Multi-cluster linker, preparation method therefor and use thereof

Abstract

The present invention relates to a multi-cluster linker, a preparation method therefor and the use thereof. Specifically, provided is a linker, which is a compound as represented by formula I or a stereoisomer thereof or a pharmaceutically acceptable salt thereof. Further provided is a targeting ligand or targeting ligand delivery conjugate using the linker.

Description

Multi-cluster linker, preparation method and application thereof

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to CN application No. 202411310792.2, filed on September 19, 2024, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to a multi-cluster linker, a preparation method and application thereof. BACKGROUND

[0004] With the continuous development of RNA interference technology, artificial synthetic oligonucleotides, such as antisense nucleic acids, small interfering nucleic acids, small activating nucleic acids, and nucleic acid aptamers, are increasingly applied to the fields of disease treatment and disease diagnosis, and become an effective drug and diagnostic reagent development technology.

[0005] However, due to the phosphate backbone in the structure of oligonucleotides, the molecules have a large number of negative charges and strong hydrophilicity, and it is difficult for them to enter human cells by free penetration to exert their pharmacological effects in the body. Therefore, designing an efficient and specific delivery method for nucleic acid drugs is the key to improving the pharmacological effects of nucleic acid drugs.

[0006] In the existing in vivo delivery technology of oligonucleotides, the targeting ligand is coupled to the oligonucleotide by a covalent bond through a linker, which is a relatively mature delivery technology at present.

[0007] The conjugate of the coupled oligonucleotide specifically binds to a specific receptor on the surface of human cells through the targeting ligand and mediates the endocytosis of the receptor, so that the oligonucleotide conjugate with the delivery targeting head is endocytosed into the cell, the delivery of the oligonucleotide is completed, and the downstream RNA interference process is started to exert the pharmacological effects of the oligonucleotide.

[0008] Designing a linker with reasonable length, rigidity, spatial structure, physicochemical properties, hydrophilicity and hydrophobicity, and in vivo degradability has a very significant influence on the pharmacological activity of the oligonucleotide conjugate.

[0009] Therefore, new linker structures need to be further researched and developed. SUMMARY

[0010] The present application provides a series of innovative linker structures, and the oligonucleotide conjugate using the series of linkers exhibits excellent physiological activity and pharmacological effects.

[0011] To this end, in a first aspect of the application, the present application provides a linker which is a compound of Formula I or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, L A -L B

[0012] Formula I

[0013] wherein:

[0014] L A is selected from

[0015] L B is selected from

[0016] Z 1 , Z 2 , Z 3 are each independently selected from

[0017] Q is selected from

[0018] R 0 is selected from -N3, -NH2, -OH, -COOH, -I, -Cl, -Br, -F, -OMs, -OTs, -OTf, -CHO,

[0019] Preferably, R 0 is selected from -N3;

[0020] More preferably, R 0 is

[0021] Preferably, Q is selected from

[0022] More preferably, Q is selected from

[0023] Most preferably, Q is selected from

[0024] m is selected from an integer from 0 to 10, preferably from an integer from 0 to 5, more preferably from an integer from 0 to 3, most preferably from 0, 1, 2;

[0025] n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably from 3, 7, 10;

[0026] n2 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 1 to 3, most preferably 2;

[0027] n3 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 3 to 5, most preferably 4;

[0028] n4 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 1 to 3, most preferably 1;

[0029] n5 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 2 to 4, most preferably 3;

[0030] n6 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 1, 2, most preferably 1 ;

[0031] x is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 2, 3;

[0032] y is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 2 to 4, most preferably 3.

[0033] In some embodiments, L A is connected to Q by forming an amide bond. B is connected to Q by forming an amide bond.

[0034] In some embodiments, Z 1 is connected to Q by forming an amide bond.

[0035] In some embodiments, a plurality of Z 1 are connected to Q by forming amide bonds.

[0036] In some embodiments, Z 2 is connected to Q by forming an amide bond.

[0037] In some embodiments, Z 3 is connected to Z 2 by forming an amide bond.

[0038] In some embodiments, Z 1 is selected from

[0039] In some embodiments, Z 1 is selected from

[0040] In some embodiments, Z 1 is selected from In some embodiments, Z 1 is In some embodiments, Z 2 is selected from

[0041] In some embodiments, Z 2 is selected from In some embodiments, Z 2 is selected from

[0042] In some embodiments, Z 2 is selected from In some embodiments, Z 3 is selected from

[0043] In some embodiments, Z 3 is selected from

[0044] In some embodiments, Z 3 is selected from

[0045] In some embodiments, Z 3 is selected from

[0046] In some embodiments, the linker is a compound according to Formula I-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0047] wherein:

[0048] m is selected from an integer from 0 to 5, preferably from an integer from 0 to 3, more preferably from 0, 1, 2;

[0049] Z 1 is selected from

[0050] Preferably, Z 1 is selected from

[0051] Preferably, Z 1 is selected from

[0052] More preferably, Z 1 is

[0053] Q is selected from

[0054] Preferably, Q is

[0055] More preferably, Q is selected from

[0056] n1 is selected from an integer from 1-20, preferably from an integer from 5-10, more preferably from an integer from 7-10, most preferably from 7, 10;

[0057] n2 is selected from an integer from 1-5, preferably from an integer from 1-3, more preferably 2;

[0058] x is selected from an integer from 1-10, preferably from an integer from 1-5, more preferably from 2, 3;

[0059] Preferably, Z 1 is connected to Q by forming an amide bond; by forming an amide bond;

[0060] Preferably, Z 1 is connected to Q by forming an amide bond;

[0061] Preferably, a plurality of Z 1 are connected to each other by forming amide bonds.

[0062] In some embodiments, the linker is a compound represented by Formula I-2, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[0063] wherein:

[0064] Z 2 is selected from

[0065] Preferably, Z 2 is selected from

[0066] Preferably, Z 2 is selected from

[0067] More preferably, Z 2 is selected from Z 3 is selected from

[0068] Preferably, Z 3 is selected from

[0069] Preferably, Z3 selected from the group consisting of

[0070] More preferably, Z is selected from the group consisting of 3 selected from the group consisting of

[0071] Q is selected from the group consisting of

[0072] Preferably, Q is selected from the group consisting of

[0073] More preferably, Q is selected from the group consisting of

[0074] n1 is selected from the group consisting of an integer from 1 to 20, preferably from 1 to 10, more preferably from 3 to 10, most preferably from 3, 7, 10;

[0075] n2 is selected from the group consisting of an integer from 1 to 5, preferably from 1 to 3, more preferably 2;

[0076] n3 is selected from the group consisting of an integer from 1 to 5, preferably from 3 to 5, more preferably 4;

[0077] n4 is selected from the group consisting of an integer from 1 to 5, preferably from 1 to 3, more preferably 1;

[0078] n5 is selected from the group consisting of an integer from 1 to 5, preferably from 2 to 4, more preferably 3;

[0079] x is selected from the group consisting of an integer from 1 to 10, preferably from 1 to 3, more preferably 2;

[0080] y is selected from the group consisting of an integer from 1 to 10, preferably from 2 to 4, more preferably 3;

[0081] Preferably, Z is selected from the group consisting of 3 is connected to by forming an amide bond;

[0082] Preferably, Z is selected from the group consisting of 2 is connected to Q by forming an amide bond;

[0083] Preferably, Z is selected from the group consisting of 3 is connected to 2 by forming an amide bond.

