block the recruitment of MDM2 by AS1411.

[0195] Figure 15 A shows a schematic diagram of GROs (such as AS1411) recruiting MDM2 depending on the interaction with NCL; Figure 15 B shows a schematic diagram of the possible mode of action of PROTAC targeting EGFR formed by GROs (such as AS1411) as MDM2 recruitment elements.

[0196] Figure 16 The figure shows the results that AS1411-Gef degrades EGFR.

[0197] Figure 17 The figure shows the results that AS1411-Gef promotes the ubiquitination of EGFR.

[0198] Figure 18 The figure shows the results that AS1411-Gef mediates the degradation of EGFR through the ubiquitin-proteasome pathway.

[0199] Figure 19 The figure shows the results that AS1411-Gef mediates the degradation of EGFR depending on MDM2 and NCL.

[0200] Figure 20The figure shows the results that AS1411-Gef can inhibit the proliferation of U87MG cells and promote the apoptosis of U87MG cells.

[0201] Figure 21 The figure shows the results that AS1411-Gef can inhibit the proliferation of U251 cells and promote the apoptosis of U251 cells.

[0202] Figure 22 The figure shows the results that AS1411-Gef can inhibit the growth of glioma in vivo and improve the survival rate of experimental animals. Detailed implementation manners

[0203] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the technical field to which the present invention pertains.

[0204] The term "alkyl" refers to a straight-chain or branched-chain hydrocarbon radical without unsaturated bonds, and the hydrocarbon radical is connected to other parts of the molecule by a single bond. Typical alkyl groups contain 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. In the present invention, C 0 alkyl refers to H, that is, C 0-10 alkyl (or C 0 -C 10 alkyl) includes H and C 1-10 alkyl (or C 1 -C 10 alkyl).

[0205] The term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by removing two hydrogen atoms from an alkane molecule, which can be straight-chain or branched-chain and is connected to other parts of the molecule by a single bond. In this article, typical alkylene has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methylene (-CH 2 -), ethylene, propylene, butylene, etc. In the present invention, C 0 alkylene refers to a single bond, that is, C 0-10 alkylene (or C 0 -C 10 alkylene) includes a single bond and C 1-10 alkylene (or C 1 -C 10 alkylene).

[0206] The term "cycloalkyl" refers to alicyclic hydrocarbons, such as those containing 1 to 4 monocyclic and / or fused rings, having 3 - 18 carbon atoms, preferably 3 - 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or adamantyl, etc.

[0207] The term "alkoxy" refers to a substituent formed by replacing the hydrogen in a hydroxyl group with an alkyl group, such as an alkoxy group containing 1 - 10 carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, etc.

[0208] The term "alkylamino" refers to a substituent formed by replacing one or two hydrogens in an amino group (-NH 2 ) with an alkyl group, such as an alkylamino group containing 1 - 10 carbon atoms, for example

[0209] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0210] The term "haloalkyl" refers to a group formed by replacing one or more hydrogens in an alkyl group with a halogen atom (e.g., fluorine, chlorine, bromine, or iodine), for example -CHF 2 、-CH 2 F、-CF 3 、-CH 2 -CF 3 、-CH 2 CH 2 -CF 3 、-CH 2 CH 2 CH 2 -CF 3 。

[0211] The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing monocyclic aryl groups and / or fused aryl groups, such as those containing 1 - 3 monocyclic or fused rings and 6 - 18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl groups described as C 6 -C 12 in the present invention refer to aryl groups containing 6 - 12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, etc.

[0212] The term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group containing 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused, spiro, or bridged ring systems. The heterocyclic group can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl). Suitable heteroaryls in the compounds of the present invention contain 1, 2, or 3 heteroatoms selected from N, O, S, and P atoms, and the heteroaryls include, for example, coumarin, including 8-coumarin, quinolinyl, including 8-quinolinyl, isoquinolinyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indazinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furanopyridyl. Suitable heterocycloalkyls in the compounds of the present invention contain 1, 2, or 3 heteroatoms selected from N, O, or S atoms, and the heterocycloalkyls include, for example, pyrrolidinyl, tetrahydrofuryl, dihydrofuran, tetrahydrothienyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathianyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxiranyl, thiiranyl, azepinyl, oxazepinyl, diazepinyl, triazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinuclidinyl. In the present invention, for an optionally substituted heterocyclic group, the position of substitution can be any suitable carbon atom or heteroatom. For example, for where the substitution position of R can be any suitable carbon atom or nitrogen atom, and it can be, for example

[0213] In the present invention, "D" refers to deuterium; "substituted with deuterium" means replacing one or more hydrogen atoms with the corresponding number of deuterium atoms.

[0214] It should be recognized that depending on the source of the chemical materials used in the synthesis, there are some variations in the natural isotope abundances in the synthesized compounds. Accordingly, the compounds of the present invention will inherently contain small amounts of deuterated isotopologues. Despite such variations, the concentrations of the stable hydrogen and carbon isotopes at natural abundance are low and insignificant compared to the degree of stable isotope substitution in the compounds of the present invention. See, e.g., Wada, E et al., Seikagaku, 1994, 66:15; Gannes, LZ et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.

[0215] In the compounds of the present invention, any atom not specified as deuterium is present at its natural isotope abundance. Unless otherwise indicated, when a position is specifically designated as “H” or “hydrogen”, that position is understood to have hydrogen with its isotopic composition at natural abundance. Similarly, unless otherwise indicated, when a position is specifically designated as “D” or “deuterium”, that position is understood to have deuterium with an abundance greater than the natural abundance of deuterium (which is 0.015%) by at least 3000-fold (i.e., at least 45% deuterium incorporation).

[0216] The term “isotope enrichment factor” as used herein refers to the ratio of the isotopic abundance of a particular isotope to its natural abundance.

[0217] In other embodiments, the compounds of the present invention have an isotope enrichment factor of at least 3500 (52.5% deuterium incorporation at each specified deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation) or at least 6633.3 (99.5% deuterium incorporation) for each specified deuterium atom.

[0218] The term “isotopologue” refers to a substance in which the chemical structure is identical to a particular compound of the present invention except for its isotopic composition.

[0219] The term “pharmaceutically acceptable salt” includes acid addition salts and base addition salts.

[0220] The term "acid addition salt" includes, but is not limited to, salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, and salts derived from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Accordingly, such salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodide, acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, and mesylate, and also salts of amino acids such as arginine salt, gluconate, galacturonate, etc. Acid addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid in a conventional manner to form the salt. The free base form can be regenerated by contacting the salt form with a base and the free base can be isolated in a conventional manner.

[0221] The term "base addition salt" refers to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid form can be regenerated by contacting the salt form with an acid and the free acid can be isolated in a conventional manner.

[0222] The term "stereoisomers" includes enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cycloalkyl groups which may be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention.

[0223] The term "solvate" refers to the physical association of a compound of the present invention with one or more solvent molecules. The physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be isolated, for example when one or more solvent molecules are incorporated into the lattice of a crystalline solid. Solvates include solution-phase and isolable solvates. Representative solvates include ethanolates, methanolates, etc.

[0224] The term "prodrug" refers to a form of a compound of formula I that is suitable for administration to a patient, has no excessive toxicity, irritation, allergic reaction, etc., and is effective for its intended purpose, including acetal, ester, and zwitterionic forms. The prodrug is converted in vivo, such as by hydrolysis in the blood, to give the parent compound.

[0225] In the present invention, the term "oligonucleotide" consists of no more than 30 (e.g., 5, 10, 15, 20, 25, 30) nucleotides (deoxyribonucleotides and / or ribonucleotides), which is single-stranded or double-stranded, especially single-stranded. The oligonucleotide may include chemical modifications, nucleic acid unit substitutions, or attachment of functional groups; wherein, the chemical modification is that at least one base is modified, and the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, isotopic labeling, phosphorothioate backbone modification, methoxy modification, and fluoro modification; the nucleic acid unit substitution is that at least one nucleic acid unit is replaced with LNA, UNA, or GNA; the functional groups include at least one of a fluorescent group, a radioactive group, biotin, digoxin, a nanoluminescent material, a nucleic acid substance, or an enzyme label.

[0226] In the present invention, for deoxyribonucleotides, A represents deoxyadenosine monophosphate, T represents deoxythymidine, C represents deoxycytidine, and G represents deoxyguanosine; for ribonucleotides, A represents adenosine, G represents guanosine, C represents cytidine, and U represents uridine.

[0227] In the present invention, the term "oligopeptide" refers to a peptide composed of 2 - 10 (e.g., 2, 3, 4, 5, 6, 8, 10) amino acids, "polypeptide" contains 11 - 50 (e.g., 15, 20, 30, 40, 50) amino acids, and "protein" contains more than 50 amino acids.

