Novel VEGFR (vascular endothelial growth factor receptor) degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a novel VEGFR (vascular endothelial growth factor receptor) degradation agent as well as a preparation method and application thereof. The VEGFR degradation agent is PROTAC with GROs (such as AS1411) as an MDM2 recruitment element, target protein VEGFR and E3 ubiquitin ligase MDM2 can be pulled close in vivo, so that the VEGFR is labeled with ubiquitin, and then the VEGFR is degraded through a ubiquitin-proteasome pathway. Experiments show that the VEGFR degradation agent can effectively degrade VEGFR 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.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a novel VEGFR degrader, a preparation method thereof, and an application thereof. Background Art

[0002] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a homodimeric glycoprotein with a molecular weight of 34-45 kDa and plays an important role in promoting angiogenesis, inhibiting apoptosis of endothelial cells, and increasing vascular permeability. VEGFR is the receptor of VEGF and includes three types: VEGFR1, VEGFR2, and VEGFR3. When VEGF binds to the receptor, the conformation of the intracellular kinase domain changes, generating kinase activity to catalyze the phosphorylation of substrate proteins, and ultimately producing a series of biological effects through the cascade reaction of signaling molecules. The VEGF / VEGFR signal participates in the occurrence and development of various diseases, including cardiovascular and cerebrovascular diseases, nervous system diseases, infectious diseases, inflammation, metabolic diseases, diabetes, and ophthalmic diseases. VEGF is overexpressed in almost all tumors and is closely related to tumor growth, metastasis, pathological grading, and prognosis. Therefore, VEGF / VEGFR has become an important target for various diseases.

[0003] In recent years, a large number of tyrosine kinase inhibitors targeting VEGFR have been developed globally. 14 small molecule VEGFR inhibitors have been approved for marketing, and more compounds are successively in the stages of clinical trials and activity tests, such as imatinib, sorafenib, tivozanib, cediranib, vorolanib, dovitinib, telatinib, WXFL-152, TEL-VEGFR2-BaF3, etc. Various types of VEGFR inhibitors can improve the survival rate of tumor patients, but such drugs often show primary or acquired drug resistance within one year during the treatment of tumors.

[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 three parts: 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. The target protein labeled 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 function repeatedly in cells. 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.

[0005] Studies have found that inhibiting the VEGFR protein requires maintaining the drug at a high concentration for a long time. High - dose administration will lead to the enrichment of the VEGFR protein due to negative feedback, thus greatly weakening the inhibitory effect. Therefore, there is a need to further develop more effective new drugs against the VEGFR protein. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides a novel VEGFR degrader and its preparation method and application. The VEGFR degrader is a PROTAC using GROs (such as AS1411) as the MDM2 recruitment element, which can bring the target protein VEGFR and the E3 ubiquitin ligase MDM2 closer in vivo, label VEGFR with ubiquitin tags, and then degrade it through the ubiquitin - proteasome pathway. Experiments show that the VEGFR degrader can effectively degrade VEGFR and produce corresponding therapeutic effects, such as inhibiting the proliferation of tumor (such as glioma) cells and promoting tumor cell apoptosis.

[0007] In the first aspect of the present invention, there is provided a VEGFR degrader and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds, and the VEGFR degrader has the following structure: Among them, GRO is a guanine-rich oligonucleotide (G-rich oligonucleotides) residue that can specifically bind to nucleolin (NCL), L is a linker structure part, VEGFRB is a vascular endothelial growth factor receptor VEGFR recognition / binding part, and p is any suitable integer within 1 - 100.

[0008] Specifically, VEGFRB can be a structural part of any suitable VEGFR small molecule inhibitor (for example, a known tyrosine kinase inhibitor targeting VEGFR, such as the quinazoline derivative used as an angiogenesis inhibitor described in WO2000047212A1) or a VEGFR antibody residue.

[0009] In particular, the VEGFR degrader has the following structure:

[0010]

[0011] Among them,

[0012] GRO is a guanine-rich oligonucleotide (G-rich oligonucleotides) residue that can specifically bind to nucleolin (NCL);

[0013] L is a linker structure part, that is, a linker;

[0014] Ring A is an 8-, 9-, 10-, 12- or 13-membered bicyclic or tricyclic ring, optionally containing 1 - 3 heteroatoms independently selected from O, N, and S;

[0015] R 1 is one or more independent substituents on Ring A, selected from: H, =O, halogen, cyano, nitro, azide, 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 alkylene)-(4 - 10-membered heterocyclic group), C 1 -C 10 haloalkyl, C 1 -C 10Halogenated alkoxy, -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)CO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)CON(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -(C 0 -C 6 alkylene)-O(C 0-10 alkyl), -(C 0 -C 6 alkylene)-S(C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO 2 (C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C 6 alkylene)-COO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-OCO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-CON(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C 6 alkylene)-CO(C 0-10 alkyl);

[0016] R 2 is one or more independent substituents on the quinazoline ring, selected from: H, halogen, cyano, nitro, azide, C 1 -C10 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 alkylene)-(4 - 10 membered heterocyclic group), C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)CO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)CON(C 0-10 alkyl), -(C 0 -C 6 alkylene)-N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -(C 0 -C 6 alkylene)-O(C 0-10 alkyl), -(C 0 -C 6 alkylene)-S(C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO 2 (C 0-10 alkyl), -(C 0 -C 6 alkylene)-SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C6 alkylene)-COO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-OCO(C 0-10 alkyl), -(C 0 -C 6 alkylene)-CON(C 0-10 alkyl)(C 0-10 alkyl), -(C 0 -C 6 alkylene)-CO(C 0-10 alkyl), or two R 2 together with the carbon atom to which they are attached form a heterocyclic ring (such as an oxygen-containing heterocyclic ring);

[0017] X 1 is selected from: a single bond, C 1 -C 6 alkylene, C 1 -C 6 alkenylene, C 1 -C 6 alkynylene, -O-(C 0 -C 6 alkylene)-, -S-(C 0 -C 6 alkylene)-, -N(R 4 )(C 0 -C 6 alkylene)-, -N(R 4 )(C(O)-(C 0 -C 6 alkylene)-, -C(O)-(C 0 -C 6 alkylene)-, -C(S)-(C 0 -C 6 alkylene)-, -CON(R 4 )(C 0 -C 6 alkylene)-, -SO 2 (C 0-10 alkyl)-;

[0018] X 2 is selected from: a single bond, C 1 -C 6 alkylene, C 1 -C 6 alkenylene, C 1 -C 6 alkynylene, phenylene, C 3 -C 10 cycloalkylene, 4- to 10-membered heteroarylene, -O-(C0 -C 6 -alkylene)-, -S-(C 0 -C 6 -alkylene)-, -N(R 5 )-(C 0 -C 6 -alkylene)-, -N(R 5 )C(O)-(C 0 -C 6 -alkylene)-, -C(O)-(C 0 -C 6 -alkylene)-, -C(S)-(C 0 -C 6 -alkylene)-, -CON(R 5 )-(C 0 -C 6 -alkylene)-, -SO 2 (C 0-10 alkyl)-;

[0019] R 3 is selected from: H, C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, C 6 -C 10 aryl, C 3 -C 10 cycloalkyl, 4- to 10-membered heterocyclic group, -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); wherein, the H on the alkyl, aryl, cycloalkyl, and heterocyclic group is optionally substituted by one or more groups selected from the following: H, =O, halogen, cyano, nitro, azide, 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 alkylene)-(4- to 10-membered heterocyclic group), C 1 -C 10 haloalkyl, -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 4 and R 5 are independently selected from: H, C 1 -C 10 alkyl; wherein the alkyl is optionally substituted by one or more groups selected from the following: halogen, cyano, -O(C 0-10 alkyl), -N(C0-10 alkyl)(C 0-10 alkyl);

[0021] Z is selected from: -O-, -NH-, -S-, -CH 2 -, single bond;

[0022] p is an integer from 1 to 100 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 40, 50, 60, 80, 100).

[0023] In a preferred embodiment of the present invention, p is 1, that is, the general formula I is

[0024] Specifically, the GROs are guanine-rich oligonucleotides containing one or more GGT motifs and having G4 structural characteristics.

[0025] Preferably, the GROs have stable G4 structural characteristics, wherein 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 biological studies [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.

[0026] In some embodiments of the present invention, detected using a fluorescent probe (such as N-methyl mesoporphyrin IX (NMM), O-phenanthroline derivative), 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.).

