CN118767154A - A multi-arm polyethylene glycol drug conjugate and its application - Google Patents

Abstract

The invention relates to a multi-arm polyethylene glycol drug conjugate, which has a plurality of end groups, can be connected with a plurality of same or different active groups, simultaneously carries a plurality of same or different drug molecules, can increase drug loading and can realize the treatment purpose of simultaneously treating a plurality of targets by once administration.

Description

A multi-arm polyethylene glycol drug conjugate and its application

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to a multi-arm polyethylene glycol drug conjugate and its application in the field of biomedicine.

Background Art

With the continuous development of molecular biology technology, the relationship between gene mutation and disease has been increasingly understood, and nucleic acid drugs have also attracted much attention due to their huge application potential in disease diagnosis and treatment. Nucleic acid drugs can directly target pathogenic genes or mRNA and cure diseases at the molecular level. Compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs can fundamentally regulate the expression of pathogenic genes, and have obvious advantages such as simple design, short R&D cycle, strong targeting specificity, wide range of treatment fields and long-term effectiveness. At present, it is widely used in the treatment of genetic diseases, tumors, viral infections and other diseases, and is one of the most promising areas in biomedical research and development today. Nucleic acid drug therapy is based on the Watson-Crick base complementary pairing principle. By specifically targeting the pathogenic mRNA gene that expresses related proteins, it indirectly regulates the expression of various proteins and achieves the purpose of curing diseases at the molecular level. It has the characteristics of rich targets, high specificity, long duration of drug efficacy, and low risk of drug interactions. There are various ways of administration, such as subcutaneous injection, intravenous drip, intrathecal injection, intravitreal injection, etc. Traditional small molecule drugs and antibody drugs need to bind to target proteins to exert their therapeutic effects. Although they have many advantages, they are limited by spatial structure and limited binding sites (related to whether the target protein has a suitable pocket structure, size, depth, polarity, etc.), and the number of druggable target proteins is limited. Nucleic acid drugs can avoid the limitation of non-druggable targets faced by small molecule drugs and antibody drugs, and can regulate intracellular and extracellular and cell membrane proteins. In theory, nucleic acid drugs only need to know the base sequence of the pathogenic target gene to design the corresponding target nucleotide sequence, and then deliver the drug to the cell through a suitable delivery system to achieve the purpose of curing the disease.

siRNA drugs work through the principle of RNA interference (RNAi): siRNA interacts with other proteins in cells to induce the formation of gene silencing complexes, which target the target mRNA complementary to siRNA through the principle of base complementary pairing, thereby promoting its degradation and silencing, thereby inhibiting the expression of pathogenic proteins. siRNA drugs can be used to treat various diseases such as rare diseases, infectious diseases, tumors, etc., and have broad application prospects.

In the prior art, patent US9320814B2 discloses a composition and method for delivering nucleic acid molecules to cells, which discloses the use of polymer-siRNA complexes to improve the therapeutic application of siRNA, wherein the polymer-siRNA complex comprises at least one block copolymer and at least one nucleic acid molecule, but only generally discloses that coupling a polymer to siRNA can improve the effect of siRNA in inhibiting mRNA.

Patent BRPI1012141B1 discloses a siRNA conjugate having the following structure: A-X-R-Y-B, wherein one of A and B is a hydrophilic polymer compound, the other is a hydrophobic polymer compound, X and Y are connecting molecules, and R is siRNA. It discloses that the hydrophilic or hydrophobic polymer compound is conjugated to siRNA through chemical bonds to improve the in vivo stability of siRNA, and provides that a single-stranded polymer-siRNA can improve the stability of siRNA in vivo.

Patent document CN114748640A discloses a pH-responsive siRNA delivery system, which directly uses polypeptide molecules and PEG molecules to modify siRNA molecules to prepare conjugated compounds. It only discloses that PEG can form a single-stranded connection with siRNA, which can improve the problems of insufficient delivery efficiency, poor biodistribution, and low concentration in the target site in the prior art.

Currently, many siRNA drugs have been developed and successfully launched in the world, such as Onpattro or Givlaari. The existing technology has disclosed the immunogenicity, toxicity, stability and other issues of siRNA drugs. However, there is little disclosure on how to use siRNA drugs more efficiently, such as how to make siRNA drugs treat multiple diseases with one drug, increase their drug loading capacity and increase their efficacy.

Summary of the invention

In order to overcome the shortcomings of the prior art, the present invention designs a drug that can carry multiple siRNAs at the same time. The technical problem to be solved by the present invention is to use multi-arm polyethylene glycol as the core, and simultaneously link multiple siRNA drugs on a multi-arm polyethylene glycol molecule to achieve the purpose of treating multiple diseases with one drug or increasing the siRNA loading amount.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

The first aspect of the present invention provides a multi-arm polyethylene glycol drug conjugate, wherein the multi-arm polyethylene glycol drug conjugate has a structure shown in general formula I:

in:

R is a core structure selected from a polyhydroxy structure, a polyamino structure or a polycarboxyl structure;

n, m, and l are integers from 0 to 250 (e.g., 0, 1, 10, 20, 30, 40, 44, 45, 46, 47, 50, 55, 56, 57, 60, 65, 66, 67, 68, 69, 70, 71, 75, 78, 79, 80, 81, 82, 85, 89, 90, 91, 92, 95, 100, 111, 112, 113, 114, 115, 116, 120, 150, 200, 250), n, m, and l can be the same or different, and n, m, and l can be average values or specific values;

N, M, and L are each independently selected from integers of 1 to 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and N+M+L≥3;

The polyethylene glycol in the present invention can be a polyethylene glycol mixture derivative or a single molecular weight polyethylene glycol derivative.

The coupling mode of polyethylene glycol to the conjugate can be carried out in a cleavable manner or a non-cleavable manner.

The core structure of the present invention can have three functional groups as connecting structures, and the functional groups are independently selected from hydroxyl, amino, hydrazine, azide, olefin, alkyne, carboxyl (aldehyde, ketone, ester, carboxylic acid, anhydride, acyl halide), thiol, disulfide, nitrile, epoxide, imine, nitro and halide.

A ₁ , A ₂ , and A ₃ are linking groups, independently selected from the group consisting of one or more of an amide bond, a C ₁ to C ₁₂ alkylene group, a urea group, an amine group, a carbonyl group, an ether group, an ester group, a biotin-streptavidin complex, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a -triazole-, a carbon-sulfur bond, or an ether bond; the linking groups A ₁ , A ₂ , and A ₃ of the present invention may be the same or different.