[0228] The terms "patient" or "subject", etc. are used interchangeably herein and refer to any animal or its cells treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, such as rats, mice, guinea pigs, rabbits, dogs, monkeys, or humans, especially humans.

[0229] The term "treatment" refers to preventing, curing, reversing, weakening, alleviating, minimizing, inhibiting, arresting, and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0230] The term "prevention" refers to, before the onset of a disease, avoiding, minimizing, or making it difficult for the disease to occur or develop through treatment.

[0231] The term "tumor" refers to an abnormal mass of tissue, in which the growth of the mass exceeds and is uncoordinated with that of normal tissue. Tumors can be "benign" or "malignant", depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion, and metastasis. "Benign tumors" are usually well-differentiated, characterized by slower growth than malignant tumors and remaining confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors may later give rise to malignant tumors, which may be due to additional genetic alterations in subsets of the neoplastic cells of the tumor, and these tumors are called "precancerous tumors". "Malignant tumors" are usually poorly differentiated (anaplastic) and are characterized by rapid growth, accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. In addition, malignant tumors usually have the ability to metastasize to distant sites.

[0232] The term "cancer" refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyle d.; Williams & Wilkins: Philadelphia, 1990).

[0233] The term "autoimmune disease" refers to a disease caused by the body's immune response to its own antigens, resulting in damage to its own tissues.

[0234] The term "inflammation" is the body's defensive response to a stimulus, manifested as redness, swelling, heat, pain, and dysfunction, etc.; it can be infectious inflammation caused by infection or non-infectious inflammation not caused by infection, such as inflammation caused by immune responses (such as various types of hypersensitivity reactions, inflammation caused by some autoimmune diseases). The term "inflammatory disease" refers to a disease with inflammation.

[0235] The term "diseases related to pathogen infection" mainly refers to diseases caused by pathogen infection, including symptoms of body damage and infection response manifestations caused by pathogen invasion. Pathogens can be microorganisms (such as viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi, etc.), parasites (protozoa, worms, etc.) or other vectors. In particular, the pathogen is a virus, for example, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Epstein - Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, cowpox virus), Papovaviridae (such as papillomavirus (HPV)), Parvoviridae (such as B19 virus), Hepadnaviridae (such as hepatitis B virus), Polyomaviridae (such as polyomavirus), Reoviridae (such as reovirus, rotavirus), Picornaviridae (such as enterovirus, foot - and - mouth disease virus), Caliciviridae (such as Norwalk virus, hepatitis E virus), Togaviridae (such as rubella virus), Arenaviridae (such as lymphocytic choriomeningitis virus), Retroviridae (HIV - 1, HIV - 2, HTLV - 1), Flaviviridae (such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus type 1 (HPIV), HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV - 229E, HCoV - OC43, HCoV - NL63, HCoV - HKU1, SARS - CoV, MERS - CoV, SARS - CoV - 2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus).

[0236] The term "cardiovascular disease" refers to a class of diseases involving the heart or blood vessels.

[0237] The term "metabolic disease" refers to a disease caused by the accumulation or deficiency of certain metabolites such as sugar, fat, protein, purine, calcium, copper, etc. when the biochemical process in the body is disordered.

[0238] The term "fibrosis" refers to a pathological process in which parenchymal cells of an organ undergo necrosis due to inflammation, and the extracellular matrix in the tissue increases abnormally and deposits excessively. In severe cases, it causes the destruction of tissue structure and organ sclerosis.

[0239] All publications, patents, and published patent specifications cited herein are incorporated herein by reference in their entirety.

[0240] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0241] The sequences involved in the embodiments are shown in the following table:

[0242] Table 1 Sequence names and numbers

[0243] Name Sequence Number AS1411 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′ SEQ ID NO:1 iSN04 5’-AGATTAGGGTGAGGGTGA-3’ SEQ ID NO:2 CRO 5′-CCTCCTCCTCCTTCTCCTCCTCCTCC-3′ SEQ ID NO:3 NC 5’-GGAATTCCCGGTGCGCCGATCGCCGGATATAACTT-3’ SEQ ID NO:4 siNC 5’-UUCUCCGAACGUGUCACGUTT-3’ SEQ ID NO:5 siNCL 5’-GGAUGACGACGACGACGAAGATT-3’ SEQ ID NO:6 siMDM2 5’-GCUUGGCCUACAGUCAUCUTT-3’ SEQ ID NO:7

[0244] Example 1: NCL can bind to MDM2

[0245] (1) Co-Immunoprecipitation (Co-IP) is a classic method for studying protein-protein interactions based on the specific interaction between an antibody and an antigen. After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), incubate with an antibody against NCL overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. Wash the NCL and its interacting protein complex bound to the magnetic beads with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and continue for 10 minutes. Adsorb the magnetic beads with a magnetic stand, take the supernatant to a new tube. Then perform a western blot experiment to detect whether NCL binds to MDM2. The steps of the western blot experiment are as follows: Separate the protein samples by SDS-PAGE electrophoresis, transfer the separated proteins to a PVDF membrane, block with TBST buffer containing 5% skim milk at room temperature for 1 hour, then incubate overnight at 4°C with the primary antibody against MDM2 (purchased from proteintech, catalog number 27883-1-AP) or the primary antibody against NCL (purchased from Cell Signaling Technology, catalog number 14574S). After washing with TBST, incubate the membrane with an HRP-labeled secondary antibody (purchased from Abbkine, catalog number AS014) at room temperature for 1 hour, and visualize the protein band blot using an enhanced chemiluminescence detection kit (purchased from Abbkine, catalog number RM00021P). Figure 1The results of A indicate that NCL can bind to MDM2.

[0246] (2) Mix 2 μg / mL recombinant human NCL (rhNCL, purchased from ACROBiosystems, NUL-H5253) and 2 μg / mL recombinant human MDM2 (rhMDM2, purchased from R&D Systems, E3-202-050), incubate at 4°C for 7 hours, then add the antibody against NCL and continue to incubate overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802), rotate and incubate at room temperature for 1.5 hours. Wash the magnetic beads 3 times with TBST buffer, add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, take the supernatant and transfer it to a new tube. Then perform an immunoblotting experiment to detect whether NCL binds to MDM2. Figure 1 The results of B indicate that NCL can bind to MDM2.

[0247] Example 2: AS1411 is a GRO with stable G4 structural characteristics

[0248] GROs contain one or more GGT motifs and have stable G4 structural characteristics. GROs are guanine-rich oligonucleotides that can specifically bind to NCL. The GROs described in the present invention can be one of DNA aptamer AS1411, AS1411 derivatives / analogues, all of which have the above-mentioned stable G4 structural characteristics and the property of specifically binding to NCL. NCL is a multifunctional shuttle protein that shuttles between the cell nucleus and cytoplasm. NCL is highly expressed in a variety of diseased cells (such as tumors, etc.) and specifically translocates to the cell surface, affecting multiple processes such as cell proliferation, apoptosis, and metastasis. However, NCL is not expressed on the surface of normal cells, making NCL a surface molecular marker and potential therapeutic target for diseased cells (such as tumors, etc.). NCL on the membrane surface of diseased cells (such as tumors, etc.) can also act as a receptor for a variety of ligand molecules, mediating the entry of ligand molecules into diseased cells (such as tumors, etc.).

[0249] (1) Both AS1411 (SEQ ID NO:1) and iSN04 (SEQ ID NO:2) are oligonucleotides that specifically bind to NCL. We detected whether AS1411 and iSN04 have stable G4 structural characteristics. The experimental procedure is as follows: Add 5 μM NC (negative control, SEQ ID NO:4), AS1411 or iSN04 to water with a total volume of 100 μL, heat at 95 °C for 7 minutes, immediately incubate on ice for 3 minutes, and then add 2 μL of N-methyl mesoporphyrin IX (NMM) (purchased from MCE, catalog number HY-133821) to make its final concentration 1 μM, and incubate in the dark at room temperature for 1 hour. Detect the fluorescence intensity with a microplate reader, emission wavelength 550 - 700 nm, excitation wavelength 399. The results are as Figure 2 shown. No G4 structural characteristics were detected for iSN04, but AS1411 has stable G4 structural characteristics (fluorescence signal intensity exceeds 4000).