[0027] In one embodiment of the present invention, when detected using N-methyl mesoporphyrin IX (NMM) as a fluorescent probe, in the presence or absence of K + AS1411 can form stable G4 structural characteristics in the presence. Specifically, the detection method may include the following steps: adding the GRO to be tested in the presence or absence of K +In the solution, heat at 90 - 99 °C for 5 - 10 minutes, incubate on ice, then add NMM, incubate in the dark at room temperature, and detect the fluorescence intensity (as described in Example 2 of the present invention).

[0028] Specifically, the GROs include chemical modification on 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 a fluorescent group, a radioactive group, a therapeutic drug, biotin, digoxin, a nanoluminescent material, a nucleic acid substance, or an enzyme label.

[0029] 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.

[0030] Preferably, the GROs are one of the DNA aptamer AS1411, AS1411 derivatives / analogues.

[0031] 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 include one or more GGT motifs, have a G4 structural feature, and can specifically bind to NCL.

[0032] 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.

[0033] Specifically, the sequence of the AS1411 is as shown in SEQ ID NO: 1: 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

[0034] In some preferred embodiments of the present invention, the GROs are AS1411.

[0035] In some other embodiments of the present invention, the GROs are selected from: GRO29A, GRO15A, AT11.

[0036] Specifically, L can be any suitable linking moiety that links the GRO to the vascular endothelial growth factor receptor VEGFR recognition / binding moiety.

[0037] 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.

[0038] In some embodiments of the present invention, L is linked to the 3′-end of the GRO.

[0039] In some embodiments of the present invention, L is linked to the 5′-end of the GRO.

[0040] In some embodiments of the present invention, the GRO moiety in the general formula I has the following structure:

[0041]

[0042] Specifically, L has the following structure: Wherein,

[0043] 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 )-(C0 -C 6 (alkylene)-;

[0044] L 3 is a divalent group linked to the A ring and is selected from: a single bond, -O-(C 0 -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 heteroalkyl group;

[0045] L 2 is a single bond or a divalent saturated or unsaturated straight-chain or branched-chain C1-C50 hydrocarbon chain (such as a C1-C20 alkyl chain), and 0-6 methylene units in the hydrocarbon chain (such as an 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)-,

[0046] 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)S R 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 , -SORL0 、-SO 2 R L0 、-OSO 2 R L0 , C 1 -C 10 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)-(4-10 membered heterocyclyl);

[0047] R L0 , R L1 and R L2 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 alkylene)-(4-10 membered heterocyclic group), wherein the 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 10The H in aryl or 4-10 membered heterocyclic group may optionally be substituted by one or more groups selected from the following: halogen, cyano, nitro, azide, hydroxy, amino, mercapto, carboxyl, C 1 -C 10 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)-(4-10 membered heterocyclic group).

[0048] Specifically, each -CY- independently is 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.

[0049] In some embodiments of the present invention, each -CY- independently is selected from the following:

[0050]

[0051]

[0052] 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 1-8cycloalkyl), N(C 1-8 cycloalkyl)(C 1-8 alkyl), OH, NH 2 、SH, SO 2 (C 1-8 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 2N(C 1-8 alkyl) 2 NHSO 2 NH(C 1-8 Alkyl), NHSO 2 N(C 1-8 alkyl) 2 or NHSO 2 NH 2 or, R L4 , R L5 Together with the atoms to which it is attached, it forms a cycloalkylene or heterocyclylene.

[0053] More specifically, R L4 , R L5 Independently selected from: -CH 3 , -OH,

[0054] or, R L4 , R L5 Together with the atoms to which they are attached, they form a three-membered to six-membered cycloalkylene group (e.g. ) or a four-membered to six-membered heterocycloalkylene group (such as ).

[0055] In some embodiments of the present invention, R L1 For H.

[0056] In some embodiments of the present invention, R L4 For H.

[0057] In some embodiments of the present invention, R L4 For OH.

[0058] In some embodiments of the present invention, R L5 For H.

[0059] In some embodiments of the present invention, R L5 For OH.

[0060] In one embodiment of the present invention, L 2 The following scheme (1) is adopted: L 2 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)-、

[0061] 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.

[0062] More specifically, L 2 can be selected from: C1-C20 linear 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 6Alkylene)-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)-、

[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 straight-chain alkylene, -(C 0 -C 6 Alkylene)-(CH 2 CH 2 O) m2 -(C 1 -C 6 Alkylene)-、-(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)-, wherein one methylene unit in the alkylene is independently replaced by a group capable of being linked 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 linked to L 3 , and the y end is linked 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 1-3 methylene units are independently replaced by the following groups: -CY-, Optionally, L 2 further 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] In some specific embodiments of the present invention, L 2 is selected from:

[0077] 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

[0078] L 1 can be single-bonded or O, that is, GRO is connected to L via a phosphodiester bond (that is 2 ).

[0079] Specifically, the oligonucleotide is composed of A and / or T.

[0080] 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.

[0081] In some embodiments of the present invention, L 2 is a double-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-T pairs, for example, a double-stranded oligonucleotide residue composed of 6 A-T pairs.

[0082] In some embodiments of the present invention, L 2 is a single-stranded DNA oligonucleotide residue consisting of 6 A's.

[0083] In some embodiments of the present invention, L 2 is a double-stranded DNA oligonucleotide consisting of 6 A-T pairs.

[0084] In some embodiments of the present invention, L 2 is a double-stranded DNA oligonucleotide consisting of 10 A-T pairs.

[0085] In some embodiments of the present invention, L 2 is a double-stranded DNA / RNA hybrid oligonucleotide, where the DNA single strand consists of 6 T's and the RNA single strand consists of 6 A's.

[0086] In another embodiment of the present invention, L 2 adopts the following scheme (4): L 2 is an oligopeptide residue consisting of 2-10 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid residues.

[0087] In some embodiments of the present invention, L 3 is a single bond.

[0088] In some embodiments of the present invention, L 3 is -C(O)-.

[0089] In some embodiments of the present invention, Z is -O-.

[0090] Specifically, the vascular endothelial growth factor receptor VEGFR recognition / binding portion is the structural portion of a VEGFR small molecule inhibitor, and the small molecule inhibitor can be, but is not limited to, imatinib, sorafenib, tivozanib, cediranib, vorolanib, dovitinib, telatinib, WXFL-152, TEL-VEGFR2-BaF3.

[0091] In some embodiments of the present invention, the vascular endothelial growth factor receptor VEGFR recognition / binding portion is the structural portion of a quinazoline derivative small molecule inhibitor (such as the quinazoline derivative used as an angiogenesis inhibitor described in WO2000047212A1).

[0092] Specifically, ring A can be saturated or unsaturated, aromatic or non-aromatic; in some embodiments of the present invention, ring A is a 9- or 10-membered bicyclic ring, optionally containing 1-3 heteroatoms independently selected from O, N, and S, such as

[0093] In some embodiments of the present invention, part is

[0094] In some preferred embodiments of the present invention, part is wherein R 1a 、R 1b has the above definition of R 1 .

[0095] Specifically, R 1a is selected from: H, hydroxy, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl; in one embodiment of the present invention, R 1a is methyl.

[0096] Specifically, R 1b is selected from: H, hydroxy, halogen, C 6 -C 6 alkyl, C 6 -C 6 haloalkyl; in one embodiment of the present invention, R 1b is halogen, such as F.

[0097] In some embodiments of the present invention, part is wherein R 2a 、R 2b has the above definition of R 2 .

[0098] Specifically, R 2a is selected from: H, -O(C 0-6 alkyl); in one embodiment of the present invention, R 2a is methoxy.

[0099] In some embodiments of the present invention, R 2b is

[0100] Specifically, X 1 is selected from: single bond, C 1 -C 6 alkylene, -O-(C 0 -C6 - (alkylene)-, -S-(C 0 -C 6 - (alkylene)-; In some embodiments of the present invention, X 1 is -O-(C 0 -C 6 - (alkylene)-.

[0101] Specifically, X 2 is selected from: a single bond, C 1 -C 6 - (alkylene), C 1 -C 6 - (alkenylene), -O-(C 0 -C 6 - (alkylene)-, -S-(C 0 -C 6 - (alkylene)-, -N(R 5 )(C 0 -C 6 - (alkylene)-.

[0102] Specifically, R 3 is selected from: a 4- to 8-membered heterocyclic group, -N(C 0-10 (alkyl)(C 0-10 (alkyl), -O(C 0-10 (alkyl), -S(C 0-10 (alkyl), -SO 2 (C 0-10 (alkyl); wherein the H on the alkyl and heterocyclic group is optionally substituted by one or more groups selected from the following: H, =O, halogen, C 1 -C 6 (alkyl), -O(C 0-6 (alkyl).