[0084] In some embodiments, Z is selected from the group consisting of 2 selected from the group consisting of

[0085] Preferably, Z is selected from the group consisting of 2 selected from the group consisting of

[0086] Preferably, Z 2 is selected from

[0087] More preferably, Z 2 is selected from

[0088] Z 3 is selected from

[0089] Preferably, Z 3 is selected from

[0090] Preferably, Z 3 is selected from

[0091] More preferably, Z 3 is selected from

[0092] Q is selected from

[0093] Preferably, Q is selected from

[0094] More preferably, Q is

[0095] n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably from 3, 7, 10;

[0096] n2 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 2;

[0097] n4 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 1;

[0098] n5 is selected from an integer from 1 to 5, preferably from an integer from 2 to 4, more preferably 3;

[0099] x is selected from an integer from 1 to 10, preferably from an integer from 1 to 3, more preferably 2.

[0100] In some embodiments, Z 2 is selected from

[0101] Preferably, Z 2 is selected from

[0102] Preferably, Z 2 is selected from

[0103] More preferably, Z 2 is

[0104] Z 3 is selected from

[0105] More preferably, Z 3 is selected from

[0106] More preferably, Z 3 is selected from

[0107] More preferably, Z 3 is selected from

[0108] Q is selected from

[0109] More preferably, Q is

[0110] More preferably, Q is

[0111] n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably 7;

[0112] n3 is selected from an integer from 1 to 5, preferably from an integer from 3 to 5, more preferably 4;

[0113] n5 is selected from an integer from 1 to 5, preferably from an integer from 2 to 4, more preferably 3;

[0114] y is selected from an integer from 1 to 10, preferably from an integer from 2 to 4, more preferably 3.

[0115] In some embodiments, the linker is a compound selected from the group consisting of:

[0116] In a second aspect of the application, the present application provides a targeting ligand comprising a linking unit, said linking unit being a structure according to Formula I’ or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, A -L B’

[0117] Formula I’

[0118] wherein:

[0119] L Aselected from the group consisting of

[0120] L B’ selected from the group consisting of

[0121] Q’ is selected from the group consisting of

[0122] Preferably, Q’ is selected from the group consisting of

[0123] More preferably, Q’ is selected from the group consisting of

[0124] Most preferably, Q’ is selected from the group consisting of

[0125] Z 3 , Z 2 , Z 1 , m, n1, n6, x, y are each independently as described in any of the technical solutions of the first aspect.

[0126] In some embodiments, L A is connected to L B’ by forming an amide bond.

[0127] In some embodiments, Z 1 is connected to Q’ by forming an amide bond.

[0128] In some embodiments, a plurality of Z 1 are connected by forming amide bonds.

[0129] In some embodiments, Z 2 is connected to Q’ by forming an amide bond.

[0130] In some embodiments, Z 3 is connected to Z 2 by forming an amide bond.

[0131] In some embodiments, the linking group is a structure represented by Formula I’-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0132] wherein:

[0133] Q’ is selected from the group consisting of

[0134] Preferably, Q’ is selected from the group consisting of

[0135] More preferably, Q’ is selected from the group consisting of

[0136] Z 1 each of m, n1, x is independently described in any of the technical solutions of the first aspect.

[0137] In some embodiments, Z 1 is connected to by forming an amide bond.

[0138] In some embodiments, Z 1 is connected to Q’ by forming an amide bond.

[0139] In some embodiments, a plurality of Z 1 are connected by forming amide bonds.

[0140] In some embodiments, the connecting group is a structure represented by Formula I’-2, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[0141] wherein:

[0142] Q’ is selected from

[0143] Preferably, Q’ is selected from

[0144] More preferably, Q’ is selected from

[0145] Z 3 , Z 2 , n1, x, y are each independently described in any of the technical solutions of the first aspect.

[0146] In some embodiments, Z 3 is connected to by forming an amide bond.

[0147] In some embodiments, Z 2 is connected to Q’ by forming an amide bond.

[0148] In some embodiments, Z 3 is connected to Z 2 by forming an amide bond.

[0149] In some embodiments, the connecting unit is a structure selected from the following, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0150] In some embodiments, the targeting ligand further comprises a ligand unit, which is connected to the connecting unit.

[0151] In some embodiments, the ligand unit is a structure according to Formula II or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0152] wherein:

[0153] R 1 , R 2 , R 3 , R 4 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl;

[0154] Preferably, R 1 , R 3 , R 4 are hydrogen;

[0155] Preferably, R 2 is selected from the group consisting of C1-C6 alkyl;

[0156] More preferably, R 2 is methyl;

[0157] s1, s2, s3, s4 are each independently selected from an integer between 1 and 10;

[0158] Preferably, s1 is selected from an integer between 1 and 5, preferably from 2, 3, 4, more preferably 3;

[0159] Preferably, s2 is selected from an integer between 1 and 5, preferably from 1, 2, 3, more preferably 1;

[0160] Preferably, s3 is selected from an integer between 1 and 5, preferably from 1, 2, 3, more preferably 1;

[0161] Preferably, s4 is selected from an integer between 1 and 5, preferably from 3, 4, 5, more preferably 4.

[0162] In some embodiments, the ligand unit is a structure selected from the group consisting of:

[0163] In some embodiments, the targeting ligand is a compound selected from the group consisting of:

[0164] In a third aspect of the application, the present application provides a targeted ligand delivery conjugate comprising a linker group, said linker group being a structure according to Formula I” or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, L A’ -L B’

[0165] Formula I”

[0166] wherein:

[0167] L A’ selected from

[0168] L B’ selected from

[0169] Q’ is selected from

[0170] Preferably, Q’ is selected from

[0171] More preferably, Q’ is selected from

[0172] Most preferably, Q’ is selected from

[0173] Z 3 , Z 2 , Z 1 , m, n1, n6, x, y are each independently as described in any of the technical solutions of the first aspect.

[0174] In some embodiments, L A’ is connected to L B’ by forming an amide bond.

[0175] In some embodiments, Z 1 is connected to Q’ by forming an amide bond.

[0176] In some embodiments, a plurality of Z 1 are connected by forming amide bonds.

[0177] In some embodiments, Z 2 is connected to Q’ by forming an amide bond.

[0178] In some embodiments, Z 3 is connected to Z 2 by forming an amide bond.

[0179] In some embodiments, the linking group is a structure represented by Formula I”-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0180] wherein:

[0181] Q’ is selected from

[0182] Preferably, Q’ is selected from

[0183] More preferably, Q’ is selected from

[0184] Z 1 , m, n1, x are each independently as described in any of the technical solutions of the first aspect.

[0185] In some embodiments, Z 1 is connected to Q’ by forming an amide bond. by forming an amide bond.

