Kidney-targeting delivery ligand, conjugate, composition and use thereof

Abstract

Provided are a kidney-targeting delivery ligand, a conjugate, a composition, and a use thereof, relating to the technical field of oligonucleotide drugs. Provided is a delivery ligand suitable for kidney targeting. The delivery ligand and an active substance (such as an oligonucleotide) are covalently linked by means of a linker group to obtain an oligonucleotide conjugate. The oligonucleotide conjugate has affinity for a cell receptor present on a kidney target cell and can selectively and effectively reduce or inhibit the expression of a kidney target gene in a subject (such as human or animal). The provided oligonucleotide conjugate or pharmaceutical composition can effectively treat and / or prevent pathological conditions or diseases caused by aberrant expression of a specific gene in kidney tissue cells.

Description

Kidney-targeting delivery ligands, conjugates, compositions and uses thereof

[0001] Cross-reference to related applications

[0002] The present disclosure claims priority to Chinese Patent Application No. 202410536410.1, filed on April 30, 2024, entitled “Kidney-targeting double-stranded oligonucleotide conjugates and uses thereof,” and to Chinese Patent Application No. 202410748954.4, filed on June 11, 2024, entitled “Kidney-targeting delivery ligands, conjugates, compositions and uses thereof,” the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure is in the field of oligonucleotide pharmaceuticals and relates specifically to compounds and methods for the in vivo delivery of RNAi agents, e.g., double-stranded oligonucleotide molecules, to extrahepatic cells, in particular to kidney-targeting delivery ligands, conjugates, compositions and uses thereof. BACKGROUND

[0004] The kidney plays an important role in the transport and excretion of different substances in the body and controls the intermediate metabolism of a variety of substances in the body. At present, there are at least one hundred million people worldwide suffering from various chronic kidney diseases. Despite the existence of various drugs and treatment methods, such as immunosuppressants, anti-inflammatory agents or diuretics, there is still room for improvement in the targeted delivery of drugs for the treatment of kidney diseases to the kidney and their long-lasting effectiveness.

[0005] Oligonucleotide-based drugs, such as double-stranded RNAi agents, have excellent therapeutic potential, but the extrahepatic delivery thereof has long been a challenge, in particular to achieve as high an affinity and selectivity for the kidney as possible has not been satisfactorily achieved. Therefore, there is still a need for new conjugate molecules or delivery carrier molecules to effectively direct oligonucleotide-containing drug molecules to extrahepatic, in particular kidney, cells. SUMMARY

[0006] The present disclosure relates to polypeptide molecules suitable for kidney targeting, which can serve as carrier molecules for the delivery of oligonucleotides. The present disclosure further relates to conjugate molecules comprising said polypeptides and at least one active substance, such as an oligonucleotide, covalently bound via a linker. Furthermore, the present disclosure relates to the use of said peptides and conjugates for kidney targeting, as well as pharmaceutical compositions comprising said peptides or conjugates.

[0007] The present disclosure finds that a peptide with a special amino acid sequence and its oligonucleotide conjugate covalently linked with at least one targeting ligand has selectivity for kidney cells, and the oligonucleotide conjugate has affinity to cell receptors present on kidney target cells. The conjugate of the present disclosure can selectively and effectively reduce or inhibit the expression of a target gene in the kidney of a subject (e.g., a human or an animal).

[0008] The present disclosure also describes a pharmaceutical composition comprising the conjugate, the conjugate comprising an oligonucleotide molecule capable of inhibiting the expression of a target gene, the composition further comprising at least one pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can selectively and effectively reduce or inhibit the expression of a target gene in kidney cells in vivo.

[0009] In a first aspect of the present disclosure, the present disclosure provides a targeting delivery ligand having targeting activity for a kidney cell surface receptor, the targeting delivery ligand comprising an amino acid sequence represented by formula (I), or an amino acid sequence variant having a deletion, substitution or addition of 1-6 amino acid residues of the amino acid sequence and having the same targeting activity, or an amino acid sequence variant having at least 60% sequence homology with the amino acid sequence and having the same targeting activity.

[0010] Phe-Ser-AA1-Cha-Ala-Gly-AA2-Ile-Asp-AA3-Ile (I)

[0011] wherein AA1, AA2 are selected from any amino acid; and AA3 is selected from arginine or alkylated arginine.

[0012] wherein Phe represents phenylalanine; Ser represents serine; Cha represents cyclohexylalanine; Ala represents alanine; Gly represents glycine; Ile represents isoleucine; and Asp represents aspartic acid.

[0013] According to an embodiment of the present disclosure, AA1, AA2 are independently selected from proline or hydroxyproline; and AA3 is selected from arginine or N-alkyl substituted arginine derivative.

[0014] According to an embodiment of the present disclosure, the targeting delivery ligand comprises any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0015] In a second aspect of the present disclosure, the present disclosure provides use of the targeting delivery ligand of the first aspect of the present disclosure for targeted delivery of an active pharmaceutical molecule to kidney cells.

[0016] In a third aspect of the present disclosure, the present disclosure provides a conjugate comprising a structure represented by formula (200), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0017] wherein Nu represents an active drug molecule.

[0018] a is an integer selected from 1-5.

[0019] each M is conjugated to a different position of Nu.

[0020] each M is independently selected from

[0021] In some optional embodiments of the present disclosure, Nu is selected from a double-stranded oligonucleotide.

[0022] In some optional embodiments of the present disclosure, a is selected from 1, and one of the M is conjugated to the 3' end of the sense strand or the 5' end of the sense strand of the double-stranded oligonucleotide.

[0023] In some optional embodiments of the present disclosure, a is selected from 2, and two of the M are conjugated to the 3' end of the sense strand and the 5' end of the sense strand of the double-stranded oligonucleotide, respectively.

[0024] In some optional embodiments of the present disclosure, the conjugate is targeted for delivery to a kidney cell.

[0025] In a fourth aspect of the present disclosure, the present disclosure provides a composition comprising the conjugate of the third aspect of the present disclosure.

[0026] In a fifth aspect of the present disclosure, the present disclosure provides use of any of the following in the manufacture of a medicament for preventing and / or treating a disease:

[0027] (I) the targeting delivery ligand of the first aspect of the present disclosure; and / or

[0028] (II) the conjugate of the third aspect of the present disclosure; and / or

[0029] (III) the composition of the fourth aspect of the present disclosure.

[0030] In some optional embodiments of the present disclosure, the disease is selected from a kidney-derived disease.

[0031] In some alternative embodiments of the present disclosure, the renal disease includes, but is not limited to, hypertension, hypertension-related renal impairment, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia renal impairment, hepatitis B virus-related renal impairment, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremic syndrome, or other systemic lupus erythematosus (SLE)-related kidney disease.

[0032] In a sixth aspect of the present disclosure, the present disclosure provides use of any of the following in the manufacture of a medicament for reducing expression or activity of a target gene in a kidney cell:

[0033] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0034] (II) the conjugate of the third aspect of the present disclosure; and / or

[0035] (III) the composition of the fourth aspect of the present disclosure.

[0036] In a seventh aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any of the following and a pharmaceutically acceptable carrier or excipient:

[0037] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0038] (II) the conjugate of the third aspect of the present disclosure; and / or

[0039] (III) the composition of the fourth aspect of the present disclosure.

[0040] In an eighth aspect of the present disclosure, the present disclosure provides a method for reducing expression or activity of a target gene in a kidney cell, characterized in that the method comprises contacting the kidney cell with any of the following:

[0041] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0042] (II) the conjugate of the third aspect of the present disclosure; and / or

[0043] (III) the composition of the fourth aspect of the present disclosure; and / or

[0044] (IV) the pharmaceutical composition of the seventh aspect of the present disclosure.

[0045] In a ninth aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate for inhibiting gene expression in a kidney cell, comprising:

[0046] (a) a single- or double-stranded oligonucleotide molecule having the following features:

[0047] (i) an antisense strand comprising 17-35 nucleotides, wherein at least 15 nucleotides are complementary or substantially complementary to a mRNA sequence of a gene in a kidney cell;

[0048] (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand;

[0049] (b) a targeting ligand having affinity for a receptor present on the surface of a kidney cell, wherein the targeting ligand is a polypeptide; optionally, the polypeptide has at least 4 amino acid residues;

[0050] and,

[0051] (c) a linker group, wherein the oligonucleotide molecule is covalently linked to the targeting ligand via the linker group;

[0052] the polypeptide fragment included in the targeting ligand is -dXa-Ser-dXb-X2-dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; wherein X2 is selected from any unnatural amino acid; dXa, dXb, dXc are selected from any D-amino acid; Arg(Ak) is selected from arginine or alkylated modified arginine; Xd is selected from Hyp or Pro.

[0053] In some embodiments, the targeting ligand is selected from a polypeptide comprising any of the amino acid sequence segments shown in A1) - A2):

[0054] A1) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-;

[0055] A2) -DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

[0056] In some embodiments, the receptor is selected from a natriuretic peptide receptor.

[0057] In some alternative embodiments, the targeting ligand linked by the linker is a ligand unit selected from any of the structures shown in B1) - B2):

[0058] B1) La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa;

[0059] B2) La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Laa.

[0060] Any one of La and Laa is selected from a linker, the other is a terminal blocking group, the linker is a linking group and comprises at least one of a triazole group or a PEG unit or an acyl group. In some alternative embodiments, La is a linker, the Linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-.

[0061] In some alternative embodiments, the Ligand Unit is selected from any one of the structures shown in Formula C1) - C2), or a pharmaceutically acceptable salt thereof:

[0062] C1) -La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc;

[0063] C2) -La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Lc;

[0064] wherein La is a linker independently selected from any bond, -NH-, an amide group or a linking group comprising at least one PEG unit.

[0065] In some alternative embodiments, La is a linking group comprising at least 2 PEG units (e.g. La is a linking group comprising 2, 3, 4, 5 or 6 PEG units).

[0066] In some alternative embodiments, La is selected from an amide group or -triazole-(PEG)n-, for example n is 1-3, m is 0 or 1, and Z is -CO- (carbonyl) or -NH-;

[0067] wherein Lc is a terminal blocking group. In some alternative embodiments, Lc is a C-terminal blocking group, the Lc is selected from an amino group or an alkyl substituted amine group, preferably -NHCH3.

[0068] In a tenth aspect of the present disclosure, the present disclosure provides a polypeptide capable of delivering an oligonucleotide to a kidney cell, the polypeptide comprising any one of the amino acid sequence segments shown in A1) - A2):

[0069] A1) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-;

[0070] A2) -DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

[0071] In an eleventh aspect of the present disclosure, the present disclosure provides a ligand unit molecule capable of delivering an oligonucleotide to a kidney cell, which is selected from any one of the compounds represented by D1) - D2) :

[0072] D1) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-CONHCH3;

[0073] D2) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-CONH2.

[0074] In a twelfth aspect of the present disclosure, the present disclosure provides a use of the polypeptide of the tenth aspect or the ligand unit molecule of the eleventh aspect for delivering an oligonucleotide molecule to a kidney cell.

[0075] The present disclosure provides a delivery ligand suitable for kidney targeting, which is covalently linked to an active substance (e.g., an oligonucleotide) via a linker group to obtain an oligonucleotide conjugate, which has affinity for a cell receptor present on a kidney target cell, and can selectively and effectively reduce or inhibit the expression of a target gene in the kidney of a subject (e.g., a human or an animal). The oligonucleotide conjugate or pharmaceutical composition provided by the present disclosure can effectively treat and / or prevent pathological conditions or diseases caused by abnormal expression of a specific gene in kidney tissue cells. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 shows the inhibitory activity of a target gene in the kidney of a mouse administered with the siRNA conjugate described in Test Example 1.

[0077] Figure 2 shows the inhibitory activity of a target gene in the kidney cortex of a mouse administered with the siRNA conjugate described in Test Example 2.

[0078] Figure 3 shows the inhibitory activity of a target gene in the kidney of a mouse administered with the siRNA conjugate described in Test Example 3.

[0079] Figure 4 shows the level of alanine aminotransferase (ALT) in the serum of ICR mice administered with the siRNA conjugate R309001.

[0080] Figure 5 shows the level of aspartate aminotransferase (AST) in serum of ICR mice after administration of siRNA conjugate R309001.

[0081] Figure 6 shows the level of urea (UREA) in serum of ICR mice after administration of siRNA conjugate R309001.

[0082] Figure 7 shows the HE staining of kidney tissue sections of ICR mice in each group after administration of siRNA conjugate R309001;▲represents renal tubular vacuolar degeneration of the kidney, ♀ represents female mice, and ♂ represents male mice.

[0083] Figure 8 shows the pathological score of liver tissue of ICR mice in each group after administration of siRNA conjugate R309001.

[0084] Figure 9 shows the HE staining of liver tissue sections of ICR mice in each group after administration of siRNA conjugate R309001. DETAILED DESCRIPTION

[0085] The technical solutions in the embodiments of the present application will be described clearly and completely below, and those skilled in the art can implement the process parameters by referring to the content herein and making appropriate improvements.

[0086] TERMINOLOGY

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. The publications, patent applications, patents, and other references noted herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0088] In the present disclosure, the term “comprising” or “including” is an open-ended expression, which is used in the present disclosure to mean the phrase “including but not limited to”, and is used interchangeably with it, indicating that it includes the content indicated by the present disclosure, but does not exclude other aspects.

[0089] In the present disclosure, the term “optionally”, “optional” or “optional” generally means that the event or condition described subsequently can but does not necessarily occur, and the description includes both the case where the event or condition occurs and the case where the event or condition does not occur.

[0090] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. The siRNA mediates the RNA transcript targeting cleavage by forming a silencing complex (RISC) of the RISC pathway. Specifically, the siRNA directs the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins.

