Novel BRD4 degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a novel BRD4 degradation agent as well as a preparation method and application thereof. The BRD4 degradation agent is PROTAC with GROs (such as AS1411) as an MDM2 recruitment element, target protein BRD4 and E3 ubiquitin ligase MDM2 can be pulled close in vivo, so that the BRD4 is labeled with ubiquitin, and then the BRD4 is degraded through a ubiquitin-proteasome pathway. Experiments show that the BRD4 degradation agent can effectively degrade BRD4 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 BRD4 degrader, a preparation method thereof, and an application thereof. Background Art

[0002] The bromodomain and extra-terminal (BET) family is a class of proteins that can specifically recognize acetylated lysine to regulate gene transcription. Bromodomain-containing protein 4 (BRD4) is a member of the BET family and can bind to acetylated histones or non-histones, thereby regulating gene replication and transcription and affecting processes such as the cell cycle, cell differentiation, and signal transduction. The N-terminus of BRD4 contains two tandem highly conserved bromodomains, namely BD1 and BD2, which can specifically bind to lysine acetylation residues. The C-terminus of BRD4 contains an extra-terminal domain that interacts with histone modification factors and is mainly responsible for regulating gene transcription. Studies have found that BRD4 plays an important role in cancers, metabolism, autoimmune, and inflammatory diseases, and has become an emerging therapeutic target for various diseases.

[0003] In recent years, a large number of BRD4 inhibitors have been developed and can be classified into two categories according to their binding modes, namely monovalent inhibitors and bivalent inhibitors. Monovalent BRD4 inhibitors can bind to the BD1 or BD2 domain and are mainly classified into: triazoles, isoxazoles, quinolinones, pyridones, and tetrahydroquinolines, etc., according to the characteristics of their chemical structures. Bivalent inhibitors can bind to both the BD1 and BD2 domains simultaneously. However, monovalent inhibitors can only inhibit a single bromodomain of BRD4, with relatively single structural modification, and the inhibition efficiency and disease treatment effect are limited. Although bivalent inhibitors improve the inhibition efficiency and disease treatment effect, they mainly connect two monovalent inhibitors through a flexible linker, with a relatively large molecular weight and poor drug-likeness. Moreover, inhibiting BRD4 protein requires maintaining the drug at a high concentration for a long time, and high-dose administration will cause BRD4 protein enrichment due to negative feedback, thus greatly weakening the inhibition effect and generating drug resistance. Therefore, there is a need to further develop more effective novel drugs against BRD4 protein.

[0004] Targeted protein degradation is a brand-new and breakthrough drug development strategy that utilizes the inherent protein degradation pathways within cells to directly degrade pathogenic target proteins. This novel 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 polyubiquitin 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 through multiple cycles within the cell. Based on such a special mechanism of action of PROTAC, PROTAC drugs have important advantages for overcoming drug resistance and developing drugs for undruggable targets. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention provides a novel BRD4 degrader and its preparation method and application. The BRD4 degrader is a PROTAC using GROs (such as AS1411) as the MDM2 recruitment element, which can bring the target protein BRD4 and the E3 ubiquitin ligase MDM2 closer in vivo, enabling BRD4 to be tagged with ubiquitin and then degraded through the ubiquitin-proteasome pathway. Experiments show that the BRD4 degrader can effectively degrade BRD4 and produce corresponding therapeutic effects, such as inhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells.

[0006] In the first aspect of the present invention, there is provided a BRD4 degrader and its pharmaceutically acceptable salts, stereoisomers, esters, prodrugs, solvates or deuterated compounds, and the BRD4 degrader has the following structure:

[0007]

[0008] Wherein,

[0009] GRO is a guanine-rich oligonucleotides residue capable of specifically binding to nucleolin (NCL);

[0010] L is a linker;

[0011] represents a double bond or a single bond,

[0012] X 1 is C or N;

[0013] Ring A is an aromatic ring or a heterocyclic ring;

[0014] Ring B is an aromatic ring or a heterocyclic ring;

[0015] R 1 is one or more independent substituents on the benzene ring, selected from: H, C 1 -C 10 alkyl, -(C 0 -C 6 alkylene)-(C 3 -C 10 cycloalkyl), -(C 0 -C 6 alkylene)-(C 6 -C 10 aryl), -(C 0 -C 6 alkylene)-(4- to 10-membered heterocyclic group), halogen, cyano, nitro, azide, C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO 2 (C 0-10 alkyl), -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl);

[0016] R 2 is one or more independent substituents on Ring A, selected from: H, C 1-C 10 alkyl, -(C 0 -C 6 alkylene)-(C 3 -C 10 cycloalkyl), -(C 0 -C 6 alkylene)-(C 6 -C 10 aryl), -(C 0 -C 6 alkylene)-(4- to 10-membered heterocyclic group), halogen, cyano, nitro, azide, C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO 2 (C 0-10 alkyl), -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl);

[0017] R 3 is one or more independent substituents on the B ring and is selected from: H, C 1 -C 10 alkyl, -(C 0 -C 6 alkylene)-(C 3 -C 10 cycloalkyl), -(C 0 -C 6 alkylene)-(C 6 -C 10 aryl), -(C 0 -C 6(alkylene)-(4- to 10-membered heterocyclic group), halogen, cyano, nitro, azide, C 1 -C 10 haloalkyl, C 1 -C 10 haloalkoxy, -N(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl)CO(C 0-10 alkyl), -N(C 0-10 alkyl)CON(C 0-10 alkyl), -N(C 0-10 alkyl)SO 2 (C 0-10 alkyl), -O(C 0-10 alkyl), -S(C 0-10 alkyl), -SO(C 0-10 alkyl), -SO 2 (C 0-10 alkyl), -SO 2 N(C 0-10 alkyl)(C 0-10 alkyl), -COO(C 0-10 alkyl), -OCO(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -CO(C 0-10 alkyl);

[0018] R 4 is selected from: single bond, C 1 -C 6 alkylene, -N(C 0-10 alkyl)-;

[0019] 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).

[0020] In a preferred embodiment of the present invention, p is 1, that is, the general formula I is

[0021] Specifically, the GROs are guanine-rich oligonucleotides containing one or more GGT motifs and having G4 structural characteristics.

[0022] Preferably, the GROs have stable G4 structural features, and the G4 structural feature signals can be detected by the methods described in the prior art (see, for example, Yuan Yu, 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 using circular dichroism (CD), nuclear magnetic resonance (NMR), ultraviolet spectroscopy, molecular fluorescence spectroscopy, single molecule fluorescence resonance energy transfer (FRET), etc.

[0023] In some embodiments of the present invention, fluorescence probes (such as N-methyl mesoporphyrin IX (NMM), O-phenanthroline derivative) are used for detection, and the GROs have stable G4 structural feature signals (see, for example, Zhang Suge, Sun Hongxia, Tang Yalin. Research progress on DNA G-quadruplex recognition probes [J]. Chemical Bulletin. 2016, 79(5): 387-394.).

[0024] In one embodiment of the present invention, when N-methyl mesoporphyrin IX (NMM) is used as a fluorescence probe for detection, stable G4 structural features can be formed by AS1411 in the presence or absence of K + Specifically, the detection method may include the following steps: adding the GRO to be tested into a solution with or without K + and heating at 90-99 °C for 5-10 minutes, incubating on ice, then adding NMM, incubating in the dark at room temperature, and detecting the fluorescence intensity (such as described in Example 1 of the present invention).

[0025] Specifically, the GROs include chemical modification, nucleic acid unit replacement or connection of functional groups on the GROs; these modifications or functional groups can be used to improve the stability of PROTAC, provide detection signals, or form compositions with other substances. Among them, the chemical modification is that at least one base is modified, and the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, isotopic labeling, phosphorothioate backbone modification, methoxy modification, and fluoro modification; the nucleic acid unit replacement is that at least one nucleic acid unit is replaced with LNA, UNA or GNA; the functional groups include at least one of fluorescent groups, radioactive groups, therapeutic drugs, biotin, digoxin, nanoluminescent materials, nucleic acid substances or enzyme labels.

[0026] Preferably, the GROs have 4 to 100 (such as 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 80, 100) nucleotides.

[0027] Preferably, the GROs are one of the DNA aptamer AS1411, AS1411 derivatives / analogues.

[0028] Among them, the AS1411 derivatives / analogues are GROs with a homology of more than 60% (such as 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) to AS1411, and contain one or more GGT motifs, have stable G4 structural characteristics, and can specifically bind to NCL GROs.