[0103] More specifically, in the definition of R 3 , the heterocyclic group is a nitrogen-containing heterocyclic group, which can be saturated or unsaturated. For example,

[0104] In some embodiments of the present invention, R 2b is wherein a is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, 6), and R 6 and R 7 are independently selected from: H, C 1 -C 6 (alkyl), or R 6 and R 7 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocycle (e.g., ), the H on the heterocycle is optionally substituted by one or more groups selected from the following: H, ═O, halogen, C 1 -C 6 alkyl.

[0105] In some embodiments of the present invention, the moiety is

[0106] In some embodiments of the present invention, L 3 is a single bond or C(O).

[0107] In some embodiments of the present invention, the VEGFR degrader has the following structure:

[0108]

[0109] More specifically, the VEGFR degrader has the following structure:

[0110]

[0111] Specifically, in Formulas II and III, GRO, L 1 and L 2 are as described above.

[0112] Specifically, in Formulas II and III, L 1 is connected to any base, sugar or phosphate backbone at the 3'-end, 5'-end or in the middle of GRO.

[0113] In some embodiments of the present invention, L 1 is a single bond or O, that is, GRO is connected to L through a phosphoester bond (i.e., 2 ).

[0114] In some embodiments of the present invention, L 2 adopts the above-mentioned scheme (1) and / or (2), especially scheme (1), for example

[0115] In one embodiment of the present invention, the VEGFR degrader has the following structure:

[0116]

[0117] In one embodiment of the present invention, the VEGFR degrader has the following structure:

[0118]

[0119] In other embodiments of the present invention, the VEGFR degrader has the following structure:

[0120]

[0121]

[0122]

[0123] In a second aspect of the present invention, there is provided a method for preparing the VEGFR degrader described in the first aspect, which includes the step of coupling a small molecule ligand moiety with GROs through L.

[0124] In some embodiments of the present invention, the preparation method includes the following steps: first prepare connect it to a solid support, and then synthesize an oligonucleotide through solid-phase synthesis to obtain the target product;

[0125] L' is an optionally suitable linking group;

[0126] R F is a protected hydroxyl group.

[0127] 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 as shown.

[0128] Specifically, in the solid-phase synthesis, L' contains (for example )

[0129] In some embodiments of the present invention, R F is -ODMTr.

[0130] In some embodiments of the present invention, the solid support is controlled pore glass beads (CPG), and the pore size of the CPG depends on the length of the synthesized oligonucleotide.

[0131] In some embodiments of the present invention, the preparation method includes the following steps: first prepare react and connect with GROs; or,

[0132] first prepare GRO-L'-R F , and then connect it with a small molecule ligand; or,

[0133] separately prepare GRO-L”R F ' and then couple the two;

[0134] Among them, L', L", L''' are optionally suitable linking groups, and R F , R F ', R F " are optionally suitable reactive groups.

[0135] In the third aspect of the present invention, a pharmaceutical composition comprises the VEGFR 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.

[0136] 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.

[0137] Specifically, the pharmaceutical composition can be administered by any suitable route, 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.).

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Specifically, in the pharmaceutical composition, the PROTAC can be used alone or in combination with other types of active ingredients.

[0142] Specifically, the amount of the active ingredient in the unit dose formulation can be varied 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 desired, the composition may further comprise other suitable therapeutic agents.

[0143] In a fourth aspect of the present invention, there is provided a delivery system for a PROTAC, which comprises the above-mentioned VEGFR degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof and a carrier, and can deliver the VEGFR degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof to target cells or target tissues etc. without NCL expression on the cell surface.

[0144] 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 carriers (such as lentivirus, adenovirus, adeno-associated virus vectors), non-viral carriers (such as lipid nanoparticles (LNP), polymeric nanocarriers, inorganic nanocarriers, protein carriers, exosomes, etc.).

[0145] In some embodiments of the present invention, the carrier is a lipid nanoparticle (LNP).

[0146] In a fifth aspect of the present invention, there is provided the use of the above-mentioned VEGFR degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the preparation of a drug for preventing and / or treating VEGFR-related diseases.

[0147] Specifically, the diseases are those for which degradation of VEGFR can be beneficial for their prevention and / or treatment, such as, but not limited to, tumors, autoimmune diseases, inflammatory diseases, diseases related to pathogen infections, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, fibrotic diseases, ocular diseases, etc.

[0148] Specifically, the tumors include, but are not limited to: lung cancer, malignant melanoma, brain tumors, tumors of the digestive organs, uterine cancer, testicular cancer, maxillary cancer, throat cancer, tongue cancer, oral cancer, various sarcomas, osteosarcoma, hematological malignancies, nervous system tumors, glioblastoma multiforme, glioblastoma, glioma, skin cancer, skin appendage cancer and skin metastases, medulloblastoma, blastoma, liposarcoma, neuroendocrine tumors, synovial sarcoma, gastrinoma, carcinoid tumors, 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, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, thyroid cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, prostate cancer, salivary gland adenocarcinoma, renal cancer, vulvar cancer, anal cancer, penile cancer, esophageal cancer, biliary tract tumors and head and neck cancer.

[0149] Specifically, the hematological malignancies include: leukemia, lymphoma, multiple myeloma (MM).

[0150] 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 lymphoblastic 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.

[0151] Specifically, 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 ( 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, particularly diffuse large B-cell lymphoma (DLBCL).

[0152] In some embodiments of the present invention, the tumor is glioma.

[0153] 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.

[0154] 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, Herxheimer reaction, encephalitis, myelitis, meningitis, and malaria, etc.

[0155] 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.

[0156] Specifically, the pathogen can be a microorganism, a parasite (protozoa, worms, etc.) or other agents.

[0157] Specifically, the microorganism can be selected from one or more of: virus, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi, etc.

[0158] 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 variola 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).

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Specifically, the ocular diseases include but are not limited to: ischemic retinopathy, iris neovascularization, intraocular neovascularization, age-related macular degeneration, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic retinal ischemia or proliferative diabetic retinopathy, etc.

[0164] In particular, the diseases are selected from: ovarian cancer, breast cancer, colorectal cancer, renal cancer, lung cancer, sarcoma, glioblastoma, glioma.

[0165] In the sixth aspect of the present invention, a method for treating VEGFR-related diseases is provided, which includes the step of administering to a subject in need thereof the VEGFR 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.

[0166] Specifically, the diseases are as described in the fifth aspect of the present invention.

[0167] Specifically, the subject is a mammal, especially a human.

[0168] Specifically, the administration can be carried out by any suitable administration route, such as gastrointestinal 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.

[0169] In the present invention, the VEGFR includes VEGFR1, VEGFR2, VEGFR3, preferably VEGFR2.

[0170] The present invention provides a PROTAC for degrading VEGFR prepared by using GROs as a novel recruitment element of MDM2, and its preparation method and application. The inventors found a new function of GROs in recruiting MDM2 through research, and used it to prepare a PROTAC targeting VEGFR, which can effectively degrade VEGFR and produce corresponding curative effects (such as inhibiting the proliferation of tumor cells and promoting apoptosis of tumor cells), and has very good application prospects and research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1 The figure shows the result that NCL can bind to MDM2.

[0172] Figure 2 The figure shows the result that AS1411 is a GRO with stable G4 structural features.

[0173] Figure 3 The figure shows the result of the weak G4 structural features of iSN04.

[0174] Figure 4 A shows the figure of the result that CRO cannot capture NCL and MDM2; Figure 4 B shows the figure of the result that AS1411 captures NCL and MDM2 in a concentration-dependent manner; Figure 4 C shows the figure of the result that only high-concentration iSN04 can capture NCL, but iSN04 cannot capture MDM2.

[0175] Figure 5 A shows the figure of the result that CRO can neither bind to NCL nor recruit MDM2; Figure 5 B shows the figure of the result that AS1411 recruits 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.

[0176] Figure 6 A shows the figure of the result that AS1411 does not affect the interaction between NCL and MDM2; Figure 6 B shows the figure of the result that iSN04 blocks the binding between NCL and MDM2.

[0177] Figure 7 The figure shows the result that AS1411 can specifically recognize glioma cells in vitro.

[0178] Figure 8 The figure shows the result that AS1411 can enter glioma cells in vitro.

[0179] Figure 9 The figure shows the result that AS1411 can penetrate and enter the interior of glioma cell spheres.

[0180] Figure 10 The figure shows the result that AS1411 can specifically target glioma in vivo without entering normal brain tissue.