[0250] (2) Guanine-rich DNA sequences can form more obvious G4 structural characteristics in the presence of metal ions (usually sodium and potassium ions). Add 5 μM NC, AS1411 or iSN04 to 100 mM K + solution with a total volume of 100 μL, mix well, heat at 95 °C for 7 minutes, immediately incubate on ice for 3 minutes, and then add 2 μL of N-methylmesoporphyrin IX (NMM) (purchased from MCE, catalog number HY-133821) to make its final concentration 1 μM, and incubate in the dark at room temperature for 1 hour. Detect the fluorescence intensity with a microplate reader, emission wavelength 550 - 700 nm, excitation wavelength 399. The results are as Figure 3 shown. iSN04 has weak G4 structural characteristics (fluorescence signal intensity 1000 - 2000), but the G4 structural characteristics of AS1411 are more stable (fluorescence signal intensity exceeds 10000).

[0251] The above results indicate that iSN04 can form weak G4 structures only in the presence of K + , and cannot form G4 structural characteristics without K + , but AS1411 can form stable G4 structural characteristics with or without the presence of K + .

[0252] Example 3: AS1411 recruits MDM2 depending on its interaction with NCL

[0253] (1) The procedure of the pull-down experiment is to immobilize a substance with known identity (bait) on a carrier and use it to capture binding proteins (prey) and proteins that interact with the binding proteins from a complex mixture. This experiment is used to demonstrate the possible interaction between the bait and a certain protein or protein complex. After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), incubate them with 5'-biotinylated CRO (cytosine-rich oligonucleotides, SEQ ID NO:3, negative control), AS1411 or iSN04 at different concentrations (0 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM) at 4 °C for 6 hours, and then add streptavidin agarose gel beads (purchased from cytiva, catalog number 17511301) and continue to incubate at 4 °C overnight. After washing the biotinylated CRO, AS1411 or iSN04 bound to the gel beads and the proteins they captured multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100 °C and maintain for 10 minutes. After centrifugation, take the supernatant, which is the pull-down product. Immunoblotting experiments were used to detect the pull-down product. Figure 4 The results of A show that CRO can neither capture NCL nor MDM2; Figure 4 The results of B show that AS1411 can capture NCL and MDM2 in a concentration-dependent manner; Figure 4 The results of C show that high-concentration iSN04 can capture NCL, but iSN04 cannot capture MDM2 within the concentration range used.

[0254] (2) Mix 6 μg / mL recombinant human NCL and 6 μg / mL recombinant human MDM2 and incubate at 4 °C for 7 hours, then add 400 nM of 5'-biotinylated CRO, AS1411 or iSN04, incubate at 4 °C for 6 hours, add streptavidin agarose gel beads (purchased from cytiva, catalog number 17511301) and continue to incubate at 4 °C overnight. Wash the gel beads 4 times with TBST buffer, add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100 °C and maintain for 10 minutes. After centrifugation, take the supernatant. Then immunoblotting experiments were used to detect the pull-down product. Figure 5 The results of A show that CRO can neither bind to NCL nor recruit MDM2. Figure 5 The results of B show that AS1411 recruits a large amount of MDM2 depending on its interaction with NCL. Figure 5The results of C indicated that iSN04 could only bind to NCL, but could not recruit MDM2 depending on its interaction with NCL.

[0255] (3) After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), they were incubated with different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) of AS1411 or iSN04 at 4 °C for 6 hours, and then 5 μL of NCL antibody was added and incubated overnight at 4 °C. Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) were added and incubated with rotation at room temperature for 1.5 hours. The NCL bound to the magnetic beads and the proteins captured by it were washed with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015) was added, and heated to 100 °C for 10 minutes. The magnetic beads were adsorbed with a magnetic stand, and the supernatant was taken into a new tube. Then, immunoblotting (western blot) was performed to detect whether NCL bound to MDM2 and whether this binding was affected by AS1411 or iSN04. Figure 6 The results of A indicated that AS1411 did not affect the interaction between NCL and MDM2. The same experiment was carried out using derivatives / analogues of AS1411, and the experimental results were similar to those of AS1411. However, Figure 6 The results of B indicated that iSN04 blocked the binding between NCL and MDM2.

[0256] Example 4: AS1411 can target glioma cells

[0257] (1) NCL is highly expressed in a variety of tumor cells (including glioma cells, etc.) and specifically translocates to the tumor cell membrane surface, and NCL is not expressed on the surface of normal cells. Therefore, we examined the binding ability of AS1411 to glioma cells U87MG and U251 and human normal mammary epithelial cells MCF 10A. The three types of cells were respectively seeded in 6-well plates (purchased from Corning, catalog number 3335) and cultured overnight. 200 nM Cy5-labeled NC or AS1411 was added respectively and incubated for 6 h. After washing with PBS, flow cytometry (BD, model BD FACSCanto SORP) was used for analysis to observe the binding of NC or AS1411 to the three types of cells. The results were as Figure 7 shown in A-7C. Compared with NC, AS1411 could significantly bind to glioma cells U87MG and U251 cells, but did not bind to human normal mammary epithelial cells MCF 10A. It indicated that AS1411 could specifically recognize glioma cells in vitro.

[0258] (2) Glioma cells U87MG, U251 and human normal breast epithelial cells MCF 10A were respectively inoculated on cell culture slides (purchased from BIOLOGIX, product number: 07 - 2101) and cultured overnight. 200 nM Cy5-labeled NC or AS1411 was added respectively and incubated for 6 h. After washing with PBS, it was sealed with an anti-fluorescence quenching mounting medium (purchased from Beyotime, product number P0126), and observed under a confocal fluorescence microscope (ZEISS, model 980) for the entry of NC or AS1411 into cells. The results were as Figure 8 shown in Figure A - 8C. NC could neither enter U87MG and U251 cells nor enter MCF 10A cells; AS1411 was significantly enriched in U87MG and U251 cells, but AS1411 could not enter MCF 10A cells. It was shown that AS1411 could specifically enter glioma cells.

[0259] (3) Glioma U87MG or U251 cells were inoculated in a 96-well spherical microporous plate (purchased from Corning, product number 4520) and cultured for 3 days. 200 nM Cy5-labeled NC or AS1411 was added respectively and incubated for 6 h. After washing with PBS, it was placed under a confocal fluorescence microscope (ZEISS, model 980) to observe the distribution of NC or AS1411. The results were as Figure 9 shown in Figures A and 9B. NC could not penetrate the 3D cell spheres formed by U87MG and U251, but AS1411 had a relatively strong penetration ability for the 3D cell spheres formed by U87MG and U251.

[0260] (4) An in-situ glioma mouse model was constructed using U87MG-LUC-GFP cells, and the process was as follows: The tumor injection area in the brain of nude mice was located by a brain stereotaxic apparatus (RWD, model 68803). 1 mm forward from the midpoint of the bregma, then 1.8 mm to the right, and the injection depth was 3 mm. 3×10 7 U87MG-LUC-GFP cells were injected. In the control group, nude mice were not injected with U87MG-LUC-GFP cells. 7 days after modeling, 0.2 μmol / kg Cy5-labeled AS1411 was injected via the tail vein, and in vivo imaging of mice (PerkinElmer, model IVIS Spectrum) was performed at 5 min, 15 min, 30 min, 60 min, and 90 min after injection. The results were as Figure 10 shown. AS1411 could specifically target glioma in vivo, but could not enter the normal brain tissue of control nude mice, indicating that AS1411 could be used for in vivo imaging, diagnosis and detection of glioma.

[0261] (5) An orthotopic glioma mouse model was constructed using U87MG-LUC-GFP cells as follows: The tumor injection area in the brain of nude mice was located by a stereotaxic apparatus (RWD, model 68803). 1 mm forward from the midpoint of the bregma and then 1.8 mm to the right, with an injection depth of 3 mm. 3×10 7 U87MG-LUC-GFP cells were injected. Seven days after modeling, 0.2 μmol / kg of Cy5-labeled AS1411 or NC sequence was injected via the tail vein, and in vivo imaging of the mice (PerkinElmer, model IVIS Spectrum) was performed at 1, 2, 4, and 8 h after injection respectively to observe the in vivo distribution of AS1411 and the NC sequence in the orthotopic glioma mouse model. The results are as Figure 11 shown in Figure A. AS1411 was significantly enriched in gliomas, while the NC sequence could not be enriched in gliomas. This indicates that AS1411 can be used for in vivo imaging, diagnosis, and detection of gliomas.

[0262] (6) Cy5-labeled AS1411 or NC sequence was injected into nude mice with orthotopic gliomas constructed using U87MG-LUC-GFP cells. After 1 h, the brain tissues were taken, fixed with formalin, dehydrated with a sucrose gradient, and then embedded with OCT embedding medium (purchased from SUKURA, catalog number 4583). Sections were made using a cryostat (Leica, model CM1950). The sections were sealed with an anti-fluorescence quenching mounting medium (purchased from Beyotime, catalog number P0126) and observed under a confocal fluorescence microscope (ZEISS, model 980) to observe the cell distribution of NC or AS1411. The results are as Figure 11 shown in Figure B. NC was not significantly distributed in either glioma cells or non-tumor cell regions in the brain tissue; AS1411 was significantly enriched in glioma cells and not distributed in non-tumor cell regions in the brain tissue, indicating that AS1411 can be used for in vivo imaging, diagnosis, and detection of gliomas.