[0029] 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.

[0030] Specifically, the sequence of the AS1411 is as shown in SEQ ID NO: 1: 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

[0031] In some preferred embodiments of the present invention, the GROs are AS1411.

[0032] In some other embodiments of the present invention, the GROs are selected from: GRO29A, GRO15A, AT11.

[0033] Specifically, L can be connected to any base, sugar or phosphate backbone at the 3′ end, 5′ end or in the middle of the GRO.

[0034] In some embodiments of the present invention, L is connected to the 3′ end of the GRO.

[0035] In some embodiments of the present invention, L is connected to the 5′ end of the GRO.

[0036] In some embodiments of the present invention, the GRO moiety in Formula I has the following structure:

[0037]

[0038] Specifically, L has the following structure: Wherein,

[0039] L 1 is a divalent group linked to GRO and is selected from: a single bond, -O-(C 0 -C 6 alkylene)-, -S-(C 0 -C 6 alkylene)-, -N(R L1 )-(C 0 -C 6 alkylene)-, -N(R L2 )C(O)-(C 0 -C 6 alkylene)-, -OP(O)(OR L1 )O-(C 0 -C 6 alkylene)-, -C(O)-(C 0 -C 6 alkylene)-, -C(S)-(C 0 -C 6 alkylene)-, -CON(R L1 )-(C 0 -C 6 alkylene)-;

[0040] L 3 is selected from: a single bond, -O-(C 0 -C 6 alkylene)-, -S-(C 0 -C 6 alkylene)-, -N(R L3 )-(C 0 -C 6 alkylene)-, -N(R L3 )CO-(C 0 -C 6 alkylene)-, a 4- to 10-membered heteroalkyl group;

[0041] L 2is a single bond or divalent saturated or unsaturated straight-chain or branched 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)-,

[0042] amino acid residue, nucleotide residue, oligonucleotide residue, oligopeptide residue, where m2 is an integer selected from 1-10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), and each -CY- is independently an optionally substituted divalent ring selected from: arylene, cycloalkylene, heterocyclylene; the H in the hydrocarbon chain can be optionally substituted by one or more groups selected from: halogen, cyano, nitro, azide, -OR L0 , -C(O)R L0 , -C(S)R L0, -C(O)OR L0 , -C(S)SR L0 , -OC(O)R L0 , -OC(S)R L0 , -OC(S)SR L0 , -C(O)N(R L0 ) 2 , -OC(O)N(R L0 ) 2 , -N(R L0 )C(O)OR L0 , -N(R L0 )SO 2 R L0 , -SO 2 N(R L0 ) 2 , -OSO 2 N(R L0 ) 2 , -N(R L0 )C(O)R L0 , -N(R L0 ) 2 , -SR L0 , -SOR L0 , -SO 2 R L0 , -OSO 2 R L0 , C 1 , -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);

[0043] R L0 , R L1 , R L2 , and R L3 , independently selected from: H, 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- to 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 10 aryl, 4- to 10-membered heterocyclic group, the H in which 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- to 10-membered heterocyclic group).

[0044] Specifically, each -CY- is independently selected from the following optionally substituted divalent rings: phenylene, bicyclic arylene, tricyclic arylene, monocyclic cycloalkylene, bicyclic cycloalkylene, tricyclic cycloalkylene, monocyclic heteroarylene, bicyclic heteroarylene, tricyclic heteroarylene, monocyclic heterocycloalkylene, bicyclic heterocycloalkylene, tricyclic heterocycloalkylene.

[0045] In some embodiments of the present invention, each -CY- is independently selected from the following:

[0046]

[0047]

[0048] wherein, R L4 , R L5 are independently selected from: H, OH, halogen, C 1-8 alkyl, O(C 1-8 alkyl), S(C 1-8 alkyl), NH(C 1-8 alkyl), N(C 1-8 alkyl) 2 , C 3-11 cycloalkyl, C 3-11 heterocycloalkyl, O(C 1-8 cycloalkyl), S(C 1-8 cycloalkyl), NH(C 1-8 cycloalkyl), N(C 1-8 cycloalkyl)(C 1-8 alkyl), OH, NH 2 , SH, SO 2 (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 1-8alkyl), CO 2 H, CN, CF 3 , CHF 2 , CH 2 F, NO 2 , SF 5 , SO 2 NH(C 1-8 alkyl), SO 2 N(C 1-8 alkyl) 2 , S(=O)N(C 1-8 alkyl) 2 , C(=O)NH(C 1-8 alkyl), C(=O)N(C 1-8 alkyl) 2 , N(C 1-8 alkyl)C(=O)NH(C 1-8 alkyl), N(C 1-8 alkyl)C(=O)N(C 1-8 alkyl) 2 , NHC(=O)NH(C 1-8 alkyl), NHC(=O)N(C 1-8 alkyl) 2 , NHC(=O)NH 2 , N(C 1-8 alkyl)SO 2 NH(C 1-8 alkyl), N(C 1-8 alkyl)SO 2 N(C 1-8 alkyl) 2 , NHSO 2 NH(C 1-8 alkyl), NHSO 2 N(C 1-8 alkyl) 2 or NHSO 2 NH 2 ; or, R L4 , R L5 together with the atom to which it is attached forms a cycloalkylidene or heterocycloalkylidene group.

[0049] More specifically, R L4 , R L5 are independently selected from: -CH 3 ,

[0050] or, R L4 , R L5 together with the atom to which it is attached forms a three- to six-membered cycloalkylidene group (such as ) or a four- to six-membered heteroalkyl ring (such as

[0051] In some embodiments of the present invention, R L1 is H.

[0052] In some embodiments of the present invention, R L3 is H.

[0053] In some embodiments of the present invention, R L4 is H.

[0054] In some embodiments of the present invention, R L4 is OH.

[0055] In some embodiments of the present invention, R L5 is H.

[0056] In some embodiments of the present invention, R L5 is OH.

[0057] In one embodiment of the present invention, L 2 adopts the following scheme (1): L 2 is a C1-C20 straight-chain alkylene group, and 0-6 methylene units in the alkylene group are independently substituted by the following groups: -O-, -S-, -S-S-, -C(O)-, -C(O)O-, -OC(O)-, -N(R L2 )-, -C(O)N(R L2 )-, -N(R L2 )C(O)-,

[0058] 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.

[0059] More specifically, L 2 can be selected from: C1-C20 straight-chain alkylene group, -(CH 2 CH 2 O) m2 -CH 2 -, -(CH 2 CH 2 O) m2 -CH2 CH 2 -、-CH 2 -(CH 2 CH 2 O) m2 -CH 2 -、-CH 2 CH 2 -(CH 2 CH 2 O) m2 -CH 2 -、-CH 2 CH 2 -(CH 2 CH 2 O) m2 -CH 2 CH 2 -、-(C 1 -C 10 -alkylene)-O-(C 1 -C 10 -alkylene)-、-(C 1 -C 10 -alkylene)-NH-(C 1 -C 10 -alkylene)-、-(C 1 -C 10 -alkylene)-C(O)NH-(C 1 -C 10 -alkylene)-、-(C 1 -C 10 -alkylene)-NHC(O)-(C 1 -C 10 -alkylene)-、-(C 1 -C 6 -alkylene)-O-(C 1 -C 6 -alkylene)-C(O)NH-(C 1 -C 6 -alkylene)-、-(C 1 -C 6 -alkylene)-O-(C 1 -C 6 -alkylene)-NHC(O)-(C 1 -C 6 -alkylene)-、

[0060]

[0061]

[0062] Among them, 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.

[0063] In some specific embodiments of the present invention, L 2 is selected from:

[0064]

[0065] 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).

[0066] In some embodiments of the present invention, is wherein the J ring is a 4- to 10-membered saturated heterocyclic ring.

[0067] 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,

[0068] In some embodiments of the present invention, G is a sugar residue, such as can be

[0069] 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

[0070] 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)-,

[0071] 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.

[0072] In some embodiments of the present invention, -CY- is selected from:

[0073]

[0074] In some specific embodiments of the present invention, L 2 is selected from:

[0075] In another embodiment of the present invention, L 2 adopts the following scheme (3): L 2 is an oligonucleotide residue, such as a DNA oligonucleotide residue, an RNA oligonucleotide residue, a DNA / RNA hybrid oligonucleotide residue, which can be single-stranded or double-stranded. In this case, L 1 can be a single bond or O, that is, GRO is connected to L through a phosphodiester bond (that is 2 ).