[0186] In some embodiments, Z 1 is connected to Q’ by forming an amide bond.

[0187] In some embodiments, a plurality of Z 1 are connected by forming an amide bond.

[0188] In some embodiments, the linking group is a structure represented by Formula I”-2 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0189] wherein:

[0190] Q’ is selected from

[0191] Preferably, Q’ is selected from

[0192] More preferably, Q’ is selected from

[0193] Z 3 , Z 2 , n1, x, y are each independently as described in any of the technical solutions of the first aspect.

[0194] In some embodiments, Z 3 is connected to Q’ by forming an amide bond. by forming an amide bond.

[0195] In some embodiments, Z 2 is connected to Q’ by forming an amide bond.

[0196] In some embodiments, Z 3 is connected to Z 2 by forming an amide bond.

[0197] In some embodiments, the linking group is a structure selected from the following or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0198] In some embodiments, the targeting ligand delivery conjugate further comprises a ligand unit, which is attached to the linker.

[0199] In some embodiments, the ligand unit is as described in any of the second aspect.

[0200] In some embodiments, the targeting ligand delivery conjugate further comprises a delivered molecule, which is attached to the ligand unit and the linker.

[0201] In some embodiments, the delivered molecule includes, but is not limited to, an RNAi agent, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid, a natural or modified nucleic acid oligonucleotide, a natural or modified nucleic acid polynucleotide, a peptide, a nucleic acid aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, a polyethylene glycol, a hapten, digoxin, biotin, a radioactive atom or molecule, or a fluorophore.

[0202] In some embodiments, the delivered molecule is an RNAi agent.

[0203] In some embodiments, the delivered molecule is a small interfering RNA (siRNA), which comprises a sense strand and an antisense strand, and the sequence of the sense strand and the antisense strand of the siRNA is the sense strand and the antisense strand sequence of duplex 1 described in Table 1.

[0204] In some embodiments, the targeting ligand delivery conjugate is selected from:

[0205] wherein R z represents the delivered molecule.

[0206] In a fourth aspect of the present application, the present application provides a composition or a pharmaceutical composition comprising the linker described in any of the first aspect, or the targeting ligand described in any of the second aspect, or the targeting ligand delivery conjugate described in any of the third aspect; and optionally a pharmaceutically acceptable excipient.

[0207] In a fifth aspect of the present application, the present application provides use of the linker described in any of the first aspect, or the targeting ligand described in any of the second aspect, or the targeting ligand delivery conjugate described in any of the third aspect, or the composition or the pharmaceutical composition described in any of the fourth aspect, in the manufacture of a medicament or a drug for delivering a target molecule.

[0208] In a sixth aspect of the present application, the present application provides a method of delivering a molecule to be delivered to a target tissue and / or cell, comprising administering to a subject one or more targeting ligand delivery conjugates described in any of the technical solutions of the third aspect.

[0209] In a seventh aspect of the present application, the present application provides use of the linker described in any of the technical solutions of the first aspect, or the targeting ligand described in any of the technical solutions of the second aspect, or the targeting ligand delivery conjugate described in any of the technical solutions of the third aspect, or the composition or pharmaceutical composition described in any of the technical solutions of the fourth aspect, in the manufacture of a medicament for treating and / or preventing a disease.

[0210] In an eighth aspect of the present application, the present application provides the targeting ligand described in any of the technical solutions of the second aspect, or the targeting ligand delivery conjugate described in any of the technical solutions of the third aspect, or the composition or pharmaceutical composition described in any of the technical solutions of the fourth aspect, for use in treating and / or preventing a disease.

[0211] In a ninth aspect of the present application, the present application provides a method of treating and / or preventing a disease, comprising administering to a subject in need thereof an effective amount of the targeting ligand described in any of the technical solutions of the second aspect, or the targeting ligand delivery conjugate described in any of the technical solutions of the third aspect, or the composition or pharmaceutical composition described in any of the technical solutions of the fourth aspect.

[0212] In some embodiments, the disease is a disease mediated by expression of a target gene.

[0213] In some embodiments, the ligand is an αvβ6 integrin ligand.

[0214] In some embodiments, the cell is selected from the group consisting of type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells, epithelial tumors (carcinomas), macrophages, endothelial cells, fibroblasts, smooth muscle cells, granulocytes, T cells, immune cells.

[0215] Definitions of terms used in connection with the present application: unless otherwise indicated, the initial definition of a group or term provided herein applies throughout the specification; for terms not specifically defined herein, the meaning given to them by one of ordinary skill in the art in light of the disclosure and context should prevail.

[0216] In this invention, a variable is selected from integers of “XY”, which means that the variable is selected from any integer value within the range including the endpoints X and Y. For example, “m is selected from integers from 0 to 10” means that m is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0217] In this invention, the options of multiple variables represented by the same symbol in the same or different structural formulas are independent of each other; that is, their respective options can be the same or different. For example, if Z 2 Selected from Z 3 Selected from And when n2 is selected from integers from 1 to 3, it represents that Z 2 The variables n2 and Z 3 The options for the variable n2 in the two n2 variables are independent of each other, meaning that the options for these two n2 variables can be the same (e.g., both being the integer 1) or different (e.g., Z). 2 In Z, n2 is an integer 1, and Z 3 In this context, n2 is an integer 3.

[0218] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C1-C6 alkyl refers to an alkyl group having 1 to 6 member atoms, such as 1 to 4 member atoms, or 1 to 3 member atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. C1-C6 alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl.

[0219] In the sequence of this invention, unless otherwise specified, uppercase letters C, G, U, A, and T represent the base composition of nucleotides, which are unmodified nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the identifier m is a 2'-methoxynucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the identifier f is a 2'-fluoronucleotide; lowercase letter s indicates that the two nucleotides adjacent to the identifier f on the left and right are linked by a thiophosphate group; VP indicates that the nucleotide adjacent to its right is a vinylphosphonate modified nucleotide; invAb indicates a reverse debasement residue.

[0220] As will be understood by those skilled in the art, the structure of the 2'-fluoronucleotide is as follows:

[0221] The structure of 2'-methoxynucleotide is shown below:

[0222] The structural formula of invAb is shown below:

[0223] C6-NH2 (i.e., C6-amino) has the formula and as will be understood by those skilled in the art, the phosphate group is the 5' phosphate of the sense strand.

[0224] The term "pharmaceutically acceptable" means that which is generally compatible with pharmaceutical practice and / or with the physical and chemical make-up of other ingredients in a pharmaceutical dosage form and physiologically acceptable to the recipient.

[0225] The terms "salt" and "pharmaceutically acceptable salt" refer to the acid and / or base addition salts of the aforementioned compounds or stereoisomers thereof, and also include zwitterionic salts (inner salts), and further include quaternary ammonium salts, such as alkyl ammonium salts. These salts can be prepared in the final isolation and purification step of the compounds. They can also be prepared by contacting the compounds, or the stereoisomers thereof, with a suitable acid or base, either neat or in a suitable solvent, in an amount that is equivalent to the amount of the acid or base. These salts can be formed in solution or in a solid state, and can be recovered by filtration or by evaporation of the solvent. The salts of the present application can be hydrochloric, sulfuric, citric, benzenesulfonic, hydrobromic, hydrofluoric, phosphoric, acetic, propionic, succinic, oxalic, malic, succinic, fumaric, maleic, tartaric, or trifluoroacetic acid salts.