[0091] In the present disclosure, the terms "sequence" and "nucleotide sequence" refer to a series of nucleobases or nucleotides. As used in the present disclosure, a "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes the purine bases adenine and guanine, and the pyrimidine bases cytosine, thymine, and uracil. Nucleobases can be further modified. The synthesis of modified nucleobases, including phosphoramidite compounds of modified nucleobases, is known in the art.

[0092] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides, including a sense strand and an antisense strand, through partial or complete base pairing, and the length of the sense strand and the antisense strand can be the same or different, as long as there is at least a region of partial base pairing to form a duplex region. An oligonucleotide having a double-stranded structure belongs to the double-stranded oligonucleotide of the present disclosure. In the present disclosure, the nucleotides constituting the double-stranded oligonucleotide can be modified or unmodified nucleotides, and when referring to modified nucleotides, the modification referred to in the present disclosure does not specifically refer to the modification site, unless otherwise specified. In the present disclosure, the double-stranded oligonucleotide can be modified in addition to the modification of the nucleotides, and the linkage between the nucleotides, and the double-stranded oligonucleotide containing the modified linkage between the modified nucleotides also belongs to the double-stranded oligonucleotide of the present disclosure. In the present disclosure, the double-stranded oligonucleotide can further contain a compound molecule or a modifier acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as a ligand to form a conjugate, in addition to the nucleotide portion.

[0093] In the present disclosure, the term "antisense strand (or referred to as guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or referred to as passenger strand)" refers to an iRNA strand that contains a sequence that is substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, for example, within 5, 4, 3, or 2 nucleotides of the 5'- and / or 3' terminus of the iRNA. It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest.

[0094] In the present disclosure, the term "ligand" or "conjugate group" refers to an atom or group of atoms that is bound to an oligonucleotide or other oligomer. Generally, a conjugate group modifies one or more properties of the compound to which it is attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties. The term "linked" as used herein when referring to the linkage between two molecules means that the two molecules are directly or indirectly linked by a covalent bond or that the two molecules are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

[0095] In the present disclosure, the term "targeting ligand" refers to a polypeptide that has affinity for a receptor present on the surface of a kidney cell; the term "ligand unit" refers to a targeting ligand linked to a linker; the term "ligand unit molecule" refers to a compound that is reacted with an oligonucleotide in a molecular form.

[0096] In the present disclosure, the term "linked" or "conjugated" when referring to the linkage between two compounds or molecules means that the two molecules are linked by a covalent bond or are associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise specified, the terms "linked" and "conjugated" as used in the present disclosure can refer to the linkage between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0097] In the present disclosure, a linking group is one or more atoms that link one molecule or portion of a molecule to a second molecule or second portion of a molecule. A linking group can comprise any number of atoms or functional groups. In some embodiments, a linking group is used solely to link two biologically active molecules.

[0098] Unless otherwise specified, the symbols as used in the present disclosure mean that any group or groups can be attached thereto, consistent with the scope of the application described in the present disclosure.

[0099] In the present disclosure, The point at which a group is attached by a covalent bond.

[0100] According to common knowledge in the art, a peptide is a compound which is produced by the linkage of two or more amino acids by amide bonds. Here, the individual amino acids are linked in a certain order (sequence) into a chain. An amino acid is a compound which carries at least one amino group and at least one carboxyl group. Both natural (in vivo produced proteinogenic amino acids), non-natural amino acids or prepared amino acids which can exist in organisms are included.

[0101] In the peptides of the present disclosure, the amino acid units can be present in the D- or L-form, except where specifically noted.

[0102] As used herein, the term "standard amino acid" refers to the following twenty amino acids: alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0103] As used herein, the term "non-standard amino acid" refers to an amino acid other than a "standard amino acid" as defined herein. "Non-standard amino acids" include, but are not limited to, N-formylmethionine, hydroxyproline, selenomethionine, isovaline, citrulline (Cit), ornithine, a-methyl-aspartate (aMeD), a-methyl-leucine (aMeL), N-methylalanine, N-methyl-glycine (NMe G), N-methyl-leucine (NMe L), O-cyclohexyl-alanine (Cha), N-ethylalanine, N,N-epsilon-dimethyllysine (K(Me)2), dimethylarginine (R(Me)2), n-alkylated L-a amino acids, and other amino acid analogs or mimetics that function in a similar manner to naturally occurring amino acids.

[0104] As used herein and as understood by one of skill in the art, a polyethylene glycol (PEG) unit refers to the repeating unit of formula (CH2CH2O). It will be understood that in the chemical structures disclosed herein, a PEG unit can be depicted as (CH2CH2O), (OCH2CH2), or (CH2OCH2). It will further be understood that the number indicating the number of repeating PEG units can be placed on either side of the parentheses indicating the PEG unit. It will be further understood that terminal PEG units can be capped with an atom (e.g., a hydrogen atom) or some other moiety.

[0105] In the present disclosure, the term "pharmaceutical composition" or "composition" can refer to the use for the treatment of a disease, as well as for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the auxiliaries that constitute one or more accessory ingredients. Typically, a composition is prepared by uniformly and intimately bringing the active siRNA into association with a liquid auxiliary, a finely divided solid auxiliary, or both.

[0106] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients constituting a formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved or approvable by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0107] In the present disclosure, the terms "pharmaceutically acceptable carrier or adjuvant" can include any and all solvents, solid diluents, or other liquid adjuvants, etc. that are suitable for the particular target dosage form. Except insofar as any conventional adjuvant is incompatible with the siRNA of the present disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated to be within the scope of this disclosure.

[0108] In the present disclosure, "aliphatic ring" refers to a structure having a cyclic carbon skeleton. The term "5-8 membered aliphatic ring" refers to a monocyclic structure having 5 to 8 carbon atoms in the cyclic carbon skeleton. The term "saturated aliphatic ring" refers to a cyclic carbon skeleton consisting of carbon-carbon single bonds only.

[0109] In the present disclosure, "5-8 membered aliphatic heterocycle" refers to an aliphatic ring in which one or more carbon atoms are replaced by a heteroatom. The term "saturated aliphatic heterocycle" refers to an aliphatic ring in which one or more carbon atoms are replaced by a heteroatom.

[0110] In the present disclosure, the terms "treat," "treating," or "treatment" can be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Herein, a therapeutic benefit is achieved with regard to the underlying disorder by observing an improvement in one or more of the physiological symptoms associated with the underlying disorder, notwithstanding the fact that the subject can still be afflicted with the underlying disorder.

[0111] In the present disclosure, the terms "prevent" and "prevention" are used interchangeably and refer to a method of obtaining a beneficial or desired result, including, but not limited to, prophylactic benefit. To obtain "prophylactic benefit", a conjugate, RNAi agent or composition can be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0112] In the present disclosure, the term "administration" generally refers to the introduction or delivery of a pharmaceutical preparation of the present disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting a cell, organ or tissue with the drug can be employed. The administration can include, without limitation, intravenous, intraarterial, intranasal, intraabdominal, intramuscular, subcutaneous or oral. The daily dose can be divided into one, two or more doses of suitable form for administration at one, two or more times during a certain period of time.

[0113] As used herein, the term "modulating gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is up- or down-regulated such that the expression, level or activity is greater or less than that observed in the absence of the modulating agent. For example, the term "modulating" can mean "inhibiting", but the use of the term "modulating" is not limited to this definition.

[0114] In addition to any conventional excipients, the use of ranges which are incompatible with the siRNA of the present disclosure, for example any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner, are also within the scope of the present disclosure.

[0115] Targeted delivery ligand

[0116] In a first aspect of the present disclosure, the present disclosure provides a targeted delivery ligand having a targeting activity to a kidney cell surface receptor, the targeted delivery ligand comprising an amino acid sequence represented by Formula (I), or an amino acid sequence variant having a deletion, substitution or addition of 1-6 amino acid residues (preferably, having a deletion, substitution or addition of 1, 2 or 3 amino acid residues) from the amino acid sequence and having the same targeting activity, or an amino acid sequence variant having at least 60%, at least 70%, at least 80%, at least 90% or more sequence homology with the amino acid sequence and having the same targeting activity.

[0117] Phe-Ser-AA1-Cha-Ala-Gly-AA2-Ile-Asp-AA3-Ile (I)

[0118] In some alternative embodiments of the present disclosure, the targeting delivery ligand is an amino acid sequence variant of the amino acid sequence having 1-3 amino acid residues deleted, substituted or added and having the same targeting activity.

[0119] wherein AA1, AA2 are selected from any amino acid; AA3 is selected from arginine or alkylated arginine.

[0120] wherein Phe represents phenylalanine; Ser represents serine; Cha represents cyclohexylalanine; Ala represents alanine; Gly represents glycine; Ile represents isoleucine; Asp represents aspartic acid.

[0121] In some alternative embodiments of the present disclosure, the targeting delivery ligand is a polypeptide, a polypeptide variant or a polypeptide derivative comprising the amino acid sequence Phe-Ser-AA1-Cha-Ala-Gly-AA2-Ile-Asp-AA3-Ile.

[0122] The polypeptide variant is selected from: an amino acid sequence comprising 1-3 amino acid residues deleted, substituted or added to the amino acid sequence and having the same targeting activity as the polypeptide, or, an amino acid sequence comprising at least 60% sequence homology to the amino acid sequence and having the same targeting activity as the polypeptide.

[0123] wherein AA1, AA2 are independently selected from proline or hydroxyproline; AA3 is selected from arginine or N-alkyl substituted arginine derivative.

[0124] wherein the polypeptide derivative is selected from the above polypeptide or polypeptide variant substituted with a substituent at the N-terminus and / or C-terminus, and the polypeptide derivative has the targeting activity to the cell surface receptor of the kidney.

[0125] In some alternative embodiments of the present disclosure, the substituent is selected from a terminal protecting group or a covalent linking group for linking an oligonucleotide.

[0126] In some alternative embodiments of the present disclosure, the targeting delivery ligand comprises the amino acid sequence DPhe-Ser-AA1-Cha-DAla-Gly-AA2-Ile-Asp-AA3-Ile in the direction from the amino terminus to the carboxyl terminus.

[0127] DPhe represents D-phenylalanine; Ser represents L-serine; Cha represents L-cyclohexylalanine; DAla represents D-alanine; Gly represents glycine; Ile represents L-isoleucine; Asp represents L-aspartic acid.

[0128] In some alternative embodiments of the present disclosure, the targeting delivery ligand comprises a structure represented by Formula (100), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0129] wherein R1is selected from H or hydroxyl; R2is selected from H or hydroxyl; and R3is selected from H or C1-C3alkyl.

[0130] In some alternative embodiments of the present disclosure, the targeting delivery ligand comprises a structure represented by Formula (101), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0131] According to embodiments of the present disclosure, the targeting delivery ligand comprises any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0132] In some alternative embodiments of the present disclosure, the targeting delivery ligand comprises a structure represented by Formula (102), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0133] wherein Lz1is selected from H, C1-C3alkyl, or wherein each of g1', g2', g3' is independently selected from an integer from 1 to 10, and Ly' is selected from -COOH or -NH2.

[0134] Lz2is selected from -NH2, -NH-C1-C3alkyl, or wherein each of g1", g2", g3" is independently selected from an integer from 1 to 10, and Ly" is selected from -COOH or -NH2.

[0135] Meanwhile, when Lz1is selected from Lz2is selected from -NH2or -NH-C1-C3alkyl; and when Lz2is selected from Lz1is selected from H, C1-C3alkyl.

[0136] In some alternative embodiments of the present disclosure, the targeting delivery ligand comprises a structure represented by Formula (103), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0137] According to embodiments of the present disclosure, the targeting delivery ligand comprises any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0138] In some alternative embodiments of the present disclosure, the targeted delivery ligand specifically binds to a renal cell surface receptor.

[0139] In some alternative embodiments of the present disclosure, the renal cell surface receptor is selected from the group consisting of a Lina peptide receptor.

[0140] Use of a targeted delivery ligand

[0141] In a second aspect of the present disclosure, the present disclosure provides use of a targeted delivery ligand as described in the first aspect of the present disclosure for targeted delivery of an active pharmaceutical molecule to a renal cell.

[0142] In some alternative embodiments of the present disclosure, the active pharmaceutical molecule is selected from the group consisting of a double-stranded oligonucleotide, a single-stranded oligonucleotide, or a small molecule drug.

[0143] In some alternative embodiments of the present disclosure, the double-stranded oligonucleotide is selected from the group consisting of an siRNA.

[0144] In some alternative embodiments of the present disclosure, the single-stranded oligonucleotide is selected from the group consisting of an ASO.

[0145] According to embodiments of the present disclosure, the active pharmaceutical molecule is selected from the group consisting of a double-stranded oligonucleotide.

[0146] According to embodiments of the present disclosure, the active pharmaceutical molecule is selected from the group consisting of an siRNA.

[0147] siRNA conjugate

[0148] In a third aspect of the present disclosure, the present disclosure provides a conjugate comprising a structure represented by formula (200), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0149] wherein, Nu represents an active pharmaceutical molecule.

[0150] a is selected from an integer from 1 to 5.

[0151] a M are respectively conjugated to different positions of Nu.

[0152] each M is independently selected from the group consisting of

[0153] each Ly is independently selected from the group consisting of or -S-S-.

[0154] each Lz is independently selected from the group consisting of wherein, g1, g2, g3 are each independently selected from an integer from 1 to 10, Z is selected from the group consisting of

[0155] In some optional embodiments of the present disclosure, g1 is selected from 2, g2 is selected from 2, g3 is selected from 2.