[0076] Specifically, the oligonucleotide is composed of A and / or T.

[0077] 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.

[0078] 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.

[0079] In some embodiments of the present invention, L 2 is a single-stranded DNA oligonucleotide residue, which is composed of 6 A.

[0080] In some embodiments of the present invention, L 2 is a DNA double-stranded oligonucleotide, which is composed of 6 A-T pairs.

[0081] In some embodiments of the present invention, L 2 is a DNA double-stranded oligonucleotide, which is composed of 10 A-T pairs.

[0082] In some embodiments of the present invention, L 2Double-stranded oligonucleotides for DNA / RNA hybridization, where the DNA single strand consists of 6 Ts and the RNA single strand consists of 6 As.

[0083] In another embodiment of the present invention, L 2 Adopts the following scheme (4): L 2 Is an oligopeptide residue composed of 2 - 10 (such as 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid residues.

[0084] In some embodiments of the present invention, L 3 Is a single bond.

[0085] In some embodiments of the present invention, L 3 Is -N(H)-.

[0086] In some embodiments of the present invention, L 3 Is

[0087] In some embodiments of the present invention, L 3 Is a 4 - 8 membered saturated nitrogen - containing heterocyclic group, such as

[0088] Specifically, R 1 Is selected from: H, C 1 -C 6 Alkyl, halogen (such as Cl), cyano, nitro, hydroxy, amino, C 1 -C 6 Halogenated alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Halogenated alkoxy; in some embodiments of the present invention, R 1 Is a halogen, such as Cl.

[0089] Specifically, the A ring is a benzene ring or a 5 - 6 membered nitrogen - containing heterocycle, for example In some embodiments of the present invention, the A ring is In some embodiments of the present invention, the A ring is

[0090] Specifically, R 2 Is selected from: H, C 1 -C 6 Alkyl, halogen, C 1 -C 6 Halogenated alkyl, C 1 -C 6 Alkoxy, C 1 -C 6 Halogenated alkoxy; in some embodiments of the present invention, R 2 Is C1 -C 3 alkyl, such as methyl.

[0091] In some embodiments of the present invention, is

[0092] Specifically, ring B is a benzene ring or a 5- or 6-membered heteroaromatic ring, such as In some embodiments of the present invention, ring B is In some embodiments of the present invention, ring B is

[0093] Specifically, R 3 is selected from: H, C 1 -C 6 alkyl, halogen, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy; in some embodiments of the present invention, R 3 is C 1 -C 3 alkyl, such as methyl.

[0094] In some embodiments of the present invention, is wherein, R 3 ' has the above definition of R 3 .

[0095] In some embodiments of the present invention,

[0096] In some embodiments of the present invention, part is

[0097] Specifically, R 4 is selected from: methylene, -N(H)-, -N(CH 3 )-; in some embodiments of the present invention, R 4 is methylene.

[0098] In some embodiments of the present invention, the BRD4 degrader has the following structure:

[0099]

[0100] Specifically, in formula II, the partial configuration is

[0101] In some embodiments of the present invention, the BRD4 degrader has the following structure:

[0102]

[0103] Specifically, in Formulas II and III, GRO, L 1 and L 2 are as described above.

[0104] 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.

[0105] In some embodiments of the present invention, L 1 is a single bond or O, that is, GRO is connected to L through a phosphate ester bond (i.e., 2 ).

[0106] In some embodiments of the present invention, L 2 adopts the above-mentioned scheme (1) and / or (2), especially scheme (1), for example

[0107] More specifically, the BRD4 degrader has the following structure:

[0108]

[0109] In one embodiment of the present invention, the BRD4 degrader has the following structure:

[0110]

[0111] In other embodiments of the present invention, the BRD4 degrader has the following structure:

[0112]

[0113]

[0114]

[0115]

[0116] In the second aspect of the present invention, a method for preparing the BRD4 degrader described in the first aspect is provided, which includes the step of coupling a small molecule ligand moiety with GROs through L.

[0117] 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;

[0118] L' is an optionally suitable linking group;

[0119] R F is a protected hydroxyl group.

[0120] 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.

[0121] Specifically, in the solid-phase synthesis, L' contains (for example ).

[0122] In some embodiments of the present invention, R F is -ODMTr.

[0123] In some embodiments of the present invention, the solid-phase carrier is controlled pore glass beads (CPG), and the pore size of the CPG is determined according to the length of the synthesized oligonucleotide.

[0124] In some embodiments of the present invention, the preparation method includes the following steps: first prepare and react and link with GROs; or,

[0125] first prepare GRO-L'-R F , and then link with a small molecule ligand; or,

[0126] separately prepare GRO-L”R F ' and then couple the two;

[0127] wherein, L', L”, L”' are optionally suitable linking groups, and R F , R F ', R F ” are optionally suitable reactive groups.

[0128] In the third aspect of the present invention, a pharmaceutical composition comprises the BRD4 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.

[0129] 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.

[0130] Specifically, the pharmaceutical composition can be administered by any suitable route, such as gastrointestinal 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.) routes.

[0131] 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.

[0132] When preparing injections, any commonly used carriers in the art can be used, such as: water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyethoxylated isostearyl alcohol, and fatty acid esters of polyethylene sorbitan, etc. In addition, commonly used solubilizers and buffers can also be added.

[0133] 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, 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.

[0134] Specifically, in the pharmaceutical composition, the PROTAC can be used alone or in combination with other types of active ingredients.

[0135] Specifically, the amount of the active ingredient in the unit dosage preparation can be changed or adjusted between 0.1 mg and 1000 mg (e.g., 0.1, 1, 5, 10, 20, 40, 50, 100, 200, 400, 500, 1000 mg), depending on the specific application and potency of the active ingredient. If necessary, the composition can also contain other suitable therapeutic agents.

[0136] In the fourth aspect of the present invention, a delivery system for a BRD4 degrader is provided, which comprises the above-mentioned BRD4 degrader and a carrier, and can deliver the BRD4 degrader to target cells or target tissues without NCL expression on the cell surface, etc.

[0137] Specifically, the above-mentioned carrier can be any carrier suitable for delivering nucleic acid drugs into target tissues or target cells, etc., such as those disclosed in the prior art (see, for example, Wang Junfeng, Tan Manman, Wang Ying, etc. Research progress on modification and delivery of nucleic acid drugs [J]. Journal of Zhejiang University (Medical Sciences), 2023, 52(04): 417-428. which is incorporated herein by reference), for example, viral vectors (such as lentivirus, adenovirus, adeno-associated virus vector), non-viral vectors (such as lipid nanoparticles (LNP), polymer nanocarriers, inorganic nanocarriers, protein carriers, exosomes, etc.).

[0138] In some embodiments of the present invention, the carrier is a lipid nanoparticle (LNP).

[0139] In the fifth aspect of the present invention, there is provided the use of the BRD4 degrader described in the first aspect or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof in the preparation of a drug for preventing and / or treating BRD4-related diseases.

[0140] Specifically, the diseases are those that can benefit from the prevention and / or treatment by degrading BRD4, such as, but not limited to, tumors, autoimmune diseases, inflammatory diseases, diseases related to pathogen infections, neurodegenerative diseases, cardiovascular diseases, metabolic diseases, fibrotic diseases, etc.

[0141] 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, gliomas, glioblastoma multiforme, skin cancer, skin appendage cancer and skin metastatic cancer, 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, lung squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, thyroid cancer, bladder cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, prostate cancer, salivary gland cancer, kidney cancer, vulvar cancer, anal cancer, penile cancer, esophageal cancer, biliary tract tumors and head and neck cancer.

[0142] Specifically, the hematological malignancies include: leukemia, lymphoma, multiple myeloma (MM).

[0143] Specifically, the leukemia can be chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL), chronic myelogenous leukemia (CML) (e.g., B-cell CML, T-cell CML), acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), acute monocytic leukemia, etc.

[0144] Specifically, the lymphoma can be Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström macroglobulinemia)), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma and T-cell NHL, such as precursor T lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sézary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma, NK / T-cell lymphoma, especially diffuse large B-cell lymphoma (DLBCL).

[0145] In some embodiments of the present invention, the tumor is selected from: breast cancer, lung cancer, liver cancer, kidney cancer, acute myeloid leukemia (AML), multiple myeloma (MM).

[0146] 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.

[0147] Specifically, the inflammatory diseases include but are not limited to: osteoarthritis, acute gout, multiple sclerosis, inflammatory bowel disease (such as 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 (such as acute pancreatitis), postoperative syndrome, sarcoidosis, Herxheimer reaction, encephalitis, myelitis, meningitis, and malaria, etc.