[0226] The term "stereoisomer" refers to compounds having the same molecular formula and sequence of bonded atoms but differing in the spatial arrangement of the atoms. Stereoisomers include enantiomeric and diastereomeric forms.

[0227] The term "treatment" generally refers to obtaining a desired pharmacologic and / or physiologic effect. The term "treatment" as used herein covers any treatment of a disease or condition in a patient, and includes: (a) preventing the disease or condition from occurring in a subject that is predisposed or does not yet experience symptoms of the disease or condition; (b) inhibiting the disease or condition, i.e., arresting its development; or (c) relieving the disease or condition, i.e., causing regression of the disease or condition.

[0228] The term "subject" refers to a vertebrate animal. In certain embodiments, the vertebrate animal is a mammal. Mammals include, but are not limited to, domestic and farm animals such as cows, horses, sheep, pigs, goats, and the like; companion animals such as dogs, cats, and the like; primates including humans; mice and rats. In certain embodiments, the mammal is a human.

[0229] The term "effective amount" means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A "therapeutically effective amount" of a substance / molecule of the present application can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount of the substance / molecule that has therapeutic benefit beyond any toxic or deleterious effects. In the context of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent, preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer.

[0230] Obviously, according to the above-mentioned content of the present application, other various forms of modification, replacement or change can be made according to the common technical knowledge and usual means in the art without departing from the above-mentioned basic technical idea of the present application.

[0231] The above-mentioned content of the present application will be further explained in detail through the following specific embodiments in the form of examples. However, it should not be understood that the above-mentioned subject matter of the present application is limited to the following examples. Any technology realized based on the above-mentioned content of the present application belongs to the scope of the present application. DETAILED DESCRIPTION

[0232] Unless otherwise specified, the reagents and raw materials used in the following examples are commercially available reagents or raw materials for those skilled in the art, and the instruments used in the following examples are instruments commonly used or operated by those skilled in the art.

[0233] The present application will be further explained and described below in combination with specific embodiments.

[0234] Example 1: Synthesis of oligonucleotide sequence

[0235] The siRNA sequence of the present application is synthesized by using the solid-phase phosphoramidite triester method. The relevant monomers come from Wuhu Huarenhe Sugar Wisdom. Carrier: 2000A, 500 mol synthesis column; coupling time: 4 min; ammonolysis condition: 55°C, concentrated ammonia (25% ammonia solution)

[0236] The sequence synthesis was performed using a 0.2 μmol 48-channel synthesizer. The phosphoramidite monomers were used at a concentration of 0.05 M and the activator was used at a concentration of 0.3 M BTT. Step 1 - Deprotection, the DMT group on the CPG-bound nucleoside was removed with trichloroacetic acid to expose the 5' hydroxyl group for the next coupling. Step 2 - Activation, the monomer was activated with an excess of tetrazole before being loaded onto the synthesis column. Step 3 - Coupling, the phosphoramidite tetrazole undergoes a nucleophilic reaction with the CPG-bound nucleotide, and after coupling the tetrazole is removed, extending the oligonucleotide chain. Step 4 - Capping, just before use, a mixture of acetic anhydride and N-methylimidazole is mixed with a small amount of the 5' hydroxyl group that did not participate in the coupling reaction to form an ester bond. Step 5 - Oxidation, the trivalent phosphorus is oxidized to pentavalent phosphorus with 0.05 M iodine solution. The above steps 1 to 5 are repeated, and when the oligonucleotide chain is extended to the desired length, the synthesis is complete. Cleavage and deprotection - carrier (CPG) is added to concentrated ammonia solution (25% ammonia solution), and the temperature is raised to 55°C for overnight reaction to ammonolysis.

[0237] wherein the 5' end of the sense strand is conjugated to a C6-NH2 intermediate of the sense strand-C6-NH2 intermediate has the structure obtained by using 5'-TFA-Amino-Modifier C6 phosphoramidite monomer (cas no. 133975-85-6) in the solid-phase synthesis of the sense strand, the structure of the above phosphoramidite monomer is

[0238] After HPLC purification, freeze-drying and quality inspection, the salt was exchanged with sodium acetate alcohol, desalted with a 3KD ultrafiltration tube, and the amount of the sense strand and the antisense strand was determined by spectrophotometer after desalting. The sense strand with a C6 amino linker at the end was used for further coupling. The sequence information is shown in Table 1.

[0239] Table 1 Sequence information

[0240] Example 2: Preparation of linker and conjugate

[0241] The present application at least relates to the following linker and the preparation method thereof.

[0242] In addition, the present application also exemplarily provides an oligonucleotide conjugate prepared by using the above linker and the preparation method thereof. The conjugate involved in the present application is shown in Table 2-1, and the abbreviations of the modified monomers are shown in Table 2-2.

[0243] Table 2-1 Conjugate information

[0244] Table 2-2 Modified monomer abbreviation table

[0245] 1. Preparation of conjugate 1

[0246] 1.1 Synthesis of TA14 linker

[0247] Step 1. Synthesis of compound TA14-2

[0248] To a solution of compound TA14-1 (25 g, 60.16 mmol) in water (300 mL) was added Fmoc-Cl (18.68 g, 72.19 mmol) and sodium bicarbonate (7.58 g, 90.24 mmol) and stirred at room temperature for 60 min until LC-MS detected the starting material completely disappeared. The reaction solution was directly prepared by MPLC to give compound TA14-2 ((30 g, 47.04 mmol, 78.19% yield). LCMS (E+) m / z: 638.5 [M+H] +

[0249] Step 2. Synthesis of compound TA14-3

[0250] To a solution of compound TA14-2 (30 g, 47.04 mmol) in dichloromethane (300 mL) was added trifluoroacetic acid (300 mL) and stirred at room temperature for 10 min until LC-MS detected the starting material completely disappeared. The reaction solution was directly prepared by MPLC to give compound TA14-3 (28.5 g, 46.40 mmol, 98.65% yield, 95% purity, TF). LCMS (E+) m / z: 470.3 [M+H] +

[0251] Step 3. Synthesis of compound TA14-4

[0252] To a solution of compound TA14-3 (28.5 g, 46.40 mmol, 95% purity, TF) in DMF (300 mL) was added 2-[2-(propargyloxy)ethoxy]ethanamine (23.25 g, 162.40 mmol), HATU (61.75 g, 162.40 mmol), and DIPEA (23.99 g, 185.60 mmol, 32.33 mL) and stirred at room temperature for 2 h until LC-MS detected the starting material completely disappeared. The reaction solution was directly prepared by MPLC to give compound TA14-4 (30 g, 35.50 mmol, 76.52% yield). LCMS (E+) m / z: 845.5 [M+H] +

[0253] Step 4. Synthesis of compound TA14

[0254] To a solution of compound TA14-4 (30 g, 35.50 mmol) in DMF (300 mL) was added succinic anhydride (10.13 g, 88.76 mmol) and DIPEA (91.77 g, 710.07 mmol, 123.68 mL) and stirred at 45 °C for 20 hours until LC-MS indicated the starting material was completely consumed. The reaction was directly prepared by MPLC to give TA14 (5.1 g, 6.92 mmol, 19.49% yield). LCMS (E+) m / z: 737.7 [M+H] +

[0255] 1.2 Synthesis of conjugate intermediate 1

[0256] The oligonucleotide amine compound sense strand-C6-NH2 was dissolved in sodium borate buffer (sodium borate 250 mM, pH = 9.4) to prepare a 4 mM concentration solution (100 μL, 400 nmol). To the solution was added a mixture of fragment TA14 (10 μL, 2000 nmol, 5 equivalents, 200 mM DMA solution), HATU (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution) and DIPEA (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution), mixed well, and then the reaction was placed at 25 °C for 0.5 hours.

