Polypeptide ligand for mediating targeted natriuretic peptide receptor and application thereof

Abstract

The invention provides a polypeptide ligand for mediating a targeted natriuretic peptide receptor and application of the polypeptide ligand, and belongs to the technical field of oligonucleotide drug delivery. The invention discloses a polypeptide ligand suitable for targeting a linatide receptor, the ligand can be covalently linked with an active substance (such as oligonucleotide) through a linking group to obtain an oligonucleotide conjugate, and the targeting delivery capacity of the oligonucleotide conjugate in a target tissue (such as a kidney) is improved. The expression of a target gene of a subject (such as a human or an animal) can be selectively and effectively reduced or inhibited, and the compound can be used for treating and / or preventing pathological conditions or diseases caused by abnormal expression of a specific gene in a target tissue cell.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of oligonucleotide drug delivery, and particularly relates to a polypeptide ligand for mediating targeted natriuretic peptide receptor and use thereof. BACKGROUND

[0002] The natriuretic peptide (NP) family mainly contains three natriuretic peptide molecules, namely atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). ANP and BNP are mainly highly expressed in the atrium of the heart, and compared with ANP and BNP, CNP has a lower expression level in the heart and is more abundant in the brain, chondrocytes and endothelial cells. Different natriuretic peptides have similar extracellular domains, bind to ligands in the form of homodimers, and need to exert their active effects through the binding of single transmembrane receptors. So far, three natriuretic peptide receptors have been found, namely natriuretic peptide receptor A (NPR-A), natriuretic peptide receptor B (NPR-B) and natriuretic peptide receptor C (NPR-C). The natriuretic peptide receptors are expressed in various tissues and organs, NPR-A is mainly expressed in the kidney, adrenal gland, terminal ileum, adipose tissue, aorta and lung tissue, NPR-B is mainly expressed in fibroblasts, and NPR-C is mainly expressed in the atrium, mesentery, placenta, lung, kidney and vein tissue, as well as aortic smooth muscle and endothelial cells. Different natriuretic peptide receptors can bind to the same ligand, for example, ANP, BNP and CNP can all bind to NPR-C. The natriuretic peptide receptors and their ligands provide alternative solutions in the field of drug delivery, mainly including the structural modification of endogenous polypeptides and the high-throughput screening of new structural types of small molecule agonists and antagonists. However, there are few reports on using ligands of various NPRs as drug molecule delivery carriers. Exemplarily, as the natural ligand of NPR, ANP / BNP can be used as a delivery carrier, for example, coupling an active drug to ANP, entering cardiomyocytes through NPR-mediated endocytosis, achieving heart targeting and reducing systemic toxicity. NPR-C is the most abundant type of NPR receptor, accounting for more than 95% in the kidney (also expressed in the lung, kidney, vascular smooth muscle cell line and fibroblast line), and the high-affinity ligand of NPR-C can be used as an effective kidney delivery system to deliver other drug active molecules including oligonucleotides to endothelial cells to regulate metabolic-related gene expression. Therefore, developing new ligand molecules for targeting various NPRs to deliver oligonucleotides is expected to be an effective strategy for an extrahepatic delivery system. SUMMARY

[0003] The present disclosure relates to polypeptide ligands suitable for targeting natriuretic peptide receptors, which can deliver oligonucleotide drugs into cells as carrier molecules for delivering oligonucleotides. The present disclosure also relates to oligonucleotide conjugates formed by covalently linking at least one polypeptide ligand to a pharmaceutically active molecule, such as an oligonucleotide. Furthermore, the present disclosure relates to the use of the polypeptide ligands and oligonucleotide conjugates for natriuretic peptide receptor targeted delivery.

[0004] In a first aspect of the present disclosure, the present disclosure provides a polypeptide ligand mediating targeting of natriuretic peptide receptors, the polypeptide ligand comprising a polypeptide consisting of 4-20 amino acid residues and a linker or bond for linking the polypeptide to a pharmaceutically active molecule; the natriuretic peptide receptor is selected from at least one of natriuretic peptide receptor A, natriuretic peptide receptor B or natriuretic peptide receptor C.

[0005] In some alternative embodiments, the natriuretic peptide receptor is selected from natriuretic peptide receptor C.

[0006] In some alternative embodiments, the polypeptide ligand comprises 5-15 amino acid residues; further, the polypeptide ligand comprises 7-13 amino acid residues. In some alternative embodiments, the pharmaceutically active molecule can be selected from oligonucleotides.

[0007] The polypeptide ligand comprises consecutive three amino acid residues in the amino acid sequence -Arg-Ile-Asp-Arg- or -Ile-Asp-Arg-Ile-, or consecutive three amino acid residues in an amino acid sequence having 1 amino acid residue difference from any of the above sequences; wherein each amino acid residue is independently a natural or modified amino acid residue, which can be in D-form or L-form; wherein the modified amino acid residue refers to one or more of side chain modification, terminal group modification or chiral isomerization; the modification is substitution of a functional group or H of the side chain of the amino acid residue with a modifying group selected from one of substituted or unsubstituted C1-C6 alkyl (preferably C1-C3 alkyl), cycloalkyl, aryl or heteroaryl; optionally, the modification is an alkylation modification, and the modifying group is selected from methyl, ethyl or propyl.

[0008] In some alternative embodiments, the Arg comprises an alkylated modified arginine residue, such as a methylated modified arginine.

[0009] In some alternative embodiments, the polypeptide ligand comprises the amino acid sequence -Arg-Ile-Asp-Arg-.

[0010] In some alternative embodiments, the polypeptide ligand comprises the amino acid sequence -Ile-Asp-Arg-Ile-.

[0011] In some alternative embodiments, the polypeptide ligand comprises the amino acid sequence -Ile-Asp-Arg(Me)-Ile-; Arg(Me) is a methylated arginine residue.

[0012] In some alternative embodiments, the polypeptide ligand comprises -Arg-Ile-Asp-Arg-Ile-.

[0013] In some alternative embodiments, the polypeptide ligand has an amino acid sequence as shown below (left to right direction represents from N-terminus to C-terminus): - P1-Ile-Asp-Ra-Ile-P2- wherein, Ra is selected from Arg or a methylated arginine residue Arg(Me), or a dimethylated arginine residue; P1 represents an amino acid sequence consisting of 5-10 amino acid residues, and P2 represents any amino acid residue or is absent.

[0014] In some alternative embodiments, P1 represents an amino acid sequence consisting of 6-7 amino acid residues.

[0015] Further, the polypeptide ligand further comprises an amino acid sequence -Cha-Z-Gly-, Z is any amino acid residue.

[0016] In some alternative embodiments, the polypeptide ligand comprises 4 consecutive amino acid residues in an amino acid sequence that differs from the amino acid sequence -Ile-Asp-Arg-Ile-Gly- by no more than 1 or 2 residue difference.

[0017] In some alternative embodiments, the polypeptide ligand comprises 4 consecutive amino acid residues in an amino acid sequence that differs from the amino acid sequence -AA1-Ile-Asp-Arg-Ile- by no more than 1 residue difference, wherein AA1 is selected from any one of the amino acid residues of Arg, Pro or Hyp; preferably, the polypeptide comprises an amino acid sequence of -Arg-Ile-Asp-Arg-Ile-, -Pro-Ile-Asp-Arg-Ile- or -Hyp-Ile-Asp-Arg-Ile-; further, the polypeptide ligand comprises a structure as shown below: -Arg-Ile-Asp-Arg-Ile-NH2.

[0018] In some alternative embodiments, the polypeptide ligand comprises 4 or 5 contiguous amino acid residues in an amino acid sequence that differs from the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly- by no more than 1 residue difference; preferably, the polypeptide ligand comprises any 4 contiguous amino acid residues in the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly-; further, the polypeptide ligand comprises any 5 contiguous amino acid residues in the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly-; wherein the AA1 is selected from any one of the amino acid residues of Arg, Pro or Hyp.

[0019] In some alternative embodiments, the polypeptide ligand comprises 5 or 6 contiguous amino acid residues in an amino acid sequence that differs from the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-AA2- by no more than 1 residue difference, wherein AA2 is selected from a Gly residue or is absent; preferably, the polypeptide ligand comprises any 5 contiguous amino acid residues in the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-Gly-; further, the polypeptide ligand comprises any 6 contiguous amino acid residues in the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-Gly-; wherein the AA1 is selected from any one of the amino acid residues of Arg, Pro or Hyp.

[0020] In some alternative embodiments, the polypeptide ligand comprises the amino acid sequence -Gly-Arg-Ile-Asp-Arg-Ile-Gly-.

[0021] In some alternative embodiments, the polypeptide ligand comprises 6 or 7 contiguous amino acid residues in an amino acid sequence that differs from the amino acid sequence -Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2- by no more than 1 residue difference, wherein the AA0 is selected from any one of the amino acid residues of Gly, Ala or Ser, the AA1 is selected from any one of the amino acid residues of Arg, Pro or Hyp, and AA2 is selected from a Gly residue or is absent; preferably, the polypeptide ligand comprises 7 contiguous amino acid residues in the amino acid sequence -Cha-AA0-Gly-Gly-AA1-Ile-Asp-Arg-Ile-AA2-; further preferably, the polypeptide ligand comprises 7 contiguous amino acid residues in the amino acid sequence -Cha-AA0-Gly-Gly-AA1-Ile-Asp-Arg-Ile-AA2- and comprises at least three contiguous amino acid residues in the -Ile-Asp-Arg-Ile- sequence.

[0022] In some alternative embodiments, the polypeptide ligand comprises 7 or 8 consecutive amino acid residues in an amino acid sequence that differs from the amino acid sequence -X1-Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2- by no more than 1 residue difference, wherein AA0 is selected from any one of the amino acid residues Gly, Ala or Ser, AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp, and AA2 is selected from a Gly residue or is absent; X1 is a chemical bond or a sequence of 1-5 amino acid residues, preferably selected from: a chemical bond, Cys, Ser, Ala, Gly, or any one of the amino acid sequences -A1-A2-A3-A4-A5-, wherein A1 is selected from Arg or is absent, A2 is selected from Arg, Ser or is absent, A3 is selected from Phe, Ser or is absent, A4 is selected from Ser or is absent, and A5 is selected from Cys, Ser, Ala, Gly, Pro, Hyp or is absent; further optionally, Pro and Hyp are in the D-configuration. Preferably, the polypeptide comprises 8 consecutive amino acid residues in the amino acid sequence -X1-Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2-, and comprises at least three consecutive amino acid residues in the -Ile-Asp-Arg-Ile- sequence.

[0023] wherein each amino acid residue is independently a natural or modified amino acid residue, which can be in the L or D configuration.

[0024] wherein the Arg in the -Ile-Asp-Arg-Ile- sequence is selected from an unmodified Arg or a methylated Arg (i.e. Arg(Me)), the methylation modification comprising a methyl group attached to the N atom at the end of the side chain of arginine, which can be mediated by protein arginine methyltransferases (PRMTs).

[0025] In some alternative embodiments, the polypeptide ligand of the present disclosure has the structure shown below (left to right represents from N-terminus to C-terminus): Z1-X1-Cha-AA0-Gly-AA1-Ile-Asp-Ra-Ile-AA2-Z2 wherein Ra is selected from Arg or a methylated arginine Arg(Me), or a dimethylated arginine.

[0026] Z1, Z2 represent a capping group of the terminal amino acid (carboxyl terminal or amino terminal) of the polypeptide or a covalent attachment site to an active molecule.

[0027] In some alternative embodiments, Z1 is selected from any of H, an amino protecting group, or a linking site, and Z2 is selected from any of H, a carboxyl protecting group, or a linking site, or at least one of Z1 and Z2 is a linking site.

[0028] In some alternative embodiments, Z1 is a capping group at the amino terminus (N-terminus) of the polypeptide, which can be preferably selected from H, an acyl group, an acetyl group, an acetyl group of a hydroxyl substituent (HOCH2CO-), or a linking site; and Z2 is a capping group at the carboxyl terminus (C-terminus) of the polypeptide, which is selected from a covalent linking site, an amide, or a substituted amide, preferably selected from -CONH2 or -CONHCH3.