[0263] (7) Seven days after establishing the orthotopic glioma nude mouse model using U87MG-LUC-GFP cells, the brain tissues were collected. After fixation with formalin and dehydration in a sucrose gradient, they were embedded with OCT embedding medium (purchased from SUKURA, product number 4583). Sections were made using a cryostat (Leica, model CM1950). They were permeated with a permeation solution (purchased from Beyotime, product number P0095) for 15 min, blocked with a blocking solution (purchased from Beyotime, product number P0252) for 1 h, and incubated overnight at 4 °C with the primary antibody against NCL (purchased from Cell Signaling Technology, product number 14574S) and the primary antibody against the vascular endothelial cell marker CD31 (purchased from Abcam, product number ab28364). After washing with PBS, they were incubated with a fluorescently labeled secondary antibody (purchased from Abcam, product number ab150077 or ab150080) for 1 h at room temperature, sealed with an anti-fluorescence quenching mounting medium (purchased from Beyotime, product number P0126), and observed under a confocal fluorescence microscope (Zeiss, model LSM980) for the expression of GFP, CD31, and NCL and the co-localization of GFP or CD31 with NCL. The results were as Figure 12 shown. There was a large amount of expression of CD31 and NCL in glioma tissues. NCL co-localized with CD31 and GFP respectively. The expression of CD31 and NCL was less in non-tumor sites, indicating that there was a large amount of angiogenesis in glioma tissues, and both glioma cells and vascular endothelial cells highly expressed NCL.

[0264] Example 5: AS1411 can penetrate the blood-tumor barrier

[0265] The blood-brain barrier (BBB) is composed of tightly connected endothelial cells that line the inner layer of brain capillaries and play an important role in maintaining the homeostasis of the central nervous system. The BBB functions by facilitating the transport of essential nutrients such as amino acids, monocarboxylic acids, nucleosides, and vitamins, while preventing the entry of harmful substances into the brain. However, this selective permeability also poses significant challenges and limits the ability of therapeutic drugs to enter brain tumors. Studies have widely demonstrated that brain tumors can alter the BBB to form a highly heterogeneous vascular structure, namely the blood-tumor barrier (BTB). In the BTB, brain tumor-associated endothelial cells exhibit different phenotypic and functional characteristics from normal endothelial cells in the healthy BBB. These brain tumor-associated endothelial cells are less prone to apoptosis, are resistant to cytotoxic therapies, and actively participate in the abnormal reorganization of the BTB.

[0266] (1) We established in vitro blood-tumor barrier and blood-brain barrier models. Specifically, as Figure 13As shown in A: We separately seeded glioma cells U87MG or normal glial cells and brain microvascular endothelial cells bEnd.3 in a co-culture system. Among them, the brain microvascular endothelial cells bEnd.3 were seeded in the upper chamber of the co-culture system, and the glioma cells U87MG or normal glial cells were seeded in the lower chamber of the co-culture system. After culturing for 5 days, a blood-tumor barrier and a blood-brain barrier model were formed respectively. After the model was constructed, 1 μM of Cy5-labeled AS1411 was added to the upper chamber to detect the ability of AS1411 to penetrate the bEnd.3 cell layer. The results are as Figure 13 shown in B. Compared with the bEnd.3 cell layer (blood-brain barrier) co-cultured with normal glial cells, AS1411 more easily penetrated the bEnd.3 cell layer (blood-tumor barrier) co-cultured with glioma cells U87MG. This is consistent with the above data that AS1411 can specifically target gliomas in vivo but cannot enter normal brain tissue.

[0267] (2) We co-cultured normal glial cells, glioma cells U87MG, glioma cells U87MG treated with NCL siRNA (siNCL, SEQ ID NO:6), or glioma cells U87MG treated with negative control siRNA (siNC, SEQ ID NO:5) with brain microvascular endothelial cells bEnd.3 for 5 days to detect the NCL level in bEnd.3 cells. The results are as Figure 13 shown in C. After co-culturing with glioma cells U87MG, the NCL level in bEnd.3 cells increased significantly; after co-culturing with glioma cells U87MG treated with siNCL, the NCL level in bEnd.3 cells decreased; after co-culturing with glioma cells U87MG treated with siNC, the NCL level in bEnd.3 cells still increased significantly. This indicates a positive correlation between NCL in glioma cells and NCL in brain microvascular endothelial cells.

[0268] (3) We co-cultured glioma cells U87MG treated with siNCL or glioma cells U87MG treated with siNC with brain microvascular endothelial cells bEnd.3 for 5 days and added 1 μM of Cy5-labeled AS1411. The results are as Figure 13 shown in D. After knocking down NCL in glioma cells U87MG, the ability of AS1411 to penetrate the bEnd.3 cell layer was weakened.

[0269] (4) Brain tumor cells can transfer pro-angiogenic factors to the vascular endothelial cells of the blood-brain barrier through extracellular vesicles (EVs), promoting the transformation of the normal blood-brain barrier (BBB) into a blood-tumor barrier (BTB). We collected EVs from normal glial cells, glioma cells U87MG, glioma cells U87MG treated with siNCL, or glioma cells U87MG treated with siNC, added them to the culture medium of brain microvascular endothelial cells bEnd.3 and incubated for 6 h, and then added 200 nM Cy5-labeled AS1411 and co-incubated for 6 h. The results are as Figure 13 shown in Figure E. After incubation of EVs from glioma cells U87MG with bEnd.3 cells, the binding ability of AS1411 to bEnd.3 cells was significantly enhanced; after incubation of EVs from glioma cells U87MG with knocked-down NCL with bEnd.3 cells, the binding ability of AS1411 to bEnd.3 cells decreased.

[0270] The above results indicate that brain tumor cells transfer NCL to the vascular endothelial cells of the blood-brain barrier through extracellular vesicles, promoting the transformation of the normal blood-brain barrier into a blood-tumor barrier. AS1411 penetrates the blood-tumor barrier by binding to NCL on the surface of vascular endothelial cells of the blood-tumor barrier and specifically targets brain tumor cells. Therefore, AS1411 can be used for in vivo imaging, diagnosis, and detection of brain tumors.

[0271] Example 6: Silencing NCL can block the recruitment of MDM2 by AS1411

[0272] (1) After lysing glioma U87MG cells with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), 5 μL of NCL antibody was added and incubated at 4 °C overnight. Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) were added and incubated with rotation at room temperature for 1.5 hours. The NCL bound to the magnetic beads and the proteins it captured were washed with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015) was added, and heated to 100 °C for 10 minutes. The magnetic beads were adsorbed with a magnetic stand, and the supernatant was taken into a new tube. Then, immunoblotting experiments were performed. The results are as Figure 14 shown in Figure A. NCL can interact with MDM2, but not with EGFR.

[0273] (2) Incubate glioma cells U87MG with 500 nM biotin-labeled negative control CRO (Bio-CRO) or AS1411 (Bio-1411) for 6 hours. After cell lysis, incubate the supernatant with streptavidin agarose gel beads (purchased from Cytiva, catalog number 17511301) overnight at 4°C. After washing the biotin-labeled CRO or AS411 bound to the gel beads and the proteins they captured multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. After centrifugation, take the supernatant, which is the pull-down product. Detect the pull-down product by immunoblotting experiment. The results are as Figure 14 shown in B, AS1411 can capture NCL and MDM2.

[0274] (3) Transfect negative control siRNA (siNC) or NCL siRNA (siNCL) into glioma cells U87MG. After 48 hours, incubate the cells with 500 nM biotin-labeled AS1411 for 6 hours. Lyse the cells, and then incubate the supernatant with streptavidin agarose gel beads overnight at 4°C. After washing the biotin-labeled AS1411 bound to the gel beads and the proteins they captured multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. After centrifugation, take the supernatant, which is the pull-down product. Detect the pull-down product by immunoblotting experiment. The results are as Figure 14 shown in C, silencing NCL can block the recruitment of MDM2 by AS1411.

[0275] Example 7: Preparation of GROs-based PROTAC molecules targeting EGFR

[0276] We simulated the three-dimensional structure diagrams of AS1411 and NCL-MDM2 complex using Discovery Studio and AlphaFold2 respectively, and predicted the conformation of the interaction between AS1411 and NCL-MDM2 complex using HDOCK. The results showed that GROs recruit MDM2 depending on the interaction with NCL, and the possible mode of action is as Figure 15 shown in A; A possible mode of action of PROTAC targeting EGFR formed by GROs as a recruitment element of MDM2 is as Figure 15 shown in B.