[0156] In some optional embodiments of the present disclosure, Lz is selected from

[0157] In some optional embodiments of the present disclosure, Lz is selected from

[0158] Lx1 is selected from wherein b1, b2, b3, b4, b5 are each independently selected from an integer from 1 to 5.

[0159] In some optional embodiments of the present disclosure, b1 is selected from 6, b2 is selected from 3, b3 is selected from 2, b4 is selected from 2, b5 is selected from 2.

[0160] According to embodiments of the present disclosure, Lx1 is selected from

[0161] selected from

[0162] In some optional embodiments of the present disclosure, selected from

[0163] Lx2 is selected from wherein c1, c2, c3, c4, c5, c6, c7, c8 are each independently selected from an integer from 1 to 5.

[0164] In some optional embodiments of the present disclosure, c1 is selected from 6, c2 is selected from 3, c3 is selected from 2, c4 is selected from 2, c5 is selected from 2, c6 is selected from 2, c7 is selected from 2, c8 is selected from 2.

[0165] According to embodiments of the present disclosure, Lx2 is selected from

[0166] selected from

[0167] Lx3 is selected from wherein d1, d2, d3, d4, d5, d6, d7, d8, d9, d 10 , d 11 are each independently selected from an integer from 1 to 5.

[0168] In some optional embodiments of the present disclosure, d1 is selected from 6, d2 is selected from 3, d3 is selected from 2, d4 is selected from 2, d5 is selected from 2, d6 is selected from 2, d7 is selected from 2, d8 is selected from 2, d9 is selected from 2, d 10selected from 2, d 11 selected from 2.

[0169] According to embodiments of the present disclosure, Lx3is selected from

[0170] selected from

[0171] each Cx is independently unsubstituted or substituted 4-10 membered aliphatic ring; e1 is selected from an integer between 1-5; each e2 is independently selected from an integer between 0-5; each e3 is independently selected from an integer between 0-5; each e4 is independently selected from an integer between 1-5; each X1 is independently selected from NH, O, or S; each X2 is independently selected from e5 is selected from an integer between 1-10; each X3 is independently selected from hydroxyl or thiol; each R5 is independently H, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy.

[0172] In some optional embodiments of the present disclosure, each Cx is independently selected from

[0173] In some optional embodiments of the present disclosure, each Cx is selected from

[0174] In some optional embodiments of the present disclosure, X1 is selected from NH.

[0175] In some optional embodiments of the present disclosure, X2 is selected from

[0176] In some optional embodiments of the present disclosure, each R5 is selected from H.

[0177] In some optional embodiments of the present disclosure, e1 is selected from 1, 2, or 3.

[0178] In some optional embodiments of the present disclosure, e2 is selected from 1.

[0179] In some optional embodiments of the present disclosure, e3 is selected from 1.

[0180] In some optional embodiments of the present disclosure, e4 is selected from 2.

[0181] In some optional embodiments of the present disclosure, selected from

[0182] In some optional embodiments of the present disclosure, selected from

[0183] each Cy is independently selected from a 5-6 membered saturated oxygen-containing heterocycle; f1 is selected from an integer between 1-5; each f2 is independently selected from an integer between 0-5; each f3 is independently selected from an integer between 0-5; each f4 is independently selected from 1 or 2; each f5 is independently selected from an integer between 1-5; each Y1 is independently selected from O, S, or NH; each Y2 is independently selected from hydroxyl or thiol; each R6 is independently selected from H or C1-C6 alkoxy; and each R7 is independently selected from H, C1-C6 alkyl, or C1-C6 alkoxy.

[0184] In some optional embodiments of the present disclosure, each Cy is independently selected from a 5-6 membered saturated oxygen-containing heterocycle.

[0185] In some optional embodiments of the present disclosure, each Cy is independently selected from a 5-6 membered saturated heterocycle containing one oxygen atom.

[0186] In some optional embodiments of the present disclosure, each Cy is independently selected from

[0187] In some optional embodiments of the present disclosure, each Y1 is independently selected from O.

[0188] In some optional embodiments of the present disclosure, f1 is selected from 1, 2, or 3.

[0189] In some optional embodiments of the present disclosure, f2 is selected from 1.

[0190] In some optional embodiments of the present disclosure, f3 is selected from 0.

[0191] In some optional embodiments of the present disclosure, f5 is selected from 1.

[0192] In some optional embodiments of the present disclosure, each R6 is independently selected from H or methoxy.

[0193] In some optional embodiments of the present disclosure, each R7 is selected from H.

[0194] In some optional embodiments of the present disclosure, is selected from

[0195] In some optional embodiments of the present disclosure, is selected from

[0196] In some optional embodiments of the present disclosure, is selected from

[0197] each Ma is independently selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0198] wherein R1is selected from H or hydroxyl; R2is selected from H or hydroxyl; R3is selected from H or C1-C3alkyl; R4is selected from H or C1-C3alkyl;

[0199] or, wherein R1is selected from H or hydroxyl; R2is selected from H or hydroxyl; R3is selected from H or C1-C3alkyl; R4is selected from H or C1-C3alkyl.

[0200] when Z is selected from ,

[0201] Ma is selected from

[0202] when Z is selected from ,

[0203] Ma is selected from

[0204] In some optional embodiments of the present disclosure, Ma is selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

[0205] In some optional embodiments of the present disclosure, Ma is selected from any one of the following structures:

[0206] In some optional embodiments of the present disclosure, in the conjugate, Nu is selected from a double-stranded oligonucleotide.

[0207] In some optional embodiments of the present disclosure, a is selected from 1 or 2.

[0208] In some optional embodiments of the present disclosure, Nu is selected from a double-stranded oligonucleotide, and a is selected from 1 or 2.

[0209] In some optional embodiments of the present disclosure, a is selected from 1, one of the M is conjugated to the 3' end of the sense strand or the 5' end of the sense strand of the double-stranded oligonucleotide.

[0210] In some optional embodiments of the present disclosure, a is selected from 2, two of the M are respectively conjugated to the 3' end of the sense strand and the 5' end of the sense strand of the double-stranded oligonucleotide.

[0211] In some optional embodiments of the present disclosure, the conjugate is targeted for delivery to a kidney cell.

[0212] Compositions

[0213] In a fourth aspect of the present disclosure, the present disclosure provides a composition comprising the conjugate of the third aspect of the present disclosure.

[0214] Use for treating a disease

[0215] In a fifth aspect of the present disclosure, the present disclosure provides use of any of:

[0216] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0217] (II) the conjugate of the third aspect of the present disclosure; and / or

[0218] (III) the composition of the fourth aspect of the present disclosure.

[0219] In some optional embodiments of the present disclosure, the disease is selected from a nephrogenic disease.

[0220] In some optional embodiments of the present disclosure, the nephrogenic disease includes, but is not limited to, hypertension, hypertensive-related renal impairment, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia-related renal impairment, hepatitis B virus-related renal impairment, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremic syndrome, or other systemic lupus erythematosus (SLE)-related kidney disease.

[0221] Use for reducing the expression or activity of a target gene in a kidney cell

[0222] In a sixth aspect of the present disclosure, the present disclosure provides use of any of:

[0223] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0224] (II) the conjugate of the third aspect of the present disclosure; and / or

[0225] (III) the composition of the fourth aspect of the present disclosure.

[0226] Pharmaceutical composition

[0227] In a seventh aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any of:

[0228] (I) the targeted delivery ligand of the first aspect of the present disclosure; and / or

[0229] (I) the targeting delivery ligand of the first aspect of the disclosure; and / or

[0230] (III) the composition of the fourth aspect of the disclosure.

[0231] A method of reducing expression or activity of a target gene in a kidney cell

[0232] In an eighth aspect of the disclosure, the disclosure provides a method of reducing expression or activity of a target gene in a kidney cell, characterized in that the method comprises contacting with the kidney cell any of:

[0233] (I) the targeting delivery ligand of the first aspect of the disclosure; and / or

[0234] (II) the conjugate of the third aspect of the disclosure; and / or

[0235] (III) the composition of the fourth aspect of the disclosure; and / or

[0236] (IV) the pharmaceutical composition of the seventh aspect of the disclosure.

[0237] Oligonucleotide conjugate

[0238] In a ninth aspect of the disclosure, the disclosure provides an oligonucleotide conjugate for use in inhibiting expression of a gene in a kidney cell, comprising:

[0239] (a) a single- or double-stranded oligonucleotide molecule having the following features:

[0240] (i) an antisense strand comprising 17-35 nucleotides, wherein at least 15 nucleotides are complementary or substantially complementary to a mRNA sequence of a gene in a kidney cell;

[0241] (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand;

[0242] (b) a targeting ligand having affinity for a receptor present on the surface of a kidney cell, wherein the targeting ligand is a polypeptide; optionally, the polypeptide has at least 4 amino acid residues;

[0243] and,

[0244] (c) a linker group, wherein the oligonucleotide molecule is covalently linked to the targeting ligand via the linker group;

[0245] The targeting ligand comprises a polypeptide fragment -dXa-Ser-dXb-X2-dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; wherein X2 is selected from any unnatural amino acid; dXa, dXb, dXc are selected from any D-amino acid; Arg(Ak) is selected from arginine or alkylated modified arginine; Xd is selected from Hyp or Pro.

[0246] In some embodiments, the targeting ligand is selected from a polypeptide comprising any of the amino acid sequence segments shown in A1) - A2):

[0247] A1) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-;

[0248] A2) -DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

[0249] In some embodiments, the receptor is selected from natriuretic peptide receptor.

[0250] In some embodiments, the Linker is connected to the N- or C-terminus of an amino acid in the polypeptide.

[0251] In some embodiments, the Linker is selected from substituted or unsubstituted aliphatic chain, 3-6 membered heterocyclic ring, C6-C 10 aromatic ring, (PEG)n, disulfide bond-containing, amide group-containing or triazole group-containing group, or a combination thereof; wherein n is selected from 1-10.

[0252] In some embodiments, the Linker comprises any of the following substituents or any combination thereof:

[0253] disulfide bond, amide group, triazole group, -NH-, -C(O)-,

[0254] wherein a, b, c or d is each independently selected from an integer from 0-10, preferably an integer from 1-8.

[0255] * represents the connection point to the oligonucleotide molecule or to the targeting ligand, or the point of connection of the substituents to each other.

[0256] In some alternative embodiments, the targeting ligand connected by the linker is a ligand unit, and the ligand unit is selected from any of the structures shown in B1) - B2):

[0257] B1) La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa;

[0258] B2) La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Laa;

[0259] Any one of La and Laa is selected from a linker, the other is a terminal blocking group, the linker is a linking group and comprises at least one of a triazole group or a PEG unit or an acyl group. In some alternative embodiments, La is a linker, the Linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-.

[0260] In some alternative embodiments, the Ligand Unit is selected from any one of the structures shown in Formula C1) - C2), or a pharmaceutically acceptable salt thereof:

[0261] C1) -La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc;

[0262] C2) -La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Lc;

[0263] wherein La is a linker independently selected from any bond, -NH-, an amide group or a linking group comprising at least one PEG unit.

[0264] In some alternative embodiments, La is a linking group comprising at least 2 PEG units (e.g. La is a linking group comprising 2, 3, 4, 5 or 6 PEG units).

[0265] In some alternative embodiments, La is selected from an amide group or -triazole-(PEG)n-, for example n is 1-3, m is 0 or 1, and Z is -CO- (carbonyl) or -NH-;

[0266] wherein Lc is a terminal blocking group. In some alternative embodiments, Lc is a C-terminal blocking group, the Lc is selected from an amino group or an alkyl substituted amine group, preferably -NHCH3.

[0267] Polypeptides for targeted delivery of oligonucleotides

[0268] In a tenth aspect of the present disclosure, the present disclosure provides a polypeptide capable of delivering an oligonucleotide to a kidney cell, the polypeptide comprising any of the amino acid sequence segments shown in A1)-A2):

[0269] A1)-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-;

[0270] A2)-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

[0271] Ligand unit molecules for targeted delivery of oligonucleotides

[0272] In an eleventh aspect of the present disclosure, the present disclosure provides a ligand unit molecule capable of delivering an oligonucleotide to a kidney cell, which is selected from any of the compounds shown in D1)-D2):

[0273] D1) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-CONHCH3;

[0274] D2) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-CONH2.

[0275] Use of polypeptides and ligand unit molecules

[0276] In a twelfth aspect of the present disclosure, the present disclosure provides use of the polypeptide of the tenth aspect or the ligand unit molecule of the eleventh aspect, for delivering an oligonucleotide molecule to a kidney cell.

[0277] In order to make the objects, technical solutions and advantages of the present disclosure clearer, the following will further describe the embodiments of the present disclosure with reference to the examples.

[0278] Unless otherwise specified, the reagent ratios described in the embodiments of the present disclosure are calculated by volume ratio (v / v).

[0279] Unless otherwise specified, the raw materials and reagents used in the preparation of the compounds provided by the present disclosure are commercially available. Among them, the main reagent consumables used in the present disclosure are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0280] Table 1. Main reagent consumables

[0281] Table 2. Main instruments and equipment

[0282] Preparation Example 1: Preparation of a targeted delivery ligand

[0283] The Fmoc solid-phase polypeptide synthesis method is used to sequentially connect the amino acid monomers from the carboxyl end to the amino end according to the amino acid sequence.