Said D ₁ -D ₃ are drug molecules, independently selected from: small molecule drugs, dyes, peptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles, viruses, quantum dots or magnetic resonance imaging contrast agents;

Preferably, _D1 to _D3 are independently selected from small molecule drugs, polypeptides, antibodies, and nucleic acids, wherein the nucleic acids include nucleotide monomers or oligonucleotides; the nucleotide monomers include four deoxyribonucleotide monomers and four nucleotide monomers; the oligonucleotides are substituted oligonucleotides and non-substituted oligonucleotides, wherein the substituted oligonucleotides are phosphorodiamidate morpholino oligonucleotides, and the non-substituted oligonucleotides are selected from any one of locked nucleic acids, siRNA, microRNA, nucleic acid aptamers, peptide nucleic acids, decoy ODNs, catalytic RNAs, and CpG dinucleotides;

In an embodiment of the present invention, D ₁ -D ₃ are all different in type, or at least one of them is different in type.

In an embodiment of the present invention, _D1 - _D3 are all different in type, or at least one is different in type. In some embodiments of the present invention, the siRNA is modified by phosphate backbone, sugar ring, base, and a combination of one or more of 3' and 5' terminal thiol, azido, maleimide, alkynyl, tetrazine, cyclooctyne or biotin modification.

In some embodiments of the present invention, the targets of the siRNA include: one or more of MLPH, TOP1, TOP2, TUBB2, B7H3, KDR, Tyr, CFD, EGFR, MITF, FGF, and JAK.

Furthermore, the multi-arm polyethylene glycol drug conjugate has a structure shown in general formula II:

Further, the n, m, and l are each independently selected from an integer of 0 to 80 (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 40, 50, 60, 70, 80), preferably, n, m, and l are each independently selected from an integer of 0 to 60, more preferably, n, m, and l are each independently selected from an integer of 0 to 50;

Furthermore, the multi-arm polyethylene glycol drug conjugate has the following general formula III structure:

Wherein, siRNA ₁ , siRNA ₂ , and siRNA ₃ are double-stranded siRNAs, which may be the same or different;

Preferably, the siRNA ₁ , siRNA ₂ , and siRNA ₃ are all different double-stranded siRNAs.

Preferably, the siRNA can be unmodified siRNA and modified siRNA, wherein the modified siRNA includes phosphate backbone modification, sugar ring modification, base modification, and a combination of one or more modifications selected from the group consisting of 3′ and 5′ terminal thiol, azido, maleimide, alkynyl, tetrazine, cyclooctyne or biotin modification.

Furthermore, the targets of the siRNA include, but are not limited to, one or more of MLPH, TOP1, TOP2, TUBB2, B7H3, KDR, Tyr, CFD, KDR, EGFR, MITF or FGF.

Further, the R is Preferably, the R is a triol group, and more preferably, the R is a glycerol group. Further, the multi-arm polyethylene glycol drug conjugate has the following structure

Furthermore, the multi-arm polyethylene glycol drug conjugates A ₁ , A ₂ , and A ₃ are selected from one or more combinations of the following structures:

-(CH ₂ ) _i -, -(CH ₂ ) _i NH-, -(CH ₂ ) _i NHCO-, -CO(CH ₂ ) _i CO-, -(CH ₂ ) _i CO-, -NH(CH ₂ ) _iNHCO- ,

wherein i is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), R ₁ is a C ₁ to C ₁₀ alkane (which may be C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10), and j is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);

In a specific embodiment of the present invention, _A1 , _A2 , and _A3 are directly connected to the modified siRNA. During the preparation process, for example, when one end of PEG is a DBCO end group, and the end group of siRNA is modified with _N3 , the connection mode between siRNA and PEG is that the end group of PEG is coupled with the active group at the 3' or 5' end of siRNA, which is irrelevant to the phosphate backbone modification, sugar ring modification, and base modification of siRNA.

Preferably, A ₁ , A ₂ , and A ₃ are selected from one or more combinations of the following structures: -CH ₂ CH ₂ -SS-, -NH

(CH ₂ ) _i NHCO-, -(CH ₂ ) _i NHCO-,

Furthermore, the multi-arm polyethylene glycol drug conjugate structure is selected from the following IV to VI structures:

Among them, in structures IV to VI, siRNA1, siRNA2, and siRNA3 may be the same or different. Further, siRNA1, siRNA2, and siRNA3 are independently selected from the sequences shown in Tables 1 to 8:

Table 1MLPH siRNA sequences

Table 2 Top1 siRNA sequences

Table 3 TUBB2 siRNA sequence list

Table 4B7-H3 siRNA sequences

Table 5 KDR siRNA sequence list

Table 6 CFD siRNA sequence list

Table 7 EGFR siRNA sequence list

Table 8 TOP2A siRNA sequence list

The second aspect of the present invention further provides a pharmaceutical composition, which comprises the multi-arm polyethylene glycol drug conjugate described in the first aspect;

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, wherein the excipients are selected from: carriers, diluents, adhesives, lubricants, wetting agents;

More preferably, the dosage form of the pharmaceutical composition includes: tablets, capsules, pills, injections, emulsions, microemulsions, nanoparticles, inhalants, lozenges, gels, powders, suppositories, suspensoids, creams, jellies or sprays;

More preferably, the pharmaceutical composition can be administered orally, subcutaneously, intramuscularly, intravenously, rectally, vaginally, nasally, transdermally, subconjunctivally, intraocularly, orbitally, retro-ocularly, retinaly, choroidally or intrathecally.

The third aspect of the present invention provides a drug delivery system, comprising the multi-arm polyethylene glycol drug conjugate of the present invention. Preferably, the drug delivery system is a lipid delivery system, and more preferably, the drug delivery system is a lipid nanoparticle.

More preferably, the drug delivery system further comprises a combination of one or more of cationic lipids, steroids, auxiliary lipids and PEG lipids.

More preferably, the cationic lipid is selected from ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), octadecane-9-yl 8-((2-hydroxyethyl)(6-oxO-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3,13-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium (DDA), 1,2-dimyristoyl-3- Trimethylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearyl trimethylammonium propane (DSTAP), N-[1-(2,3-diallyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycerotrioxy-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA).

More preferably, the auxiliary lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

More preferably, the steroid is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid.