[0181] Figure 11 The figure shows the result that AS1411 can specifically accumulate in glioma in vivo.

[0182] Figure 12 The figure shows the results of a large amount of angiogenesis in gliomas and high expression of NCL in gliomas and vascular endothelial cells.

[0183] Figure 13 The figure shows the results of AS1411 being able to penetrate the blood-tumor barrier.

[0184] Figure 14 The figure shows the results of AS1411 recruiting MDM2 and silencing NCL can block the recruitment of MDM2 by AS1411.

[0185] 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 VEGFR formed by GROs (such as AS1411) as recruitment elements of MDM2.

[0186] Figure 16 The figure shows the results of AS1411-Ced degrading VEGFR.

[0187] Figure 17 The figure shows the results of AS1411-Ced promoting the ubiquitination of VEGFR.

[0188] Figure 18 The figure shows the results of AS1411-Ced mediating the degradation of VEGFR through the ubiquitin-proteasome pathway.

[0189] Figure 19 The figure shows the results of AS1411-Ced mediating the degradation of VEGFR depending on MDM2 and NCL.

[0190] Figure 20 The figure shows the results of AS1411-Ced being able to inhibit the proliferation of U87MG cells and promote the apoptosis of U87MG cells.

[0191] Figure 21 The figure shows the results of AS1411-Ced being able to inhibit the proliferation of U251 cells and promote the apoptosis of U251 cells.

[0192] Figure 22 The figure shows the results of AS1411-Ced being able to inhibit the growth of gliomas in vivo and improve the survival rate of experimental animals. Detailed implementation manners

[0193] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0194] The term "alkyl" refers to a straight-chain or branched-chain hydrocarbon radical that does not contain unsaturated bonds, and the hydrocarbon radical is connected to the rest 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, i.e., C 0-10 alkyl (or C 0 -C 10 alkyl) includes H and C 1-10 alkyl (or C 1 -C 10 alkyl).

[0195] 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 the rest of the molecule by a single bond. In this context, typical alkylene 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 methylene (-CH 2 -), ethylene, propylene, butylene, etc. In the present invention, C 0 alkylene refers to a single bond, i.e., 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).

[0196] The term "cycloalkyl" refers to an alicyclic hydrocarbon, such as a monocyclic and / or fused-ring containing 1 to 4 rings and 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.

[0197] 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.

[0198] 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

[0199] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0200] The term "haloalkyl" refers to a group formed by replacing one or more hydrogens in an alkyl group with halogen atoms (such as 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 。

[0201] The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing monoaryl groups and / or fused aryl groups, such as those containing 1 - 3 monocyclic or fused rings and 6 - 18 (such as 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl group of C 6 -C 12 in the present invention refers to an aryl group containing 6 - 12 carbon ring atoms, such as phenyl, naphthyl, biphenyl, indenyl, etc.

[0202] 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

[0203] In the present invention, "D" refers to deuterium; "substituted with deuterium" means substituting one or more hydrogen atoms with the corresponding number of deuterium atoms.

[0204] It should be recognized that depending on the source of the chemical materials used in the synthesis, there will be some variation in the natural isotope abundances in the synthesized compounds. Thus, the compounds of the present invention will inherently contain minor amounts of deuterated isotopologues. Despite such variation, the concentration of the stable hydrogen and carbon isotopes at natural abundance is low and inconsequential compared to the degree of stable isotope substitution in the compounds of the present invention. See, for example, Wada, E et al., Seikagaku, 1994, 66:15; Gannes, LZ et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.

[0205] 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 at 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).

[0206] As used herein, the term "isotope enrichment factor" refers to the ratio of the isotope abundance of a particular isotope to its natural abundance.

[0207] 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.

[0208] 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.

[0209] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0210] 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 include salts of amino acids such as arginine salts, gluconates, galacturonates, 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 separating the free base in a conventional manner.

[0211] The term "base addition salt" refers to salts formed with metals or amines, such as the 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 separating the free acid in a conventional manner.

[0212] The term "stereoisomers" includes enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cycloalkyl groups that can 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.

[0213] The term "solvate" refers to the physical association of a compound of the present invention with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, solvates 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.

[0214] 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.

[0215] In the present invention, the term "oligonucleotide" consists of within 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 nano-luminescent material, a nucleic acid substance, or an enzyme label.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] The term "prevention" refers to, before the onset of a disease, treating to avoid, minimize, or make it difficult for the disease to occur or develop.

[0221] The term "tumor" refers to an abnormal mass of tissue, where the growth of the mass exceeds and is not coordinated with the growth 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 "preneoplastic tumors". "Malignant tumors" are usually poorly differentiated (anaplastic) and are characterized by a characteristic 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.

[0222] The term "cancer" refers to malignant tumors (Stedman’s Medical Dictionary, 25th ed.; Hensyle d.; Williams & Wilkins: Philadelphia, 1990).

[0223] The term "autoimmune disease" refers to a disease caused by the body's immune reaction against its own antigens, resulting in damage to its own tissues.

[0224] 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 reactions (such as various types of hypersensitivity reactions, inflammation caused by some autoimmune diseases). The term "inflammatory disease" refers to a disease with inflammation.

[0225] The term "diseases associated with 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, 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).

[0226] The term "cardiovascular disease" refers to a group of diseases involving the heart or blood vessels.

[0227] 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 processes in the body are disordered.

[0228] 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.

[0229] All publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

[0230] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0231] The sequences involved in the following embodiments are shown in the table below:

[0232] Table 1 Sequence names and numbers

[0233] 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

[0234] Example 1: NCL can bind to MDM2

[0235] (1) Co-Immunoprecipitation (Co-IP) is a classic method for studying protein-protein interactions based on the specific interaction between antibodies and antigens. After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), incubate overnight at 4°C with an antibody against NCL. 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 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 maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, take the supernatant into 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 a primary antibody against MDM2 (purchased from proteintech, catalog number 27883-1-AP) or a 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 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). Figure 1The results of A indicate that NCL can bind to MDM2.

[0236] (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 three 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 immunoblotting to detect whether NCL binds to MDM2. Figure 1 The results of B indicate that NCL can bind to MDM2.

[0237] Example 2: AS1411 is a GRO with a stable G4 structural feature

[0238] GROs contain one or more GGT motifs and have a stable G4 structural feature. 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 having the above-mentioned stable G4 structural feature 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 various ligand molecules, mediating the entry of ligand molecules into diseased cells (such as tumors, etc.).

[0239] (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. 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).

[0240] (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 and 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. 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).

[0241] The above results indicate that iSN04 can form a weak G4 structure only in the presence of K+, and cannot form G4 structural characteristics in the absence of K+, but AS1411 can form stable G4 structural characteristics in the presence or absence of K+.

[0242] Example 3: AS1411 recruits MDM2 depending on its interaction with NCL

[0243] (1) The procedure of the pull-down experiment is to immobilize a substance with a 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), they were incubated 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 streptavidin agarose gel beads (purchased from cytiva, catalog number 17511301) were added and the incubation continued 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), SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015) was added, heated to 100°C and maintained for 10 minutes. After centrifugation, the supernatant was taken, which was the pull-down product. Immunoblotting was used to detect the pull-down product.

[0244] Figure 4 The results of A showed that CRO could neither capture NCL nor MDM2; Figure 4 The results of B showed that AS1411 could capture NCL and MDM2 in a concentration-dependent manner; Figure 4 The results of C showed that high-concentration iSN04 could capture NCL, but iSN04 could not capture MDM2 within the concentration range used.

[0245] (2) After mixing 6 μg / mL recombinant human NCL and 6 μg / mL recombinant human MDM2 and incubating them at 4°C for 7 hours, 400 nM of 5'-biotinylated CRO, AS1411, or iSN04 was added, incubated at 4°C for 6 hours, and streptavidin agarose gel beads (purchased from cytiva, catalog number 17511301) were added and the incubation continued at 4°C overnight. The gel beads were washed 4 times with TBST buffer, SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015) was added, heated to 100°C and maintained for 10 minutes. After centrifugation, the supernatant was taken. Then, immunoblotting was used to detect the pull-down product. Figure 5 The results of A showed that CRO could neither bind to NCL nor MDM2. Figure 5 The results of B showed that AS1411 recruited a large amount of MDM2 depending on its interaction with NCL. Figure 5The results of C showed that iSN04 could only bind to NCL, but could not recruit MDM2 depending on its interaction with NCL.