[0148] 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.

[0149] Specifically, the pathogen can be a microorganism, a parasite (protozoa, worms, etc.) or other vectors.

[0150] Specifically, the microorganism can be selected from one or more of the following: virus, chlamydia, rickettsia, mycoplasma, bacteria, spirochete, fungus, etc.

[0151] 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).

[0152] Specifically, the diseases associated with 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] In particular, the diseases are selected from: hematological malignancies (such as acute lymphoblastic leukemia, mixed lineage leukemia, multiple myeloma, Burkitt's lymphoma), hepatocellular carcinoma, triple-negative breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, neuroblastoma, etc.

[0157] In the sixth aspect of the present invention, a method for treating BRD4-related diseases is provided, which includes the step of administering to a subject in need thereof the BRD4 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.

[0158] Specifically, the diseases are as described in the fifth aspect of the present invention.

[0159] Specifically, the subject is a mammal, especially a human.

[0160] Specifically, the administration can be carried out by any suitable route of administration, 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.).

[0161] The present invention provides a PROTAC for degrading BRD4 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 BRD4, which can effectively degrade BRD4 and produce corresponding therapeutic effects (such as inhibiting the proliferation of tumor cells and promoting apoptosis of tumor cells), and has very good application prospects and research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 The result graph showing that NCL can bind to MDM2 is shown.

[0163] Figure 2 The result graph showing that AS1411 is a GROs with stable G4 structural characteristics is shown.

[0164] Figure 3 The result graph showing the weak G4 structural features of iSN04.

[0165] Figure 4 A shows the result graph that CRO cannot capture NCL and MDM2; Figure 4 B shows the result graph that the capture of NCL and MDM2 by AS1411 is concentration-dependent; Figure 4 C shows the result graph that only high concentration of iSN04 can capture NCL, but iSN04 cannot capture MDM2.

[0166] Figure 5 A shows the result graph that CRO can neither bind to NCL nor recruit MDM2; Figure 5 B shows the result graph that AS1411 can recruit MDM2 in large amounts 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.

[0167] Figure 6 A shows the result graph that AS1411 does not affect the interaction between NCL and MDM2; Figure 6 B shows the result graph that iSN04 blocks the binding between NCL and MDM2.

[0168] Figure 7 A shows the result graph that AS1411 can significantly bind to Hep3B cells, Figure 7 B shows the result graph that AS1411 can capture NCL and MDM2; Figure 7 C shows the result graph that silencing NCL can block the recruitment of MDM2 by AS1411.

[0169] Figure 8 A shows the schematic diagram that GROs (such as AS1411) recruit MDM2 depending on their interaction with NCL; Figure 8 B shows the possible schematic diagram of the mode of action of the PROTAC targeting BRD4 formed by GROs (such as AS1411) as the recruitment element of MDM2.

[0170] Figure 9 The result graph showing the degradation of BRD4 by AS1411-JQ1.

[0171] Figure 10 The result graph showing the promotion of the ubiquitination of BRD4 by AS1411-JQ1.

[0172] Figure 11 The result graph showing that AS1411-JQ1 mediates the degradation of BRD4 through the ubiquitin-proteasome pathway.

[0173] Figure 12 The figure shows the results of AS1411-JQ1-dependent degradation of BRD4 mediated by MDM2 and NCL.

[0174] Figure 13 The figure shows the results that AS1411-JQ1 can inhibit the proliferation of Hep3B cells in vitro and promote the apoptosis of Hep3B cells.

[0175] Figure 14 The figure shows the results that AS1411-JQ1 can inhibit the proliferation of A549 cells in vitro and promote the apoptosis of A549 cells.

[0176] Figure 15 The figure shows the results that AS1411-JQ1 can inhibit the growth of tumor cells in vivo. Detailed implementation mode

[0177] 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.

[0178] 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 other parts of the molecule by a single bond. Typical alkyl groups contain 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. In the present invention, C 0 alkyl refers to H, that is, C 0-10 alkyl (or C 0 -C 10 alkyl) includes H and C 1-10 alkyl (or C 1 -C 10 alkyl).

[0179] The term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by removing two hydrogen atoms from an alkane molecule, which can be straight-chain or branched-chain and is connected to other parts of the molecule by a single bond. In this article, typical alkylene has 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, preferably 1 to 6 carbon atoms, such as methylene (-CH 2 -), ethylene, propylene, butylene, etc. In the present invention, C 0 alkylene refers to a single bond, that is, C 0-10 alkylene (or C 0 -C 10 alkylene) includes a single bond and C 1-10 alkylene (or C 1 -C10 (alkylene).

[0180] The term "cycloalkyl" refers to alicyclic hydrocarbons, such as those containing 1 to 4 monocyclic and / or fused rings, having 3 - 18 carbon atoms, preferably 3 - 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or adamantyl, etc.

[0181] 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.

[0182] 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

[0183] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0184] The term "haloalkyl" refers to a group formed by replacing one or more hydrogens in an alkyl group with a halogen atom (e.g., fluorine, chlorine, bromine, or iodine), for example, -CHF 2 、-CH 2 F、-CF 3 、-CH 2 -CF 3 、-CH 2 CH 2 -CF 3 、-CH 2 CH 2 CH 2 -CF 3 。

[0185] 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 (e.g., 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.

[0186] The term "heterocyclic group" refers to a 3- to 18-membered non-aromatic ring group that contains 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, tetrahydropyranyl, 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

[0187] In the present invention, "D" refers to deuterium; "substituted with deuterium" means replacing one or more hydrogen atoms with the corresponding number of deuterium atoms.

[0188] It should be recognized that depending on the source of the chemical materials used in the synthesis, there may be some variation in the natural isotope abundances in the synthesized compounds. Thus, the compounds of the present invention will inherently contain small amounts of deuterated isotopologues. Despite such variation, the concentrations of the stable hydrogen and carbon isotopes of natural abundance are low and insignificant 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.

[0189] In the compounds of the present invention, any atom not specified as deuterium is present in its natural isotope abundance. Unless otherwise specified, when a position is specifically designated as "H" or "hydrogen", that position is understood to have hydrogen with its isotopic composition according to natural abundance. Similarly, unless otherwise specified, when a position is specifically designated as "D" or "deuterium", that position is understood to have deuterium with an abundance at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 45% deuterium incorporation).

[0190] As used herein, the term "isotope enrichment factor" refers to the ratio of the isotope abundance of a particular isotope to its natural abundance.

[0191] In other embodiments, the compounds of the present invention have an isotope enrichment factor of at least 3500 (52.5% deuterium incorporation at each designated 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 designated deuterium atom.

[0192] The term "isotopologue" refers to a substance in which the chemical structure differs from a particular compound of the present invention only in its isotopic composition.

[0193] The term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0194] 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. Thus, these salts include, but are not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromides, iodides, acetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, maleates, tartrates, and methanesulfonates, 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 in a conventional manner with a sufficient amount of the desired acid to form the salt. The free base form can be regenerated by contacting the salt form with a base and the free base can be isolated in a conventional manner.

[0195] The term "base addition salt" refers to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form in a conventional manner with a sufficient amount of the desired base to form the salt. The free acid form can be regenerated by contacting the salt form with an acid and the free acid can be isolated in a conventional manner.

[0196] The term "stereoisomers" includes enantiomers, diastereomers, and geometric isomers. Some compounds of the present invention have cycloalkyl groups which may be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention.

[0197] 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 crystal lattice of a crystalline solid. Solvates include solution-phase and isolable solvates. Representative solvates include ethanolates, methanolates, etc.

[0198] 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.

[0199] In the present invention, the term "oligonucleotide" consists of no more than 30 (e.g., 5, 10, 15, 20, 25, 30) nucleotides (deoxyribonucleotides and / or ribonucleotides), which is single-stranded or double-stranded, especially single-stranded. The oligonucleotide may include chemical modifications, nucleic acid unit substitutions, or attachment of functional groups; wherein, the chemical modification is that at least one base is modified, and the chemical modification includes at least one of phosphorylation, methylation, amination, thiolation, isotopic labeling, phosphorothioate backbone modification, methoxy modification, and fluoro modification; the nucleic acid unit substitution is that at least one nucleic acid unit is replaced with LNA, UNA, or GNA; the functional groups include at least one of a fluorescent group, a radioactive group, biotin, digoxin, a nanoluminescent material, a nucleic acid substance, or an enzyme label.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] The term "tumor" refers to an abnormal mass of tissue in which the growth of the mass exceeds and is uncoordinated with that of normal tissue. Tumors can be "benign" or "malignant", depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion, and metastasis. "Benign tumors" are usually well-differentiated, characterized by slower growth than malignant tumors and remaining confined to the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant sites. In some cases, certain "benign" tumors may later give rise to malignant tumors, which may be due to additional genetic alterations in subsets of the neoplastic cells of the tumor, and these tumors are called "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.