[0029] Further optionally, Z1 is at the amino terminus of the polypeptide and is selected from a covalent linking site; and Z2 is at the carboxyl terminus of the polypeptide and is selected from an amide group.

[0030] X1 is a chemical bond or a short sequence of 1-5 amino acid residues, preferably selected from a chemical bond, Cys, Ser, Ala, Gly, or the amino acid sequence -A1-A2-A3-A4-A5-; wherein A1 is selected from Arg or absent, A2 is selected from Arg, Ser, or absent, A3 is selected from Phe, Ser, or absent, A4 is selected from Ser or absent, and A5 is selected from Cys, Ser, Ala, Gly, Pro, Hyp, or absent, further optionally Pro or Hyp is selected from the D-configuration.

[0031] wherein AA0 is selected from the amino acid residues Gly, Ala, or Ser, AA1 is selected from Arg, Pro, or Hyp, and AA2 is selected from a Gly residue or absent.

[0032] In some alternative embodiments, X1 is a short sequence of 3 amino acid residues -A3-A4-A5-; further preferably, wherein -A3-A4- is -Phe-Ser-; and A5 is selected from Pro or Hyp, optionally selected from the D-configuration.

[0033] In some alternative embodiments, the polypeptide ligand of the present disclosure comprises an amino acid sequence as shown below (left to right direction represents from N-terminus to C-terminus): -(L / D)Phe-Ser-AA0-Cha-AA1-Gly- AA2-Ile-Asp-AA3-Ile- wherein AA0 is selected from Pro or Hyp in the D- or L-configuration (preferably the D-configuration), AA1 is selected from Ala or Ser in the D- or L-configuration, and AA2 is selected from Pro or Hyp in the L-configuration, and AA3 is selected from Arg or Arg(Me).

[0034] In some alternative embodiments, the polypeptide ligand comprises or has any one of the following amino acid sequences (based on N-C terminal orientation): - Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 1) - Ser-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 2) - Ala-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 3) - Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 4) - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg-Ile-; (SEQ ID NO. 5) - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; (SEQ ID NO. 6) - Phe-Ser-Hyp-Cha-Ala-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; (SEQ ID NO. 7) - Phe-Ser-Hyp-Cha-Ser-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; (SEQ ID NO. 8) - Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 9) - Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 10) - Phe-Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 11) - Phe-Ser-Pro-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 12) - Phe-Ser-Gly-Cha-Ala-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 13) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) Each amino acid residue is in the L- or D- form.

[0035] In some alternative embodiments, each Phe is in the D- form.

[0036] In some alternative embodiments, each Ala is in the D- form.

[0037] In some alternative embodiments, the polypeptide ligand comprises an amino acid sequence (based on N-C terminal orientation) of any one of the following: - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) - Phe-Ser-Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; (SEQ ID NO. 16) DPhe represents D-configuration of Phe, and the rest of the amino acids have the same meaning.

[0038] In some alternative embodiments, the polypeptide ligand is a linear peptide or a cyclic peptide, wherein the side chains of any non-adjacent amino acid residues in the polypeptide are connected to each other to form a cyclic peptide.

[0039] In some alternative embodiments, the side chains of the amino acid residues can be connected directly, or through an amide-containing linkage, a disulfide bond, or an alkylene / alkenylene-containing linker; wherein the alkylene is selected from C2-C 10 linear alkylene, and the alkenylene is selected from C4-C 10 alkenylene.

[0040] In some alternative embodiments, the two amino acid residues whose side chains are connected to each other in the cyclic peptide are separated by 1-6 amino acid residues.

[0041] In some alternative embodiments, the polypeptide ligand comprises a cyclic peptide structural unit, which has the following primary amino acid sequence: Cys-Phe-Gly-PPa-Asp-Arg-Ile-PPb-Ser-PPc-Leu-Gly-Cys, and the two Cys form a cyclic peptide unit through a disulfide bond; wherein PPa and PPb are each independently a short peptide consisting of 3 arbitrary amino acid residues, and PPc is an arbitrary amino acid residue.

[0042] In some alternative embodiments, the polypeptide ligand comprises an amino acid sequence as follows: -Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-, and has a disulfide bond between the two Cys.

[0043] In some alternative embodiments, the polypeptide ligand comprises a modified amino acid, and comprises the following structural unit: , (e.g., ), In some alternative embodiments, the polypeptide ligand comprises the following structure: wherein Ar is selected from aryl, heteroaryl, fused ring aromatic system, or a combination thereof; In some alternative embodiments, Ar is selected from substituted or unsubstituted naphthyl, diphenyl, triphenyl, cyclohexyl, indolyl, adamantyl; the substituent is selected from halogen, hydroxyl, amino, C1-C6 alkoxy, C1-C6 alkyl, phenyl, aryl, heteroaryl, alkylenearyl, alkyleneheteroaryl; R2 is O or N; R3 is any amino acid residue or modified amino acid residue.

[0044] In some alternative embodiments, the polypeptide ligand further includes a linker group for linking to a pharmaceutically active molecule; the linker group is used to link the polypeptide to the pharmaceutically active molecule; The linking group is covalently linked to amino acid residues in the polypeptide (preferably amino acid residues at the amino or carboxyl terminus of the polypeptide) and includes functional groups capable of undergoing a conjugation reaction with the active pharmaceutical molecule to form a covalently linked group; the functional group can be one or more, and is independently selected from azide, alkynyl, mercapto, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, disulfide, sulfonate, alkenyl, hydrazide, or phosphoramidyl.

[0045] In some alternative embodiments, the linking group is selected from substituted or unsubstituted straight-chain / branched C1-C14 groups. 15 The hydrocarbon chain contains one or more methylene (-CH2-) units, and one or more substituents are replaced by one or more substituents selected from the group consisting of: triazolyl, -C(O), -OC(O)-, -NHC(O)-, -NH-, O, S, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6 imenyl, C6-C 10 aryl or C3-C 10 Heterocyclic groups; In some alternative embodiments, when the linking group has substituents, the substituents are selected from one or more of the group consisting of: hydroxyl, amino, halogen, C1-C6 alkyl, C6 ... 10 Aryl, C5-C 10 Heteroaryl, C1-C5 alkoxy, C1-C5 alkylphenyl, nitro, -CO NH2, -C(O)C1-C6 alkyl, -SO2NH2 or a combination of the above substituents.

[0046] In some alternative embodiments, the active pharmaceutical molecule is selected from double-stranded oligonucleotides.

[0047] In some alternative embodiments, the polypeptide ligand comprises the following structure: R1is selected from -Ar-(L)i-R5, wherein L is -(CH2)n1-(R4)n2, n1 is an integer from 0 to 6, each R4 is independently O or S, n2 is 0 or 1, and i is selected from an integer from 0 to 10; R2, R3 are as defined above; R5is selected from azido, alkynyl, thiol, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, disulfide, sulfonate, alkenyl, hydrazide, or phosphoramidite.

[0048] In some alternative embodiments, the polypeptide ligand is selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof: ; ; ; .

[0049] In a second aspect of the present disclosure, the present disclosure provides use of the polypeptide ligand of the first aspect for mediating targeting of a lina peptide receptor as a delivery vehicle for a pharmaceutically active molecule.

[0050] In some alternative embodiments, the pharmaceutically active molecule is selected from a small molecule compound, an antibody, or an oligonucleotide.

[0051] In some alternative embodiments, the polypeptide ligand for mediating targeting of a lina peptide receptor is used as a delivery vehicle for delivering an oligonucleotide into a cell.

[0052] In some alternative embodiments, the cell is selected from a cell in heart, kidney, lung, liver, adrenal gland, fat, cerebral cortex, or tumor tissue.

[0053] In some alternative embodiments, the oligonucleotide is an siRNA having a sense strand and an antisense strand, each strand optionally having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence complementary or substantially complementary to the sense strand and a target mRNA; and optionally, the polypeptide ligand is linked to the 5' end and / or the 3' end of the sense strand.

[0054] In a third aspect of the present disclosure, the present disclosure provides an oligonucleotide conjugate comprising a covalently linked oligonucleotide molecule and the polypeptide ligand of the first aspect of the present disclosure; In some alternative embodiments, the conjugate is formed by covalent conjugation reaction of the oligonucleotide and the polypeptide ligand of the first aspect of the present disclosure, the covalent conjugation reaction comprising an addition reaction, a coupling reaction, or a substitution reaction.

[0055] wherein the conjugate contains one or more polypeptide ligand units; In some alternative embodiments, the oligonucleotide is linked to the amino- or carboxy-terminal position of the polypeptide via a covalent linker, for example, the polypeptide ligand unit is linked to the oligonucleotide via a covalent linker at the amino-terminus of the polypeptide.

[0056] In some alternative embodiments, the conjugate has the following structure: Nu-[-La-(-Lb-PP) n3 ] n4 wherein n3 and n4 are independently selected from an integer from 1 to 3.

[0057] La and Lb are linkers, wherein the site of La directly linked to the oligonucleotide is selected from a phosphodiester bond, a phosphorothioate bond or a disulfide bond; the linker is as defined in the preceding claims.

[0058] La is a substituted or unsubstituted 4-10 membered saturated or unsaturated aliphatic ring, 3-6 membered heterocyclic ring, C6-C 10 aromatic ring; Lb is a C1-C20 alkylene, (PEG) n group containing a disulfide bond, amide group or triazole group, or a combination thereof; wherein n is selected from an integer from 1 to 20 PP is a polypeptide, the polypeptide is as defined in the preceding claims.

[0059] The polypeptide simultaneously has the amino acid sequences -Ile-Asp-Ra-Ile- and -Cha-Z-Gly-, wherein Ra is selected from Arg or Arg(Me), and Z is selected from any neutral amino acid in D or L configuration, preferably any of Gly, Ala or Ser.

[0060] In some alternative embodiments, the polypeptide comprises the following amino acid sequence: -DPhe-Ser-AA0-Cha-AA1-Gly- AA2-Ile-Asp-AA3-Ile-; wherein AA0 is selected from Gly, DPro or DHyp (D configuration), AA1 is selected from Gly, DAla or DSer, AA2 is selected from Pro or Hyp, and AA3 is selected from Arg or Arg(Me).

[0061] In some alternative embodiments, the oligonucleotide is a double-stranded oligonucleotide siRNA, and the sense and antisense strands each comprise 15-25 nucleotides; each of the nucleotides is a modified or unmodified nucleotide.

[0062] In some alternative embodiments, the double-stranded oligonucleotide is delivered to a target tissue / target cell of the kidney, heart, lung, liver, adrenal gland, fat (preferably subcutaneous fat or gonadal fat), or cerebral cortex.

[0063] In some alternative embodiments, the double-stranded oligonucleotide is selected from functional oligonucleotide molecules having therapeutic effects on kidney-related diseases or symptoms.

[0064] In some alternative embodiments, the target tissue is a tissue of adipose tissue or a kidney organ, preferably a proximal tubular epithelial tissue of the kidney; and the target cell is an epithelial cell of the proximal tubular epithelial tissue.

[0065] In a fourth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising one or more of the polypeptide ligand of the first aspect of the present disclosure or the oligonucleotide conjugate of the third aspect of the present disclosure, a pharmaceutically acceptable salt, metabolite, or prodrug thereof, and a pharmaceutically acceptable excipient.

[0066] In a fifth aspect of the present disclosure, the present disclosure provides use of one or more of the polypeptide ligand of the first aspect of the present disclosure or the oligonucleotide conjugate of the third aspect of the present disclosure, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the fourth aspect of the present disclosure, in the manufacture of a medicament for treating and / or preventing a disease associated with dysregulation of gene expression.

[0067] The use comprises administering to a subject in need thereof an effective amount of the polypeptide ligand of the first aspect of the present disclosure or the oligonucleotide conjugate of the third aspect of the present disclosure, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the fourth aspect of the present disclosure.