[0246] (3) After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), incubate them with different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 20 μM) of AS1411 or iSN04 at 4 °C for 6 hours, then add 5 μL of NCL antibody and continue to incubate overnight at 4 °C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate and incubate at room temperature for 1.5 hours. Wash the NCL 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 keep for 10 minutes. Adsorb the magnetic beads with a magnetic stand, take the supernatant into a new tube. Then perform immunoblotting (western blot) to detect whether NCL binds to MDM2 and whether this binding is affected by AS1411 or iSN04. Figure 6 The results of A showed 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 showed that iSN04 blocked the binding between NCL and MDM2.

[0247] Example 4: AS1411 can target glioma cells

[0248] (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. Seed the three types of cells into 6-well plates (purchased from Corning, catalog number 3335) respectively and culture overnight. Add 200 nM Cy5-labeled NC or AS1411 respectively and incubate for 6 h. After washing with PBS, use a flow cytometer (BD, model BD FACSCanto SORP) for analysis to observe the binding of NC or AS1411 to the three types of cells. The results are 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.

[0249] (2) Glioma cells U87MG, U251 and normal human mammary epithelial cells MCF 10A were respectively inoculated on cell culture slides (purchased from BIOLOGIX, product number: 07 - 2101) and cultured overnight. 200 nM of Cy5-labeled NC or AS1411 was added respectively and incubated for 6 h. After washing with PBS, the slides were sealed with an anti-fluorescence quenching mounting medium (purchased from Beyotime, product number P0126) and observed under a confocal fluorescence microscope (ZEISS, model 980) to check the entry of NC or AS1411 into the 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. This indicates that AS1411 can specifically enter glioma cells.

[0250] (3) Glioma U87MG or U251 cells were inoculated in 96-well spherical microporous plates (purchased from Corning, product number 4520) and cultured for 3 days. 200 nM of 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 relatively strong penetrability into the 3D cell spheres formed by U87MG or U251.

[0251] (4) 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 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. In the control group, nude mice were not injected with U87MG-LUC-GFP cells. Seven days after modeling, 0.2 μmol / kg of Cy5-labeled AS1411 was injected via the tail vein, and in vivo imaging of the 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 9 shown. AS1411 could specifically target glioma in vivo but could not enter the normal brain tissue of the control nude mice, indicating that AS1411 can be used for in vivo imaging, diagnosis, and detection of glioma.

[0252] (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 A. AS1411 was significantly enriched in gliomas, while the NC sequence could not be enriched in gliomas. It indicates that AS1411 can be used for in vivo imaging, diagnosis, and detection of gliomas.

[0253] (6) Cy5-labeled AS1411 or NC sequence was injected into the orthotopic glioma nude mice 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 B. NC had no obvious distribution in either glioma cells or non-tumor cell areas in the brain tissue; AS1411 was significantly enriched in glioma cells and had no distribution in the non-tumor cell areas in the brain tissue, indicating that AS1411 can be used for in vivo imaging, diagnosis, and detection of gliomas.

[0254] Seven days after establishing an orthotopic glioma nude mouse model using U87MG-LUC-GFP cells, the brain tissue was collected. After fixation with formalin and dehydration using a sucrose gradient, it was embedded with OCT embedding medium (purchased from SUKURA, catalog number 4583). Sections were made using a cryostat (Leica, model CM1950). It was permeated with permeation solution (purchased from Beyotime, catalog number P0095) for 15 minutes, blocked with blocking solution (purchased from Beyotime, catalog number P0252) for 1 hour, and incubated overnight at 4°C with the primary antibody against NCL (purchased from Cell Signaling Technology, catalog number 14574S) and the primary antibody against the vascular endothelial cell marker CD31 (purchased from Abcam, catalog number ab28364). After washing with PBS, it was incubated with a fluorescently labeled secondary antibody (purchased from Abcam, catalog number ab150077 or ab150080) for 1 hour at room temperature, sealed with an anti-fluorescence quenching mounting medium (purchased from Beyotime, catalog 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.

[0255] Example 5: AS1411 can penetrate the blood-tumor barrier

[0256] The blood-brain barrier (BBB) consists 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 harmful substances from entering 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 compared to normal endothelial cells in the healthy BBB. These brain tumor-associated endothelial cells are less prone to apoptosis, are resistant to cytotoxic therapy, and actively participate in the abnormal reorganization of the BTB.

[0257] (1) We constructed in vitro blood-tumor barrier and blood-brain barrier models. Specifically, as Figure 13As shown in A: We inoculated glioma cells U87MG or normal glial cells and brain microvascular endothelial cells bEnd.3 into a co-culture system respectively. Among them, brain microvascular endothelial cells bEnd.3 were inoculated into the upper chamber of the co-culture system, and glioma cells U87MG or normal glial cells were inoculated into 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 μΜ 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 co-cultured with normal glial cells, AS1411 was more likely to penetrate the bEnd.3 cell layer (blood-tumor barrier) co-cultured with glioma cells U87MG. This is consistent with the data that AS1411 can specifically target gliomas in vivo but cannot enter normal brain tissue mentioned above.

[0258] (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, and detected 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. It shows that there is a positive correlation between NCL in glioma cells and NCL in brain microvascular endothelial cells.

[0259] (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 μΜ 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.

[0260] (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 incubated for another 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.

[0261] 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.

[0262] Example 6: Silencing NCL can block the recruitment of MDM2 by AS1411

[0263] (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 rack, and the supernatant was taken into a new tube. Then, immunoblotting experiments were performed.

[0264] The results are as Figure 14 shown in Figure A. NCL can interact with MDM2, but does not bind to VEGFR2.

[0265] (2) Incubate glioma U87MG cells with 500 nM biotin-labeled negative control CRO (Bio-CRO) or AS1411 (Bio-AS1411) 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 assay.

[0266] The results are as Figure 14 shown in B. AS1411 can capture NCL and MDM2.

[0267] (3) Transfect negative control siRNA (siNC) or NCL siRNA (siNCL) into glioma U87MG cells. 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 assay.

[0268] The results are as Figure 14 shown in C. Silencing NCL can block the recruitment of MDM2 by AS1411.

[0269] Example 7: Preparation of GROs-based PROTAC molecules targeting VEGFR.

[0270] We respectively simulated the three-dimensional structure diagrams of AS1411 and NCL-MDM2 complex using Discovery Studio and AlphaFold2, 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 VEGFR formed by GROs as a recruitment element of MDM2 is as Figure 15 shown in B.

[0271] In this example, AS1411, a typical representative of GROs, was used as the E3 ligase recruitment element, and Cediranib (Ced), a VEGFR inhibitor, was used as an example of the target protein ligand to prepare a PROTAC for degrading VEGFR (hereinafter simply referred to as AS1411-Ced).

[0272] The preparation steps are as follows:

[0273] (1) Synthesis of Compound 2

[0274]

[0275] Under a nitrogen atmosphere at 25 °C, a solution of acetic anhydride (33 mL, 1060.8 mmol) was added to a solution of Compound 1 (10.2 g, 156 mmol) in pyridine (8.3 mL). The mixture was stirred at 90 °C for 2 hours, and LCMS showed that the reaction was complete. The mixture was triturated with 500 mL of water at 25 °C for 10 min, filtered, and the filter cake was dried to obtain Compound 2 as a white solid (10.2 g, yield: 83.69%).

[0276] LCMS: m / z = 235.2 [M+H] + t R = 1.024 min. Purity: 84.47% (254 nm).

[0277] (2) Synthesis of Compound 3

[0278]

[0279] Under a nitrogen atmosphere at 25 °C, DMF (3 mL) was added to a solution of Compound 2 (10.2 g, 43.55 mmol, 1.0 eq) in SOCl 2 (100 mL). The mixture was stirred at 80 °C for 2 hours. LCMS showed that the reaction was complete. The reaction mixture was filtered and concentrated under reduced pressure to obtain Compound 3 as a yellow solid (10.5 g, crude product).

[0280] LCMS: m / z = 253.0 [M+H] + ,t R = 1.340 min. Purity: crude

[0281] (3) Synthesis of Compound 4

[0282]

[0283] Under a nitrogen atmosphere at 25 °C, NH 3 / MeOH (300 mL, 10%). The mixture was then stirred at 80 °C for 10 minutes. LCMS showed that the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give Compound 4 as a yellow solid (4.3 g, yield: 50.57%).

[0284] LCMS: m / z = 211.0 [M+H] + , t R = 1.123 min. Purity: 94.94% (254 nm).