[0284] The amino acid monomer of Ile is Fmoc-Ile-OH;

[0285] The amino acid monomer of Arg is Fmoc-Arg(Pbf)-OH;

[0286] The amino acid monomer of Arg(Me) is Fmoc-N-Me-Arg(Pbf)-OH;

[0287] The amino acid monomer of Asp is Fmoc-Asp(OtBu)-OH;

[0288] The amino acid monomer of Pro is Fmoc-Pro-OH;

[0289] The amino acid monomer of DPro is Fmoc-D-Pro-OH;

[0290] The amino acid monomer of Hyp is Fmoc-Hyp(tBu)-OH;

[0291] The amino acid monomer of DHyp is Fmoc-D-Hyp(tBu)-OH;

[0292] The amino acid monomer of Gly is Fmoc-Gly-OH;

[0293] The amino acid monomer of DAla is Fmoc-D-Ala-OH;

[0294] The amino acid monomer of Cha is Fmoc-Cha-OH;

[0295] The amino acid monomer of Ser is Fmoc-Ser(tBu)-OH;

[0296] The amino acid monomer of DPhe is Fmoc-D-Phe-OH.

[0297] In this preparation example, the specific preparation method of the targeted delivery ligand includes the following steps:

[0298] (1-1) Take the polypeptide synthesis resin Fmoc-Linker-MBHA Resin (degree of substitution about 0.5 mmol / g, total 1.0 mmol reaction sites) 2.0 g, use N, N-dimethylformamide (DMF) to swell for 20 minutes; then, add 20% Pip / DMF mixed solution (i.e. the volume ratio of Piperazine and DMF is 1:4) of 3 times the volume of resin, and blow nitrogen for 30 minutes, dry and wash (wash with 2 times the volume of resin DMF for 5 times), to obtain H2N-Linker-MBHA Resin.

[0299] (1-2) Take 3.0 mmol of amino acid monomer Fmoc-Ile-OH, 6.0 mmol of N, N-diisopropyl ethylamine (DIPEA, CAS number 7087-68-5), 2.85 mmol of benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, CAS number 94790-37-1), and an appropriate amount of solvent DMF to react for 30 minutes, dry and wash (wash with 2 times the volume of resin DMF for 3 times), to obtain Fmoc-Lys(Boc)-Liner-MBHA Resin; then, add 20% Pip / DMF mixed solution of 3 times the volume of resin, blow nitrogen for 30 minutes, dry and wash (wash with 2 times the volume of resin DMF for 5 times), to obtain H2N-Ile(Boc)-Liner-MBHA Resin;

[0300] Then, step (1-2) needs to be repeated every time an amino acid monomer is connected, so as to obtain the following amino acid sequence with a hydroxyl protecting group:

[0301] DPhe-Ser(tBu)-DHyp(tBu)-Cha-DAla-Gly-Hyp(tBu)-Ile-Asp(OtBu)-Arg(Me)(Pbf)-Ile-Liner-MBHA Resin;

[0302] DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(OtBu)-Arg(Pbf)--Ile-Liner-MBHA Resin;

[0303] DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(OtBu)-Arg(Pbf)--Ile-Liner-MBHA Resin;

[0304] DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(OtBu)-Arg(Me)(Pbf)-Ile-Liner-MBHA Resin.

[0305] (1-3) Take 3.0 mmol N3-PEG2-CH2CH2COOH (CAS No. 1312309-63-9), 6.0 mmol DIPEA, 2.85 mmol HBTU, and an appropriate amount of solvent DMF, and react for 30 minutes. Dry and wash (methyl alcohol washing 3 times), and obtain the following compounds, respectively:

[0306] N3-PEG2-CH2CH2CO-DPhe-Ser(tBu)-DHyp(tBu)-Cha-DAla-Gly-Hyp(tBu)-Ile-Asp(EDANS)-Arg(Me)(Pbf)-Ile-Liner-MBHA Resin;

[0307] N3-PEG2-CH2CH2CO-DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(EDANS)-Arg(Pbf)-Ile-Liner-MBHA Resin;

[0308] N3-PEG2-CH2CH2CO-DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(EDANS)-Arg(Pbf)-Ile-Liner-MBHA Resin;

[0309] N3-PEG2-CH2CH2CO-DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(EDANS)-Arg(Me)(Pbf)-Ile-Liner-MBHA Resin.

[0310] (1-4) Cleavage: 6 times the volume of resin of cleavage solution (cleavage solution is prepared by trifluoroacetic acid, anisole, 1,2-ethanedithiol, phenol and water with a volume of 87.5:5:2.5:2.5:2.5), shaking bed shaking for 2 hours, filtering off the resin, precipitating the filtrate with anhydrous ether, and washing the precipitate with anhydrous ether 3 times, finally putting the precipitate in a vacuum drying oven, drying at room temperature for 24 hours, HPLC purification, and obtaining the targeted delivery ligand with a purity of more than 95%, respectively.

[0311] LD106: N3-PEG2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)- Ile-CONHCH3

[0312] LD110: N3-PEG2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile- CONH2

[0313] LD117: N3-PEG2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile- CONHCH3

[0314] LD118: N3-PEG2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg(Me)- Ile-CONHCH3

[0315] Preparation 2: Synthesis of compound NM041

[0316] In this preparation example, the synthesis process of compound NM041 is as shown below:

[0317] (2-1) Synthesis of compound NM041-2

[0318] Compound NM041-1 (25.0 g, 1.0 eq, 1,3,4,6-tetraacetyloxy-alpha-D-glucopyranose, CAS No. 4292-12-0) was dissolved in 250 ml of toluene, warmed to 110 °C, and tributyltin hydride (17.7 g, 1.0 eq, CAS No. 688-73-3) and azobisisobutyronitrile (1 g, 0.1 eq, CAS No. 78-67-1) were added, and refluxed at 110 °C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25 °C, ethyl acetate (100 ml) and potassium fluoride (10.59 g in 30 ml of water, 3.0 eq) were added, stirred at 25 °C for 2 h, filtered and the organic phase was separated, the aqueous phase was washed with ethyl acetate twice (50 ml x 2), the organic phases were combined, 50 ml of saturated sodium chloride aqueous solution was added to the organic phase and washed once, dried over anhydrous sodium sulfate and suction filtered, and concentrated under reduced pressure to obtain compound NM041-2 (18.7 g, yield 87.9%) as a light yellow oil. MS ESI (m / z) = 333.1 [M+H] + .

[0319] (2-2) Synthesis of compound NM041-3

[0320] Compound NM041-2 (18.7 g, 1 eq) was dissolved in 50 ml of anhydrous methanol, and sodium methoxide (0.288 g, 0.1 eq) was added, and stirred at 25°C for 1 hour. After the reaction was completed, the pH of the reaction solution was adjusted to 6-7 with 4 mol / L hydrogen chloride solution in 1,4 dioxane in an ice bath, concentrated, and then washed with acetonitrile twice, and dried in vacuum to obtain compound NM041-3 (9.4 g, yield 100%) as a white solid.

[0321] (2-3) Synthesis of compound NM041-4

[0322] Compound NM041-3 (9.4 g, 1.0 eq) was dissolved in 150 ml of acetonitrile, and benzaldehyde dimethyl acetal (30 ml, 3 eq) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq) were added, and stirred at 25°C for 5 hours, and then 3 ml of triethylamine was added, and stirred at 25°C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml of water was added to the reaction solution, and extracted with ethyl acetate twice (100 ml x 2), and the organic phase was combined, dried with anhydrous sodium sulfate, and filtered under suction, and concentrated under reduced pressure, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 57 / 43, v / v) to obtain compound NM041-4 (8.6 g, yield 59.5%) as a white solid. MS ESI (m / z) = 252.2 [M+H] + .

[0323] (2-4) Synthesis of compound NM041-5

[0324] Compound NM041-4 (4.3 g, 1.0 eq) was dissolved in 40 ml of N,N-dimethylformamide (DMF), and sodium hydride (2.7 g, 68 mmol, 4 eq) was added under ice bath, and reacted for 30 minutes under ice bath, and then 3-bromopropynyl (8.1 g, 4 eq) was added, and stirred at 25°C for 2 hours, and then quenched by adding 20 ml of water. After the reaction was completed, the reaction solution was extracted with ethyl acetate three times (50 ml x 3), and the organic phase was combined, and washed with saturated sodium chloride solution five times (20 ml x 5), and the organic phase was dried with anhydrous sodium sulfate and filtered, and concentrated to obtain compound NM041-5 (yield 100%) as a brown oil. MS ESI (m / z) = 329.2 [M+H] + .

[0325] (2-5) Synthesis of compound NM041-6

[0326] Compound NM041-5 (5.6 g, 1.0 eq) was dissolved in 30 ml of dichloromethane (DCM), 300 ml of 70 mass% aqueous acetic acid solution was added, and the reaction was carried out at 70°C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to obtain compound NM041-6 (yield 100%) as a yellow oil. MS ESI (m / z) = 241 [M+H] + .

[0327] (2-6) Synthesis of compound NM041-7

[0328] Compound NM041-6 (5.6 g, 1.0 eq) was dissolved in 50 ml of pyridine, 4,4'-dimethoxytrityl chloride (10.2 g, 1.3 eq, abbreviated as DMTrCl, CAS No. 40615-36-9) was added under ice bath, nitrogen was replaced for 3 times, and stirred at 25°C for 3 hours, and then 50 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, extracted with ethyl acetate for 3 times (50 ml x 3), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain compound NM041-7 (5 g, yield 39.6%) as a light yellow solid. MS ESI (m / z) = 543.1 [M+H]+.

[0329] (2-7) Synthesis of compound NM041

[0330] Compound NM041-7 (2.0 g, 1.5 eq) was dissolved in 20 ml of anhydrous dichloromethane, DCI (347.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy) phosphine (1.22 g, 1.1 eq, CAS No. 102691-36-1) were added respectively, nitrogen was replaced for 3 times, and stirred at 25°C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate solution was added to the reaction solution, extracted with dichloromethane for 3 times (20 ml x 3), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM041 (2 g, yield 73.09%) as a white powder. MS ESI (m / z) = 743.2 [M+H]+.

[0331] 1H NMR (400 MHz, Acetonitrile-d3) δ 7.53 - 7.47 (m, 2H), 7.36 (tt, J = 9.6, 3.4 Hz, 6H), 7.28 - 7.22 (m, 1H), 6.94 - 6.86 (m, 4H), 4.45 (d, J = 2.4 Hz, 1H), 4.35 (dt, J = 6.1, 1.9 Hz, 2H), 4.18 (ddd, J = 11.2, 5.4, 2.6 Hz, 1H), 3.81 (s, 7H), 3.79 - 3.20 (m, 8H), 3.07 (dt, J = 10.5, 7.0 Hz, 1H), 2.79 (q, J = 2.6 Hz, 1H), 2.72 (dt, J = 5.1, 2.4 Hz, 1H), 2.69 - 2.63 (m, 1H), 2.48 - 2.34 (m, 1H), 2.17 (d, J = 1.0 Hz, 2H), 1.08 (dd, J = 6.8, 4.1 Hz, 10H), 0.90 (d, J = 6.8 Hz, 2H).

[0332] Preparation Example 3: Synthesis of compound NM064

[0333] In this preparation example, the synthesis process of compound NM064 is as follows:

[0334] (3-1) Synthesis of compound NM064-2

[0335] Compound NM064-1 (13 g, 1.0 eq, methyl B-D-glucopyranoside, CAS No. 709-50-2) was dissolved in 150 ml of acetonitrile, benzaldehyde dimethyl acetal (30 ml, 3.0 eq, CAS No. 1125-88-8) and DL-10-camphorsulfonic acid (1.5 g, 0.1 eq, CAS No. 5872-08-2) were added respectively, stirred at 25 °C for 5 hours, 3 ml of triethylamine was added, and stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated, 100 ml of water was added, extracted with ethyl acetate twice (100 ml x 2), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, and purified by column normal phase purification (eluent: ethyl acetate / petroleum ether = 57 / 43, volume ratio v / v) to obtain compound NM064-2 (11.5 g, yield 60.8%) in the form of white solid. MS ESI (m / z) = 283 [M+H] + .

[0336] (3-2) Synthesis of compound NM064-3

[0337] Compound NM064-2 (5 g, 1.0 eq) was dissolved in 40 ml of DMF, and sodium hydride (2.7 g, 4 eq) was added under ice bath. The reaction was carried out for 30 minutes under ice bath, and 3-bromopropyne (8.1 g, 4 eq) was added. The reaction was stirred at 25°C for 2 hours, and 20 ml of water was added for quenching. After the reaction was completed, the reaction solution was extracted with 50 ml of ethyl acetate three times (50 ml x 3), and the combined organic phase was washed with 20 ml of saturated sodium chloride aqueous solution five times (20 ml x 5). The organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated to obtain brown oil of NM064-3 (6.3 g, yield 100%). MS ESI (m / z) = 359 [M+H] + .

[0338] (3-3) Synthesis of compound NM064-4

[0339] Compound NM064-3 (6.3 g, 1.0 eq) was dissolved in 30 ml of DCM, and 300 ml of 70 mass% acetic acid aqueous solution was added. The reaction was carried out at 70°C for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to obtain yellow oil of compound NM064-4 (4.78 g, yield 100%). MS ESI (m / z) = 271 [M+H] + .

[0340] (3-4) Synthesis of compound NM064-5

[0341] Compound NM064-4 (4.78 g, 1.0 eq) was dissolved in 50 ml of pyridine, and DMTrCl (7.8 g, 1.3 eq) was added under ice bath. Nitrogen was replaced three times, and the reaction was stirred at 25°C for 3 hours. The reaction was quenched with 50 ml of methanol. After the reaction was completed, the reaction solution was concentrated, 50 ml of water was added, and the mixture was extracted with 50 ml of ethyl acetate three times (50 ml x 3). The combined organic phase was dried over anhydrous sodium sulfate and filtered, and concentrated. The product was purified by column chromatography (normal phase) (eluent: ethyl acetate / petroleum ether = 16 / 84, v / v) to obtain yellowish solid of compound NM064-5 (6.7 g, yield 66.3%). MS ESI (m / z) = 573 [M+H] + .