[0277] In this example, AS1411, a typical representative of GROs, was used as the E3 ligase recruitment element, and Gefitinib (Gef), an EGFR inhibitor, was used as an example of the target protein ligand to prepare a PROTAC for degrading EGFR (hereinafter simply referred to as AS1411-Gef).

[0278] The preparation steps are as follows:

[0279] (1) Synthesis of compound A-3

[0280]

[0281] To a solution of compound A-1 (5.130 g, 27.54 mmol) in ACN (100 mL) was added compound A-2 (4.27 g, 30.72 mmol) and K 2 CO 3 (7.64 g, 55.28 mmol). The mixture was stirred at 90 °C for 4 hours. The mixture was concentrated, diluted with water (100 mL), and extracted with DCM (100 mL * 3). The organic layer was dried with Na 2 SO 4 , concentrated, and purified by DCM:MeOH = 0 - 10% to obtain the target compound A-3 (white solid, 4.7 g, 70%)

[0282] LCMS: m / z = 245.3 [M + H] + t R = 5.347 min. Purity: 100.00% (214 nm).

[0283] 1 HNMR (400 MHz, CDCl3) δ 4.88 (s, 1H), 3.92–3.71 (m, 2H), 3.55–3.29 (m, 4H), 2.71–2.57 (m, 2H), 2.55–2.35 (m, 4H), 1.74 (m, 2H), 1.46 (s, 9H).

[0284] (2) Synthesis of compound INT-A

[0285]

[0286] At 0 °C, TEA (3.7 g, 36.56 mmol) and MsCl (2.492 g, 21.75 mmol) were added to a solution of compound A-3 (4.463 g, 18.27 mmol) in DCM (40 mL). The mixture was stirred at 0 °C for 1 h. LCMS showed that the target MS was found. Water (20 mL) was added to the mixture at low temperature, and the mixture was extracted with DCM (3 × 25 mL). The organic layers were combined, washed with brine (3 × 30 mL), dried over Na 2 SO 4 dried, filtered, and concentrated in vacuo to give the target compound INT-A (yellow oil, 5.681 g, yield: 96%)

[0287] LCMS: m / z = 323.3 [M+H] + t R = 5.843 min. Purity: 100.00% (214 nm).

[0288] 1 1H NMR (400 MHz, CDCl3) δ 4.31 (t, J = 6.4 Hz, 2H), 3.48–3.34 (m, 4H), 3.01 (s, 3H), 2.47 (t, J = 6.8 Hz, 2H), 2.42–2.32 (m, 4H), 1.93 (m, 2H), 1.46 (s, 9H).

[0289] (3) Synthesis of compound B-3

[0290]

[0291] At 0 °C, DCC (39 g, 189.16 mmol) was slowly added to a solution of compound B-1 (10 g, 75.69 mmol) and compound B-2 (33.5 g, 181.99 mmol) in DCM (120 mL). The mixture was stirred at room temperature overnight. The reaction mixture was filtered and purified by SGC (DCM: 100%) to give the target compound B-3 (white solid, 25 g, 71%).

[0292] LCMS: m / z = N / A [M+H] + t R = 7.159 min. Purity: 100.000% (214 nm).

[0293] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 2.95 (t, J = 7.2 Hz, 4H), 2.07 (p, J = 7.4 Hz, 2H).

[0294] 19 FNMR(400MHz, DMSO-d 6 ) δ 153.64 (m, 4F), 158.32 (m, 2F), 162.90 (m, 4F).

[0295] (4) Synthesis of compound INT-B

[0296]

[0297] At 0 °C, a solution of TEA (1 mL, 7.19 mmol) and a solution of compound B-4 (1.081 g, 2.58 mmol) in DCM (10 mL) were slowly added to a solution of compound B-3 (2.4 g, 5.17 mmol) in DCM (10 mL). The mixture was stirred at room temperature for 10 hours. LCMS showed that the target MS was found. The mixture was concentrated and purified by SGC (MeOH:DCM = 0%-10%) to obtain the target compound INT-B (colorless oil, 1.4 g, crude).

[0298] LCMS: m / z = 700.5 [M+H] + t R = 1.145 min. Purity: 100.00% (214 nm).

[0299] (5) Synthesis of compound 2

[0300]

[0301] To a solution of compound 1 (2.5 g, 7.82 mmol) in DMF (40 mL) was added K 2 CO 3 (2.5 g, 18.09 mmol) and compound INT-A (4.338 g, 13.61 mmol). Under a nitrogen atmosphere, the mixture was stirred at 80 °C for 16 hours. LCMS showed that the target MS was found. The reaction mixture was concentrated, diluted with water (200 mL), and extracted with ethyl acetate (100 mL * 3). The organic layer was concentrated and purified by silica gel column chromatography, washed with EA:PE = 20-100% to obtain the target compound 2 (white solid, 3.29 g, 77%).

[0302] LCMS: m / z = 546.5 [M+H] + t R = 3.853 min. Purity: 100.00% (214 nm).

[0303] 1 HNMR(400MHz, CDCl 3)δ8.66(s,1H),7.90–7.88(m,1H),7.61–7.53(m,1H),7.50(s,1H),7.25(s,1H),7.20–7.11(m,2H),4.17(t,J=6.4Hz,2H),3.99(s,3H),3.53–3.39(m,4H),2.60(t,J=7.2Hz,2H),2.50–2.42(m,4H),2.14–2.09(m,2H),1.46(s,9H).

[0304] 19 FNMR(400MHz,CDCl 3 )δ120.94(s,1F).

[0305] (6) Synthesis of Compound 3

[0306]

[0307] To a solution of Compound 2 (1.008 g) in DCM (10 mL) was added TFA (10 mL). The mixture was stirred at 0 °C for 1 h under a nitrogen atmosphere. LCMS indicated that the target MS was found. The reaction mixture was concentrated under vacuum to give the target compound 3 (brown oil, 1.4 g, TFA salt), which was directly used in the next step.

[0308] LCMS: m / z = 446.3 [M+H] + t R = 0.758 min. Purity: 100.00% (214 nm).

[0309] (7) Synthesis of Compound 4

[0310]

[0311] To a solution of Compound 3 (700 mg, TFA salt) in DCM (10 mL) was added TEA (5 mL). The mixture was stirred at 0 °C for 5 min under a nitrogen atmosphere. Then a solution of INT-B (1.4 g, crude) in DCM (10 mL) was slowly added. The reaction mixture was stirred at room temperature overnight and monitored by LCMS. The reaction mixture was concentrated and purified by SGC (MeOH:DCM = 0%-10%) to give the target compound 4 (white solid, 670 mg, crude).

[0312] LCMS: m / z = 659.2 [M+H-DMTr] + t R = 11.10 min. Purity: 95.03% (214 nm).

[0313] (8) Synthesis of Compound 5

[0314]

[0315] To a solution of Compound 4 (657 mg, crude) in DCM (20 mL) were added DMAP (205 mg, 1.68 mmol) and succinic anhydride (144 mg, 1.44 mmol). The reaction mixture was stirred at room temperature for 8 h. LCMS indicated that the target MS was found. TEA (0.5 mL) was added to the mixture, followed by stirring for 1 h. The mixture was concentrated and purified by reverse-phase chromatography, eluting with TEA:acetonitrile = 5%-95% (TEA (aq, 5 mL / 5 L)) to give the target compound 5 (white solid, 349.4 mg, TEA salt).

[0316] LCMS: m / z = 1059.7 [M-H] - t R = 7.635 min. Purity: 100.00% (214 nm).

[0317] 1 HNMR (400 MHz, DMSO-d 6 ) δ 9.58 (s, 1H), 8.50 (s, 1H), 8.12 (m, 1H), 7.85–7.76 (m, 2H), 7.44 (m, 1H), 7.35–7.26 (m, 4H), 7.20 (m, 6H), 6.87 (d, J = 8.4 Hz, 4H), 5.30 (m, 1H), 4.19 (t, J = 5.6 Hz, 3H), 3.94 (s, 3H), 3.80–3.64 (m, 7H), 3.55 (d, J = 9.2 Hz, 1H), 3.40 (m, 6H), 3.24–3.19 (m, 1H), 2.98 (d, J = 6.4 Hz, 1H), 2.73–2.58 (m, 3H), 2.31 (m, 7H), 2.20 (m, 2H), 2.08–1.92 (m, 3H), 1.77–1.56 (m, 2H), 1.01 (t, J = 7.2 Hz, 4H).