[0285] (4) Synthesis of Compound 6

[0286]

[0287] At 0 °C under a nitrogen atmosphere, DBAD (1.84 mg, 6.18 mmol) was added to a solution of Compound 4 (4.3 g, 20.43 mmol), Compound 5 (2.77 g, 21.44 mmol) and PPh3 (6.9 g, 26.55 mmol) in DCM (70 mL). The mixture was then stirred at 25 °C for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated. The residue was quenched with water (200 mL) and extracted with ethyl acetate (200 mL × 2). The organic layer was washed with brine (200 mL), dried over anhydrous Na 2 SO 4 and filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (ethyl acetate in PE = 0 to 100%) to give Compound 6 as a yellow solid (4.5 g, yield: 63.8%).

[0288] LCMS: m / z = 322.2 [M+H] + , t R = 1.085 min. Purity: 100% (254 nm).

[0289] (5) Synthesis of Compound 8

[0290]

[0291] At 25 °C under a nitrogen atmosphere, Compound 7 (2.6 g, 15.72 mmol) and DMAP (3.9 g, 28.58 mmol) were added to a solution of Compound 6 (4.5 g, 14.29 mmol) in DMSO (50 mL). The mixture was stirred at 80 °C for 3 hours. LCMS showed that the reaction was complete. The mixture was concentrated, the residue was quenched with water (200 mL), and extracted with ethyl acetate (200 mL × 2). The organic layer was washed with brine (200 mL × 1), dried over anhydrous Na 2 SO4 Dry, filter and concentrate in vacuo. Purify the crude product by silica gel column chromatography (ethyl acetate in PE = 0 to 100%) to obtain Compound 8 (2 g, crude) as a yellow solid.

[0292] LCMS: m / z = 451.4 [M+H] + , t R = 1.085 min. Purity: 88.20% (254 nm).

[0293] (6) Synthesis of Compound 10

[0294]

[0295] Under a nitrogen atmosphere at 0 °C, add NaH (266 mg, 6.66 mmol, 60% dispersed in mineral oil) to a solution of Compound 8 (2 g, 4.44 mmol, 1.0 eq, Cediranib) in DMF (20 mL). After 30 minutes, add Compound 9 (1.4 g, 6.66 mmol), and stir the mixture at 25 °C for 3 hours. LCMS shows that the reaction is complete. Concentrate the mixture. Quench the residue with water (200 mL) and extract with ethyl acetate (200 mL × 2). Wash the organic layer with brine (200 mL), dry over anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. Purify the crude product by silica gel column chromatography (ethyl acetate in PE = 0 to 100%) to obtain Compound 10 (3.3 g, crude) as a yellow solid.

[0296] LCMS: m / z = 579.4 [M+H] + , t R = 1.440 min. Purity: 74.40% (254 nm).

[0297] (7) Synthesis of Compound 11

[0298]

[0299] Under a nitrogen atmosphere at 0 °C, add NaOH (230 mg, 5.8 mmol) to a mixture of Compound 10 (3.3 g, crude) in THF (20 mL) and H 2 O (10 mL). Stir the mixture at 25 °C for 3 hours. LCMS shows that the reaction is complete. Concentrate the mixture. Extract the residue with ethyl acetate (200 mL × 2). Wash the organic layer with brine (100 mL), dry over anhydrous Na 2 SO 4Dry, filter and concentrate in vacuo. Purify the crude product by silica gel column chromatography (ethyl acetate in PE = 0 to 100%) to give compound 11 as a yellow solid (1 g, 31.06%).

[0300] LCMS: m / z = 565.2 [M+H] + , t R = 1.362 min. Purity: 100% (254 nm).

[0301] (8) Synthesis of compound 12

[0302]

[0303] To a solution of compound 11 (950 mg, 1.68 mmol) in DCM (15 mL) was added DIEA (869 mg, 6.73 mmol). The mixture was stirred at 25 °C for 10 minutes under an argon atmosphere. Then 2,2,2-trifluoroacetic acid perfluorophenyl ester (941 mg, 3.36 mmol) was added, and the mixture was stirred overnight at 25 °C under an argon atmosphere. LCMS showed that the reaction was complete. The mixture was extracted with DCM and diluted with water. The organic layer was washed with saturated sodium chloride and dried over anhydrous Na 2 SO 4 After filtration, the organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with MeOH:DCM = 15% to give the product (white solid, 1100 mg, 1.51 mmol, yield: 89.7%).

[0304] LCMS: m / z = 731.2 [M+H] + t R = 1.638 min. Purity: 77.8% (254 nm).

[0305] (9) Synthesis of compound 14

[0306]

[0307] To a solution of compound 12 (1100 mg, 1.51 mmol) in MeCN (25 mL) was added DIEA (780 mg, 6.04 mmol). The mixture was stirred at 25 °C for 10 minutes under an argon atmosphere. Then (3S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (949 mg, 2.27 mmol) was added. The mixture was stirred overnight at 25 °C under an argon atmosphere. LCMS showed that the reaction was complete. The mixture was diluted with water (100 mL) and extracted with DCM (100 mL × 3). The organic layer was washed with brine (100 mL) and dried over anhydrous Na 2SO 4 Dry. After filtration, the organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with MeOH:DCM = 15%, to give the product (white solid, 800 mg, 0.83 mmol, yield: 54.8%).

[0308] LCMS: m / z = 966.5 [M+H] + t R = 1.438 min. Purity: 95% (254 nm).

[0309] (10) Synthesis of Compound 15

[0310]

[0311] To a solution of compound 14 (800 mg, 0.83 mmol) in DCM (15 mL) was added DMAP (405 mg, 3.32 mmol). The mixture was stirred at 25 °C for 10 min under an argon atmosphere. Then dihydrofuran-2,5-dione (124 mg, 1.24 mmol) was added to the system and the mixture was stirred at 25 °C for 5 h under an argon atmosphere. LCMS showed that the reaction was complete. The mixture was diluted with water (100 mL) and extracted with DCM (100 mL×3). The organic layer was washed with brine (100 mL) and dried over anhydrous Na 2 SO 4 Dry. After filtration, the organic layer was concentrated under reduced pressure. The residue was purified by reverse-phase chromatography [ACN / water (T ethyl acetate: 0.02%): 5% - 30%] to give the desired product (white solid, 700 mg, 0.657 mmol, yield: 79%).

[0312] LCMS: m / z = 1064 [M-H] - t R = 2.015 min. Purity: 100% (214 nm).

[0313] 11H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 7.60 (s, 1H), 7.39 (s, 1H), 7.36–7.14 (m, 10H), 7.09–6.98 (m, 1H), 6.88 - 6.85 (m, 4H), 6.31 - 6.33 (m, 1H), 5.35 - 5.25 (m, 1H), 4.26 (t, J = 6.4 Hz, 2H), 4.16 - 4.22 (m, 1H), 4.07 - 4.14 (m, 2H), 3.99 (s, 3H), 3.79–3.66 (m, 7H), 3.55 (d, J = 11.6 Hz, 1H), 3.20 - 3.32 (m, 1H), 3.02 - 3.07 (m, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.46 - 2.37 (m, 9H), 2.27 - 2.18 (m, 3H), 2.03 - 1.96 (m, 4H), 1.72 - 1.62 (m, 6H), 1.58 - 1.48 (m, 2H), 1.43 - 1.33 (m, 2H).

[0314] (11) Synthesis of Compound 15-CPG

[0315]

[0316] 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 15 (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 CAN. 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, and the mixture was shaken at room temperature for 1 hour. Then the mixture was filtered and washed three times with ACN (2 mL). The mixture was freeze-dried to obtain 15-CPG (1000 mg) as a white powder.

[0317] (12) Synthesis of Compound AS1411-Ced

[0318]

[0319] 15-CPG was synthesized by a K-AH-8 solid-phase synthesizer in a synthesis column (60 mg * 8). The solid-phase synthesis includes four steps: detritylation, coupling, capping, and oxidation. After the reaction was completed, a mixed solution of 1.5 mL of ammonia water and TEA (ammonia water:TEA = 10:1) was added to the CPG in each synthesis column, and it was heated in an oven at 55 °C for 3 hours. Then the supernatant was collected and washed with water (1 mL * 3). The crude product was purified by reversed-phase ion-pair high-performance liquid chromatography (Waters 2489, 3767) (chromatographic column: XBridge PrepC18 10 μm OBDTM) (mobile phase A: 0.1% T ethyl acetate + 2% HFIP aqueous solution, mobile phase B: methanol) to obtain AS1411-Ced, a white powder (6.66 mg, purity = 96.03%).