[0257] After the reaction was completed, ethanol precipitation was performed: 10% of the total volume of 5 M sodium chloride solution was added to the solution, followed by the addition of 3 times the total volume of anhydrous ethanol, and after uniform shaking, the reaction was frozen in dry ice for 2 hours, and then centrifuged at 12000 rpm for half an hour, the supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a conjugate intermediate 1 solution, and LC-MS confirmed that the reaction conversion rate was 90%.

[0258] 1.3 Synthesis of conjugate 1

[0259] The conjugate intermediate 1 was dissolved in sodium bicarbonate buffer (sodium bicarbonate 250 mM, pH = 8.5) to prepare a 1 mM concentration solution (100 μL, 100 nmol). To the solution was added SM6.1 (30 μL, 3000 nmol, 30 equivalents, 100 mM DMA solution) (ArrowHead, WO2023070082A2), copper sulfate (10 μL, 1000 nmol, 10 equivalents, 100 mM aqueous solution) and sodium ascorbate (10 μL, 10000 nmol, 100 equivalents, 1000 mM aqueous solution), mixed well, and then the reaction was placed at 60 °C for 15 minutes.

[0260] After the reaction was completed, ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride solution was added to the solution, and then 3 times the total volume of anhydrous ethanol was added, and after uniform shaking, the reaction was frozen in dry ice for 2 hours, and then centrifuged at a speed of 12000 rpm for half an hour, the supernatant was discarded, and the remaining precipitate was dissolved with deionized water. The obtained solution was confirmed by LCMS to have a reaction conversion rate of 80%. After reverse phase chromatography purification, the pure product was obtained. After freeze-drying and quality inspection, the product was salted with sodium acetate alcohol, desalted with a 3KD ultrafiltration tube, and the molar mass of the positive strand was determined by spectrophotometry after desalting. The conjugate 1 duplex was obtained by mixing the positive strand and the negative strand at a ratio of 1:1 and annealing.

[0261] 2. Preparation of conjugate 2

[0262] 2.1 Synthesis of L1 linker

[0263] Step 1. Synthesis of compound L1-2

[0264] Compound L1-1 (4.9 g, 17.76 mmol) was dissolved in tetrahydrofuran (50 mL) and water (50 mL), and sodium carbonate (11.3 g, 106.52 mmol) was added. The reaction system was kept in an ice bath, and 9-fluorenylmethyl-N-succinimidyl carbonate (9.0 g, 26.64 mmol) was added, and the reaction was carried out at room temperature for 16 h. After the reaction was completed, tetrahydrofuran was removed by reduced pressure concentration, extracted with dichloromethane (20 mL), and the crude product of the aqueous phase was separated by reverse phase preparation (0.5% TFA) to obtain oily compound L1-2 (5.0 g, 10.04 mmol, yield: 56.5%). MS-ESI calculated value [M+H] + 499.1, found 499.1.

[0265] Step 2. Synthesis of compound L1-3

[0266] Compound L1-2 (5.0 g, 10.04 mmol) was dissolved in DMF (150 mL) and the reaction system was thermostated with an ice-salt bath, N,N-diisopropylethylamine (6.5 g, 50.20 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (15.3 g, 40.16 mmol) were added. After stirring for 10 minutes, 2-[2-(propargyloxy)ethoxy]ethanamine (5.7 g, 40.16 mmol) was added. The reaction temperature was controlled and the reaction was carried out in an ice-salt bath for 2 hours. After the reaction was completed, water (100 mL) was added to the reaction system, and ethyl acetate was extracted 3 times (300 mL*3), the organic phase was combined and concentrated, and the crude product was separated by reverse phase preparation (0.5% TFA) to obtain compound L1-3 (3.5 g, 4.01 mmol, yield: 39.9%) as an oil. MS-ESI calculated value [M+H] + 874.4, found 874.4

[0267] Step 3. Synthesis of compound L1-5

[0268] Compound L1-4 (15.00 g, 79.69 mmol) was dissolved in acetic anhydride (30 mL) and refluxed for 2 hours. The acetic anhydride was removed by concentration under reduced pressure to obtain an oil, and the crude product was washed 4 times (dichloromethane / petroleum ether 1:10) to obtain compound L1-5 (5.0 g, 29.41 mmol, yield: 36.9%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 2.45 (t, J = 7.4 Hz, 4H), 1.67 (q, J = 7.2 Hz, 4H), 1.41 - 1.31 (m, 6H).

[0269] Step 4. Synthesis of compound L1

[0270] Compound L1-3 (3.15 g, 3.61 mmol) was dissolved in a mixed solvent of DMF (40 mL) and N,N-diisopropylethylamine (10 mL), and L1-5 (1.53 g, 9.02 mmol) was added, and then the reaction system was thermostated at 45 degrees Celsius for 16 hours. After the reaction was completed, the crude product was separated by reverse phase preparation (0.5% TFA) to obtain compound L1 (1.0 g, 1.21 mmol, yield: 33.7%) as a white solid. MS-ESI calculated value [M+H] + 822.4, found 822.3.

[0271] 2.2 Synthesis of conjugate intermediate 2

[0272] Dissolve oligonucleotide amine compound sense strand-C6-NH2 into sodium borate buffer (sodium borate 250 mM, pH = 9.4) to prepare a 4 mM concentration solution (100 μL, 400 nmol). Add a mixed solution of linker L1 (10 μL, 2000 nmol, 5 equivalents, 200 mM DMA solution), HATU (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution) and DIPEA (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution) to the solution, mix well, and then place the reaction solution in a 25 degree water bath for 0.5 hours.

[0273] After the reaction is completed, perform ethanol precipitation: add a total volume of 10% of a 5 M sodium chloride solution to the solution, then continue to add a total volume of 3 times anhydrous ethanol, mix well, then place the reaction in dry ice for 2 hours, then centrifuge at a speed of 12000 rpm for half an hour, discard the supernatant, and dissolve the remaining precipitate in deionized water to obtain a conjugate intermediate 2 solution, and LC-MS confirms that the reaction conversion rate is 80%.