More preferably, the lipid-PEG conjugate is selected from the polyethylene glycol lipid selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

The fourth aspect of the present invention provides a use of the drug conjugate described in the first aspect in the preparation of a targeted drug, wherein the drug is used for diagnosing and/or treating any disease caused by abnormally high expression of pathogenic proteins or pathogenic genes.

Furthermore, the diseases include: tumors, infectious diseases, hyperlipidemia, cancer, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, eye diseases and rare diseases, etc.

Preferably, the cancer includes, but is not limited to, lymphoma, B cell tumor, T cell tumor, myeloid/monocytic tumor, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.

More preferably, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia;

More preferably, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia;

More preferably, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

Preferably, the autoimmune diseases include but are not limited to allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, myasthenia gravis, multiple sclerosis, urticaria, psoriasis, dermatomyositis, Sjögren's syndrome, pain or neurological disorders, etc.

Preferably, the inflammatory disease includes acute inflammation and chronic inflammation.

More preferably, the inflammatory disease includes but is not limited to degenerative inflammation, exudative inflammation, proliferative inflammation, specific inflammation, etc., including but not limited to severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, post-streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2-induced vascular leakage syndrome or radiographic (contrast) contrast agent allergies, etc.

Preferably, the neurodegenerative diseases include but are not limited to Alzheimer's disease, progressive blindness or extraocular muscle palsy, multiple system atrophy, frontotemporal dementia, Huntington's chorea, cortical basal degeneration, spinocerebellar ataxia, motor neuron disease, hereditary motor sensory neuropathy, etc.

Preferably, the eye disease includes, but is not limited to, macular degenerative diseases such as all stages of age-related macular degeneration (AMD) including dry and wet (non-exudative and exudative) forms, diabetic retinopathy and other ischemia-related retinopathies, choroidal neovascularization (CNV), uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), corneal neovascularization, and retinal neovascularization.

More preferably, the age-related macular degeneration (AMD) includes non-exudative (e.g., intermediate dry AMD or geographic atrophy (GA)) and exudative (e.g., wet AMD (choroidal neovascularization (CNV)) AMD, diabetic retinopathy (DR), endophthalmitis and uveitis, and non-exudative AMD may include hard drusen, soft drusen, geographic atrophy and/or pigment agglomeration, etc.

Beneficial effects of the present invention:

The multi-arm polyethylene glycol drug conjugate provided by the present invention has targeting properties, and can be connected with a targeting group according to specific treatment needs, so that the drug can enter the pathogenic cells in a targeted manner to achieve the purpose of precise treatment. Compared with the single-arm drug conjugate, the multi-arm polyethylene glycol drug conjugate of the present invention can increase the drug loading capacity. The multi-arm polyethylene glycol drug conjugate of the present invention can also carry multiple drug molecules at the same time, and can achieve the effect of the combination of multiple drug molecules, thereby realizing the therapeutic purpose of treating multiple targets simultaneously with a single administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the NMR image of intermediate 3;

Figure 2 shows the NMR image of intermediate 4;

FIG3 shows the NMR image of intermediate 5;

FIG4 shows the NMR image of intermediate 6;

FIG5 shows the NMR image of intermediate 7;

FIG6 shows the NMR image of intermediate 8;

FIG7 shows the NMR image of intermediate 11;

FIG8 shows the NMR image of intermediate 12;

FIG9 shows the NMR image of norbornene-2-carboxamidoethylamine;

FIG10 shows the NMR image of intermediate 13;

FIG11 shows the NMR image of intermediate 14;

FIG12 shows the NMR image of compound 1;

FIG. 13 is a graph showing the gene expression levels of each group of drugs in Example 18.

DETAILED DESCRIPTION

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

The term siRNA in the present invention refers to a short (usually less than 30 nucleotides in length, more usually between about 21 to about 25 nucleotides in length) double-stranded RNA molecule.

The term _C1-12 straight chain/branched alkyl in the present invention includes methyl, ethyl, _C3 straight chain/branched alkyl, _C4 straight chain/branched alkyl, C5 straight chain/branched alkyl, _C6 straight chain/branched alkyl, _C7 straight chain/branched alkyl, _C8 straight chain/branched alkyl, _C9 straight chain/branched alkyl, _C10 straight chain/branched alkyl, _C11 straight chain/branched alkyl _{, C12} straight chain/branched alkyl.

The term "N ₃ " in the present invention refers to an azide group.

The term "TCO" in the present invention refers to a trans-cyclooctene group.

The term "Tetrazine" in the present invention and the term "MTZ" in the present invention both refer to a tetrazine group.

The term "MAL" in the present invention refers to a maleimide group.

The term "hot melt cold precipitation" in the present invention refers to the hot melt cold precipitation method, which is a commonly used method for crystallization using the difference in solubility of the solute under different temperature conditions. In the crystallization process, impurities with lower content are less likely to reach saturated precipitation than target products with higher content.

The term "THF" in the present invention refers to tetrahydrofuran, also known as oxacyclopentane and 1,4-butylene oxide, which is a colorless transparent liquid soluble in water, ethanol, ether, acetone, benzene, etc. It is mainly used as a solvent, chemical synthesis intermediate, and analytical reagent.

The term "deaerated water" in the present invention refers to purified water that has been treated by "appropriate methods" to remove air dissolved in the water.

The term _C1-10 straight chain/branched alkyl in the present invention includes methyl, ethyl, _C3 straight chain/branched alkyl, _C4 straight chain/branched alkyl, C5 straight chain/branched alkyl, _C6 straight chain/branched alkyl, _C7 straight chain/branched alkyl, _C8 straight chain/branched alkyl _{, C9} straight chain/branched alkyl, _C10 straight chain/branched alkyl.

The term Streptavidin in the present invention (CAS No.: 9013-20-1) refers to streptavidin, which is a 60-kDa extracellular protein of Streptomyces avidinii, has four high-affinity biotin binding sites, has an isoelectric point close to neutral, and does not contain carbohydrate side chains. It is often used as a multifunctional affinity tag, and has the structural formula:

The term Biotin in the present invention is "biotin", also known as vitamin B7, which is a member of the vitamin B group. It is mainly a cofactor of various carbonic acid enzymes in the human body. It is an indispensable nutrient for fatty acid synthesis, gluconeogenesis, amino acid decomposition and other processes. It has a strong affinity with streptavidin, and its dissociation constant ^Kd≈10-14 mol/L, even higher than antigen-antibody, is one of the strongest non-covalent bonds in nature. The complex they form is very stable and can connect molecules, labels or supports that need to be coupled and fixed together like "biological glue". It is convenient and not easy to dissociate, so it is widely used in biological research.