[0320] UPLC-MS (WATERS ACQUITY PREMIER): AS1411-Ced-UPLC, m / z = 8997.77465 [M] - (deconvolution);

[0321] t R = 12.044 min (260 nm). Mass error < 50 ppm.

[0322] HPLC: AS1411-Ced-HPLC, t R = 11.663 min (260 nm), purity: 96.034%.

[0323] Example 8: AS1411-Ced can degrade VEGFR

[0324] (1) Glioma U87MG cells were incubated with PBS or 500 nM AS1411-Ced (prepared in Example 7) for 6 hours. After cell lysis, they were incubated overnight at 4 °C with an antibody against VEGFR2 (purchased from Cell Signaling Technology, catalog number 2479S). Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) were added and incubated with rotation at room temperature for 2 hours. The VEGFR2 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 it was heated to 100 °C for 10 minutes. The magnetic beads were adsorbed with a magnetic rack, and the supernatant was taken into a new tube. Then an immunoblotting experiment was carried out. The results are as Figure 16As shown in A, in the presence of AS1411-Ced, the antibody against VEGFR2 can capture VEGFR2, NCL, and MDM2, indicating that AS1411-Ced can form a MDM2-NCL-PROTAC-VEGFR2 quaternary complex with NCL, MDM2, and VEGFR2.

[0325] (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-Ced for 6 hours. After cell lysis, incubate with the antibody against VEGFR2 (purchased from Cell Signaling Technology, catalog number 2479S) at 4 °C overnight. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. Wash the VEGFR2 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, take the supernatant to a new tube. Then perform immunoblotting experiments. The results are as Figure 16 As shown in B, in the presence of AS1411-Ced, the antibody against VEGFR2 cannot capture MDM2 after knocking down NCL, indicating that NCL is located between MDM2 and VEGFR2 in the quaternary complex induced by AS1411-Ced.

[0326] (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-Ced for 6 hours. After cell lysis, incubate with the antibody against VEGFR2 (purchased from Cell Signaling Technology, catalog number 2479S) at 4 °C overnight. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. Wash the VEGFR2 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, take the supernatant to a new tube. Then perform immunoblotting experiments. The results are as Figure 16As shown in C, in the presence of AS1411-Ced, the antibody against VEGFR2 can still capture NCL after knocking down MDM2, indicating that NCL is located between MDM2 and VEGFR2 in the quaternary complex induced by AS1411-Ced.

[0327] (4) Glioma U87MG cells were incubated with 0, 50, 100, 200, 500 nM of AS1411-Ced. After 18 hours, cell samples were collected and total proteins were extracted using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B). The degradation of VEGFR2 protein was detected by immunoblotting. The procedure was as follows: Protein samples were separated by SDS-PAGE electrophoresis, and the separated proteins were transferred to a PVDF membrane. After blocking with TBST buffer containing 5% skim milk at room temperature for 1 hour, the primary antibody against VEGFR2 (purchased from Cell Signaling Technology, catalog number 2479S) was incubated overnight at 4°C. After washing with TBST, the membrane was incubated with an HRP-labeled secondary antibody (purchased from: Abclonal, catalog number: AS014) at room temperature for 1 hour. The protein band blots were visualized using an enhanced chemiluminescence detection kit (purchased from: Abclonal, catalog number: RM00021P). The results were as Figure 16 shown in D. AS1411-Ced can dose-dependently reduce the protein level of VEGFR2.

[0328] (5) Glioma U87MG cells were incubated with 200 nM AS1411-Ced. At time points of 0, 3, 6, 12, 18, 24 hours, corresponding cell samples were collected for protein extraction and subsequent immunoblotting to detect the degradation of VEGFR2 protein. The results were as Figure 16 shown in E. AS1411-Ced can time-dependently reduce the protein level of VEGFR2.

[0329] (6) Glioma U87MG cells were incubated with the solvent PBS or 200 nM AS1411, 200 nM cediranib (Ced, for short), AS1411 + Ced (a combination of 200 nM AS1411 and 200 nM cediranib), or 200 nM AS1411-Ced (prepared in Example 7). After 18 hours, cell samples were collected for protein extraction and subsequent immunoblotting to detect the degradation of VEGFR2 protein. The results were as Figure 16 shown in F. Only AS1411-Ced can reduce the protein level of VEGFR2.

[0330] (7) Glioma U251 cells were incubated with 0, 10, 50, 100, 200, 500 nM of AS1411-Ced. After 18 hours, cell samples were collected, and total proteins were extracted using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B). The degradation of VEGFR2 protein was detected by immunoblotting. The results are as Figure 16 shown in Figure G. AS1411-Ced could dose-dependently reduce the protein level of VEGFR2.

[0331] (8) Glioma U251 cells were incubated with 200 nM AS1411-Ced. At time points of 0, 3, 6, 12, 18, 24 hours, corresponding cell samples were collected for protein extraction and subsequent immunoblotting to detect the degradation of VEGFR2 protein. The results are as Figure 16 shown in Figure H. AS1411-Ced could time-dependently reduce the protein level of VEGFR2.

[0332] (9) Glioma U251 cells were incubated with solvent PBS or 200 nM AS1411, Ced, AS1411 + Ced, or AS1411-Ced. After 18 hours, cell samples were collected for protein extraction and subsequent immunoblotting to detect the degradation of VEGFR2 protein. The results are as Figure 16 shown in Figure I. Only AS1411-Ced could reduce the protein level of VEGFR2.

[0333] Example 9: AS1411-Ced Promotes the Ubiquitination of VEGFR

[0334] Glioma U87MG cells were incubated with solvent PBS or 200 nM of AS1411-Ced for 6 hours. After cell lysis, they were incubated overnight at 4°C with an antibody against VEGFR2 (purchased from Cell Signaling Technology, catalog number 2479S). 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 VEGFR2 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 it was 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, the ubiquitination level of VEGFR2 was detected by immunoblotting. The results are as Figure 17 shown. AS1411-Ced promoted the ubiquitination of VEGFR2.

[0335] Example 10: AS1411-Ced mediates the degradation of VEGFR through the ubiquitin-proteasome pathway

[0336] Glioma U87MG cells were incubated with solvent PBS or 200 nM AS1411-Ced for 18 hours, and 10 μM 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 can block the degradation of VEGFR2 by AS1411-Ced, indicating that the degradation of VEGFR2 by AS1411-Ced depends on the ubiquitin-proteasome pathway.

[0337] Example 11: AS1411-Ced depends on MDM2 and NCL to mediate the degradation of VEGFR

[0338] (1) Negative control siRNA (siNC) or NCL siRNA (siNCL) was transfected into glioma U87MG cells for 48 hours, and then the cells were incubated with solvent PBS or 200 nM AS1411-Ced for 18 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 can reduce the degradation of VEGFR2 by AS1411-Ced, indicating that AS1411-Ced depends on NCL to mediate the degradation of VEGFR2.

[0339] (2) Negative control siRNA (siNC) or MDM2 siRNA (siMDM2) was transfected into glioma U87MG cells, and then the cells were incubated with solvent PBS or 200 nM AS1411-Ced for 18 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 B. Silencing MDM2 can reduce the degradation of VEGFR2 by AS1411-Ced, indicating that AS1411-Ced depends on MDM2 to mediate the degradation of VEGFR2.

[0340] Example 12: AS1411-Ced inhibits the proliferation and promotes apoptosis of glioma in vitro

[0341] (1) Glioma U87MG cells were seeded in 96-well cell culture plates, and the culture medium containing solvent PBS or 200 nM AS1411, Ced, AS1411 + Ced, or AS1411 - Ced was changed daily during the experiment. A cell counting kit-8 (CCK-8) (purchased from MCE, catalog number HY-K0301) was used for cell proliferation experiments, and the absorbance values at 450 nm were measured using an enzyme-linked immunosorbent assay (ELISA) reader on the first, second, and third days. The results are as Figure 20 shown in Figure A. AS1411 - Ced can inhibit the proliferation of glioma U87MG cells.

[0342] (2) Glioma U87MG cells were seeded in six-well plates for cell apoptosis experiments. The culture medium containing solvent PBS or 200 nM AS1411, Ced, AS1411 + Ced, or AS1411 - Ced was changed daily during the experiment. 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 20 shown in Figure B. AS1411 - Ced can promote the apoptosis of U87MG cells.