[0206] The term "cancer" refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyle d.; Williams & Wilkins: Philadelphia, 1990).

[0207] The term "autoimmune disease" refers to a disease caused by the body's immune reaction to its own antigens, resulting in damage to its own tissues.

[0208] 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.

[0209] 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).

[0210] The term "cardiovascular disease" refers to a class of diseases involving the heart or blood vessels.

[0211] The term "metabolic disease" refers to a disease caused by the accumulation or deficiency of certain metabolites such as sugar, fat, protein, purine, calcium, copper, etc. when the biochemical process in the body is disordered.

[0212] 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 abnormally increases and is excessively deposited. In severe cases, it causes tissue structure damage and organ sclerosis.

[0213] The disclosures of all publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

[0214] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0215] The sequences involved in the embodiments are shown in the following table:

[0216] Table 1 Sequence names and numbers

[0217] 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

[0218] Example 1: NCL can bind to MDM2

[0219] Co-Immunoprecipitation (Co-IP) is a classic method for studying protein-protein interactions based on the specific interaction between an antibody and an antigen.

[0220] (1) After lysing hepatoma cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), incubate with the antibody against NCL overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and incubate with rotation at room temperature for 2 hours. Wash the 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 to a new tube. Then perform an immunoblotting experiment (western blot) to detect whether NCL binds to MDM2. The steps of the immunoblotting experiment are as follows: Separate the protein samples by SDS-PAGE electrophoresis, transfer the separated proteins to a PVDF membrane, block with TBST buffer containing 5% skim milk at room temperature for 1 hour, then incubate overnight at 4°C with the primary antibody against MDM2 (purchased from proteintech, catalog number 27883-1-AP) or the primary antibody against NCL (purchased from Cell Signaling Technology, catalog number 14574S). After washing with TBST, incubate the membrane with the HRP-labeled secondary antibody (purchased from Abiotech, catalog number AS014) at room temperature for 1 hour, and visualize the protein band blot using an enhanced chemiluminescence detection kit (purchased from Abiotech, catalog number RM00021P). Figure 1 The results of A indicate that NCL can bind to MDM2.

[0221] (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) and incubate with rotation at room temperature for 1.5 hours. Wash the magnetic beads 3 times with TBST buffer, add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. Adsorb the magnetic beads with a magnetic stand, take the supernatant to a new tube. Then perform an immunoblotting experiment to detect whether NCL binds to MDM2. Figure 1 The results of B indicate that NCL can bind to MDM2.

[0222] Example 2: AS1411 is a GRO with a stable G4 structural feature

[0223] GROs contain one or more GGT motifs and have the characteristic of a stable G4 structure. GROs are guanine-rich oligonucleotides that can specifically bind to NCL. The GROs described in the present invention are one of the DNA aptamers AS1411, AS1411 derivatives / analogues, all of which have the above-mentioned stable G4 structure characteristics and the property of specifically binding to NCL. NCL is a multifunctional shuttle protein that shuttles between the cell nucleus and the 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 used as a surface molecular marker and potential therapeutic target for diseased cells (such as tumors, etc.). The NCL on the membrane surface of diseased cells (such as tumors, etc.) can also act as a receptor for a variety of ligand molecules, mediating the entry of ligand molecules into diseased cells (such as tumors, etc.).

[0224] (1) Both AS1411 and iSN04 are oligonucleotides that specifically bind to NCL. We detected whether AS1411 and iSN04 have the characteristic of a stable G4 structure. 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 structure characteristic was detected for iSN04, but AS1411 has a stable G4 structure characteristic (fluorescence signal intensity exceeds 4000).

[0225] (2) Guanine-rich DNA sequences can form more obvious G4 structure characteristics in the presence of metal ions (usually sodium and potassium ions). Add 5 μM NC (negative control, SEQ ID NO: 4), 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-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 3As shown, iSN04 has weak G4 structural features (fluorescence signal intensity of 1000 - 2000), but the G4 structural features of AS1411 are more stable (fluorescence signal intensity exceeding 10000).

[0226] The above results indicate that iSN04 can form weak G4 structures only in the presence of K + and cannot form G4 structural features without K + , while AS1411 can form stable G4 structural features with or without the presence of K + .

[0227] Example 3: AS1411 recruits MDM2 by relying on its interaction with NCL

[0228] (1) The process of the pull-down experiment is to immobilize a substance of 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.

[0229] After lysing the liver cancer cells Hep3B with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), they were incubated with 5'-biotinylated CRO (cytosine-rich oligonucleotides, negative control), AS1411, or iSN04 at different concentrations (0 μM, 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 incubated overnight at 4°C. 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 is the pull-down product. Immunoblotting experiments were used to detect the pull-down product. Figure 4 The results of A show that CRO can neither capture NCL nor MDM2; Figure 4 The results of B show that AS1411 can capture NCL and MDM2 in a concentration-dependent manner; Figure 4 The results of C show that high-concentration iSN04 can capture NCL, but iSN04 cannot capture MDM2 within the concentration range used.

[0230] (2) Mix 6 μg / mL of recombinant human NCL and 6 μg / mL of recombinant human MDM2 and incubate at 4 °C for 7 hours. Subsequently, add 400 nM of 5'-biotinylated CRO, AS1411 or iSN04 and incubate at 4 °C for 6 hours. Then add streptavidin agarose gel beads (purchased from Cytiva, catalog number 17511301) and continue to incubate overnight at 4 °C. Wash the gel beads 4 times with TBST buffer, add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100 °C and maintain for 10 minutes. Take the supernatant after centrifugation. Then perform immunoblotting experiments to detect the pulled-down products. Figure 5 The results of A indicate that CRO can neither bind to NCL nor recruit MDM2. Figure 5 The results of B indicate that AS1411 recruits a large amount of MDM2 depending on its interaction with NCL. Figure 5 The results of C indicate that iSN04 can only bind to NCL, but cannot recruit MDM2 depending on its interaction with NCL.

[0231] (3) After lysing the 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. Subsequently, add 5 μL of 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) and rotate and incubate at room temperature for 1.5 hours. Wash the NCL and the proteins it captured 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. Use a magnetic stand to adsorb the magnetic beads, and take the supernatant to a new tube. Then perform an immunoblotting experiment (western blot) to detect whether NCL binds to MDM2 and whether this binding is affected by AS1411 or iSN04. The steps of the immunoblotting experiment are as follows: Separate the protein samples by SDS-PAGE electrophoresis, transfer the separated proteins to a PVDF membrane, block it with TBST buffer containing 5% skim milk at room temperature for 1 hour, then incubate overnight with the primary antibody against MDM2 (purchased from proteintech, catalog number 27883-1-AP) or the primary antibody against NCL (purchased from Cell Signaling Technology, catalog number 14574S) at 4 °C. After washing with TBST, incubate the membrane with the HRP-labeled secondary antibody (purchased from: Abbkine, catalog number: AS014) at room temperature for 1 hour, and visualize the protein band blots using an enhanced chemiluminescence detection kit (purchased from: Abbkine, catalog number: RM00021P). Figure 6 The results of A show that AS1411 does 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 show that iSN04 blocks the binding of NCL to MDM2.

[0232] (4) Since NCL is highly expressed in various tumor cells (including hepatoma cells, etc.) and specifically distributed on the surface of tumor cells, we examined the binding ability of AS1411 to the hepatoma cells Hep3B. Incubate Hep3B cells with 500 nM Cy5-labeled CRO and AS1411 for 2 hours. Use a flow cytometer to detect their binding abilities to Hep3B cells respectively, and the results are as Figure 7 shown in A. The results show that AS1411 can significantly bind to Hep3B cells.

[0233] (5) Incubate hepatoma cells Hep3B with 1 μM biotin-labeled negative control CRO (Bio-CRO) or AS1411 (Bio-AS1411) for 12 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 AS1411 bound to the gel beads and the proteins captured by them multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. After centrifugation, take the supernatant, which is the pull-down product. Detect the pull-down product by immunoblotting experiment. The results are as Figure 7 shown in B. AS1411 can capture NCL and MDM2.