[0068] The use comprises delivering the oligonucleotide by systemic or local administration, preferably the administration is selected from the group consisting of intravenous injection, subcutaneous injection, intrathecal injection, intramuscular injection, and aerosol spray.

[0069] In a sixth aspect of the present disclosure, the present disclosure provides a kit comprising one or more of the polypeptide ligand of the first aspect of the present disclosure or the oligonucleotide conjugate of the third aspect of the present disclosure, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the fourth aspect of the present disclosure.

[0070] In a seventh aspect of the present disclosure, the present disclosure provides a method of treating and / or preventing a disease associated with dysregulation of gene expression, the method comprising administering to a subject in need thereof an effective amount of the polypeptide ligand of the first aspect of the present disclosure or the oligonucleotide conjugate of the third aspect of the present disclosure, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the fourth aspect of the present disclosure.

[0071] In some alternative embodiments, the method comprises delivering the oligonucleotide by systemic or local administration, preferably the administration is selected from the group consisting of intravenous injection, subcutaneous injection, intrathecal injection, intramuscular injection and aerosol spray.

[0072] In an eighth aspect of the present disclosure, the present disclosure provides a ligand of the polypeptide of the first aspect of the present disclosure.

[0073] In a ninth aspect of the present disclosure, the present disclosure provides a method of delivering an oligonucleotide or a small molecule active drug into a mammalian host, comprising administering to the host a pharmaceutically effective amount of the oligonucleotide conjugate of the third aspect of the present disclosure or the pharmaceutical composition of the fourth aspect of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 Relative expression levels of target genes of interest in mice after administration of the siRNA conjugate described in this example. DETAILED DESCRIPTION

[0075] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0076] Terminology 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.

[0077] In the present disclosure, the term "comprising" or "including" is an open-ended expression that is used in the present disclosure to mean "including but not limited to", and is used interchangeably with "consisting of", to indicate that the listed steps or options are essential, but also to allow for the inclusion of further steps or options.

[0078] In the present disclosure, the term "optionally", "optional" or "optional" generally means that the event or circumstance subsequently described can or can not occur, and the description includes instances in which the event or circumstance occurs, and instances in which it does not.

[0079] In the present disclosure, the term "unsubstituted" means that the designated group bears no substituents.

[0080] In the present disclosure, the terms "substituted", "substituted" and "substitution" are used interchangeably, meaning that any one or more hydrogen atoms in the given structure are replaced with a specified substituent (for example: C 1-6 alkyl, C 1-6 alkoxy or halogen), provided that the designated atom's normal valence is not exceeded and that the substitution results in a stable compound. Unless otherwise indicated, a substituted group can have a substituent at each substitutable position of the group. When more than one position in the given structure can be substituted with one or more substituents selected from a specified group, then those selected substituents can be the same or different at each position.

[0081] In the present disclosure, the term "each independently selected from" can be used interchangeably with "each independently selected from" and "independently selected from", and should be interpreted broadly, meaning that the specific options expressed between the same symbols in different groups do not affect each other, and also meaning that the specific options expressed between the same symbols in the same group do not affect each other.

[0082] In the present disclosure, "effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more of the beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying the onset of a condition, including biochemical, histological and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presenting during development of the condition. For therapeutic use, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the target gene, target mRNA or target protein of the present disclosure, or ameliorating one or more symptoms of the condition, reducing the dosage of other medications required to treat the condition, enhancing the effect of another medication, and / or delaying the progression of the condition associated with the target gene, target mRNA or target protein of the present disclosure in a patient.

[0083] In the present disclosure, the term "chemical modification" or "modification" includes all changes to nucleic acids by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0084] In the present disclosure, the bond " in the structural formula of "compound", "ligand" and "vector" means that the configuration is not specified. If there is chiral isomerism in the chemical structure, the bond " can be ", ", ", or simultaneously contain ", ". ​​​​​"Two configurations. Although all of the above structural formulas are drawn in certain isomer forms for simplicity, the present disclosure can include all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates, and enantiomers.

[0085] In the present disclosure, the term "stereoisomer" refers to a compound having the same chemical constitution, but different spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0086] 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 targeted cleavage of RNA transcripts of the RISC pathway by forming a silencing complex (RNA-induced silencing complex, RISC). Specifically, the siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins.

[0087] In the present disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing of some or all of the bases, the two oligonucleotides comprising a sense strand and an antisense strand, the sense strand and the antisense strand can be the same or different in length, as long as there is at least a region of partial base pairing to form a duplex region, the oligonucleotide with a double-stranded structure is a double-stranded oligonucleotide according to the present disclosure. In the present disclosure, the nucleotides constituting the double-stranded oligonucleotide can be modified or unmodified nucleotides, 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, the connecting bonds between the nucleotides can also be modified, the double-stranded oligonucleotide containing modified connecting bonds between the nucleotides also belongs to the double-stranded oligonucleotide according to the present disclosure. In the present disclosure, the double-stranded oligonucleotide can contain molecules or modifications of compounds acceptable in the art in addition to the nucleotide portion, in order to improve the properties of the double-stranded oligonucleotide, such as the formation of conjugates with ligands.

[0088] In the present disclosure, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence. The term "sense strand (or 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, e.g., 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 of the molecule or within the terminal regions, with the most tolerated mismatches existing within the terminal regions, e.g., 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.

[0089] In the present disclosure, "targeting delivery ligand," "delivery ligand," "ligand," "targeting delivery carrier," "delivery carrier," and "carrier" are used interchangeably and generally refer to any compound or molecule that is capable of covalently or otherwise chemically associating with a biologically active material, such as an oligonucleotide. In certain embodiments, the ligand is capable of directly or indirectly interacting with another compound, e.g., a receptor, which can be present on the surface of a cell, or alternatively can be an intracellular and / or intercellular receptor, and the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.

[0090] 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).

[0091] 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.

[0092] 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.

[0093] Unless otherwise indicated, the symbols as used in the present disclosure refer to any group or groups that can be attached thereto, consistent with the scope of the application as described in the present disclosure.

[0094] In the present disclosure, “ ” indicates the site of attachment of a group by a covalent bond.

[0095] According to the 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 protein- generating amino acids), unnatural amino acids or prepared amino acids which can exist in organisms are included.

[0096] In the peptides of the present disclosure, the amino acid units can be present in the D- or L-form, unless otherwise specified.

[0097] As used in the present disclosure, 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.

[0098] As used in the present disclosure, the term “non-standard amino acid” refers to an amino acid other than “standard amino acid” as defined in the present disclosure. “Non-standard amino acids” include, but are not limited to, N-formylmethionine, hydroxyproline (Hyp), selenomethionine, isovaline, citrulline (Cit), ornithine, alpha-methyl-aspartate (aMeD), alpha-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-alpha amino acids, and other amino acid analogs or mimetics that function in a similar manner to naturally occurring amino acids.

[0099] In the present disclosure, the term “cyclic peptide” means that two cysteines in a polypeptide chain are connected by a disulfide bond, such that an intramolecular ring is formed to form a cyclic polypeptide chain. In the present disclosure, the disulfide bond between the two cysteines in the “cyclic peptide” is represented by “︵”.

[0100] In the present disclosure, "double-stranded oligonucleotide" and "conjugate" can be obtained by the methods of preparation that are conventional in the art, such as methods of solid-phase synthesis and liquid-phase synthesis. Among them, solid-phase synthesis has been commercialized as a subscription service. Methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers having corresponding modifications are also well known to those skilled in the art.

[0101] In the present disclosure, the term "pharmaceutical composition" or "composition" can refer to the use for the treatment of a disease, and also 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 auxiliary ingredient(s) that make up the one or more additional ingredients. Typically, the compositions are prepared by uniformly and intimately bringing the active siRNA into association with a liquid auxiliary ingredient, a finely divided solid auxiliary ingredient, or both.

[0102] 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 the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" in the present disclosure means approved or approvable by a regulatory agency of the Federal or a state government or the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0103] In the present disclosure, the term "pharmaceutically acceptable carrier or adjuvant" can include any and all solvents, solid diluents or other liquid excipients, 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 an undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this disclosure.

[0104] In the present disclosure, the terms "treatment," "relief," or "amelioration" can be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Herein, a therapeutic benefit is achieved with regard to the underlying disorder by either eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder, thereby improving the subject's condition, although the subject can still be afflicted with the underlying disorder.

[0105] 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", the 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.

[0106] 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.

[0107] As used in the present disclosure, 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.

[0108] In addition to any conventional excipients, the use of ranges that 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.

[0109] The present disclosure is further illustrated below by specific examples, but it should be understood that these examples are merely set forth to provide a more detailed description of the present disclosure and are not intended to limit the present disclosure in any form.

[0110] Unless otherwise specified, the siRNA sequences used in the present disclosure are all synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Suzhou Baisin Biotechnology Co., Ltd.; the PCR primers used in the present disclosure are all synthesized by Beijing Qikexin Biotechnology Co., Ltd.; and the experimental animals C57BL / 6J mice used in the present disclosure are all purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0111] Unless otherwise specified, the CPG carrier (loading 80 umol / g) used in the present disclosure is purchased from Beijing Coupling Technology Co., Ltd. Among them, the CPG carrier is recorded as ; Representing glass balls with controllable pore size (Controlled Pore Glass, CPG).

[0112] Unless otherwise specified, the reagents, reagent consumables and instrument equipment used in the present disclosure are all from the market. Among them, the main reagents are shown in Table 1, the main reagent consumables are shown in Table 2, and the main instrument equipment is shown in Table 3.

[0113] Table 1 Main reagents Table 2 Main reagent consumables Table 3 Main instrument equipment In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure will be further described below in combination with specific examples, but the present disclosure is not limited to the following examples.

[0114] Preparation of compound LD146 The synthetic route of compound LD146 is as follows: (1-1) Synthesis of compound LD146-7 (1-1-1) Synthesis of compound LD146-2 Dissolve 11-azido-3,6,9-trioxadodecanol (20 g, 91.32 mmol, 1.0 eq) in DCM (200 mL), add triethylamine (18.44 g, 182.64 mmol, 2.0 eq), reduce to 0°C, add p-toluenesulfonyl chloride (19.08 g, 100.46 mmol, 1.1 eq), stir for 3 hours, after the reaction is completed, add a purified aqueous solution (150 mL) to wash, dry with anhydrous sodium sulfate, and filter, concentrate, and purify by column chromatography (elution gradient, ethyl acetate: petroleum ether = 40:60) to obtain 30.3 g of compound LD146-2, with a yield of 89.0%. MS ESI (M+H) = 374 m / z + .

[0115] (1-1-2) Synthesis of compound LD146-3 ​Compound LD146-2 (13 g, 34.9 mmol, 1.1 eq), potassium carbonate (13.86 g, 95.2 mmol, 3.0 eq), nitrogen replacement for 3 times, stirred at 100 °C for 6 hours. After the reaction was completed, it was cooled to room temperature, washed with ethyl acetate (200 mL) and purified water (200 mL), the organic phase was washed with saturated sodium chloride aqueous solution for 3 times, dried by adding anhydrous sodium sulfate, concentrated, column chromatography (elution gradient, ethyl acetate: petroleum ether = 40:60), to obtain 13.2 g of compound LD146-3, with a yield of 94.0%. MS ESI ( m / z ) = 404 [M+H] + .

[0116] Synthesis of compound LD146-4 Compound LD146-3 (13 g, 32.26 mmol, 1.0 eq) was dissolved in methanol (65 mL), 1M NaOH aqueous solution (65 mL) was added, stirred for 1 hour, after the reaction was completed, 1M HCl aqueous solution was added to adjust the pH to neutral, concentrated, dichloromethane was added, and filtered, concentrated to obtain 10.4 g of yellow solid compound LD146-4, with a yield of 83.2%. MS ESI ( m / z ) = 390 [M+H] + .