[0342] (3-5) Synthesis of compound NM064

[0343] Compound NM064-5 (2.0 g, 1.0 eq) was dissolved in 20 ml of anhydrous dichloromethane, 4,5-dicyanoimidazole (330.4 mg, 0.8 eq, abbreviated as DCI, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.16 g, 1.1 eq, CAS No. 102691-36-1) were added respectively, replaced with nitrogen for 3 times, stirred at 25 °C for 2 hours. After the reaction was completed, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, extracted with 20 ml of dichloromethane for 3 times (20 ml x 3), the organic phase was combined, dried with anhydrous sodium sulfate and filtered, concentrated, purified by column chromatography (C18 column, eluent: acetonitrile / water = 72 / 28, v / v), vacuum dried for 12 hours to obtain compound NM064 (2 g, yield 74.07%) in white powder. MS ESI (m / z) = 774 [M+H] + .

[0344] 1 H NMR (400 MHz, DMSO-d6) δ 7.45 - 7.39 (d, J = 7.8 Hz, 2H), 7.36 - 7.18 (tt, J = 14.5, 8.5 Hz, 7H), 6.95 - 6.84 (d, J = 7.5 Hz, 4H), 5.01 - 4.96 (s, 1H), 4.43 - 4.28 (s, 4H), 3.81 - 3.70 (s, 8H), 3.65 - 3.35 (m, 12H), 3.28 - 3.18 (dt, J = 14.5, 7.2 Hz, 1H), 3.06 - 2.97 (t, J = 9.5 Hz, 1H), 2.75 - 2.68 (m, 1H), 1.07 - 0.95 (q, J = 7.4, 6.8 Hz, 10H), 0.84 - 0.78 (d, J = 6.6 Hz, 2H).

[0345] Preparation Example 4: Synthesis of compound NM014 and compound NM014A

[0346] In this preparation example, the synthesis process of compound NM014 and compound NM014A is as shown below:

[0347] (4-1) Synthesis of compound NM014-2

[0348] Compound NM014-1 (50 g, 1.0 eq.) was added into 100 ml acetonitrile (ACN), replaced with nitrogen, the temperature was reduced to 0 °C, then trifluoromethylsilicane trimethylsilicate (44.0 g, 2.0 eq., TMSOTf, CAS No. 27607-77-8) was added dropwise, followed by triethylsilane (34.5 g, 3.0 eq., CAS No. 617-86-7), the temperature was slowly increased to 25 °C, and the reaction was carried out at 25 °C for 12 hours. After the reaction was completed, 200 ml purified water was added to the reaction solution, and 200 ml ethyl acetate was used for extraction twice, the organic phase was combined, dried with anhydrous sodium sulfate, and filtered, concentrated, and purified by column chromatography (eluent: n-heptane / ethyl acetate = 4 / 1, v / v) to obtain compound NM014-2 (41 g, yield 93.1%). ESI-MS (m / z) = 445.3 [M+H] + .

[0349] (4-2) Synthesis of compound NM014-3

[0350] Compound NM014-2 (41 g, 1.0 eq.) was added into 410 ml monomethylamine methanol, and the reaction was carried out at 25 °C for 12 hours. After the reaction was completed, the reaction solution was directly concentrated, and purified by column chromatography (eluent: dichloromethane / methanol = 10 / 1 was used first to remove impurities, and then dichloromethane / methanol = 5 / 1 was used for elution, v / v) to obtain compound NM014-3 (7.5 g, yield 60.6%). ESI-MS (m / z) = 135.2 [M+H] + .

[0351] (4-3) Synthesis of compound NM014-4

[0352] Compound NM014-3 (5.16 g, 1.0 eq.) was added into 30 ml pyridine, and the temperature was reduced to 0 °C, then DMTrCl (14.32 g, 1.1 eq.) was added in batches at 0 °C, and the reaction was carried out at 25 °C for 6 hours, and 10 ml methanol was added for quenching. After the reaction was completed, the reaction solution was directly concentrated, diluted with 50 ml ethyl acetate, washed with 30 ml saturated sodium bicarbonate once and 30 ml purified water twice, the organic phase was separated, and the organic phase was concentrated and purified by column chromatography (gradient elution of n-heptane / ethyl acetate = 5 / 1 to n-heptane / ethyl acetate = 1 / 1, v / v) to obtain compound NM014-4 (9.0 g, yield 53.6%). ESI-MS (m / z) = 437.3 [M+H] + .

[0353] (4-4) Synthesis of compound NM014-5 and compound NM014-5A

[0354] Compound NM014-4 (9.0 g, 1.0 eq.) was dissolved in 90 ml of DMF, cesium carbonate (7.4 g, 1.1 eq.) was added, and stirring was performed at 25 °C for 10 min. The temperature was lowered to 0 °C, bromopropargyl (2.5 g, 1.0 eq.) was added dropwise at 0 °C, and the temperature was slowly raised to 25 °C after the dropwise addition was completed. The reaction was performed at 25 °C for 16 h. After the reaction was completed, 100 ml of ethyl acetate was added to the reaction solution, washing was performed with 10 ml of purified water three times (10 ml x 3), the organic phase was separated, anhydrous sodium sulfate was added to the organic phase, and drying and filtration were performed. The organic phase was concentrated, and column chromatography normal phase purification (eluent: n-heptane / ethyl acetate = 5 / 1, v / v, impurities were removed first, and then the polarity was maintained) was performed, to obtain a front peak compound NM014-5 (2.7 g, yield 27.6%) and a rear peak compound NM014-5A (2.3 g, yield 23.7%). Among them, the ESI-MS (m / z) of the front peak product compound NM014-5 was 475.4 [M+H]+, and the ESI-MS (m / z) of the rear peak compound NM014-5A was 475.4 [M+H] + .

[0355] Synthesis of compound NM014

[0356] The front peak compound NM014-5 (2.57 g, 1.0 eq.) was added to 26 ml of dichloromethane, stirring was continuously performed, DCI (0.83 g, 1.3 eq.) was added, nitrogen replacement was performed, at this time, the reaction system was white turbidity, the temperature was lowered to 0 °C, bis(diisopropylamino)(2-cyanoethoxy) phosphine (2.44 g, 1.5 eq.) was added dropwise at 0 °C, the temperature was slowly raised to 25 °C after the dropwise addition was completed, and stirring was performed at 25 °C for 3 h. After the reaction was completed, the reaction system was diluted with 50 ml of dichloromethane, the organic phase was washed with 20 ml of saturated sodium bicarbonate once, anhydrous sodium sulfate was added to the organic phase, and drying and filtration were performed. The organic phase was concentrated, and column chromatography normal phase purification (eluent: n-heptane / 0.1 volume% triethylamine ethyl acetate solution = 4 / 1, v / v) was performed, to obtain compound NM014 (907 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0357] 1H NMR (400 MHz, DMSO-d6) δ 0.88 - 0.94 (d, J = 6.8 Hz, 4H), 1.05 - 1.14 (m, 8H), 1.11 - 1.22 (m, 1H), 1.18 - 1.26 (m, 1H), 2.52 - 2.60 (t, J = 5.9 Hz, 1H), 2.72 - 2.80 (m, 1H), 2.89 - 3.00 (td, J = 5.1, 10.7 Hz, 1H), 3.15 - 3.28 (ddd, J = 2.8, 10.2, 23.1 Hz, 1H), 3.41 - 3.59 (m, 4H), 3.71 - 3.78 (d, J = 2.0 Hz, 6H), 3.75 - 3.84 (m, 1H), 3.88 - 3.99 (dq, J = 2.7, 18.2 Hz, 1H), 3.99 - 4.07 (m, 1H), 4.14 - 4.21 (m, 1H), 4.18 - 4.30 (m, 1H), 4.27 - 4.38 (m, 1H), 6.84 - 6.93 (m, 4H), 7.17 - 7.35 (m, 7H), 7.36 - 7.44 (ddd, J = 1.3, 4.0, 8.2 Hz, 2H).

[0358] Synthesis of compound NM014A

[0359] The latter peak compound NM014-5A (2.3 g, 1.0 eq.) was added to 23 ml of dichloromethane, continuously stirred, DCI (0.74 g, 1.3 eq.) was added, and nitrogen was replaced. At this time, the reaction system was white turbidity, and the temperature was lowered to 0°C. Bis (diisopropylamino) (2-cyanoethoxy) phosphine (2.19 g, 1.5 eq.) was added dropwise at 0°C. After the dropwise addition was completed, the temperature was slowly increased to 25°C, and stirred at 25°C for 3 hours. After the reaction was completed, the reaction system was diluted with 50 ml of dichloromethane, and the organic phase was washed once with 20 ml of saturated sodium bicarbonate, dried over anhydrous sodium sulfate and filtered. The organic phase was concentrated and purified by column chromatography (eluent: n-heptane / 0.1 volume% triethylamine in ethyl acetate solution = 4 / 1, v / v) to obtain compound NM014A (520 mg). ESI-MS (m / z) = 698.2 [M+Na] + .

[0360] 1H NMR (400 MHz, DMSO-d6) δ 1.10 - 1.20 (dd, J = 6.7, 8.6 Hz, 12H), 2.74 - 2.82 (m, 2H), 2.96 - 3.04 (dd, J = 4.4, 10.2 Hz, 1H), 3.11 - 3.19 (dd, J = 3.3, 10.2 Hz, 1H), 3.38 - 3.43 (q, J = 2.3 Hz, 1H), 3.55 - 3.66 (m, 2H), 3.71 - 3.75 (s, 6H), 3.75 - 3.85 (m, 3H), 3.85 - 3.94 (m, 1H), 4.01 - 4.07 (dd, J = 4.4, 9.4 Hz, 1H), 4.09 - 4.20 (m, 2H), 4.22 - 4.31 (m, 1H), 4.43 - 4.52 (dq, J = 4.1, 12.0 Hz, 1H), 6.86 - 6.93 (m, 4H), 7.19 - 7.35 (m, 7H), 7.38 - 7.43 (m, 2H).

[0361] Preparation Example 5: Preparation of compound NM119

[0362] In this preparation example, the synthetic route of compound NM119 is as shown below:

[0363] (5-1) Synthesis of compound NM119-2

[0364] Compound NM119-1 (5.5 g, 28.1 mmol, 1.0 eq, CAS No. 2408968-41-0), saturated aqueous sodium bicarbonate solution (15 ml) were added to a solution of 1,4-dioxane (50 ml), and a solution of chloroformic acid-9-fluorenylmethyl ester (7.9 g, 42.1 mmol, 1.5 eq, English name Fmoc-Cl, CAS No. 28920-43-6) in 1,4-dioxane (10 ml) was added dropwise under ice bath. After the dropwise addition was completed, the reaction was stirred at 25°C for 12 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, 50 ml of ethyl acetate and 30 ml of water were added for extraction, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and suction filtered, and purified by column chromatography (normal phase, eluent: methanol / dichloromethane = 6 / 94, v / v) to obtain compound NM119-2 (6.7 g, yield 65%) as a light yellow solid. The compound was used directly in the next reaction without purification. MS ESI (m / z) = 382 [M+H] + .

[0365] (5-2) Synthesis of compound NM119-3

[0366] Compound NM119-2 (6.7 g, 17.5 mmol, 1.0 eq) was dissolved in 40 ml of pyridine, and DMTrCl (6.5 g, 26.25 mmol, 1.5 eq) was added under ice bath, and the reaction was carried out at 25 °C for 12 hours, and 20 ml of methanol was added for quenching. After the reaction was completed, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate three times (20 ml each time), and purified by column chromatography (normal phase) (eluent: ethyl acetate / petroleum ether = 28 / 72, v / v) to obtain compound NM119-3 (7.6 g, yield 65%) as a light yellow solid. MS ESI (m / z) = 683 [M+H] + .

[0367] Synthesis of compound NM119-4

[0368] Compound NM119-3 (3.7 g, 5.42 mmol, 1.0 eq) was dissolved in 15 ml of acetonitrile, and 10 ml of tetrahydropyrrole was added, and stirred at 25 °C for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and purified by column chromatography (normal phase) (eluent: methanol / dichloromethane containing 1% ammonia = 5 / 95, v / v) to obtain compound NM119-4 (1.8 g, yield 90%) as a white solid. MS ESI (m / z) = 462 [M+H] + .

[0369] Synthesis of compound NM119-6

[0370] Compound NM119-4 (2.25 g, 15.2 mmol, 1.0 eq), compound NM119-5 (1.5 g, 15.3 mmol, 1.0 eq), HATU (2.38 g, 22.95 mmol, 1.3 eq), and N,N-diisopropylethylamine (1.95 g, 22.95 mmol, 3.0 eq, abbreviated as DIEA, CAS No. 7087-68-5) were dissolved in 15 ml of DMF, and stirred at room temperature for 2-3 hours. After the reaction was completed, 100 ml of water was added to the reaction solution, extracted with ethyl acetate twice (100 ml each time), and the organic phase was combined, dried, and purified by reverse phase (eluent: acetonitrile / water = 60 / 40, v / v) to obtain compound NM119-6 (2 g, yield 62.5%) as a white solid. MS ESI (m / z) = 771.3 [M+Na] + .

[0371] Synthesis of compound NM119

[0372] Compound NM119-6 (2.0 g, 2.68 mmol, 1 eq) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (250 mg, 2.11 mmol, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (900 mg, 2.98 mmol, 1.1 eq) were added respectively, replaced with nitrogen for 3 times, stirred at room temperature for 2 hours, added 20 ml of saturated aqueous sodium bicarbonate solution, extracted with dichloromethane for 3 times (20 ml each time), separated the organic phase, and the organic phase was dried and concentrated, and purified by reversed phase (eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound NM119 (1.53 g, yield 61.2%) in white powder. MS ESI (m / z) = 949.5 [M+H] + .