[0234] (6) Transfect negative control siRNA (siNC, SEQ ID NO:5) or NCL siRNA (siNCL, SEQ ID NO:6) into hepatoma cells Hep3B. After 48 hours, incubate the cells with 1 μM biotin-labeled AS1411 for 12 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 captured by them multiple times with IP lysis buffer (purchased from Thermo Scientific, catalog number 87788), add SDS-PAGE protein loading buffer (purchased from Beyotime, catalog number P0015), heat to 100°C and maintain for 10 minutes. After centrifugation, take the supernatant, which is the pull-down product. Detect the pull-down product by immunoblotting experiment. The results are as Figure 7 shown in C. Silencing NCL can block the recruitment of MDM2 by AS1411.

[0235] Example 4: Preparation of GROs-based PROTAC molecules targeting BRD4

[0236] 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 (such as AS1411) recruit MDM2 depending on the interaction with NCL, and the possible mode of action is as Figure 8 shown in A; the possible mode of action of PROTAC targeting BRD4 formed by GROs (such as AS1411) as the recruitment element of MDM2 is as Figure 8 shown in B.

[0237] In this example, AS1411, a typical representative of GROs, was used as the E3 ligase recruitment element, and JQ1, a BRD4 inhibitor, was used as an example of the target protein ligand to prepare a PROTAC for degrading BRD4 (hereinafter simply referred to as AS1411-JQ1).

[0238] The preparation steps are as follows:

[0239] (1) Synthesis of Compound 1-1

[0240]

[0241] A solution of HCl in 1,4-dioxane (4.375 mL, 17.5 mmol) was added to a solution of Compound 1 (800 mg, 1.75 mmol) in 1,4-dioxane (5 mL). The mixture was stirred under an argon atmosphere at 25 °C for 4 hours. Then the mixture was concentrated to dryness and used directly in the next step (white solid, 700 mg, yield: 100%).

[0242] LCMS: m / z = 401.0 [M+H] + t R = 0.540 min. Purity: 97.42% (214 nm).

[0243] (2) Synthesis of Compound 1-2

[0244]

[0245] HATU (996 mg, 2.62 mmol) was added to a solution of Compound 1-1 (700 mg, 1.75 mmol) in anhydrous DMF (10 mL), and the resulting yellow mixture was stirred at room temperature for about 30 minutes. Then Compound 2 (381 mg, 2.1 mmol) and DIPEA (1.35 g, 10.48 mmol) were slowly added at low temperature. The mixture was stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate (20 mL), washed with brine (10 mL * 3), dried over MgSO 4 and concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel (gradient elution; 0 - 20% ethyl acetate / hexane as the eluent) to give the target compound as a white solid (880 mg, 1.664 mmol, yield: 95%).

[0246] LCMS: m / z = 528.0 [M+H] + t R = 1.54 min. Purity: 100% (254 nm).

[0247] (3) Synthesis of Compound 1-3

[0248]

[0249] To a mixture of compound 1-2 (880 mg, 1.67 mmol) in 1,4-dioxane (20 mL) and water (6 mL) was added LiOH·H 2 O (350 mg, 8.33 mmol). The mixture was stirred at room temperature for 3 hours and monitored by LCMS. The mixture was concentrated to remove dioxane. The residue was adjusted to pH 5 and filtered to give the desired product (850 mg, yield: 100%).

[0250] LCMS: m / z = 514 [M+H] + t R = 1.43 min. Purity: 100% (254 nm).

[0251] (4) Synthesis of compound 1-4

[0252]

[0253] To a solution of compound 1-3 (850 mg, 1.65 mmol) in DMF (15 mL) was added DIPEA (856 mg, 6.62 mmol). The mixture was stirred under an argon atmosphere at 25 °C for 10 minutes. Then 2,2,2-trifluoro-pentafluorophenyl acetate (926 mg, 3.31 mmol) was slowly added at low temperature. The mixture was stirred under an argon atmosphere at 25 °C overnight. LCMS showed that the reaction was complete. Then the mixture was diluted with DCM (100 mL), washed with water (50 mL * 3), brine (25 mL), and dried over anhydrous Na 2 SO 4 2. The mixture was concentrated and purified by silica gel column chromatography, eluting with MeOH:DCM = 15%, to give the desired white solid product (942 mg, 1.34 mmol, yield: 84%).

[0254] LCMS: m / z = 680.0 [M+H] + t R = 1.987 min. Purity: 97% (254 nm).

[0255] (5) Synthesis of compound 1-5

[0256]

[0257] (3S,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-ol (648 mg, 1.54 mmol) was added to a solution of Compound 1-4 (700 mg, 1.03 mmol) in DMF (15 mL). The mixture was stirred for 10 minutes at 25 °C under an argon atmosphere. Then DIPEA (664 mg, 5.15 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 then diluted with DCM (100 mL), washed with water (50 mL * 3) and brine (25 mL), and dried over anhydrous Na 2 SO 4 The mixture was concentrated and purified by silica gel column chromatography, eluting with MeOH:DCM = 15%, to give the desired white solid product (700 mg, yield: 74.3%).

[0258] LCMS: m / z = 915.0 [M+H] + t R = 1.963 min. Purity: 96.6% (254 nm).

[0259] (6) Synthesis of Compound 1-6

[0260]

[0261] DMAP (192 mg, 1.57 mmol) was added to a solution of Compound 1-5 (360 mg, 0.393 mmol) in DMF (5 mL). The mixture was stirred for 10 minutes at 25 °C under an argon atmosphere. Then dihydrofuran-2,5-dione (59 mg, 0.59 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 concentrated and diluted with water (50 mL), and the pH was slowly adjusted to 7 with HCl (1 M) at low temperature. The mixture was extracted with EA (100 mL * 4), concentrated and purified by reverse phase chromatography [ACN / water (TEA 0.02%): 5% - 80%] to give the desired product (white solid, 400 mg, yield: 100%).

[0262] LCMS: m / z = 1015 [M+H] + t R = 1.604 min. Purity: 100% (214 nm).

[0263] 11H NMR (400 MHz, DMSO) δ 8.16 (m, 1H), 7.44 (m, 4H), 7.25 (m, 9H), 6.87 (m, 4H), 5.25 - 5.37 (s, 1H), 4.50 (t, J = 7.2 Hz, 1H), 4.19 (s, 1H), 3.84–3.63 (m, 7H), 3.54 (m, 1H), 3.48–3.38 (m, 1H), 3.28–3.17 (m, 4H), 3.04 (m, 4H), 2.57 (m, 4H), 2.53 (s, 1H), 2.49–2.43 (m, 4H), 2.39 (s, 3H), 2.17 (m, 4H), 1.60 (m, 3H), 1.55–1.09 (m, 6H), 1.06–0.85 (m, 6H).

[0264] (7) Synthesis of Compound 1-6-CPG

[0265]

[0266] At room temperature, to a solution of Compound 1-6 (300 mg, 0.296 mmol) in CH 3 CN (12.0 mL) were added HATU (120 mg, 0.312 mmol), DIPEA (120 μL), and lcaa-CPG (1000 Å, 1500 mg), and the mixture was shaken for 12 hours. After completion of the reaction, the CPG was washed with CH 3 CN, and CAP A (acetic anhydride:tetrahydrofuran = 1:9, v / v, 6.0 mL) and CAP B (n-methylimidazole:pyridine:acetonitrile = 15:10:75, v / v / v, 6.0 mL) were added. The mixture was shaken at room temperature for 1 hour. Then the mixture was filtered and washed 3 times with CH 3 CN (2 mL). 1-6-CPG was obtained as a white powder (1540 mg) after freeze-drying the mixture.

[0267] (8) Synthesis of Compound AS1411-JQ1

[0268]

[0269] 1-6-CPG was placed in a synthesis column (30 mg * 8), and the final product was synthesized by a K-A H-8 solid-phase synthesizer. Solid-phase synthesis includes four steps: detritylation, coupling, capping, and oxidation. After the reaction was completed, 1.5 mL of ammonia water was added to the CPG in each synthesis column, and it was heated in an oven at 65 °C for 16 hours. Then the supernatant was collected and washed with water (1 mL * 3). The crude product was purified by a protein purification system (Sepure, SDA) (column: Mono Q, 1.0 mL, method: mobile phase A: 40 mM aqueous NaOH solution, mobile phase B: 40 mM NaOH + 2.0 M aqueous NaCl solution) to obtain white powder AS1411-JQ1 (10.97 mg, purity = 95.70%).