[0117] Synthesis of compound LD146-6 Compound LD146-4 (10.4 g, 26.74 mmol, 1.0 eq) was dissolved in DMF (100 mL), HATU (15.2 g, 40.1 mmol, 1.5 eq), DIEA (6.89 g, 53.47 mmol, 2.0 eq) were added, stirred for 10 minutes, compound LD146-5 (p-aminophenylacetic acid methyl ester, 4.85 g, 29.4 mmol, 1.1 eq) was added, nitrogen replacement for 3 times, stirred at room temperature for 3 hours. After the reaction was completed, it was washed with ethyl acetate (100 mL) and purified water (100 mL), the organic phase was washed with saturated sodium chloride aqueous solution for 3 times, dried by adding anhydrous sodium sulfate, concentrated, column chromatography (elution gradient, ethyl acetate: petroleum ether = 60:40), to obtain 11.6 g of compound LD146-6, with a yield of 81.0%. MS ESI ( m / z ) = 537 [M+H] + .

[0118] Synthesis of compound LD146-7 Compound LD146-6 (11.6 g, 21.6 mmol, 1.0 eq) was dissolved in methanol (116 mL), 1M NaOH aqueous solution (116 mL) was added, stirred for 1 hour, after the reaction was completed, 1M HC1 aqueous solution was added to adjust the pH to neutral, concentrated, extracted twice with ethyl acetate, and dried, concentrated to obtain 10.4 g of yellow solid compound LD146-7. Yield 92.0%. MS ESI ( m / z ) = 523 [M+H] + .

[0119] Synthesis of compound LD146-13 Synthesis of compound LD146-9 Compound LD146-8 (1.6 g, 4.03 mmol, 1.0 eq) was dissolved in dichloromethane (16 mL), HATU (2.3 g, 6.04 mmol, 1.5 eq), DIEA (1.56 g, 12.09 mmol, 3.0 eq) were added, stirred for 10 minutes, L-isoleucine hydrochloride (0.74 g, 4.43 mmol, 1.1 eq) was added, replaced with nitrogen for 3 times, stirred at room temperature for 3 hours. After the reaction was completed, purified water (20 mL) was added for washing, anhydrous sodium sulfate was added for drying, concentrated to obtain 3 g of crude compound LD146-9 for the next step. MS ESI ( m / z ) = 510 [M+H] + .

[0120] Synthesis of compound LD146-10 Compound LD146-9 (3 g, 1.0 eq) crude was dissolved in DMF (5.8 mL), tetrahydro pyrrole (0.75 g, 10.56 mmol, 2.0 eq) was added, stirred for 1 hour, after the reaction was completed, concentrated to obtain 6 g of crude compound LD146-10 for the next step. MS ESI ( m / z ) = 288 [M+H] + .

[0121] Synthesis of compound LD146-12 Fmoc-L-aspartic acid beta-t-butyl ester (2.57 g, 6.25 mmol, 1.2 eq) was dissolved in DMF (16 mL), HATU (2.98 g, 7.84 mmol, 1.5 eq), DIEA (1.35 g, 10.45 mmol, 2.0 eq) were added, stirred for 10 min, compound LD146-10 (3 g, 1.0 eq) crude was added, replaced by nitrogen for 3 times, stirred at room temperature for 3 hours. After the reaction was completed, column chromatography purification (elution gradient, acetonitrile: water = 50:50) to obtain 520 mg of compound LD146-12, with a yield of 12.2%. MS ESI ( m / z ) = 681 [M+H] + .

[0122] Synthesis of compound LD146-13 Compound LD146-12 (520 mg, 0.76 mmol, 1.0 eq) was dissolved in DCM (5.2 mL), tetrahydro-pyrrole (108 mg, 1.53 mmol, 2.0 eq) was added, stirred for 1 hour, after the reaction was completed, concentrated, added with 3 mL of ethyl acetate, filtered, and the filter cake was dried to obtain 200 mg of compound LD146-13, with a yield of 57.3%. MS ESI ( m / z ) = 458 [M+H] + .

[0123] Synthesis of compound LD146 Synthesis of compound LD146-14 Compound LD146-7 (600 mg, 1.2 mmol, 1.05 eq) was dissolved in DMF (12 mL), HATU (649 mg, 1.71 mmol, 1.5 eq), DIEA (441 mg, 3.42 mmol, 3.0 eq) were added, stirred for 10 min, compound LD146-17 (289 mg, 29.4 mmol, 1.1 eq) was added, replaced by nitrogen for 3 times, stirred at room temperature for 3 hours. After the reaction was completed, added with 20 mL of ethyl acetate and 20 mL of purified water for washing, the organic phase was washed with saturated sodium chloride aqueous solution for 3 times, added with anhydrous sodium sulfate for drying, concentrated, and column chromatography purification (elution gradient, acetonitrile: water = 60:40) to obtain 580 mg of compound LD146-14, with a yield of 69.1%. MS ESI ( m / z ) = 733 [M+H] + .

[0124] (1-3-2) Synthesis of compound LD146-15 Compound LD146-14 (580 mg, 0.79 mmol, 1.0 eq) was dissolved in methanol (5.8 mL), 1 M NaOH aqueous solution (5.8 mL) was added, stirred for 1 hour, after the reaction was completed, 1 M HCl aqueous solution was added to adjust the pH to neutral, concentrated, dichloromethane was added, and filtered, concentrated to obtain 400 mg of yellow solid compound LD146-15. Yield 70.3%. MS ESI ( m / z ) = 719 [M+H] + .

[0125] (1-3-3) Synthesis of compound LD146-16 Compound LD146-15 (170 g, 0.24 mmol, 1.0 eq) was dissolved in DMF (1.7 mL), HATU (134.8 mg, 0.35 mmol, 1.5 eq) and DIEA (61 mg, 0.47 mmol, 2.0 eq) were added, stirred for 10 minutes, compound LD146-13 (108 mg, 0.24 mmol, 1.0 eq) was added, replaced with nitrogen for 3 times, stirred at room temperature for 3 hours. After the reaction was completed, column chromatography purification (elution gradient, acetonitrile: water = 70:30) to obtain 160 mg of compound LD146-16, yield 58.4%. MS ESI ( m / z ) = 1160 [M+H] + .

[0126] (1-3-4) Synthesis of compound LD146 Compound LD146-16 (22 mg, 0.019 mmol, 1.0 eq) was dissolved in DCM (2 mL), 1 drop of trifluoroacetic acid was added, stirred for 1 hour, after the reaction was completed, concentrated, added dichloromethane 2 mL for 3 times, freeze-dried to obtain 20 mg of compound LD146, yield 96%. MS ESI ( m / z ) = 1103 [M+H] + .

[0127] Preparation Example 2 Preparation of compound LD147 The synthesis route of compound LD147 is as follows: (2-1) Synthesis of compound LD147-10 (2-1-1) Synthesis of compound LD147-10-3 Fmoc-Pbf-arginine (5.0 g, 7.7 mmol, 1.0 eq) was dissolved in dichloromethane (50 mL) in reaction kettle A, HATU (4.4 g, 11.58 mmol, 1.5 eq) was added and stirred for 20 minutes, L-isoleucine hydrochloride (1.41 g, 8.49 mmol, 1.1 eq), DIEA (4.97 g, 38.53 mmol, 5.0 eq) and dichloromethane (50 mL) were added in reaction kettle B, stirred for 20 minutes, the mixture in reaction kettle B was added to reaction kettle A, stirred at 25°C for 2 hours. After the reaction was completed, a purified water solution (100 mL) was added and washed once, dried over anhydrous sodium sulfate, and filtered, concentrated, and column chromatography purified (elution gradient, acetonitrile: water = 7:3) after adding a small amount of DMF to obtain 5.8 g of yellow solid compound LD147-10-3, with a yield of 98.9%. MS ESI (m / z) = 761 [M+H] m / z ). + .

[0128] (2-1-2) Synthesis of compound LD147-10-4 Compound LD147-10-3 (5.8 g, 7.63 mmol, 1.0 eq) was dissolved in dichloromethane (58 mL), piperidine (1.29 g, 15.18 mmol, 2.0 eq) was added, and nitrogen was replaced for 3 times, stirred at 25°C for 1 hour. After the reaction was completed, it was concentrated and column chromatography (elution gradient, MeOH: DCM = 12:88) to obtain 3.47 g of white solid compound LD147-10-4, with a yield of 84.6%. MS ESI (m / z) = 539 [M+H] m / z ). + .

[0129] (2-1-3) Synthesis of compound LD147-10-6 Fmoc-L-Aspartic acid beta-t-butyl ester (2.91 g, 7.09 mmol, 1.1 eq) was dissolved in DMF (35 mL), HATU (3.67 g, 9.67 mmol, 1.5 eq), DIEA (1.66 g, 12.9 mmol, 2 eq) were added, stirred for 10 min, compound LD147-10-4 (3.47 g, 6.45 mmol, 1.0 eq) was added, stirred at 25 °C for 3 h. After the reaction was completed, purified water solution (50 mL) was added, extracted with ethyl acetate (2 x 50 mL), the organic phase was washed with saturated brine (3 x 50 mL), dried over anhydrous sodium sulfate, and filtered, concentrated, and purified by column chromatography (elution gradient, ethyl acetate: petroleum ether = 100:0) to give 5.6 g of yellow solid compound LD147-10-6, with a yield of 93.3%. MS ESI ( m / z ) = 839 [M+H] + .

[0130] Synthesis of compound LD147-10 Compound LD147-10-6 (5.6 g, 6.02 mmol, 1 eq) was dissolved in dichloromethane (56 mL), tetrahydro-pyrrole (854 mg, 12.03 mmol, 2 eq) was added, replaced with nitrogen for three times, the reaction liquid was stirred at 25 °C for 1 h under nitrogen atmosphere. After the reaction was completed, concentrated, and purified by column chromatography (elution gradient, methanol:dichloromethane = 8:92) to give 3.6 g of white solid LD147-10, with a yield of 84.3%. MS ESI ( m / z ) = 710 [M+H] + .

[0131] Synthesis of compound LD147 Synthesis of compound LD147-3 Compound LD146-2 (1.94 g, 4.34 mmol, 1.0 eq) was dissolved in DMF (10 mL), compound LD147-2 (886.6 mg, 5.21 mmol, 1.2 eq), potassium carbonate (1.8 g, 13.0 mmol, 3.0 eq) were added, replaced with nitrogen for 3 times, stirred at 100 °C for 16 hours. After the reaction was completed, it was cooled to room temperature, saturated aqueous ammonium chloride solution (50 mL) was added, extracted with ethyl acetate (30 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 1: 1) to obtain 1.34 g of compound LD147-3, with a yield of 76.1%. MS ESI ( m / z ) = 406.2 [M+H] + .

[0132] Synthesis of compound LD147-4 Compound LD147-3 (1.34 g, 3.31 mmol, 1.0 eq) was dissolved in tetrahydrofuran (7 mL), water (7 mL), lithium hydroxide (158.1 mg, 6.62 mmol, 2.0 eq) were added, and stirred at 25 °C for 2 hours. After the reaction was completed, 1M aqueous HCl solution was added to pH = 2~3, and the solvent was concentrated to obtain 1.3 g of crude compound LD147-4. MS ESI ( m / z ) = 392.3 [M+H] + .

[0133] Synthesis of compound LD147-5 The crude compound LD147-4 (1.3 g, 3.3 mmol, 1.0 eq) was dissolved in DMF (13 mL), HATU (1.91 g, 4.95 mmol, 1.5 eq), DIEA (1.39 g, 9.9 mmol, 3.0 eq) were added, stirred at 25 °C for 30 minutes, methyl p-amino phenylacetate (1.3 g, 3.99 mmol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25 °C for 3 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (70 mL) was added, extracted with ethyl acetate (30 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated, purified by column chromatography (elution gradient, petroleum ether: ethyl acetate = 9: 1) to obtain 1.41 g of compound LD147-5, with a yield of 79.2%. MS ESI ( m / z) = 539.4 [M+H] + .