[0274] 2.3 Synthesis of conjugate 2

[0275] Dissolve the conjugate intermediate 2 in sodium bicarbonate buffer (sodium bicarbonate 250 mM, pH = 8.5) to prepare a 1 mM concentration solution (100 μL, 100 nmol). Add target head SM6.1 (30 μL, 3000 nmol, 30 equivalents, 100 mM DMA solution), copper sulfate (10 μL, 1000 nmol, 10 equivalents, 100 mM aqueous solution) and sodium ascorbate (10 μL, 10000 nmol, 100 equivalents, 1000 mM aqueous solution) to the solution, mix well, and then place the reaction in a 60 degree water bath for 15 minutes.

[0276] After the reaction is completed, perform ethanol precipitation: add a total volume of 10% of a 5 M sodium chloride solution to the solution, then continue to add a total volume of 3 times anhydrous ethanol, mix well, then place the reaction in dry ice for 2 hours, then centrifuge at a speed of 12000 rpm for half an hour, discard the supernatant, and dissolve the remaining precipitate in deionized water to obtain a solution, and LCMS confirms that the reaction conversion rate is 85%. After reverse phase chromatography purification, the pure product is obtained. After freeze-drying and quality inspection, the product is salt-exchanged with sodium acetate alcohol precipitation, desalted with a 3 KD ultrafiltration tube, and the desalted product is quantified by spectrophotometer to determine the molar mass of the sense strand. The sense strand and the antisense strand are mixed in a 1:1 ratio to obtain the conjugate 2 target duplex after annealing.

[0277] 3. Synthesis of conjugate 3

[0278] 3.1 Synthesis of L2 linker

[0279] Step 1. Synthesis of compound L2-2

[0280] Into a 100 mL single necked flask, was placed compound L2-1 (4.00 g, 11.26 mmol), N,N-diisopropylethylamine (5.82 g, 45.03 mmol), 2-(7-azobenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate (9.41 g, 24.76 mmol) and DMF (35 mL). After the mixture was stirred at room temperature for 10 min, compound L2-1' (5.51 g, 28.14 mmol) was added. After the addition, the reaction mixture was stirred at room temperature for 2 h until LC-MS indicated the starting material was consumed completely. The reaction mixture was directly subjected to MPLC to give compound L2-2 (5.70 g, 79.40% yield). LCMS (E+) m / z: 638.7 [M+H] + .

[0281] Step 2. Synthesis of compound L2-3

[0282] Into a 500 mL single necked flask, was placed intermediate L2-2 (5.60 g, 8.78 mmol), followed by dichloromethane (88 mL) and trifluoroacetic acid (88 mL) successively. After the addition, the reaction mixture was stirred at room temperature for 2.5 h until LC-MS indicated the reaction was completed. The reaction mixture was concentrated to give compound L2-3 (4.6 g) as a crude product.

[0283] Step 3. Synthesis of compound L2-4

[0284] Into a 100 mL single necked flask, was placed compound L2-3 (4.55 g, 8.66 mmol), N,N-diisopropylethylamine (4.48 g, 34.63 mmol), 2-(7-azobenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate (6.91 g, 18.18 mmol) and DMF (30 mL). After the mixture was stirred at room temperature for 10 min, compound L2-3' (4.72 g, 18.18 mmol) was added. After the addition, the reaction mixture was stirred at room temperature for 2 h until LC-MS indicated the starting material was consumed completely. The reaction mixture was directly subjected to MPLC to give compound L2-4 (6.10 g, 69.89% yield). LCMS (E+) m / z: 1008.5 [M+H] + .

[0285] Step 4. Synthesis of compound L2-5

[0286] Into a 250 mL single neck flask was placed intermediate L2-4 (6.00 g, 5.95 mmol), followed by dichloromethane (60 mL) and trifluoroacetic acid (60 mL) sequentially. After addition, the reaction was stirred at room temperature for 2.5 hours, which was monitored by LC-MS. After the reaction was completed, the reaction mixture was concentrated to give the crude compound L2-5 (4.60 g).

[0287] Step 5. Synthesis of compound L2-6

[0288] Into a 250 mL single neck flask was placed compound L2-5 (4.50 g, 5.74 mmol), N,N-diisopropylethylamine (7.42 g, 57.41 mmol), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (10.91 g, 28.71 mmol) and DMF (60 mL). After the mixture was stirred at room temperature for 10 minutes, compound L2-5' (4.11 g, 28.71 mmol) was added. After addition, the reaction was stirred at room temperature for 2 hours until the starting material was consumed by LC-MS. The reaction mixture was directly subjected to MPLC to give compound L2-6 (6.50 g, 88.14% yield). LCMS (E+) m / z: 1284.5 [M+H] + .

[0289] Step 6. Synthesis of compound L2

[0290] Into a 100 mL reaction flask was placed compound L2-6 (3.40 g, 2.65 mmol) and dissolved in DMF (16 mL). Then triethylamine (16 mL) and L2-6' (604.05 mg, 5.29 mmol) were added. After addition, the reaction was stirred at 45 degrees for 16 hours, which was monitored by LC-MS. After the reaction was completed, the reaction mixture was directly subjected to MPLC to give compound L2 (858.00 mg, 27.56% yield). LCMS (E+) m / z: 1177.1 [M+H] + .

[0291] 1H NMR (600 MHz, Methanol-d4) δ 4.33 - 4.29 (m, 2H), 4.19 (d, J = 1.8 Hz, 10H), 4.08 - 3.98 (m, 2H), 3.71 - 3.60 (m, 16H), 3.55 (q, J = 5.3 Hz, 8H), 3.46 - 3.42 (m, 2H), 3.40 - 3.32 (m, 6H), 3.31 - 3.21 (m, 4H), 2.88 (q, J = 2.4 Hz, 4H), 2.42 (t, J = 7.3 Hz, 2H), 2.37 (t, J = 7.1 Hz, 2H), 2.31 (q, J = 5.2, 3.1 Hz, 8H), 2.14 - 2.00 (m, 2H), 1.98 - 1.76 (m, 8H).

[0292] 3.2 Synthesis of conjugate intermediate 3

[0293] The oligonucleotide amine compound sense strand-C6-NH2 was dissolved into sodium borate buffer (sodium borate 250 mM, pH = 9.4) to prepare a 4 mM concentration solution (100 μL, 400 nmol). A mixed solution of linker L2 (10 μL, 2000 nmol, 5 equivalents, 200 mM DMA solution), HATU (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution) and DIPEA (5 μL, 2000 nmol, 5 equivalents, 400 mM DMA solution) was added to the solution, mixed uniformly, and then the reaction solution was placed in a 25 degree reaction for 0.5 hours.

[0294] After the reaction was completed, ethanol precipitation was performed: 5M sodium chloride solution was added to the solution to a total volume of 10%, and then 3 times the total volume of anhydrous ethanol was added. After uniform shaking, the reaction was frozen in dry ice for 2 hours, and then centrifuged at a speed of 12000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved with deionized water to obtain a conjugate intermediate 3 solution. LC-MS confirmed that the reaction conversion rate was 75%.