[0270] UPLC-MS (WATERS ACQUITY PREMIER): AS1411-JQ1 - UPLC, m / z = 8947.82315 [M] - (deconvolution); t R = 10.732 min (260 nm). Mass error < 50 ppm.

[0271] HPLC: AS1411-JQ1 - HPLC, t R = 13.843 min (260 nm), purity: 95.699%.

[0272] Example 5: AS1411-JQ1 can degrade BRD4

[0273] (1) Hepatoma cells Hep3B were incubated with 1 μM AS1411 or AS1411-JQ1 (prepared in Example 4) for 2 hours. After cell lysis, it was incubated with an antibody against NCL overnight at 4 °C. Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) were added and incubated with rotation at room temperature for 2 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 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 an immunoblotting experiment was carried out. The results are shown in Figure 9A. In the presence of AS1411-JQ1, the antibody against NCL can capture NCL, MDM2, and BRD4, indicating that AS1411-JQ1 can form a MDM2-NCL-PROTAC-BRD4 quaternary complex with NCL, MDM2, and BRD4.

[0274] (2) Incubate the liver cancer cells Hep3B with 0, 200, 400, 600, 800, 1000 nM of AS1411-JQ1. After 12 hours, collect the cell samples, extract the total proteins using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B), and detect the degradation of BRD4 protein by immunoblotting assay. The results are as Figure 9 shown in Figure B. AS1411-JQ1 can dose-dependently reduce the protein level of BRD4.

[0275] (3) Incubate the liver cancer cells Hep3B with 800 nM AS1411-JQ1. At time points of 0, 2, 4, 6, 8, 12 hours, collect the corresponding cell samples for protein extraction and subsequent immunoblotting assay to detect the degradation of BRD4 protein. The results are as Figure 9 shown in Figure C. AS1411-JQ1 can time-dependently reduce the protein level of BRD4.

[0276] (4) Incubate the lung cancer cells A549 with the solvent PBS (Veh) or 800 nM AS1411, JQ1, AS1411+JQ1, AS1411-JQ1 or the positive control dBET1 (purchased from MCE, catalog number: HY-101838). After 12 hours, collect the cell samples for protein extraction and subsequent immunoblotting assay to detect the degradation of BRD4 protein. The results are as Figure 9 shown in Figure D. AS1411-JQ1 can reduce the protein level of BRD4, and the effect is comparable to that of dBET1.

[0277] (5) Incubate the lung cancer cells A549 with 0, 200, 400, 600, 800, 1000 nM of AS1411-JQ1. After 12 hours, collect the cell samples, extract the total proteins using RIPA lysis buffer (purchased from: Beyotime, catalog number: P0013B), and detect the degradation of BRD4 protein by immunoblotting assay. The results are as Figure 9 shown in Figure E. AS1411-JQ1 can dose-dependently reduce the protein level of BRD4.

[0278] (6) Incubate the lung cancer cells A549 with 600 nM AS1411-JQ1. At time points of 0, 2, 4, 6, 8, 12 hours, collect the corresponding cell samples for protein extraction and subsequent immunoblotting assay to detect the degradation of BRD4 protein. The results are as Figure 9 shown in Figure F. AS1411-JQ1 can time-dependently reduce the protein level of BRD4.

[0279] (7) Incubate lung cancer cells A549 with solvent PBS (Veh) or 600 nM AS1411, JQ1, AS1411 + JQ1, AS1411 - JQ1 or positive control dBET1 (purchased from MCE, catalog number: HY - 101838). After 12 hours, collect cell samples for protein extraction and subsequent immunoblotting experiments to detect the degradation of BRD4 protein. The results are as Figure 9 shown in Figure G. AS1411 - JQ1 can reduce the protein level of BRD4, and the effect is comparable to that of dBET1.

[0280] Example 6: AS1411 - JQ1 can promote the ubiquitination of BRD4

[0281] Incubate liver cancer cells Hep3B with solvent PBS or 800 nM AS1411 - JQ1 for 12 hours, and add 5 μM proteasome inhibitor MG132 in the last 6 hours of incubation. After cell lysis, incubate with an antibody against BRD4 (purchased from Cell Signaling Technology, catalog number 13440S) overnight at 4°C. Add Protein A / G magnetic beads (purchased from Thermo Scientific, catalog number 88802) and rotate at room temperature for 2 hours. After washing the BRD4 bound to the magnetic beads 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. Adsorb the magnetic beads with a magnetic stand, and take the supernatant to a new tube. Then, detect the ubiquitination level of BRD4 by immunoblotting experiment. The results are as Figure 10 shown. AS1411 - JQ1 promotes the ubiquitination of BRD4.

[0282] Example 7: AS1411 - JQ1 mediates the degradation of BRD4 through the ubiquitin - proteasome pathway

[0283] Incubate liver cancer cells Hep3B with solvent PBS or 800 nM AS1411 - JQ1 for 12 hours, and add 10 μM proteasome inhibitor MG132 in the last 6 hours of incubation. Collect the corresponding cell samples for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 11 shown. MG132 can block the degradation of BRD4 by AS1411 - JQ1, indicating that the degradation of BRD4 by AS1411 - JQ1 depends on the ubiquitin - proteasome pathway.

[0284] Example 8: AS1411 - JQ1 mediates the degradation of BRD4 depending on MDM2 and NCL

[0285] (1) Transfect negative control siRNA (siNC, SEQ ID NO:5) or NCL siRNA (siNCL, SEQ ID NO:6) into hepatoma cell line Hep3B for 48 hours, and then incubate the cells with solvent PBS (Veh) or 800 nM AS1411-JQ1 for 12 hours. Collect the corresponding cell samples for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 12 shown in Figure A. Silencing NCL can reduce the degradation of BRD4 by AS1411-JQ1, indicating that AS1411-JQ1 mediates the degradation of BRD4 in a NCL-dependent manner.

[0286] (2) Transfect negative control siRNA (siNC, SEQ ID NO:5) or MDM2 siRNA (siMDM2, SEQ ID NO:7) into hepatoma cell line Hep3B for 48 hours, and then incubate the cells with solvent PBS (Veh) or 800 nM AS1411-JQ1 for 12 hours. Collect the corresponding cell samples for protein extraction and subsequent immunoblotting experiments to detect protein degradation. The results are as Figure 12 shown in Figure B. Silencing MDM2 can reduce the degradation of BRD4 by AS1411-JQ1, indicating that AS1411-JQ1 mediates the degradation of BRD4 in a MDM2-dependent manner.

[0287] Example 9: In vitro inhibition of tumor cell proliferation and promotion of apoptosis by AS1411-JQ1

[0288] (1) Seed hepatoma cell line Hep3B in 96-well cell culture plates, and change the culture medium containing solvent PBS (Veh) or 800 nM AS1411, JQ1, AS1411+JQ1 or AS1411-JQ1 every day during the experiment. Use the CCK-8 kit (purchased from MCE, catalog number HY-K0301) for cell proliferation experiments, and measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader on the first, second, third, and fourth days. The results are as Figure 13 shown in Figure A. AS1411-JQ1 can inhibit the proliferation of Hep3B cells.

[0289] (2) Seed hepatoma cell line Hep3B in six-well plates for cell apoptosis experiments. Change the culture medium containing solvent PBS (Veh) or 800 nM AS1411, JQ1, AS1411+JQ1 or AS1411-JQ1 every two days during the experiment. After 6 days, perform cell apoptosis analysis using the Annexin V-FITC and PI double staining apoptosis detection kit (purchased from Beyotime, catalog number C1062L). The results are as Figure 13 shown in Figure B. AS1411-JQ1 can promote the apoptosis of Hep3B cells.

[0290] (3) Lung cancer cells A549 were inoculated into a 96-well cell culture plate, and the culture medium containing solvent PBS (Veh) or 600 nM AS1411, JQ1, AS1411 + JQ1, or AS1411 - JQ1 was replaced every day during the experiment. A CCK-8 kit (purchased from MCE, catalog number HY-K0301) was used for the cell proliferation experiment, and the absorbance values at 450 nm were detected with an enzyme-linked immunosorbent assay reader on the first, second, third, and fourth days. The results are as Figure 14 shown in Figure A, AS1411 - JQ1 can inhibit the proliferation of A549 cells.