[0373] 1 H NMR (400 MHz, DMSO-d6) δ 7.75 - 7.67 (m, 1H), 7.67 - 7.61 (d, J = 7.4 Hz, 1H), 7.61 - 7.55 (d, J = 7.7 Hz, 1H), 7.55 - 7.43 (dtt, J = 13.0, 9.2, 4.8 Hz, 3H), 7.43 - 7.18 (m, 12H), 6.98 - 6.83 (d, J = 8.4 Hz, 4H), 5.10 - 5.01 (d, J = 14.0 Hz, 1H), 3.80 - 3.71 (s, 6H), 3.71 - 3.58 (td, J = 10.1, 9.3, 4.9 Hz, 5H), 3.57 - 3.45 (dt, J = 10.0, 6.6 Hz, 2H), 2.90 - 2.82 (dd, J = 8.5, 3.2 Hz, 1H), 2.82 - 2.74 (dd, J = 8.7, 3.7 Hz, 1H), 2.74 - 2.67 (t, J = 5.9 Hz, 2H), 2.28 - 2.14 (dd, J = 15.0, 7.5 Hz, 1H), 2.08 - 1.94 (dt, J = 14.8, 6.7 Hz, 1H), 1.86 - 1.72 (dt, J = 14.8, 6.9 Hz, 1H), 1.65 - 1.29 (t, J = 47.3 Hz, 5H), 1.29 - 1.17 (dd, J = 21.2, 9.4 Hz, 3H), 1.17 - 1.10 (d, J = 6.8 Hz, 7H), 1.10 - 0.98 (d, J = 6.7 Hz, 6H).

[0374] Preparation Example 6: Preparation of compound LK005

[0375] In this preparation example, the synthesis route of compound LK005 is as shown below:

[0376] Synthesis of compound LK005-3

[0377] Compound LK005-1 (10 g, 69.9 mmol, 2-[2-(propargyloxy)ethoxy]ethanamine, CAS No. 944561-44-8) was dissolved in DMF (100 mL), compound LK005-2 (14.19 g, 69.9 mmol, BOC-4-aminobutyric acid, CAS No. 57294-38-9), benzotriazol-1-yl-oxy- tris-(dimethylamino)-phosphonium hexafluorophosphate (66.4 g, 174.75 mmol, HATU for short, CAS No. 148893-10-1), N,N-diisopropylethylamine (27.1 g, 209.7 mmol, DIEA for short, CAS No. 7087-68-5) were added successively, and stirred at 25 °C for 6 hours. After the reaction was completed, the reaction solution was diluted with ethyl acetate (400 mL), washed with saturated aqueous sodium chloride solution (5 x 150 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound LK005-3 (20.5 g, yield 87.3%) as a light yellow oil. MS ESI (m / z) = 329.27 [M+H] + .

[0378] Synthesis of compound LK005-4

[0379] Compound LK005-3 (20 g, 60.9 mmol) was dissolved in 4 M hydrogen chloride in dioxane (200 mL), and stirred at 25 °C for 2 hours. After the reaction was completed, the reaction solution was concentrated to obtain crude compound LK005-4 (15.4 g), which was used directly in the next reaction without purification. MS ESI (m / z) = 229.23 [M+H] + .

[0380] Synthesis of compound LK005-5

[0381] Compound LK005-4 (10 g, 37.9 mmol) was dissolved in DMF (100 mL), and pentane-1,5-dioic acid monomethyl ester (6.09 g, 41.69 mmol, CAS No. 1501-27-5), HATU (36.4 g, 94.75 mmol), DIEA (24.49 g, 189.5 mmol) were added successively, and stirred at 25 °C for 6 h. After the reaction was completed, the reaction solution was diluted with ethyl acetate (300 mL), washed with saturated aqueous sodium chloride solution (5 x 100 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 3 / 1, v / v) to obtain compound LK005-5 (10.5 g, yield 77.9%) as a light yellow oil. MS ESI (m / z) = 357.27 [M+H] + .

[0382] Synthesis of compound LK005-6

[0383] Compound LK005-5 (10 g, 28.1 mmol) was dissolved in tetrahydrofuran (30 mL), and 1M sodium hydroxide solution in tetrahydrofuran (28.1 mL, 28.1 mmol) was added, and stirred at 25 °C for 1 h. After the reaction was completed, the reaction solution was adjusted to pH 2-3 with 1N hydrochloric acid, and concentrated to obtain crude compound LK005-6, which was used in the next reaction without purification. MS ESI (m / z) = 343.17 [M+H] + .

[0384] Synthesis of compound LK005

[0385] Compound LK005-6 (5 g, 14.58 mmol) was dissolved in DMF (50 mL), and p-nitrophenol (4.05 g, 29.16 mmol, CAS No. 100-02-7), N,N'-dicyclohexylcarbodiimide (6.02 g, 29.16 mmol, abbreviated as DCC, CAS No. 538-75-0) were added successively, and stirred at 25 °C for 12 h. After the reaction was completed, the reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium chloride solution (5 x 50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1, v / v) to obtain compound LK005 (3.1 g, yield 44.3%) as a yellow solid. MS ESI (m / z) = 464.12 [M+H] + .

[0386] Preparation Example 7: Preparation of siRNA conjugate

[0387] (7-1) Synthesis of siRNA

[0388] (7-1-1) The sense strand (SS) or antisense strand (AS) of the siRNA to be synthesized is introduced into an ABI 394 synthesizer, and nucleotide monomers are sequentially linked in the direction of 3'-5' according to the nucleotide sequence using a universal CPG / PS as a carrier by the method of phosphoramidite nucleic acid solid-phase synthesis. Each linkage of a nucleotide monomer includes four steps of deprotection, coupling, capping, oxidation, or sulfuration. The synthesis conditions are given below.

[0389] The nucleotide monomers are prepared into an acetonitrile solution of the nucleotide monomers at a concentration of 0.1 M.

[0390] The conditions for deprotection in each step are the same. The conditions for deprotection are a temperature of 25°C, a reaction time of 70 seconds, a deprotection reagent of dichloroacetic acid in dichloromethane (3% by volume), and a molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid-phase carrier of 5:1.

[0391] The conditions for coupling in each step are the same. The conditions for coupling are a temperature of 25°C, a molar ratio of the nucleic acid sequence linked to the solid-phase carrier to the nucleotide monomer of 1:10, a molar ratio of the nucleic acid sequence linked to the solid-phase carrier to the coupling reagent of 1:65, a reaction time of 600 seconds, a coupling reagent of 5-ethylthio-1H-tetrazole in acetonitrile at a concentration of 0.5 M, and a thio reagent of a hydrogenated xanthine in acetonitrile / pyridine mixed solution (acetonitrile and pyridine at a volume ratio of 1:1) at a concentration of 0.2 mol / L.

[0392] The conditions for capping in each step are the same. The conditions for capping are a temperature of 25°C, a reaction time of 2 minutes, a capping reagent solution of a mixture of Cap1 and Cap2 at a molar ratio of 1:1, Cap1 being N-methylimidazole in pyridine / acetonitrile mixed solution at a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile being 3:5, and Cap2 being acetic anhydride in acetonitrile solution at a concentration of 20% by volume, and a molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence linked to the solid-phase carrier of 1:1:1.

[0393] The conditions for each oxidation reaction are the same. The conditions for the oxidation reaction are: temperature is 25°C; reaction time is 3 seconds; concentration of the oxidizing reagent is 0.05 M iodine water, molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9). The conditions for the sulfurization reaction are: temperature is 25°C; reaction time is 360 seconds; concentration of the sulfurizing reagent is 0.2 M pyridine solution of hydrogen xanthate, molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9).

[0394] After the last nucleoside monomer is attached, the nucleic acid sequence attached to the solid support is subjected to cleavage, deprotection, purification, desalting, and then lyophilization to obtain the sense strand or the antisense strand, wherein:

[0395] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence attached to the solid support is added to 25% ammonia water by mass, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and vacuum concentration is carried out to dryness. After ammonia water treatment, 0.4 ml / μmol of N-methylpyrrolidine is used to dissolve the product relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trifluoromethanesulfonate to remove the 2'-O-TBDMS protection on the ribose.

[0396] The purification and desalting conditions are as follows: the nucleic acid is purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 is 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (volume ratio of water to acetonitrile is 9:1); eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (volume ratio of water to acetonitrile is 9:1); the elution gradient is eluent 1:eluent 2=(100:0)-(50:50). After the product eluate is collected, desalting is carried out using a reverse phase chromatography purification column, and the desalting conditions include desalting using a dextran gel column, the filler is dextran gel G25, and elution is carried out using deionized water.

[0397] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using liquid chromatography-mass spectrometry (LC-MS), and the measured value of the molecular weight is compared with the theoretical value; if the measured value and the theoretical value are consistent, it is indicated that the sense strand and the antisense strand of the siRNA are obtained.

[0398] (7-1-2) Mixing the SS conjugate synthesized in step (7-2) and the AS synthesized in step (7-1) in an equimolar ratio, dissolving in water for injection and heating to 95°C, slowly cooling to room temperature and keeping at room temperature for 10 minutes, allowing the sense strand and the antisense strand to form a double-stranded structure by hydrogen bonding, thereby obtaining siRNA having a sense strand and an antisense strand shown in Table 4.

[0399] In the synthesis of siRNA, the compound NM041, the compound NM064, 5'-AMINO-MODIFIER C6-TFA phosphoramidite monomer (CAS No. 133975-85-6) are respectively regarded as one nucleotide monomer to participate in the synthesis of siRNA, and form (NM041), (NM064) and (NH2-C6) respectively.

[0400] wherein (NM041) represents one nucleotide, and the structural formula of (NM041) is: If (NM041) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM041) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0401] wherein (NM041)(NM041) represents two consecutive nucleotides, and the structural formula of (NM041)(NM041) is If (NM041)(NM041) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM041)(NM041) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0402] (NM064) represents one nucleotide, and the structural formula of (NM064) is: If (NM064) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NM064) is conjugated to the 3' end of the sense strand of siRNA, the structural formula of siRNA is:

[0403] (NM064)(NM064) represents two consecutive nucleotides, and the structural formula of (NM064)(NM064) is: If (NM064)(NM064) is conjugated to the 5' end of the sense strand of siRNA, the structural formula of siRNA is: If (NH2-C6) is conjugated to the 3' end of the sense strand of the siRNA, the structure of the siRNA is

[0404] If (NH2-C6) is conjugated to the 5' end of the sense strand of the siRNA, the structure of the siRNA is If (NH2-C6) is conjugated to the 3' end of the sense strand of the siRNA, the structure of the siRNA is If (NH2-C6) is conjugated to the 3' end of the sense strand of the siRNA, the structure of the siRNA is

[0405] Synthesis of siRNA conjugate (7-2)

[0406] (7-2-1) Mix 150 μL of H2O, 70 μL of 0.2M carbonate buffer solution (pH = 9.2) and 70 μL of N,N-dimethylformamide (DMF) to obtain a mixed solvent; dissolve siRNA in the mixed solvent to obtain a siRNA solution with a concentration of 1.0 eq.

[0407] (7-2-2) Dissolve 5.0 eq of the targeting delivery ligand in 70 μL of DMF to obtain a targeting delivery ligand solution;

[0408] (7-2-3) Mix the siRNA solution obtained in step (7-2-1) and the targeting delivery ligand solution obtained in step (7-2-2) to obtain a reactant mixture; wherein the molar ratio of siRNA to targeting delivery ligand is 1:6.

[0409] (7-2-4) Mix 30.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (CAS No. 760952-88-3, abbreviated as THPTA) and 6.0 eq of CuSO4·5H2O in a volume ratio of THPTA:CuSO4·5H2O = 5:1, shake for 5 min at 40°C, then take 37 μL and add to the reactant mixture obtained in step (7-2-3), and vortex to obtain an intermediate product mixture, which has a pH of 8; then, take 25.0 eq of sodium ascorbate and quickly add to the intermediate product mixture, and vortex to treat, and react for 1 h at a temperature of 40°C to obtain a product mixture;

[0410] (7-2-5) The product mixture (3 μL) obtained in step (7-2-4) above was diluted with a mixed solution of DMF and H2O (volume ratio of DMF to H2O was 1:5), and then separated and purified by HPLC treatment. In the HPLC treatment, a C18 column was used as the chromatographic column, and a gradient elution method was used with an ammonium bicarbonate buffer solution as the mobile phase. The product after HPLC treatment was subjected to freeze-drying to obtain the siRNA conjugate.

[0411] For example, the siRNA conjugate RZ899144 has the following structural formula:

[0412] In the (NM041) at the 3' end of the sense strand of the siRNA, two alkynyl groups are covalently linked to two azido groups of two LD110 through a click chemistry reaction to form a triazole group, thereby achieving covalent linkage of the siRNA and the two targeting delivery ligands LD110.

[0413] The unmodified nucleotide sequence information for forming the siRNA conjugates shown in Table 4 is shown in Table 3.

[0414] Table 3 Unmodified nucleotide sequence information for forming siRNA conjugates

[0415] Table 4 Sequence information of siRNA conjugates

[0416] Unless otherwise specified, the base composition and modification meanings described in the embodiments of the present disclosure are as follows: capital letters A, U, G, C, and T represent the base composition of nucleotides, lowercase letters m represent that the nucleotide represented by the capital letter on the left side is a 2'-O-methyl-modified nucleotide, lowercase letters f represent that the nucleotide represented by the capital letter on the left side is a 2'-fluoro-modified nucleotide, (moe) represents that the nucleotide represented by the capital letter on the left side is a 2'-O-methoxyethyl-modified nucleotide, and lowercase letters s represent that the two nucleotides on the left and right sides are connected by a phosphorothioate bond. VP represents that the 5' end of the antisense strand of the siRNA is modified as a 5'-(E)-vinylphosphonate (5'-(E)-VP) modification.