[0134] (2-2-4) Synthesis of compound LD147-6 Compound LD147-5 (680 mg, 1.26 mmol, 1.0 eq) was dissolved in tetrahydrofuran (3.5 mL), water (3.5 mL), lithium hydroxide aqueous solution (947.6 uL, 4 M, 3.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M HCl aqueous solution was added to pH = 2~3, concentrated to remove the solvent to obtain 670 mg of crude compound LD147-6. MS ESI ( m / z ) = 525.2 [M+H] + .

[0135] (2-2-5) Synthesis of compound LD147-8 The crude compound LD147-6 (670 mg, 1.28 mmol, 1.0 eq) was dissolved in DMF (7 mL), HATU (720.5 g, 1.92 mmol, 1.5 eq), DIEA (526.9 mg, 3.84 mmol, 3.0 eq) was added, stirred at 25°C for 30 minutes, compound LD146-17 (346.4 mg, 1.54 mmol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 16 hours. After the reaction was completed, saturated ammonium chloride aqueous solution (35 mL) was added, extracted with ethyl acetate (20 mL x 3), washed with saturated sodium chloride aqueous solution (10 mL x 3), dried with anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 1:1) to obtain 263 mg of compound LD147-8, with a yield of 28.3%. MS ESI ( m / z ) =735.4 [M+H] + .

[0136] (2-2-6) Synthesis of compound LD147-9 LD147-8 (263 mg, 358.1 umol, 1.0 eq) was dissolved in tetrahydrofuran (1.5 mL), water (1.5 mL), lithium hydroxide aqueous solution (268.6 uL, 4 M, 3.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M HCl aqueous solution was added to pH = 2~3, concentrated to remove the solvent to obtain 257 mg of crude compound LD147-9. MS ESI ( m / z) = 721.3 [M+H] + .

[0137] Synthesis of compound LD147-11 The crude compound LD147-9 (260 mg, 356.7 umol, 1.0 eq) was dissolved in DMF (3 mL), HATU (201.9 g, 535.2 umol, 1.5 eq) and DIEA (147.7 mg, 1.07 mmol, 3.0 eq) were added, stirred at 25°C for 30 minutes, compound LD147-10 (301.7 mg, 428.0 umol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 16 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (15 mL) was added, extracted with ethyl acetate (10 mL x 3), washed with saturated aqueous sodium chloride solution (5 mL x 3), dried over anhydrous sodium sulfate, concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 3:2) to obtain 194 mg of compound LD147-11, with a yield of 38.4%. MS ESI ( m / z ) = 1412.8 [M+H] + .

[0138] Synthesis of compound LD147 The compound LD147-11 (35 mg, 24.8 umol, 1.0 eq) was dissolved in trifluoroacetic acid (1 mL), 1 drop of water was added, and stirred at 25°C for 16 hours. After the reaction was completed, the solvent was removed by concentration, and 27 mg of crude compound LD147 was obtained by freeze-drying. MS ESI ( m / z ) = 1104.6 [M+H] + .

[0139] Preparation of compound LD148 The synthesis route of compound LD148 is as follows: Synthesis of compound LD148-3 Compound LD148-2 (960 mg, 4.72 mmol, 1.0 eq) was dissolved in DMF (10 mL), compound LD146-2 (2.11 g, 5.67 mmol, 1.2 eq), potassium carbonate (1.95 g, 14.2 mmol, 3.0 eq) were added, replaced with nitrogen for 3 times, stirred at 100 °C for 16 hours. After the reaction was completed, it was cooled to room temperature, saturated aqueous ammonium chloride solution (50 mL) was added, extracted with ethyl acetate (30 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 1: 1) to obtain 1.79 g of compound LD148-3 with a yield of 93.7%. MS ESI ( m / z ) = 405.2 [M+H] + .

[0140] Synthesis of compound LD148-4 Compound LD148-3 (1.69 g, 4.18 mmol, 1.0 eq) was dissolved in tetrahydrofuran (8.5 mL), water (8.5 mL), lithium hydroxide aqueous solution (3.14 mL, 4 M, 3.0 eq) were added, and stirred at 25 °C for 2 hours. After the reaction was completed, 1M aqueous HCl solution was added to pH = 2~3, and the solvent was concentrated to obtain 1.6 g of crude compound LD148-4. MS ESI ( m / z ) = 391.2 [M+H] + .

[0141] Synthesis of compound LD148-5 The crude compound LD148-4 (1.6 g, 4.1 mmol, 1.0 eq) was dissolved in DMF (16 mL), HATU (2.38 g, 6.15 mmol, 1.5 eq), DIEA (1.74 g, 12.3 mmol, 3.0 eq) were added, stirred at 25 °C for 30 minutes, methyl p-amino phenylacetate (827.9 mg, 4.92 mmol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25 °C for 3 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (80 mL) was added, extracted with ethyl acetate (40 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 7:3) to obtain 615 mg of compound LD148-5 with a yield of 30.0%. MS ESI (m / z ) =538.2 [M+H] + .

[0142] Synthesis of compound LD148-6 Compound LD148-5 (515 mg, 958.6 umol, 1.0 eq) was dissolved in tetrahydrofuran (2.5 mL), water (2.5 mL), lithium hydroxide aqueous solution (719.0 uL, 4 M, 3.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M HCl aqueous solution was added to pH = 2~3, concentrated to remove the solvent to obtain 500 mg of crude compound LD148-6. MS ESI ( m / z ) = 524.3 [M+H] + .

[0143] Synthesis of compound LD148-8 The crude compound LD148-6 (500 mg, 955.7 umol, 1.0 eq) was dissolved in DMF (5 mL), HATU (546.7 g, 1.43 mmol, 1.5 eq), DIEA (399.8 mg, 2.87 mmol, 3.0 eq) was added, stirred at 25°C for 30 minutes, compound LD146-17 (262.8 mg, 1.15 mmol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 16 hours. After the reaction was completed, saturated ammonium chloride aqueous solution (25 mL) was added, extracted with ethyl acetate (20 mL x 3), washed with saturated sodium chloride aqueous solution (10 mL x 3), dried with anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 3:2) to obtain 323 mg of compound LD148-8, with a yield of 38.5%. MS ESI ( m / z ) =734.3 [M+H] + .

[0144] Synthesis of compound LD148-9 Compound LD148-8 (323 mg, 440.5 umol, 1.0 eq) was dissolved in tetrahydrofuran (1.6 mL), water (1.6 mL), lithium hydroxide aqueous solution (330 uL, 4 M, 3.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M HCl aqueous solution was added to pH = 2~3, concentrated to remove the solvent to obtain 316 mg of crude compound LD148-9. MS ESI ( m / z ) = 720.3 [M+H]+ .

[0145] Synthesis of compound LD148-11 The crude compound LD148-9 (316 mg, 439.3 umol, 1.0 eq) was dissolved in DMF (3 mL), HATU (251.1 g, 658.9 umol, 1.5 eq), DIEA (183.7 mg, 1.32 mmol, 3.0 eq) were added, stirred at 25°C for 30 minutes, compound LD147-10 (375.2 mg, 527.2 umol, 1.2 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 16 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (15 mL) was added, extracted with ethyl acetate (10 mL x 3), washed with saturated aqueous sodium chloride solution (5 mL x 3), dried over anhydrous sodium sulfate, concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 3:2) to obtain 370 mg of compound LD148-11, with a yield of 59.6%. MS ESI ( m / z ) = 1411.7 [M+H] + .

[0146] Synthesis of compound LD148 Compound LD148-11 (40 mg, 28.4 umol, 1.0 eq) was dissolved in trifluoroacetic acid (1 mL), 1 drop of water was added, and stirred at 25°C for 16 hours. After the reaction was completed, the solvent was removed by concentration, and 35 mg of crude compound LD148 was obtained by freeze-drying. MS ESI ( m / z ) = 1103.5 [M+H] + .

[0147] Preparation of compound LD149 The synthesis route of compound LD149 is as follows: Synthesis of compound LD149-3 Compound LD149-2 (1 g, 4.95 mmol, 1.0 eq) was dissolved in DMF (10 mL), compound LD146-2 (2 g, 5.36 mmol, 1.1 eq), potassium carbonate (2 g, 14.4 mmol, 3.0 eq) were added, replaced with nitrogen for 3 times, stirred at 100°C for 16 hours. After the reaction was completed, it was cooled to room temperature, saturated aqueous ammonium chloride solution (50 mL) was added, extracted with ethyl acetate (30 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried by adding anhydrous sodium sulfate, concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 1:1) to obtain 1.6 g of compound LD149-3, with a yield of 74.7%. MS ESI ( m / z ) = 403.2 [M+H] + .

[0148] Synthesis of compound LD149-4 Compound LD149-3 (1.6 g, 3.97 mmol, 1.0 eq) was dissolved in methanol (8.5 mL), water (8.5 mL), lithium hydroxide aqueous solution (7.94 mL, 2 M, 4.0 eq) were added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M aqueous HCl solution was added to pH = 2~3, and the solvent was concentrated to obtain 1.6 g of crude compound LD149-4. MS ESI ( m / z ) = 389.2 [M+H] + .

[0149] Synthesis of compound LD149-6 The crude compound LD149-4 (1.5 g, 3.85 mmol, 1.0 eq) was dissolved in DMF (16 mL), HATU (2.2 g, 5.78 mmol, 1.5 eq), DIEA (1 g, 7.75 mmol, 2.0 eq) were added, stirred at 25°C for 30 minutes, methyl p-amino phenylacetate (700 mg, 4.24 mmol, 1.1 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 3 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (80 mL) was added, extracted with ethyl acetate (40 mL x 3), washed with saturated aqueous sodium chloride solution (20 mL x 3), dried by adding anhydrous sodium sulfate, concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 7:3) to obtain 1 g of compound LD149-6, with a yield of 50.0%. MS ESI ( m / z ) = 536.2 [M+H] + .

[0150] Synthesis of compound LD149-7 Compound LD149-6 (1 g, 1.86 mmol, 1.0 eq) was dissolved in methanol (2.5 mL), water (2.5 mL), lithium hydroxide aqueous solution (1.86 mL, 2M, 2.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M aqueous HCl solution was added to pH = 2~3, and the solvent was concentrated to obtain 800 mg of crude compound LD149-7. MS ESI ( m / z ) = 522.3 [M+H] + .

[0151] Synthesis of compound LD149-9 The crude compound LD149-7 (400 mg, 0.77 mmol, 1.0 eq) was dissolved in DMF (5 mL), HATU (440.7 g, 1.15 mmol, 1.5 eq), DIEA (200 mg, 1.55 mmol, 2.0 eq) was added, stirred at 25°C for 30 minutes, compound LD146-17 (200 mg, 0.85 mmol, 1.1 eq) was added, replaced with nitrogen for 3 times, stirred at 25°C for 16 hours. After the reaction was completed, saturated aqueous ammonium chloride solution (25 mL) was added, extracted with ethyl acetate (20 mL x 3), washed with saturated aqueous sodium chloride solution (10 mL x 3), dried with anhydrous sodium sulfate, filtered and concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 3:2) to obtain 340 mg of compound LD149-9, with a yield of 60.7%. MS ESI ( m / z ) = 732.3 [M+H] + .

[0152] Synthesis of compound LD149-10 Compound LD149-9 (340 mg, 464.5 umol, 1.0 eq) was dissolved in methanol (1.6 mL), water (1.6 mL), lithium hydroxide aqueous solution (464 uL, 2 M, 2.0 eq) was added, and stirred at 25°C for 2 hours. After the reaction was completed, 1M aqueous HCl solution was added to pH = 2~3, and the solvent was concentrated to obtain 300 mg of crude compound LD149-10. MS ESI ( m / z ) = 718.3 [M+H] + .