[0318] 19 FNMR (400 MHz, DMSO-d 6 ) δ 123.31 (s, 1F).

[0319] (9) Synthesis of Compound 5-CPG

[0320]

[0321] At room temperature, HATU (80 mg, 0.208 mmol), DIEA (80 μL), and lcaa-CPG (1000 Å, 1000 mg) were added to a solution of compound 5 (200 mg, 0.189 mmol) in ACN (12.0 mL), and the mixture was shaken for 12 hours. After the reaction was completed, the CPG was washed with ACN, and CAP A (acetic anhydride: tetrahydrofuran = 1:9, v / v, 4.0 mL) and CAP B (n-methylimidazole: pyridine: acetonitrile = 15:10:75, v / v / v, 4.0 mL) were added. The mixture was shaken at room temperature for 1 hour. Then the mixture was filtered and washed three times with ACN (2 mL). After freeze-drying the mixture, compound 5-CPG (white powder, 1000 mg) was obtained.

[0322] (10) Synthesis of compound AS1411-Gef

[0323]

[0324] Compound 5-CPG was placed in a synthesis column (60 mg * 8) and synthesized by a K-A H-8 solid-phase synthesizer. The solid-phase synthesis includes four steps: detritylation, coupling, capping, and oxidation. After the reaction was completed, 1.5 mL of ammonia water was added to the CPG in each synthesis column, and the mixture was heated in an oven at 65 °C for 16 hours. Then the supernatant was collected and washed with water (1 mL * 3). The crude product was purified by a protein purification system (Sepure, SDA) (column: Mono Q, 1.0 mL, method: mobile phase A: 40 mM aqueous NaOH solution, mobile phase B: 40 mM NaOH + 2.0 M aqueous NaCl solution) to obtain the target product AS1411-Gef (white powder, 11.86 mg, purity = 98.18%).

[0325] UPLC-MS (WATERS ACQUITY PREMIER): AS1411-Gef-UPLC, m / z = 8992.79650 [M] - (deconvolution); t R = 11.285 min (260 nm). Mass error < 50 ppm.

[0326] HPLC: AS1411-Gef-HPLC, t R = 11.306 min (260 nm), purity: 98.175%.

[0327] Example 8: AS1411-Gef can degrade EGFR

[0328] (1) Incubate glioma U87MG cells with PBS or 500 nM AS1411-Gef (prepared in Example 7) for 6 hours. After cell lysis, incubate with an antibody against EGFR (purchased from Cell Signaling Technology, catalog number 4267S) overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. Wash the EGFR bound to the magnetic beads and the proteins it captured with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788). Add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, and transfer the supernatant to a new tube. Then perform immunoblotting experiments. The results are as Figure 16 shown in A. In the presence of AS1411-Gef, the antibody against EGFR can capture EGFR, NCL, and MDM2, indicating that AS1411-Gef can form a MDM2-NCL-PROTAC-EGFR quaternary complex with NCL, MDM2, and EGFR.

[0329] (2) Transfect negative control siRNA (siNC) or NCL siRNA (siNCL) into glioma U87MG cells for 48 hours, and then incubate the cells with 500 nM AS1411-Gef for 6 hours. After cell lysis, incubate with an antibody against EGFR (purchased from Cell Signaling Technology, catalog number 4267S) overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. Wash the EGFR bound to the magnetic beads and the proteins it captured with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788). Add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, and transfer the supernatant to a new tube. Then perform immunoblotting experiments. The results are as Figure 16 shown in B. In the presence of AS1411-Gef, after knocking down NCL, the antibody against EGFR cannot capture MDM2, indicating that NCL is located between MDM2 and EGFR in the quaternary complex induced by AS1411-Gef.

[0330] (3) Transfect negative control siRNA (siNC) or MDM2 siRNA (siMDM2, SEQ ID NO: 7) into glioma U87MG cells for 48 hours, and then incubate the cells with 500 nM AS1411-Gef for 6 hours. After cell lysis, incubate with an antibody against EGFR (purchased from Cell Signaling Technology, catalog number 4267S) overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and incubate with rotation at room temperature for 2 hours. Wash the EGFR bound to the magnetic beads and the proteins it captured with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, and transfer the supernatant to a new tube. Then perform immunoblotting experiments. The results are as Figure 16 shown in Figure C. In the presence of AS1411-Gef, after knocking down MDM2, the antibody against EGFR can still capture NCL, indicating that NCL is located between MDM2 and EGFR in the quaternary complex induced by AS1411-Gef.

[0331] (4) Incubate glioma cells U87MG with 0, 50, 100, 200, 500 nM AS1411-Gef (prepared in Example 7). After 12 hours, collect cell samples, extract total proteins using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B), and detect the degradation of EGFR protein by immunoblotting experiments. The process is as follows: Separate protein samples by SDS-PAGE electrophoresis, transfer the separated proteins to a PVDF membrane, block with TBST buffer containing 5% skim milk at room temperature for 1 hour, then incubate the primary antibody against EGFR (purchased from Cell Signaling Technology, catalog number 4267S) overnight at 4°C. After washing with TBST, incubate the membrane with an HRP-labeled secondary antibody (purchased from: Abclonal, catalog number: AS014) at room temperature for 1 hour, and visualize the protein band blots using an enhanced chemiluminescence detection kit (purchased from: Abclonal, catalog number: RM00021P). The results are as Figure 16 shown in Figure D. AS1411-Gef can dose-dependently reduce the protein level of EGFR.

[0332] (5) Incubate glioma cells U87MG with 200 nM AS1411-Gef, and collect corresponding cell samples at time gradients of 0, 1, 3, 6, 12, 24 hours for protein extraction and subsequent immunoblotting experiments to detect the degradation of EGFR protein. The results are as Figure 16As shown in Figure E, AS1411-Gef can time-dependently reduce the protein level of EGFR.

[0333] (6) Incubate glioma cells U87MG with solvent PBS or 200 nM AS1411, 200 nM gefitinib (Gefitinib, abbreviated as Gef), AS1411 + Gef (combination of 200 nM AS1411 and 200 nM gefitinib), or 200 nM AS1411-Gef. After 18 hours, collect cell samples for protein extraction and subsequent immunoblotting experiments to detect the degradation of EGFR protein. The results are as Figure 16 shown in Figure F. Only AS1411-Gef can reduce the protein level of EGFR.

[0334] (7) Incubate glioma U251 cells with 0, 10, 50, 100, 200, 500 nM of AS1411-Gef. After 12 hours, collect cell samples, extract total proteins using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B), and detect the degradation of EGFR protein by immunoblotting experiments. The results are as Figure 16 shown in Figure G. AS1411-Gef can concentration-dependently reduce the protein level of EGFR.

[0335] (8) Incubate glioma U251 cells with 200 nM AS1411-Gef, and collect corresponding cell samples at time gradients of 0, 1, 3, 6, 12, 24 hours for protein extraction and subsequent immunoblotting experiments to detect the degradation of EGFR protein. The results are as Figure 16 shown in Figure H. AS1411-Gef can time-dependently reduce the protein level of EGFR.

[0336] (9) Incubate glioma U251 cells with solvent PBS or 200 nM AS1411, Gef, AS1411 + Gef, or AS1411-Gef. After 12 hours, collect cell samples for protein extraction and subsequent immunoblotting experiments to detect the degradation of EGFR protein. The results are as Figure 16 shown in Figure I. Only AS1411-Gef can reduce the protein level of EGFR.

[0337] Example 9: AS1411-Gef Promotes the Ubiquitination of EGFR

[0338] The glioma cells U87MG were incubated with the solvent PBS or 200 nM of AS1411-Gef for 6 hours. After cell lysis, they were incubated overnight at 4 °C with an antibody against EGFR (purchased from Cell Signaling Technology, catalog number 4267S). Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) were added and incubated with rotation at room temperature for 2 hours. After washing the EGFR bound to the magnetic beads multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015) was added, and the mixture was heated to 100 °C for 10 minutes. The magnetic beads were adsorbed with a magnetic stand, and the supernatant was transferred to a new tube. Then, immunoblotting experiments were used to detect the ubiquitination level of EGFR. The results are as Figure 17 shown, AS1411-Gef promoted the ubiquitination of EGFR.

[0339] Example 10: AS1411-Gef mediates the degradation of EGFR through the ubiquitin-proteasome pathway

[0340] The glioma cells U87MG were incubated with the solvent PBS or 200 nM of AS1411-Gef for 12 hours, and 10 μM of the proteasome inhibitor MG132 was added during the last 6 hours of incubation. The corresponding cell samples were collected for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 18 shown, MG132 could block the degradation of EGFR by AS1411-Gef, indicating that the degradation of EGFR by AS1411-Gef depends on the ubiquitin-proteasome pathway.