The term norbornene in the present invention has the structure

The term "OPA" in the present invention refers to a propionic acid residue having the structure

The term "SOPSS" in the present invention refers to 2-thiopyridine, which has the structure

The term "S-MAL" in the present invention refers to mercaptomaleimide, which has the structure

The term "DBCO" in the present invention refers to dibenzocyclooctyne, which has the structure

The term "Me" in the present invention refers to a methyl group, and has the structure -CH ₃ .

The term "t-BuOK" in the present invention refers to potassium tert-butoxide.

The term "THF" in the present invention refers to tetrahydrofuran.

The term "MsCl" in the present invention refers to methanesulfonyl chloride.

The term "TEA" in the present invention refers to triethylamine.

The term "DCM" in the present invention refers to dichloromethane.

The term "NHS" in the present invention refers to N-hydroxysulfosuccinimide.

The term "DCC" in the present invention refers to dicyclohexylcarbodiimide.

The term "treatment" in the present invention includes eradicating, removing, reversing, alleviating, changing or controlling the disease and/or condition after the onset of the disease and/or condition.

The term "prevention" in the present invention refers to the ability to avoid, minimize or make the onset or development of a disease and/or condition difficult by treatment before the disease and/or condition occurs.

The term "disease" in the present invention refers to a physical state of the subject, which is related to the disease and/or disorder described in the present invention.

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1 Synthesis of three-arm polyethylene glycol-monopropionic acid

The synthesis steps are as follows:

Steps: A three-necked round-bottom flask is vented with nitrogen, 500g of three-arm polyethylene glycol-5K (synthesized by Jiankai) and 3L of toluene are added, heated to dissolve, and about two-thirds of the toluene is evaporated, cooled, 3L of THF and 2.8g of potassium tert-butoxide are added to react at room temperature for 2h, 21.8g of tert-butyl acrylate is added dropwise, reacted at room temperature overnight, filtered the next day, and the reaction liquid is vacuum concentrated to a viscous liquid (intermediate 1 is obtained). A 3mol/L HCl dilute hydrochloric acid solution is added to the above system, acid hydrolyzed for 4 to 5 hours, 15% sodium chloride by mass is added to the solution, extracted three times with dichloromethane, the organic phases are combined, dried over anhydrous sodium sulfate, filtered and concentrated to a viscous state. 500 g of crude DEAE was separated and purified by anion exchange, and the sodium chloride eluate was collected and the pH was adjusted to 2-3 with 2 mol/L HCl solution. The organic phases were combined and dried over anhydrous sodium sulfate overnight, and then dissolved in 10.0 L of isopropanol-ethyl acetate (3:1) mixed solvent and cooled to obtain three-arm polyethylene glycol monopropionic acid (intermediate 2) with a molecular weight of 5000.

NMR(DMSO)2.45(t,CH ₂ COOCH ₃ )3.7(t,CH ₂ -CH ₂ COOCH ₃ );

The NMR image of intermediate 1 is shown in FIG12 .

Example 2 Synthesis of three-arm polyethylene glycol-methyl propionate

Steps: A three-necked round-bottom flask was vented with nitrogen, 100 g of three-arm polyethylene glycol monopropionic acid-5000 (intermediate 2) and 1 L of anhydrous methanol were added, 20 ml of concentrated sulfuric acid was slowly added dropwise, and the reaction was carried out at room temperature for three hours. The pH value of the system was adjusted to about 7.0 with an 8% sodium bicarbonate aqueous solution by mass fraction. The mixture was extracted with dichloromethane three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and dissolved by heat with 2.0 L of isopropanol-ethyl acetate (3:1) mixed solvent and cooled, and dried under vacuum to obtain three-arm polyethylene glycol monopropionic acid methyl ester (intermediate 3).

NMR(DMSO)2.45(t,CH ₂ COOCH ₃ ), 3.32(S,CH ₂ COOCH ₃ ), 3.7(t,CH ₂ -CH ₂ COOCH ₃ )

The NMR image of intermediate 3 is shown in Figure 1.

Example 3 Synthesis of three-arm polyethylene glycol-monomethyl propionate mono-methanesulfonate

Steps: A three-necked round-bottom flask was vented with nitrogen, and 90 g of three-arm polyethylene glycol monopropionate methyl ester (intermediate 3), 500 ml of dichloromethane, 2.51 g of MsCl (methylsulfonyl chloride), and 3.10 g of triethylamine were added. The reaction system was sealed overnight. The next day, 0.2 ml of ethanol was added and stirred for 15 min, concentrated to a viscous state, and 1.8 L of isopropanol-ethyl acetate (3:1) mixed solvent was heated and cooled, and vacuum dried to synthesize three-arm polyethylene glycol monopropionate methyl ester monomethanesulfonate (intermediate 4).

NMR(DMSO)2.45(t,CH ₂ COOCH ₃ )3.17(S,SO ₂ CH ₃ ), 3.7(t,CH ₂ -CH ₂ COOCH ₃ ), 4.32ppm(t,CH ₂ OOSO ₂ CH ₃ )

The NMR image of intermediate 4 is shown in FIG2 .

Example 4 Synthesis of three-arm polyethylene glycol-monopropionic acid-monoamine

Steps: a three-mouth round-bottom flask, nitrogen, 80g of three-arm polyethylene glycol monopropionate methyl ester monomethanesulfonate (intermediate 4) was added, dissolved in 400ml of degassed water, 2mol/L sodium hydroxide solution was used to adjust the pH to about 12, the reaction was carried out at room temperature for 3 hours, 1L of ammonia solution in which 50g of ammonium chloride was dissolved was added to the system, and the mixture was stirred at room temperature for 72 hours. After completion of the reaction, 300g of sodium chloride was added, and the mixture was extracted with dichloromethane three times, and the organic phases were combined, and the mixture was concentrated to dryness at 40 degrees. 1L of degassed water was added to dissolve until clarified, the pH was adjusted to 2-3 with 2mol/L dilute hydrochloric acid, 200g of sodium chloride was added, and the organic phases were combined again with dichloromethane, dried over anhydrous sodium sulfate, and concentrated to dryness at 40 degrees. Purified with a CM-DEAE ion exchange column, the washed components were collected, the pH was adjusted to 2-3 with 2mol/L dilute hydrochloric acid, and the organic phases were combined again with dichloromethane three times, dried over anhydrous sodium sulfate, and concentrated to dryness at 40 degrees. 1.6L of isopropanol-ethyl acetate (3:1) mixed solvent was heated and cooled to obtain a synthetic three-arm polyethylene glycol monopropionic acid monoamine (intermediate 5) with a molecular weight of 5000.