[0153] Synthesis of compound LD149-12 The crude compound LD149-10 (70 mg, 88.6 umol, 1.1 eq) was dissolved in DMF (3 mL), HATU (52.5 mg, 138.1 umol, 1.5 eq), DIEA (35 mg, 0.27 mmol, 2.0 eq) were added, stirred at 25 °C for 30 min, compound LD147-10 (70 mg, 98.6 umol, 1 eq) was added, replaced with nitrogen for 3 times, stirred at 25 °C for 16 h. After the reaction was completed, saturated aqueous ammonium chloride solution (15 mL) was added, extracted with ethyl acetate (10 mL x 3), washed with saturated aqueous sodium chloride solution (5 mL x 3), dried over anhydrous sodium sulfate, concentrated, purified by reverse column chromatography (elution gradient, acetonitrile: water = 3:2) to obtain 50 mg of compound LD149-12, with a yield of 36.2%. MS ESI ( m / z ) = 1409.7 [M+H] + .

[0154] Synthesis of compound LD149 The compound LD149-12 (40 mg, 28.4 umol, 1.0 eq) was dissolved in trifluoroacetic acid (1 mL), 1 drop of water was added, and stirred at 25 °C for 12 h. After the reaction was completed, the solvent was removed by concentration, and 25 mg of crude compound LD149 was obtained by freeze-drying. MS ESI ( Preparation Example 6: Preparation of LD110 ) = 1101.5 [M+H] + .

[0155] Preparation of compound NM064 The synthesis route of compound NM064 is as follows: The specific synthesis steps of NM064 were prepared according to the related examples in the domestic invention patent CN119019354A first disclosed by the applicant on November 26, 2024.

[0156] Figure 1 LD110: N3-PEG2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-CONH2 Fmoc solid-phase peptide synthesis method was used, and amino acid monomers were connected one by one from the carboxyl end to the amino end according to the amino acid sequence.

[0157] The amino acid monomer of Ile is Fmoc-Ile-OH; The amino acid monomer of Arg is Fmoc-Arg(Pbf)-OH; The amino acid monomer of Asp is Fmoc-Asp(OtBu)-OH; The amino acid monomer of Pro is Fmoc-Pro-OH; The amino acid monomer of DPro is Fmoc-D-Pro-OH; The amino acid monomer of Gly is Fmoc-Gly-OH; The amino acid monomer of DAla is Fmoc-D-Ala-OH; The amino acid monomer of Cha is Fmoc-Cha-OH; The amino acid monomer of Ser is Fmoc-Ser(tBu)-OH; The amino acid monomer of DPro is Fmoc-D-Pro-OH.

[0158] In the preparation example, the specific preparation method of the targeted delivery ligand includes the following steps: (6-1) Take the resin Fmoc-Linker-MBHA Resin (substitution degree about 0.5 mmol / g, total 1.0 mmol reaction sites) 2.0 g, and swell with N,N-dimethylformamide (DMF) for 20 minutes; then, add 3 times the resin volume of 20% Pip / DMF mixed solution (i.e. the volume ratio of Piperazine and DMF is 1:4), and blow nitrogen for 30 minutes, dry and wash (wash with 2 times the resin volume of DMF for 5 times), to obtain H2N-Linker-MBHA Resin.

[0159] (6-2) 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 resin volume of DMF for 3 times), to obtain Fmoc-Ile (Boc)-Liner-MBHA Resin; then, add 3 times the resin volume of 20% Pip / DMF mixed solution, blow nitrogen for 30 minutes, dry and wash (wash with 2 times the resin volume of DMF for 5 times), to obtain H2N-Ile(Boc)-Liner-MBHA Resin; After that, step (6-2) 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: DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(OtBu)-Arg(Pbf)--Ile-Liner-MBHA Resin; (6-3) 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 wash 3 times) to obtain the following compound: N3-PEG2-CH2CH2CO-DPhe-Ser(tBu)-DPro-Cha-DAla-Gly-Pro-Ile-Asp(EDANS)-Arg(Pbf)-Ile-Liner-MBHA Resin; (6-4) 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 ratio of 87.5:5:2.5:2.5:2.5), shake bed for 2 hours, filter out the resin, precipitate the filtrate with anhydrous ether, and wash the precipitate with anhydrous ether 3 times. Finally, the precipitate is placed in a vacuum drying oven and dried at room temperature for 24 hours. HPLC purification, respectively, to obtain more than 95% purity of the targeted delivery ligand: LD110: N3-PEG2-CH2CH2CO-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-CONH 2。

[0160] Preparation Example 7 Synthesis of siRNA conjugate (7-1) Synthesis of the sense strand (SS) By the method of phosphoramidite nucleic acid solid phase synthesis, the nucleotide sequence is connected one by one in the direction of 3'-5'. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows: The nucleotide monomers are prepared into a 0.1M nucleotide monomer acetonitrile solution.

[0161] The conditions of deprotection reaction of each step are the same. The deprotection reaction conditions: temperature is 25℃, reaction time is 70 seconds, deprotection reagent is dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0162] The conditions of each coupling reaction are the same. The conditions of the coupling reaction are as follows: the temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is 5-ethylthio-1H-tetrazole in acetonitrile with a concentration of 0.5M, and the thio reagent is xanthane in acetonitrile / pyridine mixed solution with a concentration of 0.2M.

[0163] The conditions of each capping reaction are the same. The conditions of the capping reaction are as follows: the temperature is 25°C; the reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is N-methylimidazole in pyridine / acetonitrile mixed solution with a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is acetic anhydride in acetonitrile solution with a concentration of 20% by volume; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence connected to the solid support is 1:1:1.

[0164] The conditions of each oxidation reaction are the same. The conditions of the oxidation reaction are as follows: the temperature is 25°C; the reaction time is 3 seconds; the concentration of the oxidation reagent is 0.05M iodine water, and the molar ratio of iodine to the nucleic acid sequence connected to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions of the sulfurization reaction are as follows: the temperature is 25°C; the reaction time is 360 seconds; the concentration of the thio reagent is 0.2M xanthane in pyridine solution, and the molar ratio of the thio reagent to the nucleic acid sequence connected to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0165] After the connection of the last nucleoside monomer is completed, the nucleic acid sequence connected to the solid support is sequentially subjected to cleavage, deprotection, purification, desalting, and then freeze-drying to obtain the sense strand, wherein: The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid support is added to 25% by mass ammonia water, the amount of ammonia water is 0.5ml / 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, 0.4ml / μmol N-methylpyrrolidine is used to dissolve the product relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3ml / μmol triethylamine and 0.6ml / μmol triethylamine trifluoromethanesulfonate to remove the 2'-O-TBDMS protection on the ribose.

[0166] Purification and desalination: the purification of nucleic acid was completed by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1), eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1), and the elution gradient was eluent 1:eluent 2 = (100:0)-(50:50). After the product eluate was collected, desalination was performed using a reverse-phase chromatography purification column, and the desalination conditions included desalination using a dextran gel column, with dextran gel G25 as the filler and deionized water as the eluent.

[0167] Detection: purity detection was performed using ion exchange chromatography (IEX-HPLC), and molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS). If the measured value of the molecular weight was consistent with the theoretical value, it was indicated that the siRNA sense strand was obtained.

[0168] During the synthesis of the siRNA sense strand, the compound NM064 was regarded as a nucleoside monomer to participate in the synthesis of siRNA and form a Linker structure (NM064).

[0169] The structural formula of (NM064) is: If (NM064) is conjugated to the 5' end of the siRNA sense strand, the structural formula of the siRNA is: ; (7-2) Synthesis of antisense strand (AS) The nucleoside monomers were connected one by one in the order of 3'-5' according to the nucleotide sequence by the method of phosphoramidite nucleic acid solid-phase synthesis. Each connection of a nucleoside monomer included four reactions of deprotection, coupling, capping, oxidation or sulfurization. The reaction conditions of deprotection, coupling, capping, oxidation or sulfurization, the cleavage and deprotection conditions, and the purification and desalination conditions in the solid-phase synthesis method of the antisense strand were the same as those in step (7-1) for the synthesis of the sense strand.

[0170] Detection: purity detection was performed using ion exchange chromatography (IEX-HPLC), and molecular weight detection was performed using liquid chromatography-mass spectrometry (LC-MS). If the measured value of the molecular weight was consistent with the theoretical value, it was indicated that the siRNA antisense strand was obtained.

[0171] (7-3) Synthesis of sense strand conjugate Exemplarily, the ligand LD146 was conjugated to the siRNA sense strand The specific conjugation process is as follows: (7-3-1) Mix 150 μL of H2O, 70 μL of 0.2 M carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) to obtain a mixed solvent that dissolves the RX699293 sense strand group of the unconjugated carrier to obtain a solution of the unconjugated RX699293 sense strand of the carrier at a concentration of 1.0 eq; (7-3-2) Dissolve 5.0 eq of the ligand in 70 μL of DMF, and then dissolve the ligand unit molecule solution in the sense strand solution obtained in step (7-3-1) to obtain a reactant mixture. (7-3-3) Mix 30.0 eq of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 6.0 eq of CuSO4·5H2O at a volume ratio of THPTA:CuSO4·5H2O = 5:1, shake for 5 min at 40°C, and then add 37 μL of the mixture to the reactant mixture obtained in step (7-3-2) and vortex to obtain an intermediate product mixture. The pH of the intermediate product mixture is 8. Then, 25.0 eq of sodium ascorbate is rapidly added to the intermediate product mixture and vortexed to obtain a product mixture, which is reacted at 40°C for 1 h. Dilute 3 μL of the product mixture with a mixed solvent of DMF and H2O (volume ratio of DMF:H2O = 1:5), and then purify the product mixture by HPLC. In the HPLC process, a C18 column is used, and a gradient elution method is used with an ammonium bicarbonate buffer solution as the mobile phase. The product purified by HPLC is freeze-dried to obtain the conjugate product.

[0172] The obtained conjugate is a single strand. For example, the structure of the sense strand conjugate of RG699293 is as follows: The azide group in each of the two LD146 and the two alkyne groups of the siRNA terminal linker structure (NM064) are converted into triazole groups through a click chemistry reaction to achieve covalent linkage of the two ligands LD146 and the siRNA.

[0173] (7-4) Annealing of siRNA Mix the sense strand synthesized in step (7-3) and the antisense strand synthesized in step (7-1) at an equimolar ratio, dissolve in water for injection, and heat to 95°C. Slowly cool to room temperature and maintain at room temperature for 10 min to allow the sense strand and the antisense strand to form a double-stranded structure through hydrogen bonding, thereby obtaining siRNA having the sense strand and the antisense strand shown in Table 6.

[0174] Table 4 siRNA naked sequence information Table 5 modified siRNA sequence information Table 6 siRNA conjugate nucleotide sequence information Unless otherwise specified, the base composition and modification meanings described in each embodiment of the present disclosure are as follows: capital letters A, U, G, C, T represent the base composition of nucleotides, and lowercase letters m represent that the nucleotide represented by the previous letter is a methoxy-modified nucleotide; lowercase letters f represent that the nucleotide represented by the previous letter is a fluorine-modified nucleotide; lowercase letters (moe) represent that the nucleotide represented by the previous letter is a 2'-methoxyethoxy-modified nucleotide; lowercase letters s represent that the nucleotides represented by the previous and subsequent letters are connected by a phosphorothioate bond; VP represents that the 5' end of the antisense strand in the siRNA is modified to 5'-(E)-vinylphosphonate (5'-(E)-VP) modification.

[0175] The structural formula of VPUm is .

[0176] The structural formula of 2'-O-methyl-modified nucleotide is .

[0177] The structural formula of 2'-fluorine-modified nucleotide is .

[0178] The structural formula of 2'-O-methoxyethyl-modified nucleotide is .

[0179] Wherein, Base represents a nucleoside base, and the nucleoside base is selected from A, U, G, C or T.

[0180] The structural formula of LD110 is as follows: Biological detection experiment Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Beijing Xuanjing Ruimeide Technology Co., Ltd.; PCR primer synthesis is entrusted to Beijing Qikexing Biotechnology Co., Ltd.; and experimental animals C57BL / 6J mice are purchased from Spaf Bioscience (Beijing) Biotechnology Co., Ltd.