[0295] 3.3 Synthesis of conjugate 3

[0296] The conjugate intermediate 3 was dissolved into sodium bicarbonate buffer (sodium bicarbonate 250 mM, pH = 8.5) to prepare a 1 mM concentration solution (100 μL, 100 nmol). To the solution, target head SM6.1 (40 μL, 4000 nmol, 40 equivalents, 100 mM DMA solution), copper sulfate (10 μL, 1000 nmol, 10 equivalents, 100 mM aqueous solution) and sodium ascorbate (10 μL, 10000 nmol, 100 equivalents, 1000 mM aqueous solution) were added, mixed well, and then the reaction was placed at 60 degrees for 15 minutes.

[0297] After the reaction was completed, ethanol precipitation was performed: 10% of the total volume of 5 M sodium chloride solution was added to the solution, followed by the addition of 3 times the total volume of anhydrous ethanol. After shaking well, the reaction was frozen in dry ice for 2 hours, and then centrifuged at 12000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water. The resulting solution was confirmed by LCMS to have a reaction conversion rate of 70%. After reverse phase chromatography purification, the pure product was obtained. After lyophilization and quality inspection, the product was salt exchanged with sodium acetate alcohol, desalted with a 3KD ultrafiltration tube, and the molar mass of the positive strand was determined by spectrophotometry after desalting. The positive and negative strands were mixed in a 1:1 ratio to obtain the conjugate 3 target duplex.

[0298] Test Example 1: Activity test of conjugate (such as conjugate 1, 2, 3)

[0299] Activity detection of different linkers (TA14, L1, L2) connecting reference sequence 424 and reference target head SM6.1 in wild type C57BL / 6N mice

[0300] In vivo administration: C57BL / 6N mice were purchased from Vantianlihua. In the in vivo experiment, 3-5 mice per group, male, 6-8 weeks old. Different conjugates were delivered to the lungs at a dose of 3 mg / kg by aerosolization needle airway administration, with a 50 ul volume of aerosolization each time, and treated for 3 days. After 7 days, the mice were anesthetized and sacrificed. The pre-cooled PBS was used to take the alveolar lavage fluid (BALF), and the supernatant was taken after centrifugation to measure the RAGE protein expression level. The lung tissue was taken after perfusion and frozen for detection of RAGE mRNA and protein expression levels.

[0301] RNA expression detection: After the lung tissue was taken from the body, it was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for long-term preservation. Fresh or ultra-low temperature frozen samples were cut into 20-40 mg blocks with a disposable blade and added to 1.5 ml sterile tubes, which were quickly placed in liquid nitrogen. According to the instructions of TaKaRa MiniBEST Universal RNA Extraction Kit (Takara: 9767), the prepared tissue lysis solution was added, and the magnetic beads were homogenized on the tissue homogenizer. After homogenization, follow-up RNA extraction steps were performed according to the instructions. One Step PrimeScript TM RT-PCR Kit (Takara: RR064A) was used to detect gene expression. Real-time fluorescent PCR was performed in an ABIQ5 real-time fluorescent PCR system using the ΔΔCt method. Each lung tissue sample of the mouse was repeated in three repeated wells.

[0302] Protein level expression detection: After the mouse was anesthetized, pre-cooled PBS was used to perfuse the alveolar lavage fluid (BALF), and the sample was centrifuged in a 4°C centrifuge at 12000 rpm for 15 min, and then the supernatant was taken for protein quantification (Pierce TM BCA Protein Assay Kits (Thermo-23225). The obtained lung tissue was cut into small pieces, and 30-50 mg / tube was weighed on the balance and washed twice with pre-cooled PBS. RIPA (containing PMSF) lysis solution was added, and magnetic beads were vortexed 3 times, then centrifuged at 4°C, 14000g for 5 min to precipitate tissue or cell fragments, and the supernatant was transferred to the total protein of the tissue, which was aliquoted and stored at -80°C. Mouse RAGE Quantikine ELISA kit (R&D-MRG00) was used to detect RAGE protein expression. Each lung tissue sample of the mouse was repeated in two repeated wells.

[0303] The results of the conjugate in vivo activity test are shown in Tables 3 and 4 below.

[0304] Table 3 Expression level of RAGE mRNA in C57BL / 6N mice at different time points after aerosol administration

[0305] Table 4 Expression level of RAGE protein in lung tissue and BALF of C57BL / 6N mice at different time points after aerosol administration

[0306] From the results of the above two tables, it can be seen that the in vivo activity of conjugate 2 and conjugate 3 is significantly better than that of conjugate 1. It can be seen that the conjugate linked by the linker of the present application (such as L1, L2, L3, L4, L6, L7, L8, L9 or L10, etc.) has significantly better biological activity than the conjugate linked by the control linker (such as TA14).

Claims

1. A linker which is a compound of Formula I or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, L A -L B Formula I wherein: L A selected from L B selected from Z 1 , Z 2 , Z 3 are each independently selected from Q is selected from R 0 selected from -N3、-NH2、-OH、-COOH、-I、-Cl、-Br、-F、-OMs、-OTs、-OTf、-CHO、 Preferably, R 0 selected from -N3； More preferably, R 0 is Preferably, Q is selected from More preferably, Q is selected from Most preferably, Q is selected from m is selected from an integer from 0 to 10, preferably from an integer from 0 to 5, more preferably from an integer from 0 to 3, most preferably from 0, 1, 2; n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably from 3, 7, 10; n2 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 1 to 3, most preferably 2; n3 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 3 to 5, most preferably 4; n4 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 1 to 3, most preferably 1; n5 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 2 to 4, most preferably 3; n6 is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from 1, 2, most preferably 1; x is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from 2, 3; y is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from an integer from 2 to 4, most preferably 3.

2. The linker of claim 1, wherein, Z 1 selected from Preferably, Z 1 selected from Preferably, Z 1 selected from More preferably, Z 1 is 3. The linker of any one of claims 1-2, wherein, Z 2 selected from Preferably, Z 2 selected from Preferably, Z 2 selected from More preferably, Z 2 selected from 4. The linker of any one of claims 1-3, wherein, Z 3 selected from Preferably, Z 3 selected from Preferably, Z 3 selected from More preferably, Z 3 selected from 5. The linker of any one of claims 1-4, wherein, the linker is a compound represented by Formula I-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: m is selected from an integer from 0 to 5, preferably from an integer from 0 to 3, more preferably from 0, 1, 2; Z 1 selected from Preferably, Z 1 selected from Preferably, Z 1 selected from More preferably, Z 1 is Q is selected from Preferably, Q is More preferably, Q is selected from n1 is selected from an integer from 1 to 20, preferably from an integer from 5 to 10, more preferably from an integer from 7 to 10, most preferably from 7, 10; n2 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 2; x is selected from an integer from 1 to 10, preferably from an integer from 1 to 5, more preferably from 2, 3.