[0343] (3) Glioma U251 cells were seeded in 96-well cell culture plates, and the culture medium containing solvent PBS or 200 nM AS1411, Ced, AS1411 + Ced, or AS1411 - Ced was changed daily during the experiment. A cell counting kit-8 (CCK-8) (purchased from MCE, catalog number HY-K0301) was used for cell proliferation experiments, and the absorbance values at 450 nm were measured using an enzyme-linked immunosorbent assay (ELISA) reader on the first, second, and third days. The results are as Figure 21 shown in Figure A. AS1411 - Ced can inhibit the proliferation of U251 cells.

[0344] (4) Glioma U251 cells were seeded in six-well plates for cell apoptosis experiments. The culture medium containing solvent PBS or 200 nM AS1411, Ced, AS1411 + Ced, or AS1411 - Ced was changed daily during the experiment. 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 - Ced can promote the apoptosis of U251 cells.

[0345] Example 13: Inhibition of glioma growth by AS1411 - Ced in vivo

[0346] 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, 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, with 5 mice in each group. They were respectively given PBS, AS1411 (4.5 μmol / kg), Ced (6 mg / kg), AS1411 + Ced, and AS1411-Ced (4.5 μmol / kg). The drugs were administered every other day for a total of 5 times. Ced was administered by gavage, and AS1411 and AS1411-Ced were administered via the tail vein. In vivo imaging (PerkinElmer, model IVIS Spectrum) 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 were as Figure 22 shown in A-22D. AS1411-Ced could inhibit the growth of glioma and improve the survival rate of experimental animals.

[0347] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0348] 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.

[0349] 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. A VEGFR degrading agent or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, wherein the VEGFR degrading agent has the following structure: in, GRO is a guanine-rich oligonucleotide residue that can specifically bind to nucleolin; L is a linker; Ring A is an 8-, 9-, 10-, 12- or 13-membered bicyclic or tricyclic ring, optionally containing 1-3 heteroatoms independently selected from O, N and S; R1 is one or more independent substituents on ring A selected from: H, =O, halogen, cyano, nitro, azido, 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), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -(C0-C6 alkylene)-N(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 alkyl)CO(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 alkyl)CON(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)SO2(C 0-10 alkyl), -(C0-C6 alkylene)-O(C 0-10 alkyl), -(C0-C6 alkylene)-S(C 0-10 alkyl), -(C0-C6 alkylene)-SO(C 0-10 alkyl), -(C0-C6 alkylene)-SO2(C 0-10 alkyl), -(C0-C6 alkylene)-SO2N(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-COO(C 0-10 alkyl), -(C0-C6 alkylene)-OCO(C 0-10 alkyl), -(C0-C6 alkylene)-CON(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-CO(C 0-10 alkyl); R2 is one or more independent substituents on the quinazoline ring selected from: H, halogen, cyano, nitro, azido, 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), C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -(C0-C6 alkylene)-N(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 alkyl)CO(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 alkyl)CON(C 0-10 alkyl), -(C0-C6 alkylene)-N(C 0-10 Alkyl)SO2(C 0-10 alkyl), -(C0-C6 alkylene)-O(C 0-10 alkyl), -(C0-C6 alkylene)-S(C 0-10 alkyl), -(C0-C6 alkylene)-SO(C 0-10 alkyl), -(C0-C6 alkylene)-SO2(C 0-10 alkyl), -(C0-C6 alkylene)-SO2N(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-COO(C 0-10 alkyl), -(C0-C6 alkylene)-OCO(C 0-10 alkyl), -(C0-C6 alkylene)-CON(C 0-10 Alkyl)(C 0-10 alkyl), -(C0-C6 alkylene)-CO(C 0-10 alkyl), or two R2 together with the carbon atom to which they are attached form a heterocyclic ring; X1 is selected from the group consisting of a single bond, C1-C6 alkylene, C1-C6 alkenylene, C1-C6 alkynylene, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N(R4)-(C0-C6 alkylene)-, -N(R4)C(O)-(C0-C6 alkylene)-, -C(O)-(C0-C6 alkylene)-, -C(S)-(C0-C6 alkylene)-, -CON(R4)-(C0-C6 alkylene)-, -SO2(C 0-10 alkyl)-; X2 is selected from: a single bond, C1-C6 alkylene, C1-C6 alkenylene, C1-C6 alkynylene, phenylene, C3-C 10 Cycloalkylene, 4-10 membered heterocyclylene, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N(R5)-(C0-C6 alkylene)-, -N(R5)C(O)-(C0-C6 alkylene)-, -C(O)-(C0-C6 alkylene)-, -C(S)-(C0-C6 alkylene)-, -CON(R5)-(C0-C6 alkylene)-, -SO2(C 0-10 alkyl)-; R3 is selected from: H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, C1-C 10 Halogenated alkoxy, C6-C 10 Aryl, C3-C 10 Cycloalkyl, 4-10 membered heterocyclic group, -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 wherein the H on the alkyl, aryl, cycloalkyl, or heterocyclic group is optionally substituted by one or more groups selected from the following: H, =O, halogen, cyano, nitro, azido, 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), C1-C 10 Haloalkyl, -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); R4 and R5 are independently selected from: H, C1-C 10 wherein the alkyl group is optionally substituted by one or more groups selected from the group consisting of halogen, cyano, -O(C 0-10 Alkyl), -N(C 0-10 Alkyl)(C 0-10 alkyl); Z is selected from: -O-, -NH-, -S-, -CH2-, a single bond; p is an integer from 1 to 100; Preferably, the GROs have a stable G4 structural feature.

2. The VEGFR 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 VEGFR degrading agent according to claim 1, characterized in that The GROs is AS1411.

4. The VEGFR degrading agent according to any one of claims 1 to 3, characterized in that L has the following structure: Wherein, L1 is a divalent group connected to GRO, which is 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, in which 0-6 methylene units 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 between 1 and 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 VEGFR 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-, -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 is 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 VEGFR degrading agent according to any one of claims 1 to 3, characterized in that 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. Particularly preferred 7. The VEGFR 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 VEGFR degrading agent according to any one of claims 4 to 7, characterized in that L1 is a single bond or O.

9. The VEGFR degrading agent according to any one of claims 4 to 7, characterized in that L3 is a single bond or -C(O)-.

10. The VEGFR degrading agent according to any one of claims 4 to 9, characterized in that Part of Preferably Among them, R 1a Selected from: H, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, preferably methyl; R 1b is selected from: H, hydroxyl, halogen, C6-C6 alkyl, C6-C6 haloalkyl, preferably halogen such as F; Part of Among them, R 2a Selected from: H, -O(C 0-6 Alkyl), preferably methoxy; R 2b for Preferably, X1 is selected from: a single bond, C1-C6 alkylene, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-; Preferably, X2 is selected from: a single bond, C1-C6 alkylene, C1-C6 alkenylene, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N(R5)-(C0-C6 alkylene)-; Preferably, R3 is selected from: 4-8 membered heterocyclic group, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO2(C 0-10 wherein the H on the alkyl or heterocyclic group is optionally substituted by one or more groups selected from the following: H, =O, halogen, C1-C6 alkyl, -O(C 0-6 alkyl).

11. The VEGFR degrading agent according to claim 1, characterized in that Part of Preferably, R 2b for wherein a is an integer of 1-6, R6 and R7 are independently selected from: H, C1-C6 alkyl, or R6 and R7 together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocyclic ring, wherein H on the heterocyclic ring is optionally substituted by one or more groups selected from: H, =O, halogen, C1-C6 alkyl; Preferably, L3 is a single bond or C(O).

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

13. The VEGFR degrading agent according to any one of claims 1 to 3, characterized in that The VEGFR degrading agent has the following structure: Wherein, h is selected from an integer between 0 and 10, and i is selected from an integer between 0 and 10; preferably, the VEGFR-2 degrading agent has the following structure:

14. A pharmaceutical composition comprising the VEGFR 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 a VEGFR degrader, comprising the VEGFR degrader according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof 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 VEGFR degrader 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 a VEGFR-related disease; 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, fibrotic diseases, and eye diseases; More preferably, the tumor is selected from the group consisting of lung cancer, malignant melanoma, brain tumor, tumor of digestive organs, uterine cancer, testicular cancer, palate cancer, pharyngeal cancer, tongue cancer, oral cancer, various sarcomas, osteosarcoma, blood system tumors, nervous system tumors, brain glioma, glioblastoma, glioma, skin cancer, skin appendage cancer and skin metastasis, medulloblastoma, blastoma, liposarcoma, neuroendocrine tumor, synovial cell 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, lung squamous cell carcinoma, hepatocellular carcinoma, stomach 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, prostate cancer, salivary gland cancer, kidney cancer, vulvar cancer, anal cancer, penis cancer, esophageal cancer, biliary tract tumors and head and neck cancer.

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