[0181] Method for evaluating the target gene inhibition activity of siRNA conjugate in mice 6-8 weeks old C57BL / 6J mice (all female) were randomly grouped by weight. The mice in each group were calculated the dose of the drug according to the weight, and the drug was given by abdominal subcutaneous injection. Each siRNA conjugate was respectively configured into a solution of corresponding concentration (calculated by siRNA) 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 in each group were sacrificed respectively. The necropsy of the sacrificed mice was performed, and the target tissues of each sacrificed mouse were collected. The tissues were cut into about 2 mm 3 small pieces and stored in RNA later.

[0182] mRNA expression level detection 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, instant centrifugation, room temperature standing for 10 min, 200 μL chloroform was added, and after oscillation mixing, it was placed at room temperature for 3 min. 4℃, 12000 rpm centrifugation for 10 min. 400 μL supernatant was added to a centrifuge tube containing 400 μL isopropanol, mixed well, and placed at room temperature for 10 min. 4℃, 12000 rpm centrifugation for 10 min, discard the supernatant. Add 1 mL 75% ethanol, invert the centrifuge tube, wash the precipitate. 4℃, 12000 rpm centrifugation for 5 min, remove the supernatant, dry at room temperature, and extract the total RNA.

[0183] The above 1 μg total RNA, using reverse transcription kit (Thermo Fisher Scientific Company, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 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, 80 μL RNase-Free water was added to the reverse transcription system to obtain cDNA solution. Then use real-time fluorescence quantitative PCR kit (ABI company, SYBR™ 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 target genes and the primers for internal reference genes are used to detect target genes and internal reference genes, respectively. According to the method recorded in the real-time fluorescence quantitative PCR kit instruction book, 20 μL Real-time PCR reaction system was configured for each PCR detection hole, each reaction system contained 5 μL cDNA solution obtained by the above reverse transcription reaction, 10 μL SYBR™ 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 was placed on a real-time fluorescence quantitative PCR instrument (ABI company, StepOnePlus™), and the Real-time PCR amplification was carried out by using three-step method, and the amplification program was 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 was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to calculate the expression level and inhibition rate of target gene mRNA in each test group, and the calculation method was as follows: ΔCt(test group) = Ct(test group target gene) - Ct(test group internal reference gene) ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene) ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average) Wherein, ΔCt(control group average) is the arithmetic mean of ΔCt(control group) of each of the 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.

[0184] Test group target gene mRNA relative expression level = 2 -ΔΔCt( Test group) x 100% The target gene mRNA expression level of the test group was normalized based on the control group, and the target gene mRNA expression level of the control group was defined as 100%.

[0185] Test group target gene mRNA expression inhibition rate (%) = 1 - test group target gene mRNA relative expression level Unless otherwise specified, the in vivo activity experiment data are expressed as X ± STDEV, and the experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0186] Example 1 Evaluation of the inhibitory activity of siRNA conjugates on the target gene Superoxide dismutase 1 protein (SOD1) in mice In this example, the inhibitory activity of LD110, LD146, LD147, LD148, and LD149 conjugated siRNA sequences RG699293, RG699294, RG699295, RG699296 on the target gene SOD1 in mice was evaluated using the target gene inhibitory activity evaluation method in mice.

[0187] 6-8 week old C57BL / 6j mice were randomly divided into 6 groups according to body weight, 5 mice in each group, and each group of mice was given the above siRNA conjugates 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. The drug was administered for 3 consecutive days. The day of administration was recorded as day 0 (D0), and 5 mice in each group were sacrificed on day 14 (D16) after the last administration. The animals were subjected to gross dissection, and the renal cortex was collected and cut into several 2 mm 3 RNA extraction, Real-time PCR detection was as described above, and gene expression difference was calculated by ΔΔCt method.

[0188] Table 7 Primer sequence table The results of Example 1 showed that the siRNA sequences conjugated with LD146, LD147, LD148, and LD149 all had inhibitory effects on the target gene in the renal cortex compared with the control group. ​ , Table 8 Table 8 Inhibitory activity of target genes in mice after administration of siRNA conjugates described in this example The above detailed description is merely illustrative of the content of the present application and does not represent the limitation of the content of the present application. It is conceivable for those skilled in the art that the specific structure of the present application can have other variations.

Claims

1. A polypeptide ligand mediating targeting of the lina peptide receptor, characterized in that, The polypeptide ligand comprises a polypeptide consisting of 4-20 amino acid residues and a linker or bond for linking the polypeptide to a pharmaceutically active molecule; the natriuretic peptide receptor is selected from at least one of natriuretic peptide receptor A, natriuretic peptide receptor B or natriuretic peptide receptor C; Preferably, the natriuretic peptide receptor is selected from natriuretic peptide receptor C; Optionally, the polypeptide comprises 5-15 amino acid residues; further, the polypeptide comprises 7-13 amino acid residues; Optionally, the pharmaceutically active molecule is selected from an oligonucleotide.

2. The polypeptide ligand of claim 1, wherein The polypeptide ligand comprises consecutive three amino acid residues in the amino acid sequence -Arg-Ile-Asp-Arg- or -Ile-Asp-Arg-Ile-, or consecutive three amino acid residues in an amino acid sequence that differs from any of the above sequences by 1 amino acid residue; wherein each amino acid residue is independently a natural or modified amino acid residue, which can be in D-form or L-form; wherein the modified amino acid residue refers to one or more of side chain modification, terminal group modification or chiral isomerization; the modification is substitution of a functional group or H of the side chain of the amino acid residue with a modifying group selected from one of substituted or unsubstituted C1-C6 alkyl (preferably C1-C3 alkyl), cycloalkyl, aryl or heteroaryl; optionally, the modification is an alkylation modification, and the modifying group is selected from methyl, ethyl or propyl; Optionally, the Arg comprises an alkylated arginine residue, such as a methylated arginine; Optionally, the polypeptide ligand comprises the amino acid sequence -Arg-Ile-Asp-Arg-; Optionally, the polypeptide ligand comprises the amino acid sequence -Ile-Asp-Arg- Ile-; Optionally, the polypeptide ligand comprises the amino acid sequence -Ile-Asp-Arg(Me)-Ile-; Arg(Me) is a methylated arginine residue; Optionally, the polypeptide ligand comprises -Arg-Ile-Asp-Arg-Ile-; Optionally, the polypeptide ligand has the amino acid sequence shown as follows from N-terminus to C-terminus: - P1-Ile-Asp-Ra-Ile-P2- wherein Ra is selected from Arg, methylated arginine Arg(Me), or dimethylated arginine; P1 represents an amino acid sequence consisting of 5-10 amino acid residues, and P2 represents any amino acid residue or is absent; Optionally, P1 represents an amino acid sequence consisting of 6-7 amino acid residues; Further optionally, the polypeptide ligand further comprises the amino acid sequence -Cha-Z-Gly-, Z being any amino acid residue. The polypeptide ligand comprises consecutive 4 amino acid residues in an amino acid sequence that differs from the amino acid sequence -Ile-Asp-Arg-Ile-Gly- by no more than 1 or 2 residue difference; 3. The polypeptide ligand according to claim 1 or 2, characterized in that, Optionally, the polypeptide ligand is selected from any one of the following 1) - 5): ​ 1) the polypeptide ligand comprises 4 consecutive amino acid residues in an amino acid sequence that differs by no more than 1 residue from the amino acid sequence -AA1-Ile-Asp-Arg-Ile-, wherein AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp; preferably, the polypeptide comprises an amino acid sequence of -Arg-Ile-Asp-Arg-Ile-, -Pro-Ile-Asp-Arg-Ile- or -Hyp-Ile-Asp-Arg-Ile-; further, the polypeptide ligand comprises a structure as shown below: -Arg-Ile-Asp-Arg-Ile-NH2; 2) the polypeptide ligand comprises 4 or 5 consecutive amino acid residues in an amino acid sequence that differs by no more than 1 residue from the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly-; preferably, the polypeptide ligand comprises any 4 consecutive amino acid residues in the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly-; further, the polypeptide ligand comprises any 5 consecutive amino acid residues in the amino acid sequence -AA1-Ile-Asp-Arg-Ile-Gly-; wherein AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp; 3) the polypeptide ligand comprises 5 or 6 consecutive amino acid residues in an amino acid sequence that differs by no more than 1 residue from the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-AA2-, wherein AA2 is selected from a Gly residue or is absent; preferably, the polypeptide ligand comprises any 5 consecutive amino acid residues in the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-Gly-; further, the polypeptide ligand comprises any 6 consecutive amino acid residues in the amino acid sequence -Gly-AA1-Ile-Asp-Arg-Ile-Gly-; wherein AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp; Optionally, the polypeptide ligand comprises the amino acid sequence -Gly-Arg-Ile-Asp-Arg-Ile-Gly-; 4) the polypeptide ligand comprises 6 or 7 consecutive amino acid residues in an amino acid sequence that differs by no more than 1 residue difference from the amino acid sequence -Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2-, wherein AA0 is selected from any one of the amino acid residues Gly, Ala or Ser, AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp, and AA2 is selected from a Gly residue or is absent; preferably, the polypeptide ligand comprises 7 consecutive amino acid residues in the amino acid sequence -Cha-AA0-Gly-Gly-AA1-Ile-Asp-Arg-Ile-AA2-; further preferably, the polypeptide ligand comprises 7 consecutive amino acid residues in the amino acid sequence -Cha-AA0-Gly-Gly-AA1-Ile-Asp-Arg-Ile-AA2- and comprises at least three consecutive amino acid residues in the -Ile-Asp-Arg-Ile- sequence; 5) the polypeptide ligand comprises 7 or 8 consecutive amino acid residues in an amino acid sequence that differs by no more than 1 residue difference from the amino acid sequence -X1-Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2-, wherein AA0 is selected from any one of the amino acid residues Gly, Ala or Ser, AA1 is selected from any one of the amino acid residues Arg, Pro or Hyp, and AA2 is selected from a Gly residue or is absent; X1 is a chemical bond or a sequence of 1-5 amino acid residues, preferably selected from: a chemical bond, Cys, Ser, Ala, Gly, or any one of the amino acid sequences -A1-A2-A3-A4-A5-, wherein A1 is selected from Arg or is absent, A2 is selected from Arg, Ser or is absent, A3 is selected from Phe, Ser or is absent, A4 is selected from Ser or is absent, and A5 is selected from Cys, Ser, Ala, Gly, Pro, Hyp or is absent; further optionally, Pro, Hyp are selected from the D-configuration; preferably, the polypeptide comprises 8 consecutive amino acid residues in the amino acid sequence -X1-Cha-AA0-Gly-AA1-Ile-Asp-Arg-Ile-AA2- and comprises at least three consecutive amino acid residues in the -Ile-Asp-Arg-Ile- sequence; wherein each amino acid residue is independently a natural or modified amino acid residue, which can be selected from the L or D configuration; wherein the Arg in the -Ile-Asp-Arg-Ile- sequence is selected from an unmodified Arg or a methylated modified Arg, the methylation modification comprises a methyl group attached to the N atom at the end of the side chain of arginine, which can be mediated by protein arginine methyltransferases (PRMTs).