NMR(DMSO)2.45(t,CH ₂ COOCH ₃ )), 2.98ppm(t,CH ₂ -NH-),

3.7(t,CH ₂ CH ₂ COOCH ₃ ),

The NMR image of intermediate 5 is shown in FIG3 .

Example 5 Synthesis of three-arm polyethylene glycol-methyl propionate-monoamine

Steps: The starting material is a three-arm polyethylene glycol monopropionic acid monoamine (intermediate 5) with a molecular weight of 5000. A three-necked round-bottom flask is purged with nitrogen. A three-arm polyethylene glycol monopropionic acid monoamine (intermediate 5) with a molecular weight of 5000 is added. 1L of anhydrous methanol is added. 20ml of concentrated sulfuric acid is slowly added dropwise. The reaction is carried out at room temperature for three hours. The pH value of the system is adjusted to about 7.0 with an 8% sodium bicarbonate aqueous solution. The mixture is extracted with dichloromethane three times. The organic phases are combined, dried over anhydrous sodium sulfate, filtered, concentrated, and hot-dissolved and cold-precipitated using a 2.0L isopropanol-ethyl acetate (3:1) mixed solvent. The mixture is dried in vacuo to obtain a three-arm polyethylene glycol-monopropionic acid methyl ester-monoamine (intermediate 6).

NMR (DMSO): 2.45 (t, CH ₂ COOCH ₃ ), 2.98 ppm (t, CH ₂ -NH-), 3.32 (S, CH ₂ COOCH ₃ ) 3.7 (t, CH ₂ CH ₂ COOCH ₃ )

The NMR image of intermediate 6 is shown in FIG4 .

Example 6 Synthesis of three-arm polyethylene glycol-monomethyl propionate-monotert-butoxycarbonylamine

Steps: A three-necked round-bottom flask was purged with nitrogen, and 70 g of three-arm polyethylene glycol monopropionate methyl ester monoamine (intermediate 6), 3.9 g of Boc ₂ O, 2.0 g of triethylamine, and 350 ml of dichloromethane were added. The reaction was stirred overnight. The next day, the system was concentrated until no liquid dripped out, and 1.4 L of isopropanol-ethyl acetate (3:1) mixed solvent was added for hot dissolution and cold precipitation (specific operation: hot dissolution at 30-40 degrees until all solids were dissolved, and then ice-water bath crystallization) was performed, and vacuum drying was performed to obtain three-arm polyethylene glycol monopropionate methyl ester monotert-butoxycarbonylamine (intermediate 7).

NMR (DMSO): 1.32 (S, -CH ₃ ), 2.45 (t, CH ₂ COOCH ₃ ), 3.32 (S, CH ₂ COOCH ₃ ), 3.7 (t, CH ₂ CH ₂ COOCH ₃ )

The NMR image of intermediate 7 is shown in FIG5 .

Example 7 Synthesis of three-arm polyethylene glycol-monomethyl propionate-monotert-butoxycarbonylamine-monomethanesulfonate

Steps: The starting material is a three-arm polyethylene glycol monopropionate methyl ester monotert-butoxycarbonylamine (intermediate 7) with a molecular weight of 5000, a three-necked round-bottom flask, nitrogen, 90g of a three-arm polyethylene glycol monopropionate methyl ester monotert-butoxycarbonylamine (intermediate 7) with a molecular weight of 5000, 500ml of dichloromethane, 2.51g of MsCl (methylsulfonyl chloride), 3.10g of triethylamine, and the reaction system is sealed overnight. The next day, 0.2ml of ethanol is added and stirred for 15min, concentrated to a viscous state, 1.8L of isopropanol-ethyl acetate (3:1) mixed solvent is heated and cooled, and vacuum dried to obtain a three-arm polyethylene glycol-monopropionate methyl ester-monotert-butoxycarbonylamine-monomethanesulfonate (intermediate 8).

NMR (DMSO): 1.32 (S, -CH ₃ ), 2.45 (t, CH ₂ COOCH ₃ ), 3.17 (S, CH ₂ OOSO ₂ CH ₃ ), 3.7 (t, CH ₂ CH ₂ COOCH ₃ )

The NMR image of intermediate 8 is shown in FIG6 .

Example 8 Synthesis of three-arm polyethylene glycol-monomethyl propionate-mono-tert-butoxycarbonylamine-monothiol-2-thiopyridine

Steps: A three-necked round-bottom flask was vented with nitrogen. 60g of three-arm polyethylene glycol monopropionate methyl ester monotert-butoxycarbonylamine monomethanesulfonate (intermediate 8), 4.5g of thiourea, and 600ml of ethanol were added. The mixture was heated to reflux and reacted overnight (intermediate 9 was generated). The next day, the reaction system was concentrated. 450ml of degassed water, 16g of sodium hydroxide solid, and 8g of dithiothreitol were added. The reaction was carried out overnight under nitrogen protection. The next day, the pH of the system was adjusted to 5-6 with 2mol/L dilute hydrochloric acid. The impurities were removed by extraction with ethyl acetate three times. 60g of sodium chloride was added. The organic phases were combined, filtered with anhydrous sodium sulfate, and concentrated. The three-arm polyethylene glycol monopropionate monotert-butoxycarbonylamine monothiol (intermediate 10) was obtained. 40g of 2,2′-dithiopyridine and 400ml of MeOH were added to the three-necked flask and stirred until completely dissolved. The obtained three-arm polyethylene glycol monopropionic acid mono-tert-butoxycarbonylamine monothiol was dissolved in 0.7 L of methanol, and a methanol solution of 2,2′-dithiopyridine was slowly added dropwise. The reaction was allowed to proceed overnight. The system was concentrated to dryness the next day, and 0.7 L of degassed water was added for dissolution. The mixture was extracted three times with ethyl acetate. 120 g of sodium chloride was added, and the mixture was extracted with dichloromethane. The organic phases were combined and filtered with anhydrous sodium sulfate. The mixture was concentrated to a viscous oil. 1.4 L of isopropanol-ethyl acetate (3:1) mixed solvent was added, the mixture was dissolved by heating and precipitated by cooling. The mixture was dried in vacuo to obtain the three-arm polyethylene glycol monopropionic acid mono-tert-butoxycarbonylamine monothiol-2-thiopyridine (intermediate 11).