[0341] Example 11: AS1411-Gef depends on MDM2 and NCL to mediate the degradation of EGFR

[0342] (1) Negative control siRNA (siNC) or NCL siRNA (siNCL) was transfected into the glioma cells U87MG for 48 hours, and then the cells were incubated with the solvent PBS or 200 nM of AS1411-Gef for 12 hours. The corresponding cell samples were collected for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 19 shown in A, silencing NCL could reduce the degradation of EGFR by AS1411-Gef, indicating that AS1411-Gef depends on NCL to mediate the degradation of EGFR.

[0343] (2) Transfect negative control siRNA (siNC) or MDM2 siRNA (siMDM2) into glioma cells U87MG for 48 hours, and then incubate the cells with solvent PBS or 200 nM AS1411-Gef for 12 hours. Collect the corresponding cell samples for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 19 shown in B. Silencing MDM2 can reduce the degradation of EGFR by AS1411-Gef, indicating that AS1411-Gef mediates the degradation of EGFR in a MDM2-dependent manner.

[0344] Example 12: AS1411-Gef inhibits the proliferation and promotes apoptosis of tumor cells

[0345] (1) Seed glioma cells U87MG in 96-well cell culture plates, and replace the medium containing solvent PBS or 200 nM AS1411, Gef, AS1411+Gef or AS1411-Gef every day during the experiment. Use a CCK-8 kit (purchased from MCE, catalog number HY-K0301) to perform cell proliferation experiments, and measure the absorbance at 450 nm using a microplate reader on the first, second, and third days. The results are as Figure 20 shown in A. AS1411-Gef can inhibit the proliferation of U87MG cells.

[0346] (2) Seed glioma cells U87MG in six-well plates for cell apoptosis experiments. Replace the medium containing solvent PBS or 200 nM AS1411, Gef, AS1411+Gef or AS1411-Gef every day during the experiment. After 3 days, use an Annexin V-FITC and PI double staining cell apoptosis detection kit (purchased from Beyotime, catalog number C1062L) to perform cell apoptosis analysis. The results are as Figure 20 shown in B. AS1411-Gef can promote the apoptosis of U87MG cells.

[0347] (3) Seed glioma U251 cells in 96-well cell culture plates, and replace the medium containing solvent PBS or 200 nM AS1411, Gef, AS1411+Gef or AS1411-Gef every day during the experiment. Use a CCK-8 kit (purchased from MCE, catalog number HY-K0301) to perform cell proliferation experiments, and measure the absorbance at 450 nm using a microplate reader on the first, second, and third days. The results are as Figure 21 shown in A. AS1411-Gef can inhibit the proliferation of U251 cells.

[0348] (4) Glioma U251 cells were seeded in six-well plates for apoptosis experiments. During the experiment, the culture medium containing solvent PBS or 200 nM AS1411, Gef, AS1411 + Gef, or AS1411 - Gef was changed daily. After 3 days, apoptosis analysis was performed using an Annexin V-FITC and PI double-staining apoptosis detection kit (purchased from Beyotime, catalog number C1062L). The results are as Figure 21 shown in Figure B. AS1411 - Gef can promote the apoptosis of U251 cells.

[0349] Example 12: Inhibition of glioma growth by AS1411 - Gef in vivo

[0350] A glioma orthotopic mouse model was constructed using U87MG-LUC-GFP cells as follows: The tumor injection area in the brain of nude mice was located using a stereotaxic apparatus (RWD, model 68803). 1 mm forward from the midpoint of the bregma, then 1.8 mm to the right, and the injection depth was 3 mm. 3×10 7 U87MG-LUC-GFP cells were injected. Seven days after modeling, the nude mice were divided into 5 groups of 5 each, and were respectively given PBS, AS1411 (6 μmol / kg), Gef (12 mg / kg), AS1411 + Gef (6 μmol / kg + 12 mg / kg), and AS1411 - Gef (6 μmol / kg). The drugs were administered every other day for a total of 5 times. Gef was administered by gavage, and AS1411 and AS1411 - Gef were administered via the tail vein. In vivo imaging (PerkinElmer, model IVISSpectrum) was performed on the day of each drug administration and on the 17th day. After the drug administration, the nude mice were continued to be raised. When the nude mice showed severe hunchback, reduced activity, hemiplegia, epilepsy, or a weight loss of more than 20%, the nude mice were euthanized and the time was recorded. The results are as Figure 22 shown in Figures A - 22D. AS1411 - Gef can inhibit the growth of glioma and improve the survival rate of experimental animals.

[0351] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0352] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0353] The mere listing of the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. An EGFR degrading agent or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, wherein the EGFR degrading agent has the following structure: in, GRO is a guanine-rich oligonucleotide residue that can specifically bind to nucleolin; L is a linker; p is an integer from 1 to 100; Ring A is an aliphatic ring or a heterocyclic ring; Ring C is an aromatic ring or an aromatic heterocyclic ring; B 1 , B 2 , B 3 Independently selected from: C or N; Y 1 , Y 2 , Y 3 Independently selected from: single bond, -O-, -S-, -C0-C6 alkylene-, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N-(C0-C6 alkyl), -C(O)-, -C(S)-, -C(O)-(C0-C6 alkylene)-, -C(S)-(C0-C6 alkylene)-, -C(O)-N(C0-C6 alkyl)-, -C(S)-N(C0-C6 alkyl)-, -SO2-(C0-C6 alkylene)-, -SO-(C0-C6 alkylene)-; R1 is one or more independent substituents on the benzene ring, which are selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); R2 is one or more independent substituents on the benzene ring, which are selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); R3 is one or more independent substituents on the C ring, which are selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); Preferably, the GROs have a stable G4 structural feature.

2. The EGFR degrading agent according to claim 1, characterized in that The GROs is one of the DNA aptamer AS1411 and AS1411 derivatives / analogs; preferably, the AS1411 derivatives / analogs is one of GRO29A, GRO15A, AT11, AT11-L0, AT21, AT27, GRO29-2, GRO29-3, GRO29-5, GRO29-13, GRO14C, GRO25A, GRO28A, GRO28B, GRO29-6, GRO32A, GRO32B, GRO56A, GRO A, GRO B, GRO C, GRO D, GRO E, GRO F, GROG, GRO H, GRO I, GRO J, GRO K, GRO L, GRO M; preferably, the GROs are selected from: AS1411, GRO29A, GRO15A, AT11; Preferably, the GROs include chemical modifications, nucleic acid unit replacements or attachment of functional groups on the GROs; The chemical modification is that at least one base is modified, and the chemical modification includes at least one of phosphorylation, methylation, amination, sulfhydrylation, isotopization, thiophosphate backbone modification, methoxy modification, and fluorination modification; The nucleic acid unit is replaced by at least one nucleic acid unit being replaced by LNA, UNA or GNA; The functional group includes at least one of a fluorescent group, a radioactive group, a therapeutic drug, biotin, digoxin, a nanoluminescent material, a nucleic acid substance or an enzyme marker.