[0417] The structural formula of VPUm is

[0418] The structural formula of the 2'-O-methyl-modified nucleotide is

[0419] The structural formula of the 2'-fluoro-modified nucleotide is

[0420] 2'-O-methoxyethyl modified nucleotides have the structural formula

[0421] wherein Base represents nucleobase A, U, G, C, T.

[0422] Biological detection experiment

[0423] Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Beijing Xuanjing Rui Pharmaceutical Technology Co., Ltd.; the PCR primer synthesis is entrusted to Beijing Qikexing Biotechnology Co., Ltd.; and the experimental animals C57BL / 6J mice are purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0424] Method for evaluating the target gene inhibition activity of siRNA conjugates in mice in vivo

[0425] 6-8 week old C57BL / 6J mice were randomly divided into groups according to body weight (all female). The mice in each group were calculated according to the body weight. The drug dose was given by abdominal subcutaneous injection or tail vein injection, and each siRNA conjugate was prepared into a corresponding concentration (calculated by siRNA) solution with PBS solution for administration, and the administration volume was 5 mL (calculated by siRNA) / kg (calculated by mice). The PBS control group was given 5 mL / kg (calculated by mice) of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as DO), and at the preset time after administration, 5 mice were sacrificed in each group. The cadavers of the sacrificed mice were dissected and the target tissues of each sacrificed mouse were collected, and the tissues were cut into about 2 mm 3 pieces and stored in RNA later.

[0426] Detection of mRNA expression level

[0427] The tissue samples in different experimental groups were taken from the above RNA later, 1 mL Trizol solution was added, and the tissue samples were crushed in a Tissuelyser II type automatic tissue homogenizer for 120 s, and then centrifuged, and then placed at room temperature for 10 min. 200 μL of chloroform was added, and then the mixture was mixed and placed at room temperature for 3 min. Centrifugation was performed at 4°C and 12000 rpm for 10 min. 400 μL of supernatant was added to a centrifuge tube containing 400 μL of isopropanol, mixed, and placed at room temperature for 10 min. Centrifugation was performed at 4°C and 12000 rpm for 10 min, and the supernatant was discarded. 1 mL of 75% ethanol was added, and the centrifuge tube was inverted. Centrifugation was performed at 4°C and 12000 rpm for 5 min, and the supernatant was removed. The total RNA was extracted by drying at room temperature.

[0428] Take the above 1 μg total RNA, using reverse transcription kit (Promega Company, Reverse Transcription System, A3500) and select Oligo (dT) 15 reverse transcription primer, according to the method of reverse transcription kit instruction book record configuration 20 μL reverse transcription system and complete reverse transcription reaction. After the reaction, add 80 μL RNase-Free water to the reverse transcription system to obtain cDNA solution. Then use real-time fluorescence quantitative PCR kit (ABI Company, SYBR TM Select Master Mix, Catalog number: 4472908) to detect the expression of target gene mRNA in the tissue. In this real-time fluorescence quantitative PCR method, the primers for the target gene and the primers for the internal reference gene are used to detect the target gene and the internal reference gene, respectively. According to the method recorded in the real-time fluorescence quantitative PCR kit instruction book, 20 μL Real-time PCR reaction system is configured for each PCR detection hole, and each reaction system contains 5 μL cDNA solution obtained by the above reverse transcription reaction, 10 μL SYBR TM Select Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system is placed on a real-time fluorescence quantitative PCR instrument (ABI Company, StepOnePlus TM / 7500), and the Real-time PCR amplification is carried out using a three-step method, and the amplification program is 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, and the process of denaturation, annealing and extension is repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the ΔΔCt method is used to calculate the expression level and inhibition rate of the target gene mRNA in each test group, and the calculation method is as follows:

[0429] ΔCt (test group) = Ct (test group target gene) - Ct (test group internal reference gene)

[0430] ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene)

[0431] ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average)

[0432] ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0433] Wherein, ΔCt (control group average) is the arithmetic mean of the respective ΔCt (control group) of 5 mice in the control group at the same time point. Therefore, each sample of the test group and the control group corresponds to a ΔΔCt value.

[0434] Test group target gene mRNA relative expression level = 2 -ΔΔCt( Test group target gene mRNA relative expression level = 2

[0435] The test group target gene mRNA expression level is normalized based on the control group, and the control group target gene mRNA expression level is defined as 100%.

[0436] Test group target gene mRNA expression inhibition rate (%) = 1 - test group target gene mRNA relative expression level

[0437] Unless otherwise specified, the in vivo activity experiment data are expressed as The experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0438] Test Example 1: Evaluation of the inhibitory activity of LD110 conjugated siRNA sequence on target gene human urate anion transporter 1 (URAT1) in mice

[0439] This test example uses the target gene inhibition activity evaluation method in mice to evaluate the inhibitory activity of LD110 conjugated siRNA sequence RZ891010 and unconjugated siRNA control sequence RX891001 on target gene URAT1 in mice.

[0440] 6-8 week old C57BL / 6j mice were randomly divided into 3 groups according to body weight, 5 mice in each group, and each group of mice was given the above siRNA conjugate by subcutaneous injection in the abdomen. The volume of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the experimental group was 3 mg / kg (calculated as siRNA), and the volume was 5 mL / kg; continuous administration for 3 days. The day of administration is recorded as day 0 (D0), and 5 mice in each group are sacrificed on day 7 (D9) after the last administration. The animals were subjected to gross dissection, and the renal cortex and renal medulla were collected and cut into several 2 mm 3 pieces were preserved with RNAlater. RNA extraction, Real-time PCR detection was as described above, and gene expression difference was calculated by ΔΔCt method.

[0441] Table 5 Sequence list of primers used in test example 1

[0442] The LD110-conjugated siRNA sequence RZ891010 showed higher target gene inhibition activity in the renal cortex and medulla, and better inhibition effect in the renal cortex, compared with the siRNA control sequence RX891001 without conjugation.(Figure 1, Table 6)

[0443] Table 6 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this test example

[0444] Test Example 2: Evaluation of the inhibition activity of the LD110-conjugated siRNA sequence on the target gene URAT1 in mice

[0445] This test example evaluated the long-term inhibition activity of the LD110-conjugated siRNA sequence RZ891010 on the target gene URAT1 in mice using the method for evaluating the target gene inhibition activity in mice.

[0446] The 6-8 week old C57BL / 6j mice were randomly divided into 2 groups according to body weight, 15 mice in each group, and each group of mice was administered with the above siRNA conjugate by single subcutaneous administration in the abdomen. The administration volume of each mouse in the PBS control group was 5 mL / kg, and the administration dose of each mouse in the experimental group was 20 mg / kg (calculated based on siRNA), and the administration volume was 5 mL / kg. The day of administration was recorded as D0, and 5 mice from each group were sacrificed on D7, D14, and D28 after administration, and the animals were subjected to gross dissection, and the renal cortex and medulla were collected and cut into several 2 mm 3 pieces for preservation with RNAlater. RNA extraction, Real-time PCR detection was as described above, the primers were as shown in Table 5 of Test Example 1, and the gene expression difference was calculated by the ΔΔCt method.

[0447] The results of Test Example 2 showed that the LD110-conjugated siRNA sequence RZ891010 showed better target gene inhibition effect in the renal cortex and medulla on D7, D14, and D28, and reached the maximum inhibition effect on D14 after single administration, with inhibition rates of 69.57% and 72.45% in the renal cortex and medulla, respectively.(Figure 2, Table 7)

[0448] Table 7 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this test example

[0449] Test Example 3. Evaluation of the inhibition activity of the LD110-conjugated siRNA sequence on the target gene Superoxide dismutase 1 (SOD1) in mice

[0450] The test example uses the mouse in vivo target gene inhibition activity evaluation method to evaluate the inhibition activity of the LD110 conjugated siRNA sequence RZ899144, the LD106 conjugated siRNA sequence RZ899104, and the non-carrier conjugated siRNA control sequence RZ899056 on the target target gene SOD1 in mice.

[0451] The 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, 5 mice in each group, and each group of mice was given the above siRNA conjugate by abdominal subcutaneous administration. The volume of each mouse in the PBS control group was 5 mL / kg, and the dose of each mouse in the experimental group was 3 mg / kg (calculated by siRNA), and the volume was 5 mL / kg. Continuous administration for 3 days. The day of administration is recorded as day 0 (D0), and on D9 after administration, the animals were subjected to gross dissection, and the kidney cortex, kidney medulla, liver, fat, duodenum, heart were collected and cut into several 2mm 3 The small pieces were stored in RNAlater. RNA extraction, Real-time PCR detection was as described above, and the gene expression difference was calculated by ΔΔCt method.

[0452] Table 8 Sequence list of primers used in test example 3

[0453] The results of test example 3 show that the LD110 conjugated siRNA sequence RZ899114 and the LD106 conjugated siRNA sequence RZ899104D16 both exhibit good target gene inhibition effect in the kidney cortex and medulla, and the inhibition effect in the kidney cortex is better than that in the kidney medulla. And the non-target tissues liver, heart, duodenum and thymus have no inhibition effect, indicating the specificity of kidney targeting. (Figure 3, Table 9)

[0454] Table 9 Inhibition activity of target genes in mice after administration of siRNA conjugates described in this test example

[0455] Test example 4 siRNA conjugate in vivo toxicological evaluation

[0456] This test example uses abdominal subcutaneous injection for administration to evaluate the toxicity of the LD110 conjugated siRNA conjugate R309001 in ICR mice.

[0457] 6-8 weeks old ICR mice were randomly divided into 4 groups according to body weight, including 3 test groups and 1 blank control group (PBS control group), 6 in each group (3 males and 3 females). Each group of mice was given siRNA conjugate R309001 by abdominal subcutaneous administration, and the first administration day was recorded as D0, with a frequency of once a week, a total of three times, i.e. the administration time points were D0, D7 and D14. Among them, each mouse in the PBS control group was given PBS buffer solution without siRNA conjugate each time, and the administration volume was 10 mL / kg (based on the weight of the mouse) per time; each mouse in the test group was given PBS buffer solution containing siRNA conjugate, and the administration volume was 10 mL / kg per time, and the administration doses of the three test groups were 100 mg (based on siRNA) / kg (based on the weight of the mouse) per time, 300 mg (based on siRNA) / kg (based on the weight of the mouse) per time, and 600 mg (based on siRNA) / kg (based on the weight of the mouse) per time, respectively.

[0458] During the test period, the mice were observed and recorded for clinical performance once a day; the body weight of the mice was measured on D0, D1, D3, D5, D7, D8, D14, D15, and the mice were anesthetized with an R540IE small animal anesthetic machine and at least 0.2 mL of blood was collected from the orbit without anticoagulation. The blood sample was placed at room temperature for about 60 min, centrifuged at 3000 rpm for 15 min at 4°C to obtain a serum sample. The serum sample was stored in a -20°C refrigerator for serum biochemical analysis.

[0459] The serum sample was subjected to blood biochemical detection using a fully automatic Hitachi fully automatic biochemical analyzer.

[0460] The change rate calculation formula of each serum biochemical index is: change rate = (test group - blank control group) / blank control group.

[0461] The serum biochemical results of Test Example 4 are shown (Figures 4-6, Table 10): under each administration dose, after administration of LD110 conjugated siRNA sequence R309001, the livers of male and female mice showed no obvious liver and kidney toxicity.

[0462] Table 10 Serum biochemical detection results of ICR mice on D15 after administration of siRNA conjugate R309001 of this test example

[0463] Immediately after blood sampling at D15, the mice were euthanized, and gross necropsy was performed. The abnormal gross findings and the condition of liver, kidney and other organs were recorded and saved. The liver, kidney and brain were weighed. The abnormal tissues (if any), the right middle lobe of liver (including gallbladder) and kidney (left side) were saved in 4% paraformaldehyde fixative, and the fixed tissues were sent to Wuhan Sivier Biological Technology Co., Ltd. for pathological section making, staining (hematoxylin-eosin staining) and reading.

[0464] The severity of hepatocyte degeneration in the liver tissue pathological sections was evaluated and graded. The evaluation and grading used a four-grade grading system (Peter. Mann et al., International Harmonization of Rat and Mouse Pathology Terminology and Diagnostic Criteria [M]).

[0465] The mouse kidney tissue pathological sections (representative kidney tissue pathological sections) and pathological score results showed that vacuolar degeneration of the renal tubules of the kidney occurred in the high-dose (600 mg / kg) group, and the index showed a dose-effect relationship. A few animals in the medium-dose (300 mg / kg) group showed changes, and no changes were observed in the low-dose (100 mg / kg) group. (Figure 7)

[0466] The mouse liver tissue pathological sections and pathological score results showed that compared with the blank control, the mice administered with different doses of LD110-conjugated siRNA sequence R309001 did not show more severe hepatocyte degeneration. Only some showed granular degeneration and lymphocyte infiltration, but since the blank control mice also showed the same degree of hepatocyte changes, the evaluation reason was due to the animal's own cell metabolism. (Figures 8-9)

[0467] The above specific embodiments are only illustrative of the content of the present application and do not represent a limitation of the content of the present application. Various modifications and improvements can be made by those of ordinary skill in the art without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered within the protection scope of the present disclosure.