[0291] (4) Lung cancer cells A549 were inoculated into a six-well plate for the cell apoptosis experiment. The culture medium containing solvent PBS (Veh) or 600 nM AS1411, JQ1, AS1411 + JQ1, or AS1411 - JQ1 was replaced every two days during the experiment. After 6 days, a cell apoptosis detection kit using Annexin V-FITC and PI double staining (purchased from Beyotime, catalog number C1062L) was used for cell apoptosis analysis. The results are as Figure 14 shown in Figure B, AS1411 - JQ1 can promote the apoptosis of A549 cells.

[0292] Example 10: Inhibiting the growth of tumor cells by AS1411 - JQ1 in vivo

[0293] Hepatoma cells Hep3B were subcutaneously inoculated into BALB / c nude mice at a cell amount of 5×10 6 / mouse, and the tumor growth status was observed every day. After 6 days, solvent PBS (Veh), AS1411, JQ1, AS1411 + JQ1, AS1411 - JQ1, or the positive control dBET1 (purchased from MCE, catalog number: HY-101838) was injected via the tail vein at a dose of 3 μmol / kg, and the injection cycle was once every two days. After 14 days of drug administration, the tumor volume and weight were measured. The results are as Figure 15 shown in Figures A - 15C. The results indicate that compared with PBS, AS1411, JQ1, and AS1411 + JQ1, AS1411 - JQ1 can significantly inhibit tumor growth, and AS1411 - JQ1 is superior to the positive control dBET1.

[0294] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0295] 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.

[0296] In the present invention, simply listing the steps of a method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A BRD4 degrader or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate or deuterated compound thereof, wherein the BRD4 degrader has the following structure: in, GRO is a guanine-rich oligonucleotide residue that can specifically bind to nucleolin; L is a linker; represents a double bond or a single bond, X1 is C or N; Ring A is an aromatic ring or a heterocyclic ring; Ring B is an aromatic ring or a heterocyclic ring; R1 is one or more independent substituents on the benzene ring, which are selected from: H, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), halogen, cyano, nitro, azido, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); R2 is one or more independent substituents on ring A, which are selected from: H, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), halogen, cyano, nitro, azido, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); R3 is one or more independent substituents on the B ring, which are selected from: H, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), halogen, cyano, nitro, azido, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl); R4 is selected from: a single bond, C1-C6 alkylene, -N(C 0-10 alkyl)-; p is an integer from 1 to 100; Preferably, the GROs have a stable G4 structural feature.

2. The BRD4 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 BRD4 degrading agent according to claim 1, characterized in that The GROs is AS1411.

4. The BRD4 degradation agent according to any one of claims 1 to 3, characterized in that L has the following structure: in, L1 is a divalent group connected to GRO, which 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 selected from: a single bond, -O-(C0-C6 alkylene)-, -S-(C0-C6 alkylene)-, -N(R L3 )-(C0-C6 alkylene)-, -N(R L3 )CO-(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 BRD4 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 BRD4 degradation agent according to any one of claims 1 to 3, characterized in that L2 is selected from C1-C20 straight chain alkylene, -(C0-C6 alkylene)-(CH2CH2O) m2 -(C1-C6 alkylene)-, -(C1-C 10 Alkylene)-O-(C1-C 10 Alkylene)-, -(C1-C 10 Alkylene)-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 one methylene unit in the alkylene is replaced by Substitution, G is any suitable trivalent group; Preferably, L2 is The x end is connected to L3, the y end is connected to L1, the J ring is a 4-6 membered saturated heterocyclic ring, m2 is selected from an integer between 1 and 10, h is selected from an integer between 0 and 10, and i is selected from an integer between 0 and 10; More preferably, L2 is More preferably, L2 is selected from:

7. The BRD4 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 BRD4 degrading agent according to any one of claims 4 to 7, characterized in that L1 is a single bond or O.

9. The BRD4 degradation agent according to any one of claims 4 to 7, characterized in that L3 is selected from: a single bond, -N(H)-, 10. The BRD4 degrading agent according to any one of claims 4 to 8, characterized in that Part of R3' is selected from: H, C1-C 10 Alkyl, -(C0-C6 alkylene)-(C3-C 10 Cycloalkyl), -(C0-C6 alkylene)-(C6-C 10 aryl), -(C0-C6 alkylene)-(4-10 membered heterocyclic group), halogen, cyano, nitro, azido, C1-C 10 Halogenated alkyl, C1-C 10 Haloalkoxy, -N(C 0-10 Alkyl)(C 0-10 Alkyl), -N(C 0-10 alkyl)CO(C 0-10 Alkyl), -N(C 0-10 alkyl)CON(C 0-10 Alkyl), -N(C 0-10 Alkyl)SO2(C 0-10 Alkyl), -O(C 0-10 Alkyl), -S(C 0-10 Alkyl), -SO(C 0-10 Alkyl), -SO2(C 0-10 Alkyl), -SO2N(C 0-10 Alkyl)(C 0-10 Alkyl), -COO(C 0-10 Alkyl), -OCO(C 0-10 Alkyl), -CON(C 0-10 Alkyl)(C 0-10 Alkyl), -CO(C 0-10 alkyl).

11. The BRD4 degrading agent according to claim 10, characterized in that R1 is selected from: H, C1-C6 alkyl, halogen, cyano, nitro, hydroxyl, amino, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, preferably halogen; R2 is selected from: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, preferably C1-C3 alkyl; R3 is selected from: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, preferably C1-C3 alkyl; R3' is selected from: H, C1-C6 alkyl, halogen, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, preferably C1-C3 alkyl; R4 is selected from the group consisting of methylene, -N(H)-, and -N(CH3)-, preferably methylene.

12. The BRD4 degradation agent according to any one of claims 4 to 8 and 11, characterized in that The BRD4 degradation agent has the following structure: Preferably, the BRD4 degrading agent has the following structure:

13. The BRD4 degradation agent according to any one of claims 1 to 3, characterized in that The BRD4 degradation agent has the following structure: Wherein, h is selected from an integer between 0-10, and i is selected from an integer between 0-10; preferably, the BRD4 degrading agent is selected from the following structures:

14. A pharmaceutical composition comprising the BRD4 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 BRD4 degrader, comprising the BRD4 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 BRD4 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 BRD4-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, and fibrotic diseases; More preferably, the tumor is selected from the group consisting of lung cancer, malignant melanoma, brain tumor, tumor of digestive organs, uterine cancer, testicular cancer, palate cancer, pharyngeal cancer, tongue cancer, oral cancer, various sarcomas, osteosarcoma, blood system tumors, nervous system tumors, brain glioma, glioblastoma, skin cancer, skin appendage cancer and skin metastasis, medulloblastoma, blastoma, liposarcoma, neuroendocrine tumor, synovial cell sarcoma, gastrinoma, carcinoid tumor, mesothelioma, islet cell carcinoma, nerve sheath tumors, meningiomas, melanomas, acoustic neuromas, adenocarcinomas, lymphoid malignancies, epithelial squamous cell carcinomas, small cell lung cancer, non-small cell lung cancer, squamous cell carcinomas, lung adenocarcinomas, peritoneal cancer, lung squamous cell carcinomas, hepatocellular carcinomas, gastric cancer, intestinal cancer, pancreatic cancer, glioblastomas, 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, penile cancer, esophageal cancer, biliary tract tumors, and head and neck cancer; More preferably, the autoimmune disease is selected from the group consisting of organ-specific autoimmune disease, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis; More preferably, the inflammatory disease is selected from the group consisting of 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 disease, 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; More preferably, the neurodegenerative disease is selected from the group consisting of: 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; More preferably, the pathogen infection-related disease is selected from: influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease; More preferably, the cardiovascular disease is selected from the group consisting of coronary heart disease, peripheral arterial disease, atherosclerosis, ischemic heart disease, ischemic cardiomyopathy, myocardial infarction, heart failure, angina pectoris, myocarditis, hypercholesterolemia, hypertension, ischemia-reperfusion injury, cerebrovascular ischemia (stroke), embolism (e.g., pulmonary embolism, renal embolism, hepatic embolism, gastrointestinal embolism or peripheral limb embolism) or myocardial ischemia; More preferably, the metabolic disease is selected from the group consisting of: diabetes (such as type I diabetes, type II diabetes or gestational diabetes), obesity, fatty liver (NASH or others), cachexia, hypercholesterolemia, gout; More preferably, the fibrotic disease is selected from the group consisting of: myocardial fibrosis, pulmonary fibrosis, renal fibrosis, postoperative stenosis, keloid formation, cirrhosis, biliary cirrhosis, scleroderma.

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