3. The EGFR degrading agent according to claim 1, characterized in that The GROs is AS1411.

4. The EGFR degrading agent according to any one of claims 1 to 3, characterized in that L has the following structure: in, L1 is a divalent group connected to GRO, which can be selected from: a single bond, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N(R L1 )-(C0-C6 alkylene)-, -N(R L2 )C(O)-(C0-C6 alkylene)-, -OP(O)(OR L1 )O-(C0-C6 alkylene)-, -C(O)-(C0-C6 alkylene)-, -C(S)-(C0-C6 alkylene)-, -CON(R L1 )-(C0-C6 alkylene)-; L3 is a divalent group connected to the A ring, which can be selected from: a single bond, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -C(O)-(C0-C6 alkylene)-, -C(S)-(C0-C6 alkylene)-, -N(R L3 )-(C0-C6 alkylene)-, -CON(R L3 )-(C0-C6 alkylene)-, -N(R L3 )CO-(C0-C6 alkylene)-, -SO2-(C0-C6 alkylene)-, -SO-(C0-C6 alkylene)-, 4-10 membered heterocyclylene; L2 is a single bond or a divalent saturated or unsaturated straight or branched C1-C50 hydrocarbon chain (e.g., a C1-C20 alkyl chain), wherein 0-6 methylene units in the hydrocarbon chain (e.g., an alkyl chain) are independently substituted by: -CY-, -O-, -S-, -SS-, -C(O)-, -C(S)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R L2 )-、-N(R L2 )C(O)-、-N(R L2 )C(O)O-、-N(R L2 )C(O)N(R L2 )-、-N(R L2 )-、-S(O)2-、-S(O)2N(R L2 )-、-N(R L2 )S(O)2-、-S(O)-、-S(O)N(R L2 )-、-N(R L2 )S(O)-、-P(O)(OR L2 )O-、-P(O)-、-P(O)N(R L2 )-、-P(O)(N(R L2 )2)-、-OP(O)(OR L2 )2N(R L2 )-、-P(O)(OR L2 )2N(R L2 )-、-N(R L2 )P(O)(OR L2 )O-、-N(R L2 )P(O)-、-Si(R L2 )2-, -C(=N-CN)-, Amino acid residues, nucleotide residues, oligonucleotide residues, oligopeptide residues, wherein m2 is selected from an integer selected from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), each -CY- is independently an optionally substituted divalent ring selected from the following: arylene, cycloalkylene, heterocyclylene; H in the hydrocarbon chain may be optionally substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, -OR L0 、-C(O)R L0 、-C(S)R L0 、-C(O)OR L0 、-C(S)SR L0 、-OC(O)R L0 、-OC(S)R L0 、-OC(S)SR L0 、-C(O)N(R L0 )2、-OC(O)N(R L0 )2、-N(R L0 )C(O)OR L0 、-N(R L0 )SO2R L0 、-SO2N(R L0 )2、-OSO2N(R L0 )2、-N(R L0 )C(O)R L0 、-N(R L0 )2. -SR L0 、-SOR L0 、-SO2R L0 、-OSO2R L0 、C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclyl); R L0 , R L1 , R L2 and R L3 Independently selected from: H, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), wherein the C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C0-C6 alkylene, C3-C 10 Cycloalkyl, C6-C 10 The H in the aryl group and the 4-10 membered heterocyclic group may be optionally substituted by one or more groups selected from the following: halogen, cyano, nitro, azido, hydroxyl, amino, thiol, carboxyl, C1-C 10 Alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclyl); Preferably, L is linked to the 3' end, 5' end or any base, sugar or phosphate backbone in the middle of GRO.

5. The EGFR degrading agent according to any one of claims 1 to 3, characterized in that L2 is a C1-C20 straight chain alkylene group, wherein 0-6 methylene units in the alkylene group are independently substituted by the following groups: -O-, -S-, -SS-, -C(O)-, -C(O)O-, -OC(O)-, -N(R L2 )-、-C(O)N(R L2 )-、-N(R L2 )C(O)-、 Among them, each R L2 Independently selected from: H, C1-C6 alkyl, each R L4 Independently selected from: H, OH, C1-C6 alkoxy; Preferably, L2 can be selected from: C1-C20 straight chain alkylene, -(CH2CH2O) m2 -CH2-, -(CH2CH2O) m2 -CH2CH2-, -CH2-(CH2CH2O) m2 -CH2-, -CH2CH2-(CH2CH2O) m2 -CH2-, -CH2CH2-(CH2CH2O) m2 -CH2CH2-、-(C1-C 10 Alkylene)-O-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-NH-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-C(O)NH-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-NHC(O)-(C1-C 10 -(C1-C6 alkylene)-, -(C1-C6 alkylene)-O-(C1-C6 alkylene)-C(O)NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-O-(C1-C6 alkylene)-NHC(O)-(C1-C6 alkylene)-, wherein m2 is selected from an integer between 1 and 10, g is 0 or 1, h is selected from an integer between 0 and 10, i is selected from an integer between 0 and 10, and G is any suitable trivalent group; More preferably, L2 is selected from:

6. The EGFR degrading agent according to any one of claims 1 to 3, characterized in that L2 is selected from C1-C20 straight chain alkylene, -(C0-C6 alkylene)-(CH2CH2O) m2 -(C1-C6 alkylene)-, -(C1-C 10 Alkylene)-O-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-N(C1-C6 alkyl)-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-C(O)N(C1-C6 alkyl)-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-N(C1-C6 alkyl)C(O)-(C1-C 10 C1-C6 alkylene)-, -(C1-C6 alkylene)-O-(C1-C6 alkylene)-C(O)NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-O-(C1-C6 alkylene)-NHC(O)-(C1-C6 alkylene)-, wherein one methylene unit in the alkylene is replaced by Substitution, G is any suitable trivalent group; Preferably, L2 is The x end is connected to L3, the y end is connected to L1, the J ring is a 4-6 membered saturated heterocyclic ring, m2 is selected from an integer between 1 and 10, h is selected from an integer between 0 and 10, and i is selected from an integer between 0 and 10; More preferably, L2 is More preferably, L2 is selected from 7. The EGFR degrading agent according to any one of claims 1 to 3, characterized in that L2 is a C1-C20 straight chain alkylene group, wherein 1-3 methylene units are independently substituted by the following groups: -CY-, Optionally, L2 further comprises a group selected from the following: -O-, -C(O)-, -N(R L2 )-、-C(O)N(R L2 )-、-N(R L2 )C(O)-、 Among them, each R L2 Independently selected from: H, C1-C6 alkyl, each R L4 Independently selected from: H, OH, C1-C6 alkoxy; Preferably, the -CY- is selected from: More preferably, L2 is selected from:

8. The EGFR degrading agent according to any one of claims 4 to 7, characterized in that L1 is a single bond or O.

9. The EGFR degrading agent according to any one of claims 4 to 7, characterized in that L3 is selected from: a single bond, -N(H)-, -C(O)-, L3 is -CON(R L3 )-,R L3 Selected from: H, C1-C6 alkyl, C3-C6 cycloalkyl.

10. The EGFR degrading agent according to any one of claims 4 to 8, characterized in that: Part of R3' is selected from: H, halogen, cyano, nitro, azido, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl).

11. The EGFR degrading agent according to claim 10, characterized in that R1 is selected from the group consisting of: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -O(C 1-6 Alkyl), preferably -O(C 1-3 alkyl); R2 is selected from the group consisting of: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -O(C 1-6 Alkyl), preferably H; R3 is selected from the group consisting of: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -O(C 1-6 alkyl), preferably halogen; R2' is selected from the group consisting of: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -O(C 1-6 alkyl), preferably halogen.

12. The EGFR degrading agent according to any one of claims 4 to 8 and 11, characterized in that: The EGFR degradation agent has the following structure: Preferably, the EGFR degrading agent has the following structure:

13. The EGFR degrading agent according to any one of claims 1 to 3, characterized in that: The EGFR degradation agent has the following structure: Wherein, h is selected from an integer between 1 and 10, and i is selected from an integer between 0 and 10; Preferably, the EGFR degrading agent is selected from the following structures:

14. A pharmaceutical composition comprising the EGFR degrader according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, and one or more pharmaceutically acceptable excipients.

15. A delivery system for an EGFR degrading agent, comprising the EGFR degrading agent according to any one of claims 1 to 13 and a carrier; Preferably, the vector is a viral vector or a non-viral vector; More preferably, the viral vector is a lentivirus, adenovirus or adeno-associated virus vector; More preferably, the non-viral vector is a lipid nanoparticle (LNP), a polymer nanocarrier, an inorganic nanocarrier, a protein carrier or an exosome.

16. Use of the EGFR degrading agent according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the preparation of a medicament for preventing and / or treating EGFR-related diseases; Preferably, the disease is selected from the group consisting of: tumors, autoimmune diseases, inflammatory diseases, diseases associated with pathogen infection, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, and fibrotic diseases; More preferably, the tumor is selected from the group consisting of lung cancer, malignant melanoma, brain tumor, tumor of digestive organs, uterine cancer, fallopian tube cancer, papillary thyroid cancer, testicular cancer, palate cancer, nasopharyngeal cancer, pharyngeal cancer, tongue cancer, oral cancer, adenoid cystic carcinoma, various sarcomas, retinoblastoma, ameloblastoma, osteosarcoma, hematological tumors, nervous system tumors, glioma, glioblastoma, glioma, skin cancer, skin appendage cancer and skin metastasis, medulloblastoma, blastoma, liposarcoma, neuroendocrine tumor, synovial cell sarcoma, gastrinoma, carcinoid tumor, mesothelioma Tumors, pancreatic islet cell carcinoma, schwannoma, meningioma, melanoma, acoustic neuroma, adenocarcinoma, lymphoid malignancies, epithelial squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, peritoneal cancer, lung squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, thyroid cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, bile duct cancer, prostate cancer, salivary gland cancer, kidney cancer, vulvar cancer, anal cancer, penile cancer, esophageal cancer, cardiac cancer, biliary tumors and head and neck cancer, preferably glioma.

17. Application of GROs in the preparation of in vivo imaging, diagnosis and detection reagents for brain tumors; Preferably, the GROs is as described in claim 2 or 3, preferably AS1411; Preferably, the reagent further comprises a label, preferably a fluorophore.

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