NMR (DMSO) 1.32 (S, -CH ₃ ), 7.12, 7.68, 7.75, & 8.47 ppm (4m, pyridine protons, 4H).

The NMR image of intermediate 11 is shown in FIG7 .

Example 9 Synthesis of three-arm polyethylene glycol-monopropionic acid-NHS-mono-tert-butoxycarbonylamine-monothiol-2-thiopyridine

Steps: A three-necked round-bottom flask was vented with nitrogen, and 30 g of three-arm polyethylene glycol monopropionic acid monotert-butoxycarbonylamine monothiol-2-thiopyridine (intermediate 11) was added, 1.0 g of hydroxysuccinimide, and 300 ml of dichloromethane were fully dissolved. 1.5 g of dicyclohexylcarbodiimide was added in batches and reacted overnight. The next day, diatomaceous earth was filtered, the filtrate was concentrated and evaporated to dryness, 600 ml of isopropanol-ethyl acetate (3:1) mixed solvent was added, hot dissolved and cold precipitated, and vacuum dried to obtain three-arm polyethylene glycol monopropionic acid-NHS monotert-butoxycarbonylamine monothiol-2-thiopyridine (intermediate 12).

NMR (DMSO) 1.32 (S, -CH ₃ ), 2.83 (S, CH ₂ on NHS), 7.12, 7.68, 7.75, & 8.47 ppm (4m, pyridine protons, 4H).

The NMR image of intermediate 12 is shown in FIG8 .

Example 10 Synthesis of norbornene-2-carboxamidoethylamine

Steps: A three-necked round-bottom flask was vented with nitrogen, and 5.1 g of norbornene-2-carboxylic acid, 4.72 g of NHS and 9.16 g of DCC were added. The reaction was detected by TLC. After the reaction was completed, the insoluble matter was removed by suction filtration, 4.8 g of Boc-amino-ethylamine and 3.6 g of triethylamine were added, and the reaction was concentrated to dryness after the TLC detection was completed. Boc was removed with 3 mol/L dilute hydrochloric acid, and the reaction was tracked by TLC until it was completed. The aqueous phase was extracted with a mixed solvent of DCM:IPA=10:1 by volume until no product remained, the organic phase was dried over anhydrous sodium sulfate, and concentrated to dryness to obtain a viscous oil.

NMR (DMSO) 6.00, 6.20 (CH=CH)

The NMR spectrum of norbornene-2-carboxamidoethylamine is shown in FIG9 .

Example 11 Synthesis of three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-mono-tert-butoxycarbonylamine-monothiol-2-thiopyridine

Steps: A three-necked round-bottom flask was vented with nitrogen, and 8.0 g of intermediate 12 (three-arm polyethylene glycol monopropionic acid-NHS mono-tert-butoxycarbonylamine monothiol-2-thiopyridine), 0.31 g of norbornene-2-carboxamidoethylamine, and 0.24 g of triethylamine were added. The reaction was stirred overnight. The system was concentrated to dryness the next day. The concentrated residue was dissolved in 200 ml of water, extracted three times with 50 ml of ethyl acetate, extracted three times with 50 ml of dichloromethane, filtered with anhydrous sodium sulfate, and concentrated to a viscous oil. 160 ml of a mixed solvent of isopropanol-ethyl acetate with a volume ratio of (3:1) was added, and the mixture was dissolved by heat and cooled, and vacuum dried to obtain three-arm polyethylene glycol monopropionylaminoethylamine norbornene mono-tert-butoxycarbonylamine monothiol-2-thiopyridine (intermediate 13).

NMR (DMSO), 1.37 (S, -CH ₃ ), 6.00, 6.20 (m, CH=CH), 7.12, 7.68, 7.75, & 8.47 ppm (m, pyridine protons, 4H).

The NMR image of intermediate 13 is shown in FIG10 .

Example 12 Synthesis of three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine-monothiol-2-thiopyridine

Steps: A three-necked round-bottom flask was vented with nitrogen, and 5.0 g of intermediate 13 (three-arm polyethylene glycol monopropionylaminoethylamine norbornene monotert-butoxycarbonylamine monothiol-2-thiopyridine) and 80 ml of 3 mol/L dilute hydrochloric acid were added. After 1 hour, 20 ml of ethyl acetate was extracted three times, and 20 ml of dichloromethane was extracted three times. The mixture was filtered with anhydrous sodium sulfate and concentrated to a viscous oil. 100 ml of a mixed solvent of isopropanol-ethyl acetate (volume ratio: 1:3) was added, and the mixture was dissolved by heat and cooled, and vacuum dried to obtain three-arm polyethylene glycol monopropionylaminoethylamine norbornene monoamine monothiol-2-thiopyridine (intermediate 14).

NMR (DMSO) 6.00, 6.20 (m, CH=CH), 7.12, 7.68, 7.75, & 8.47 ppm (m, pyridine protons, 4H).

The NMR image of intermediate 14 is shown in FIG11 .

Example 13 Synthesis of three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine DBCO-monothiol-2-thiopyridine

Steps: A three-necked round-bottom flask was vented with nitrogen, and 3.0 g of intermediate 14 (three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine-monothiol-2-thiopyridine), 360 mg DBCO-NHS, 122 mg of triethylamine, and 30 ml of dichloromethane were added. The mixture was stirred overnight. The next day, the system was concentrated to dryness, and the concentrated residue was dissolved in 90 ml of water, extracted three times with 50 ml of ethyl acetate, and extracted three times with 50 ml of dichloromethane. The mixture was filtered with anhydrous sodium sulfate and concentrated to a viscous oil. 60 ml of a mixed solvent of isopropanol-ethyl acetate (1:3) was added for hot dissolution and cold precipitation, and vacuum drying was performed to obtain three-arm polyethylene glycol monopropionylaminoethylamine norbornene monoamine DBCO monothiol-2-thiopyridine.

NMR (DMSO) 6.00, 6.20 (m, CH=CH), 7.0-8.0 ppm (m, pyridine protons, 4H; DBCO, 8H).

The NMR spectrum of three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine DBCO-monothiol-2-thiopyridine is shown in Figure 11.