4. The polypeptide ligand according to claims 1 to 3, characterized in that, from N- to C-terminus, the polypeptide ligand has the structure shown below: Z1-X1-Cha-AA0-Gly-AA1-Ile-Asp-Ra-Ile-AA2-Z2 wherein Ra is selected from Arg or methylated arginine Arg (Me), or dimethylated arginine; Z1, Z2 represent a capping group of the terminal amino acid of the polypeptide (carboxy terminal or amino terminal) or a covalent attachment site to an active molecule, Optionally, Z1 is selected from any of H, an amino protecting group or an attachment site, and Z2 is selected from any of H, a carboxyl protecting group or an attachment site, or at least one of Z1 and Z2 is an attachment site; Optionally, Z1 is a capping group of the amino terminal (N-terminal) of the polypeptide, which can be preferably selected from H, acyl, acetyl, acetyl of a hydroxyl substituent (HOCH2CO-) or an attachment site; Z2 is a capping group of the carboxyl terminal (C-terminal) of the polypeptide, which is selected from a covalent attachment site, an amide or a substituted amide, preferably from -CONH2 or -CONHCH3; Further optionally, Z1 is the amino terminal of the polypeptide, selected from a covalent attachment site; Z2 is the carboxyl terminal of the polypeptide, selected from an amide group; said X1 is a chemical bond or a short sequence of 1-5 amino acid residues, preferably selected from a chemical bond, Cys, Ser, Ala, Gly or the amino acid sequence -A1-A2-A3-A4-A5-; wherein A1 is selected from Arg or is absent, A2 is selected from Arg, Ser or is absent, A3 is selected from Phe, Ser or is absent, A4 is selected from Ser or is absent, A5 is selected from Cys, Ser, Ala, Gly, Pro, Hyp or is absent, further optionally Pro or Hyp is selected from the D-configuration; wherein said AA0 is selected from the amino acid residue Gly, Ala or Ser, said AA1 is selected from Arg, Pro or Hyp, AA2 is selected from a Gly residue or is absent; Optionally, X1 is a short sequence of 3 amino acid residues -A3-A4-A5-; further preferably, wherein -A3-A4- is -Phe-Ser-; A5 is selected from Pro or Hyp, optionally A5 is selected from the D-configuration.

5. The polypeptide ligand according to claims 1 to 4, characterized in that, said polypeptide ligand comprises the amino acid sequence shown below from N-terminal to C-terminal: -(L / D)Phe-Ser-AA0-Cha-AA1-Gly- AA2-Ile-Asp-AA3-Ile- wherein AA0 is selected from Pro or Hyp in the D- or L-configuration (preferably in the D-configuration), AA1 is selected from Ala or Ser in the D- or L-configuration, AA2 is selected from Pro or Hyp in the L-configuration, AA3 is selected from Arg or Arg(Me); Optionally, said polypeptide ligand comprises or has the amino acid sequence (N-C-terminal) shown below any of: -Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; -Ser-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; -Ala-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; - Phe-Ser-Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; - Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser-Pro-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser-Gly-Cha-Ala-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser- Gly-Cha-Gly-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser- Pro-Cha-Ala-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; - Phe-Ser -Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Ser-Phe-Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Arg-Ser-Phe-Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; - Arg-Ser-Ser-Gly-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-; each amino acid residue is in the L- or D- form; optionally, each Phe is in the D-form; optionally, each Ala is in the D-form.

6. The polypeptide ligand of claim 5, wherein The polypeptide ligand comprises any one of the following amino acid sequences (based on N-C terminal orientation): - DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Gly-; - DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-; - DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg(Me)-Ile-; - DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; - DPhe-Ser-DHyp-Cha-DSer-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; DPhe represents D-configuration of Phe, and the rest of the amino acids have the same meaning.

7. The polypeptide ligand according to claims 1 to 6, characterized in that, The polypeptide ligand is a linear peptide or a cyclic peptide, wherein the side chains of any non-adjacent amino acid residues in the polypeptide are connected to each other to form a cyclic peptide; Optionally, the side chains of the amino acid residues can be linked directly, or via an amide-containing, disulfide-containing, or alkylene / alkenylene-containing linker; wherein the alkylene is selected from C2-C 10 straight-chain alkylene, and the alkenylene is selected from C4-C 10 alkenylene; Optionally, the two amino acid residues whose side chains are connected to each other in the cyclic peptide are separated by 1-6 amino acid residues; Optionally, the polypeptide ligand comprises a cyclic peptide structural unit having the following primary amino acid sequence: Cys-Phe-Gly-PPa-Asp-Arg-Ile-PPb-Ser-PPc-Leu-Gly-Cys, and the two Cys form a cyclic peptide unit through a disulfide bond; wherein PPa and PPb are each independently a short peptide consisting of 3 arbitrary amino acid residues, and PPc is an arbitrary amino acid residue; Optionally, the polypeptide ligand comprises an amino acid sequence as follows: - Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-, and having a disulfide bond between the two Cys; Optionally, the polypeptide ligand comprises a modified amino acid and comprises the following structural unit: , Optionally, the polypeptide ligand comprises the following structure: wherein Ar is selected from aryl, heteroaryl, fused ring aromatic system or a combination thereof; Optionally, Ar is selected from substituted or unsubstituted C6-C. 10 Aryl, C 10 -C 14 Fused-ring aryl groups, monocyclic or polycyclic heteroaryl groups containing 1-4 heteroatoms, wherein each heteroatom is independently selected from N, O, and S; Optionally, Ar is selected from substituted or unsubstituted naphthyl, diphenyl, triphenyl, cyclohexyl, indolyl, adamantyl, benzofuranyl, benzothiophenyl, indazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, pteridinyl, pyrazinopyridinyl, pyrimidinopyrazinyl, triazolopyrimidinyl; the substituents are selected from halogen, hydroxyl, amino, C1-C6 alkoxy, C1-C6 alkyl, phenyl, aryl, heteroaryl, alkylene aryl, alkylene heteroaryl); R2 is O or N; R3 is an arbitrary amino acid residue or a modified amino acid residue.

8. The polypeptide ligand of claims 1-7, wherein, The polypeptide ligand further comprises a linking group for connecting with a pharmaceutically active molecule; the linking group is used to connect the polypeptide with the pharmaceutically active molecule; wherein the linking group is covalently connected to an amino acid residue in the polypeptide (preferably the amino acid residue at the amino terminus or the carboxyl terminus of the polypeptide), and comprises a functional group capable of undergoing conjugation reaction with a pharmaceutically active molecule to form a covalent connection; the functional group can be one or more, which are independently selected from azido, alkyne, thiol, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, disulfide, sulfonate, alkenyl, hydrazide or phosphoramidite; optionally, the linker is selected from the group consisting of substituted or unsubstituted straight / branched C1-C 15 hydrocarbyl, and one or more methylene (-CH2-) units in the hydrocarbyl chain is replaced with one or more substituents selected from the group consisting of triazolyl, -C(O), -OC(O)-, -NHC(O)-, -NH-, O, S, -S(O)2, -OP(O)2, -CONH-, -SO2NH-, C2-C6alkenylene, C6-C 10 arylene, or C3-C 10 heterocyclyl; Optionally, when the linking group is present with a substituent, the substituent is selected from one or more of the group consisting of hydroxyl, amino, halogen, C1-C6alkyl, C6-C10aryl, C5-C10heteroaryl, C1-C5alkoxy, C1-C5alkylphenyl, nitro, -CONH2, -C(O)C1-C6alkyl, -SO2NH2, or combinations thereof. 10 aryl, C5-C 10 heteroaryl, C1-C5alkoxy, C1-C5alkylphenyl, nitro, -CONH2, -C(O)C1-C6alkyl, -SO2NH2, or combinations thereof. Optionally, the pharmaceutically active molecule is selected from a double-stranded oligonucleotide.

9. The polypeptide ligand according to any one of claims 1 to 8, characterized in that, The polypeptide ligand comprises the following structure: R1 is selected from -Ar-(L)i-R5, wherein L is -(CH2)n1-(R4)n2, n1 is an integer from 0 to 6, each R4 is independently O or S, n2 is 0 or 1, and i is an integer from 0 to 10; Ar, R2, R3 are as defined above; R5 is selected from azido, alkynyl, thiol, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, disulfide, sulfonate, alkenyl, hydrazide, or phosphoramidite.

10. The polypeptide ligand of claims 1-9, wherein, The polypeptide ligand is selected from any one of the following structures, or a derivative thereof, or a tautomer thereof, or a stereoisomer thereof: ； ； ； 。 11. Use of the polypeptide ligand of any one of claims 1-10 for mediating targeting of a liragen receptor as a delivery vehicle for a pharmaceutically active molecule. Optionally, the pharmaceutically active molecule is selected from a small molecule compound, an antibody, or an oligonucleotide. Optionally, the polypeptide ligand of any one of claims 1-10 is used as a delivery vehicle for delivering an oligonucleotide into a cell. Optionally, the cell is selected from a cell in heart, kidney, lung, liver, adrenal gland, fat, cerebral cortex, or tumor tissue. Optionally, the oligonucleotide is an siRNA having a sense strand and an antisense strand, each strand optionally having 14 to 30 nucleotides, wherein the antisense strand comprises a sequence complementary or substantially complementary to the sense strand and a target mRNA; and optionally, the polypeptide ligand is linked to the 5' end and / or 3' end of the sense strand.

12. An oligonucleotide conjugate, characterized in that, The conjugate comprises a covalently linked oligonucleotide molecule and the polypeptide ligand of any one of claims 1-10. Optionally, the conjugate is formed by covalent conjugation of the oligonucleotide and the polypeptide ligand of any one of claims 1-10, the covalent conjugation comprising an addition reaction, a coupling reaction, or a substitution reaction. Optionally, the conjugate contains one or more polypeptide ligand units. Optionally, the oligonucleotide is linked to the amino-terminal or carboxyl-terminal position of the polypeptide via a covalent linker, e.g., the polypeptide ligand unit is linked to the oligonucleotide via a covalent linker at the amino-terminal of the polypeptide. Optionally, the conjugate has the following structure: Nu - [La - (-Lb-PP) n3 ] n4 wherein n3 and n4 are each independently an integer from 1 to 3; La and Lb are linkers, wherein the site of La directly linked to the oligonucleotide is selected from a phosphodiester bond, a phosphorothioate bond, or a disulfide bond; La is a substituted or unsubstituted 4-10 membered saturated or unsaturated aliphatic ring, 3-6 membered heterocyclic ring, C6-C10 aryl ring, or C5-C10cycloalkyl ring; 10 aromatic ring; Lb is C1-C 20 alkylene, (PEG) n , disulfide, amide or triazole containing groups, or a combination of the above; wherein n is an integer selected from 1-20 PP is a polypeptide, which is as defined in the preceding claims.

13. The oligonucleotide conjugate of claim 12, wherein, The polypeptide has both the amino acid sequence -Ile-Asp-Ra-Ile- and -Cha-Z-Gly-, wherein Ra is selected from Arg or Arg(Me), and Z is selected from any neutral amino acid in either D or L configuration, preferably any one of Gly, Ala, or Ser; Optionally, the polypeptide comprises the following amino acid sequence: -DPhe-Ser-AA0-Cha-AA1-Gly- AA2-Ile-Asp-AA3-Ile-; wherein AA0 is selected from Gly, DPro or DHyp (D configuration), AA1 is selected from Gly, DAla or DSer, AA2 is selected from Pro or Hyp, and AA3 is selected from Arg or Arg(Me).

14. The oligonucleotide conjugate of claims 12-13, characterized in that, The oligonucleotide is a double-stranded oligonucleotide siRNA, and the sense and antisense strands each comprise 15-25 nucleotides; each of the nucleotides is a modified or unmodified nucleotide; Optionally, the double-stranded oligonucleotide is delivered to target tissues / cells of the kidney, heart, lung, liver, adrenal gland, fat (preferably subcutaneous fat or gonadal fat), or cerebral cortex; Optionally, the double-stranded oligonucleotide is selected from functional oligonucleotide molecules having a therapeutic effect on kidney-related diseases or symptoms; Optionally, the target tissue is a tissue of adipose tissue or a kidney organ, preferably a proximal tubular epithelial tissue of the kidney; and the target cell is an epithelial cell of the proximal tubular epithelial tissue.

15. A pharmaceutical composition comprising one or more of the polypeptide ligand of any one of claims 1-10 or the oligonucleotide conjugate of any one of claims 12-14, a pharmaceutically acceptable salt, metabolite, or prodrug thereof, and a pharmaceutically acceptable excipient.

16. Use of one or more of the polypeptide ligand of any one of claims 1-10 or the oligonucleotide conjugate of any one of claims 12-14, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 15, for the manufacture of a medicament for the treatment and / or prevention of a disease associated with dysregulation of gene expression.

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