Claims

1. A targeted delivery ligand characterized in that, The targeting delivery ligand comprises an amino acid sequence shown in formula (I), or an amino acid sequence variant having 1-6 amino acid residues of deletion, substitution or addition and having the same targeting activity, or an amino acid sequence variant having at least 60% sequence homology and having the same targeting activity; Phe-Ser-AA1-Cha-Ala-Gly-AA2-Ile-Asp-AA3-Ile (I) Wherein, AA1, AA2 are selected from any amino acid; AA3 is selected from arginine or alkylated arginine; Cha represents cyclohexylalanine; The targeting delivery ligand has a targeting activity to a kidney cell surface receptor.

2. The targeted delivery ligand of claim 1, wherein, The targeting delivery ligand is a polypeptide, a polypeptide variant or a polypeptide derivative comprising an amino acid sequence Phe-Ser-AA1-Cha-Ala-Gly-AA2-Ile-Asp-AA3-Ile; Wherein, AA1, AA2 are independently selected from proline or hydroxyproline; AA3 is selected from arginine or N-alkyl substituted arginine derivative; The polypeptide variant is selected from: an amino acid sequence comprising 1-3 amino acid residues of deletion, substitution or addition and having the same targeting activity, or an amino acid sequence comprising at least 60% sequence homology and having the same targeting activity; The polypeptide derivative is selected from the above-mentioned polypeptide or polypeptide variant substituted by a substituent at the N-terminus and / or C-terminus, and the polypeptide derivative has a targeting activity to a kidney cell surface receptor.

3. The targeted delivery ligand according to any one of claims 1-2, wherein, The targeting delivery ligand is a polypeptide comprising an amino acid sequence from the amino terminus to the carboxyl terminus in the direction of DPhe-Ser-AA1-Cha-DAla-Gly-AA2-Ile-Asp-AA3-Ile, or a polypeptide variant comprising an amino acid sequence having 1-2 amino acid residues of deletion, substitution or addition different from the above-mentioned sequence; Wherein, AA1, AA2 are independently selected from proline or hydroxyproline; AA3 is selected from arginine or N-methyl substituted arginine.

4. The targeted delivery ligand according to any one of claims 1 to 3, characterized in that, The targeted delivery ligand comprises a structure represented by Formula (100), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof: Wherein, R1 is selected from H or hydroxyl; R2 is selected from H or hydroxyl; R3 is selected from H or C1-C3 alkyl.

5. The targeted delivery ligand of claim 4, wherein, The targeted delivery ligand comprises a structure represented by Formula (101), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

6. The targeted delivery ligand according to any one of claims 1 to 5, wherein, The targeted delivery ligand is selected from the group consisting of a structure represented by Formula (102), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof: wherein Lz1is selected from H, C1-C3alkyl or wherein g1', g2', g3' are each independently selected from an integer from 1 to 10, Ly' is selected from -COOH or -NH2; Lz2is selected from -NH2, -NH-Ci-C3alkyl or wherein g1", g2", g3" are each independently selected from an integer from 1 to 10, Ly" is selected from -COOH or -NH2; Also, in Lzl is selected from Lz2is selected from -NH2or -NH-Ci-C3alkyl; in case Lz2is selected from When a is greater than 1, each M is independently conjugated to a different position of Nu; Optionally, the targeted delivery ligand is selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

7. The targeted delivery ligand according to any one of claims 1 to 6, wherein, The targeting delivery ligand specifically binds to a kidney cell surface receptor; Wherein, the kidney cell surface receptor is a lina peptide receptor.

8. Use of the targeting delivery ligand according to any one of claims 1-7 for targeting delivery of an active pharmaceutical molecule to a kidney cell; Optionally, the active pharmaceutical molecule is selected from a double-stranded oligonucleotide, a single-stranded oligonucleotide or a small molecule drug; Optionally, the active pharmaceutical molecule is selected from siRNA.

9. Conjugate, characterized in that, The conjugate comprises a structure represented by formula (200), or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: Wherein, Nu represents an active pharmaceutical molecule; a is selected from an integer of 1-5; When a is greater than 1, each M is independently conjugated to a different position of Nu; each M is each independently selected from the group consisting of wherein Lx1is selected from Wherein, b1, b2, b3, b4, b5 are each independently selected from an integer of 1-5; Lx2is selected from wherein each of cl, c2, c3, c4, c5, c6, c7, c8 is independently selected from an integer from 1 to 5; Lx3is selected from wherein d1, d2, d3, d4, d5, d6, d7, d8, d9, d 10 , d 11 each independently is selected from an integer from 1-5; each Cx is independently unsubstituted or substituted 4-10 membered aliphatic ring; e1 is selected from an integer between 1-5; each e2 is independently selected from an integer between 0-5; each e3 is independently selected from an integer between 0-5; each e4 is independently selected from an integer between 1-5; each X1 is independently selected from NH, O, or S; each X2 is independently selected from each X3 is independently selected from hydroxyl or thiol; each R5 is independently H, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy; each Cy is independently selected from a 5-6 membered saturated oxygen-containing heterocycle; fl is selected from an integer from 1 to 5; each f2 is independently selected from an integer from 0 to 5; each f3 is independently selected from an integer from 0 to 5; each f4 is independently selected from 1 or 2; each f5 is independently selected from an integer from 1 to 5; each Y1 is independently selected from O, S, or NH; each Y2 is independently selected from hydroxyl or thiol; each R6 is independently selected from H or C1-C6 alkoxy; each R7 is independently selected from H, C1-C6 alkyl, or C1-C6 alkoxy; each Lyis independently selected from the group consisting of or -S-S-; each Lzis independently selected from the group consisting of wherein g1, g2, g3 are each independently selected from an integer from 1 to 10, and Z is selected from each Ma is independently selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof: or wherein R1 is selected from H or hydroxyl; R2 is selected from H or hydroxyl; R3 is selected from H or C1-C3 alkyl; R4 is selected from H or C1-C3 alkyl; R1' is selected from H or hydroxyl; R2' is selected from H or hydroxyl; R3' is selected from H or C1-C3 alkyl; R4' is selected from H or C1-C3 alkyl.

10. The conjugate according to claim 9, characterized in that, Ma is selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof:

11. The conjugate according to any one of claims 9-10, characterized in that, Nu is selected from a double-stranded oligonucleotide, and a is selected from 1 or 2; Optionally, a is selected from 1, one of the M is conjugated to the 3' end or 5' end of the sense strand of the double-stranded oligonucleotide; Optionally, a is selected from 2, both of the M are conjugated to the 3' end and 5' end of the sense strand of the double-stranded oligonucleotide, respectively.

12. The conjugate according to any one of claims 9-11, characterized in that, gl is selected from 2, g2 is selected from 2, g3 is selected from 2; Optionally, Lz is selected from 13. The conjugate according to any one of claims 9 to 12, characterized in that, bl is selected from 6, b2 is selected from 3, b3 is selected from 2, b4 is selected from 2, b5 is selected from 2.

14. The conjugate according to any one of claims 9-12, characterized in that, cl is selected from 6, c2 is selected from 3, c3 is selected from 2, c4 is selected from 2, c5 is selected from 2, c6 is selected from 2, c7 is selected from 2, c8 is selected from 2.

15. The conjugate according to any one of claims 9-12, characterized in that, d1 is selected from 6, d2 is selected from 3, d3 is selected from 2, d4 is selected from 2, d5 is selected from 2, d6 is selected from 2, d7 is selected from 2, d8 is selected from 2, d9 is selected from 2, d 10 Selected from 2, d 11 Selected from 2.

16. The conjugate according to any one of claims 9-12, characterized in that, each Cx is independently selected from Optionally, X1 is selected from NH; Optionally, X2is selected from Optionally, each R5 is selected from H; Optionally, el is selected from 1, 2, or 3; Optionally, e2 is selected from 1; Optionally, e3 is selected from 1; Optionally, e4 is selected from 2.

17. The conjugate according to any one of claims 9-12, characterized in that, each Cy is independently selected from a 5-6 membered saturated oxygen-containing heterocycle; Optionally, each Cy is each independently selected from Optionally, each Y1 is independently selected from O; Optionally, fl is selected from 1, 2, or 3; Optionally, f2 is selected from 1; Optionally, f3 is selected from 0; Optionally, f5 is selected from 1; Optionally, each R6 is independently selected from H or methoxy; Optionally, each R7 is selected from H.

18. The conjugate according to any one of claims 9-17, characterized in that, The conjugate is targeted for delivery to a kidney cell.

19. A composition characterized in that, The composition comprises the conjugate of any one of claims 9-18.

20. Use of any of the following in the manufacture of a medicament for the prevention and / or treatment of a disease: (I) the targeting delivery ligand of any one of claims 1-8; and / or (II) the conjugate of any one of claims 9-18; and / or (III) the composition of claim 19; Optionally, the disease is selected from a kidney-derived disease. Optionally, the renal disease includes, but is not limited to, hypertension, hypertension-related renal impairment, hyperglycemia, diabetes, diabetic nephropathy, hyperuricemia, gout, hyperuricemia renal impairment, hepatitis B virus-related renal impairment, myeloma kidney disease, chronic renal failure, glomerulonephritis, renal vascular disease, C3 glomerulopathy, lupus nephritis, IgA nephropathy, polycystic kidney disease, membranous nephropathy, atypical hemolytic nephropathy, uremic syndrome, or other systemic lupus erythematosus (SLE)-related renal disease.

21. Use of any of: (I) the targeted delivery ligand of any one of claims 1-8; and / or (II) the conjugate of any one of claims 9-18; and / or (III) the composition of claim 19, in the manufacture of a medicament for reducing the expression or activity of a target gene in a kidney cell.

22. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises any of: (I) the targeted delivery ligand of any one of claims 1-8; and / or (II) the conjugate of any one of claims 9-18; and / or (III) the composition of claim 19, and a pharmaceutically acceptable carrier or excipient.

23. A method of reducing the expression or activity of a target gene in a kidney cell, characterized in that, The method comprises contacting a kidney cell with any of: (I) the targeted delivery ligand of any one of claims 1-8; and / or (II) the conjugate of any one of claims 9-18; and / or (III) the composition of claim 19; and / or (IV) the pharmaceutical composition of claim 22.

24. An oligonucleotide conjugate for inhibiting gene expression in a kidney cell, comprising: (a) a single- or double-stranded oligonucleotide molecule having the following features: (i) an antisense strand comprising 17-35 nucleotides, wherein, at least 15 nucleotides are complementary or substantially complementary to the mRNA sequence of a gene in a kidney cell; (ii) a sense strand of 15-35 nucleotides in length, which is complementary or partially complementary to the antisense strand; (b) a targeting ligand having affinity for a receptor present on the surface of a kidney cell, wherein the targeting ligand is a polypeptide; and, (c) a linker group, wherein the oligonucleotide molecule is covalently linked to the targeting ligand via the linker group; the polypeptide fragment included in the targeting ligand is -dXa-Ser-dXb-X2-dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; wherein X2 is selected from any unnatural amino acid; dXa, dXb, dXc are selected from any D-amino acid; Arg(Ak) is selected from arginine or alkylated modified arginine; Xd is selected from Hyp or Pro.

25. The oligonucleotide conjugate of claim 24, wherein, the targeting ligand is selected from a polypeptide comprising any of the amino acid sequence segments shown in A1) - A2): A1) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; A2) -DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

26. The oligonucleotide conjugate of claim 25, wherein, The Linker is attached to the N- or C-terminus of an amino acid on the polypeptide; Optionally, the Linker is selected from substituted or unsubstituted aliphatic chain, 3-6 membered heterocyclic ring, C6-C 10 aromatic ring, (PEG) n , disulfide, amide or triazole containing group, or a combination of the above; wherein n is selected from 1-10; Optionally, the Linker comprises any of the following substituents or any combination thereof: disulfide bonds, amido, triazolyl, -NH-, -C(O)-, wherein a, b, c or d are each independently selected from an integer from 0 to 10, preferably an integer from 1 to 8; * represents a point of attachment to an oligonucleotide molecule or to a targeting ligand, or a point of attachment of the substituents to each other.

27. The oligonucleotide conjugate of claim 24, wherein, The targeting ligand attached to the linker is a ligand unit selected from any of the structures shown in B1 )-B2): B1 ) La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa; B2) La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Laa; Any one of La and Laa is selected from a linker, the other is a terminal blocking group, the linker is a linking group and comprises at least one of a triazole group or a PEG unit or an acyl group; Optionally, La is a linker, the Linker structure comprises -PEG2-CH2CH2CO- or triazole-PEG2-.

28. The oligonucleotide conjugate of claim 27, wherein, The ligand unit is selected from any of the structures shown in C1 )-C2), or a pharmaceutically acceptable salt thereof: C1 ) -La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc; C2) -La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Lc; wherein La is a linker, independently selected from any bond, -NH-, an amide group or a linking group comprising at least one PEG unit; Optionally, La is a linking group comprising at least 2 PEG units; Optionally, La is selected from amido or -triazolyl-(PEG)n-, for example n is 1-3, m is 0 or 1, Z is -CO- (carbonyl) or -NH-; wherein Lc is a terminal blocking group; optionally, it is a C-terminal blocking group, Lc is selected from an amino group, or an alkyl-substituted amine group, preferably -NHCH3.

29. A polypeptide capable of delivering an oligonucleotide to a kidney cell, the polypeptide comprising any of the amino acid sequence segments shown in A1 )-A2): A1 ) -DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; A2) -DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

30. A ligand unit molecule capable of delivering an oligonucleotide to a kidney cell, selected from any of the compounds shown in D1 )-D2): D1 ) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-CONHCH3; D2) N3-(PEG)2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-lle-Asp-Arg- l ie-CONH2.

31. Use of a polypeptide according to claim 29 or a ligand unit molecule according to claim 30, characterized in that the use is for delivering an oligonucleotide molecule to a kidney cell.

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