Example 14 Synthesis of three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine DBCO-monothiol (3ARM-NB-DBCO-SH)

Steps: A three-necked round-bottom flask was purged with nitrogen, 3.0 g of intermediate 14 (three-arm polyethylene glycol-monopropionylaminoethylamine norbornene-monoamine DBCO-monothiol-2-thiopyridine) was added, dissolved with 30 ml of PBS with a pH of 7.4, stirred, 277 mg of DTT was added, and the reaction was allowed to proceed overnight at room temperature. The pH of the system was adjusted to 4-5 with 1N HCl, 4.5 g of sodium chloride was added, and the mixture was extracted three times with 15 ml + 12 ml + 10 ml of dichloromethane, dried and filtered over anhydrous sodium sulfate, and the filtrate was concentrated using a rotary evaporator at 40 degrees until no liquid dripped out, 60 ml of isopropanol was added, ice-bath precipitated, filtered, and the filter cake was washed twice with isopropanol. The filter cake was dried in vacuo to obtain three-arm polyethylene glycol monopropionylaminoethylamine norbornene monoamine DBCO monothiol (3ARM-NB-DBCO-SH, compound 1).

NMR (DMSO) 6.00, 6.20 (m, CH=CH), 7.0-8.0ppm (DBCO, 8H), 1.0-2.0ppm (SH, 1H).

Example 15 Synthesis of 3ARM-NB-DBCO-(S-MAL)-TOP1-siRNA

Take 3ARM-NB-DBCO-SH (Compound 1, the product synthesized in Example 14), dissolve it with PBS at pH = 7.4; take TOP1-1630-siRNA lyophilized powder (the specific structure of TOP1-siRNA is 5'-MAL-TOP1-siRNA, purchased from Sangon Biotech (Shanghai) Co., Ltd., SEQ ID NO: 39 and SEQ ID NO: 40), centrifuge, add PBS at pH = 7.4 to dissolve, stir and react at room temperature for 0.5-2h, use an ultrafiltration centrifuge tube with MWCO10k to desalt and remove excess PEG, and the product (Compound 2, 3ARM-NB-DBCO-(S-MAL)-TOP1-siRNA) remains in a liquid state;

Example 16 Synthesis of 3ARM-NB-(DBCO-N3)-TOP2 siRNA-S-MAL-TOP1 siRNA

Take 3ARM-NB-DBCO-(S-MAL)-TOP1-siRNA solution (Compound 2), take TOP2-1668-siRNA lyophilized powder (5'-N ₃ -TOP2-siRNA, purchased from Sangon Biotech (Shanghai) Co., Ltd., SEQ ID NO: 133 and SEQ ID NO: 134), centrifuge, add PBS with H=7.4 to dissolve, stir and react at room temperature for 0.5-2h, use ultrafiltration centrifuge tube with MWCO10k for desalting, and the product (Compound 3) is retained in liquid state.

Example 17 Synthesis of 3ARM-(NB-MTZ)TUB2 siRNA-(DBCO-N3)TOP2 siRNA-S-MALTOP1 siRNA

3ARM-NB-(DBCO-N ₃ )-TOP2 siRNA-S-MAL-TOP1 siRNA (Compound 3) liquid was taken and dissolved with PBS at pH=7.4; TUBB2-siRNA freeze-dried powder (5'-MTZ-TUBB2-457 siRNA, purchased from Sangon Biotech (Shanghai) Co., Ltd., SEQ ID NO: 65 and SEQ ID NO: 66) was taken and centrifuged, and dissolved in PBS at pH=7.4, and reacted with stirring at room temperature for 0.5-2h, and desalted to remove excess unreacted siRNA TOP1, siRNA TOP2 and siRNA TUB2 using an ultrafiltration centrifuge tube with MWCO20k, and the product was stored by freeze-drying.

Example 18 Cell experiment

One day before transfection, melanoma A375 cells in logarithmic growth phase were plated in 24-well plates at 2000-2500 cells/well and cultured overnight in a 37°C 5% CO ₂ incubator using DMEM medium containing 7.5% FBS and 1% P/S.

On the day of transfection, the culture plate was removed and the culture medium was replaced with DMEM medium containing 7.5% FBS, 450 μL/well. 25 μL Opti-MEM medium was used to dilute siRNA, and the negative control group was siNC of the same concentration (purchased from Qingke Biotechnology Co., Ltd.). Another 25 μL Opti-MEM medium was used to dilute Lipofectamine ^TM RNAiMAX transfection reagent, and the mixture was allowed to stand at room temperature for 5 min; the two were mixed and allowed to stand at room temperature for 5 min.

Add 50 μL of the above mixture to each well, the final transfection system is 500 μL, and the working concentration of each siRNA is 50 nM. Incubate in a 37°C 5% CO ₂ incubator for 48 h. Collect cells, extract cell RNA, use Nanodrop ^TM to detect RNA concentration and quality, and store in a -70°C refrigerator for later use.

0.5-1 ug RNA was reverse transcribed into a cDNA template, and then the expression level of the target gene was detected using the qReal-Time PCR method. The primer sequences used were as follows: TOP1-F: CTGTAGCCCTGTACTTCATCG (SEQ ID NO: 135), TOP1-R: CTACCACATATTCCTGACCATCC (SEQ ID NO: 136), TOP2A-F: TGACAGTGAAGAAGACAGCAG (SEQ ID NO: 137), TOP2A-R: CGATTCTTGGTTTTGGCAGG (SEQ ID NO: 138), TUBB2-F: GCCAGATCTTCAGACCAGAC (SEQ ID NO: 139), TUBB2-R: ACTCCTTCCTCACCACATCC (SEQ ID NO: 140).