6. The linker of any one of claims 1-4, wherein, the linker is a compound represented by Formula I-2 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: Z 2 selected from Preferably, Z 2 selected from Preferably, Z 2 selected from More preferably, Z 2 selected from Z 3 selected from Preferably, Z 3 selected from Preferably, Z 3 selected from More preferably, Z 3 selected from Q is selected from Preferably, Q is selected from More preferably, Q is selected from n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably from 3, 7, 10; n2 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 2; n3 is selected from an integer from 1 to 5, preferably from an integer from 3 to 5, more preferably 4; n4 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 1; n5 is selected from an integer from 1 to 5, preferably from an integer from 2 to 4, more preferably 3; x is selected from an integer from 1 to 10, preferably from an integer from 1 to 3, more preferably 2; y is selected from an integer from 1 to 10, preferably from an integer from 2 to 4, more preferably 3.

7. The linker of claim 6, wherein, Z 2 selected from Preferably, Z 2 selected from Preferably, Z 2 selected from More preferably, Z 2 selected from Z 3 selected from Preferably, Z 3 selected from Preferably, Z 3 selected from More preferably, Z 3 selected from Q is selected from Preferably, Q is selected from More preferably, Q is n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably from 3, 7, 10; n2 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 2; n4 is selected from an integer from 1 to 5, preferably from an integer from 1 to 3, more preferably 1; n5 is selected from an integer from 1 to 5, preferably from an integer from 2 to 4, more preferably 3; x is selected from an integer from 1 to 10, preferably from an integer from 1 to 3, more preferably 2.

8. The linker of claim 6, wherein, Z 2 selected from Preferably, Z 2 selected from Preferably, Z 2 selected from More preferably, Z 2 is Z 3 selected from Preferably, Z 3 selected from Preferably, Z 3 selected from More preferably, Z 3 selected from Q is selected from Preferably, Q is selected from More preferably, Q is n1 is selected from an integer from 1 to 20, preferably from an integer from 1 to 10, more preferably from an integer from 3 to 10, most preferably 7; n3 is selected from an integer from 1 to 5, preferably from an integer from 3 to 5, more preferably 4; n5 is selected from an integer of 1-5, preferably from an integer of 2-4, more preferably 3; y is selected from an integer of 1-10, preferably from an integer of 2-4, more preferably 3.

9. The linker of any one of claims 1-8, wherein, the linker is a compound selected from the group consisting of 10. A targeting ligand comprising a linker unit, the linker unit being a structure represented by Formula I’ or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, L A -L B’ Formula I’ wherein: L A selected from L B’ selected from Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Most preferably, Q' is selected from Z 3 , Z 2 , Z 1 , m, n1, n6, x, y are each independently as defined in any one of claims 1 to 4.

11. The targeting ligand of claim 10, wherein, the linking group is a structure represented by Formula I'-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Z 1 each of m, n1, x is independently as defined in claim 5.

12. The targeting ligand of claim 10, wherein, the linking group is a structure represented by Formula I’-2 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Z 3 , Z 2 , n1, x, y are each independently as defined in any one of claims 6-8.

13. The targeting ligand of any one of claims 10-12, wherein, The connecting unit is a structure selected from the following or a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

14. The targeting ligand of any one of claims 10-13, wherein, the targeting ligand further comprises a ligand unit, the ligand unit being linked to the linker unit; Preferably, the ligand unit is of the structure shown in Formula II or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , R 3 , R 4 are each independently selected from the group consisting of hydrogen, Ci-C6alkyl; Preferably, R 1 , R 3 , R 4 is hydrogen; Preferably, R 2 selected from C1-C6 alkyl; More preferably, R 2 is methyl; s1, s2, s3, s4 are each independently selected from an integer of 1-10; Preferably, s1 is selected from an integer of 1-5, preferably from 2, 3, 4, more preferably 3; Preferably, s2 is selected from an integer of 1-5, preferably from 1, 2, 3, more preferably 1; Preferably, s3 is selected from an integer of 1-5, preferably from 1, 2, 3, more preferably 1; Preferably, s4 is selected from an integer of 1-5, preferably from 3, 4, 5, more preferably 4; More preferably, the ligand unit is a structure selected from the group consisting of:

15. The targeting ligand of claim 14, wherein, the targeting ligand is a compound selected from the group consisting of 16. A targeting ligand delivery conjugate comprising a linker group, the linker group being a structure represented by Formula I” or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, L A’ -L B’ Formula I” wherein: L A’ selected from L B’ selected from Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Most preferably, Q' is selected from Z 3 , Z 2 , Z 1 , m, n1, n6, x, y are each independently as defined in any one of claims 1 to 4.

17. The targeted ligand delivery conjugate of claim 16, wherein, the linking group is a structure represented by Formula I”-1 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Z 1 each of m, n1, x is independently as defined in claim 5.

18. The targeted ligand delivery conjugate of claim 16, wherein, the linking group is a structure represented by Formula I”-2 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein: Q' is selected from Preferably, Q' is selected from More preferably, Q' is selected from Z 3 , Z 2 , n1, x, y are each independently as defined in any one of claims 6-8.

19. The targeted ligand delivery conjugate of any one of claims 16-18, wherein, the linking group is a structure selected from the group consisting of 20. The targeted ligand delivery conjugate of any one of claims 16-19, wherein, the targeting ligand delivery conjugate further comprises a ligand unit, the ligand unit being linked to the linker group; Preferably, the ligand unit is as defined in claim 14.

21. The targeted ligand delivery conjugate of claim 20, wherein, the targeting ligand delivery conjugate further comprises a delivered molecule, the ligand unit and the delivered molecule being linked via the linker group; Preferably, the delivered molecule includes, but is not limited to, an RNAi agent, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid, a natural or modified nucleic acid oligonucleotide, a natural or modified nucleic acid polynucleotide, a peptide, a nucleic acid aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, a polyethylene glycol, a hapten, digoxin, biotin, a radioactive atom or molecule, or a fluorophore; Preferably, the delivered molecule is an RNAi agent; More preferably, the delivered molecule is a small interfering RNA (siRNA), the siRNA comprising a sense strand and an antisense strand, the sequence of the sense strand and the antisense strand of the siRNA being the sense strand and the antisense strand sequence of duplex 1 as described in Table 1.

22. The targeted ligand delivery conjugate of claim 21, wherein, The targeting ligand delivery conjugate is selected from the group consisting of: wherein R z represents the delivered molecule.

23. A composition or a pharmaceutical composition comprising the linker of any one of claims 1-9, or the targeting ligand of any one of claims 10-15, or the targeting ligand delivery conjugate of any one of claims 16-22; and optionally a pharmaceutically acceptable excipient.

24. Use of the linker of any one of claims 1-9, or the targeting ligand of any one of claims 10-15, or the targeting ligand delivery conjugate of any one of claims 16-22, or the composition or the pharmaceutical composition of claim 23 in the manufacture of a medicament or a drug for delivering a molecule of interest.

25. Use of the linker of any one of claims 1-9, or the targeting ligand of any one of claims 10-15, or the targeting ligand delivery conjugate of any one of claims 16-22, or the composition or pharmaceutical composition of claim 23, in the manufacture of a medicament for the treatment and / or prevention of a disease.

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