The experimental results are shown in Figure 13. It can be seen that the inhibitory effect of TOP1+TOP2A+TUBB2 3siRNA drugs on the three genes of TOP1, TOP2A and TUBB2 is better than the effects of the other three single siRNAs or double siRNAs. The results show that in A375 cells, the three siRNAs can significantly inhibit the gene expression of TOP1, TOP2 and TUB2.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (12)

1. A multi-arm polyethylene glycol drug conjugate, characterized in that the multi-arm polyethylene glycol drug conjugate has a structure shown in general formula I: Wherein, R is a core structure selected from a polyhydroxy structure, a polyamino structure or a polycarboxyl structure; n, m, l are integers ranging from 0 to 250; N, M, and L are each independently selected from integers of 1 to 24; A ₁ , A ₂ , and A ₃ are linking groups, independently selected from the group consisting of one or more of an amide bond, a C ₁ to C ₁₂ alkylene group, a urea group, an amine group, a carbonyl group, an ether group, an ester group, a biotin-streptavidin complex, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a thiol-maleimide bond, a -triazole-, a carbon-sulfur bond, or an ether bond; Said D ₁ -D ₃ are drug molecules, independently selected from: small molecule drugs, dyes, peptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles, viruses, quantum dots or magnetic resonance imaging contrast agents. 2. The drug conjugate according to claim 1, characterized in that the multi-arm polyethylene glycol drug conjugate has a structure shown in the following general formula II: The n, m, and l are each independently selected from integers of 0 to 80. Preferably, n, m, and l are each independently selected from integers of 0 to 60. More preferably, n, m, and l are each independently selected from integers of 0 to 50. 3. The drug conjugate according to claim 1, characterized in that the multi-arm polyethylene glycol drug conjugate has the following general formula III structure: Wherein, siRNA ₁ , siRNA ₂ , and siRNA ₃ are double-stranded siRNAs, which may be the same or different; Preferably, the siRNA ₁ , siRNA ₂ or siRNA ₃ includes unmodified and modified siRNAs, and the modifications include: phosphate backbone modification, sugar ring modification, base modification, and a combination of one or more of 3′, 5′ terminal thiol, azido, maleimide, alkynyl, tetrazine, cyclooctyne or biotin modification. 4. The drug conjugate according to claim 3, characterized in that the target of the siRNA includes one or more of MLPH, TOP1, TOP2, TUBB2, B7H3, KDR, Tyr, CFD, EGFR, MITF, FGF, and JAK. 5. The drug conjugate according to any one of claims 1 to 3, wherein R is Preferably, the R is a triol group, and more preferably, the R is a glycerol group. 6. The drug conjugate according to any one of claims 1 to 3, characterized in that _A1 , _A2 , and _A3 are selected from one or more combinations of the following structures: -(CH ₂ ) _i -, -(CH ₂ ) _i NHCO-, -(CH ₂ ) _i NH-, -CO(CH ₂ ) _i CO-, -(CH ₂ ) _i CO-, -NH(CH ₂ ) _iNHCO- , wherein i is an integer from 0 to 10, R ₁ is a C ₁ to C ₁₀ alkane, and j is an integer from 0 to 10; Preferably, A ₁ , A ₂ , and A ₃ are selected from one or more combinations of the following structures: -NH(CH ₂ ) _i NHCO-, -(CH ₂ ) _i NHCO-, 7. The drug conjugate according to claim 2, characterized in that the multi-arm polyethylene glycol drug conjugate structure is selected from the following IV-VI structures: Among them, siRNA ₁ , siRNA ₂ , and siRNA ₃ may be the same or different. 8. A pharmaceutical composition, characterized in that it comprises the multi-arm polyethylene glycol drug conjugate according to any one of claims 1 to 7; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, and the excipients are selected from: carrier, diluent, binder, lubricant or wetting agent; More preferably, the dosage form of the pharmaceutical composition includes: tablets, capsules, pills, injections, emulsions, microemulsions, nanoparticles, inhalants, lozenges, gels, powders, suppositories, suspensoids, creams, jellies or sprays; More preferably, the pharmaceutical composition can be administered orally, subcutaneously, intramuscularly, intravenously, rectally, vaginally, nasally, transdermally, subconjunctivally, intraocularly, orbitally, retro-ocularly, retinaly, choroidally or intrathecally. 9. A drug delivery system, characterized in that it comprises the multi-arm polyethylene glycol drug conjugate according to any one of claims 1 to 7, preferably, the drug delivery system is a lipid delivery system, and more preferably, the drug delivery system is a lipid nanoparticle. 10. The drug delivery system as claimed in claim 9, characterized in that the drug delivery system further comprises a combination of one or more of a cationic lipid, a steroid, a helper lipid and a PEG lipid; Preferably, the cationic lipid is selected from ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), octadecane-9-yl 8-((2-hydroxyethyl)(6-oxO-6-(undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3,13-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium (DDA), 1,2-dimyristoyl-3-trimethylammonio methylammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearyl trimethylammonium propane (DSTAP), N-[1-(2,3-diallyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-sn-glycerotrioxy-3-ethylphosphocholine (DOEPC), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); Preferably, the auxiliary lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE); Preferably, the steroid is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid; Preferably, the lipid polyethylene glycol conjugate is selected from the polyethylene glycol lipid selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosyl acetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). 11. Use of the multi-arm polyethylene glycol drug conjugate according to any one of claims 1 to 7 in the preparation of targeted drugs, characterized in that the drug is used for diagnosing and/or treating any disease caused by abnormally high expression of pathogenic proteins or pathogenic genes. 12. The use according to claim 11, characterized in that the diseases include: tumors, infectious diseases, hyperlipidemia, cancer, autoimmune diseases, inflammatory diseases, neurodegenerative diseases, eye diseases and rare diseases; Preferably, the cancer includes lymphoma, B cell tumor, T cell tumor, bone marrow/monocyte tumor, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; More preferably, the leukemia is selected from acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; More preferably, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; More preferably, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma or chondrosarcoma; Preferably, the autoimmune diseases include: allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, myasthenia gravis, multiple sclerosis, urticaria, psoriasis, dermatomyositis, Sjögren's syndrome, pain or neurological disorders; Preferably, the inflammatory diseases include: acute inflammation, chronic inflammation, degenerative inflammation, exudative inflammation, proliferative inflammation, and specific inflammation; More preferably, the inflammatory disease includes: severe burns, endotoxemia, septic shock, adult respiratory distress syndrome, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, post-streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergy, IL-2-induced vascular leak syndrome or radiographic contrast agent allergy; Preferably, the neurodegenerative diseases include: Alzheimer's disease, progressive blindness or extraocular muscle palsy, multiple system atrophy, frontotemporal dementia, Huntington's chorea, cortical basal degeneration, spinocerebellar ataxia, motor neuron disease, hereditary motor sensory neuropathy; Preferably, the eye diseases include: macular degenerative diseases such as all stages of age-related macular degeneration including dry and wet forms, diabetic retinopathy and other ischemia-related retinopathies, choroidal neovascularization, uveitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion, corneal neovascularization, and retinal neovascularization; More preferably, the age-related macular degeneration includes non-exudative and exudative AMD, diabetic retinopathy, endophthalmitis and uveitis. In addition, non-exudative AMD may include hard drusen, soft drusen, geographic atrophy and/or pigment agglomeration.