Kidney-targeting polypeptide-oligonucleotide conjugate and use thereof

Abstract

A polypeptide-oligonucleotide conjugate and use thereof. The polypeptide-oligonucleotide conjugate comprises (1) a polypeptide that binds to an LRP2 receptor, and (2) one or two oligonucleotide drugs for regulating the expression of a kidney target gene. The conjugate specifically delivers an siRNA drug to the kidney using the LRP2-targeting peptide.

Description

Kidney-targeted polypeptide oligonucleotide conjugate and application thereof

[0001] The present application claims priority to the Chinese patent application No. 2024111020356 filed on August 12, 2024, entitled "Kidney-targeted polypeptide oligonucleotide conjugate and application thereof", and the Chinese patent application No. 2025104191326 filed on April 03, 2025, entitled "Kidney-targeted polypeptide oligonucleotide conjugate and application thereof", both of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and specifically relates to a kidney-targeted polypeptide oligonucleotide conjugate and application thereof. BACKGROUND

[0003] In the treatment of kidney diseases, multiple targets have been identified and used to develop small molecule drugs and antibody drugs. For example, sodium-glucose co-transporter 2 (SGLT2), urate transporter 1 (URAT1), hypoxia-inducible factor proline hydroxylase (HIF-PH) and regulatory hypoxia-inducible factor (HIFs), angiotensin-converting enzyme (ACE), endothelin-1 (Endothelin-1), TGF-b, NF-κB, phosphodiesterase type 5 (PDE5), NADPH oxidase 1 (NOX1), etc. are important targets for kidney diseases.

[0004] SGLT2 is highly expressed in proximal tubular cells and is responsible for reabsorbing filtered glucose into the blood. SGLT2 inhibitors, by inhibiting this process, reduce blood glucose levels and play a role in treating diabetes. SGLT2 inhibitors such as dapagliflozin and canagliflozin have been approved for the treatment of diabetic nephropathy. However, these drugs, although effective, have some significant limitations, including the need for frequent dosing and possible side effects, and the safety concerns of frequent dosing and long-term use limit the application of these drugs.

[0005] URAT1 is mainly expressed in renal tubular epithelial cells and is involved in the reabsorption of uric acid. Similarly, URAT1 inhibitors such as lesinurad and febuxostat, although effective in reducing serum uric acid levels, their long-term safety and efficacy still need to be further verified, and long-term use of these drugs can cause side effects such as abnormal kidney function.

[0006] Hypoxia-inducible factor proline hydroxylase (HIF-PH) plays a crucial role in regulating hypoxia-inducible factors (HIFs), which are key mediators of cellular response to oxygen levels. HIF-PH is expressed in renal tubular cells, including proximal and distal tubule segments. In these cells, HIF-PH regulates the stability of HIFs, thereby affecting various functions including metabolic responses, production of erythropoietin, and response to hypoxic stress.

[0007] Angiotensin-converting enzyme (ACE) is mainly expressed in proximal tubular epithelial cells: ACE is highly expressed on the brush border of these cells, facing the tubular lumen. ACE inhibitors dilate blood vessels and lower blood pressure by inhibiting the production of angiotensin II. ACE inhibitors have been shown to reduce proteinuria in the kidney, a common symptom of many kidney diseases. In some chronic kidney diseases, such as diabetic nephropathy and hypertension-induced kidney damage, ACE inhibitors can slow the further deterioration of kidney function.

[0008] In response to these limitations, siRNA drugs, as a new emerging treatment method, show great potential. siRNA drugs can fundamentally address the etiology of diseases by specifically silencing target gene expression, with fewer side effects and longer duration of action. Through targeted siRNA delivery systems, drug administration frequency can be effectively reduced, and systemic side effects can be significantly reduced.

[0009] Low-density lipoprotein receptor-related protein 2 (LRP2), also known as megalin, is a multifunctional receptor highly expressed in the kidney. LRP2 is enriched on the surface of renal tubular cells and is involved in the reabsorption and transport of various substances. Studies have shown that LRP2 not only plays an important role in maintaining the function of renal tubules, but also can be used as a potential target for delivering drugs into the kidney.

[0010] LRP2 mediates the uptake and metabolism of various ligands, including vitamin D binding protein, steroid hormones, and polypeptides, through endocytosis and transcellular transport mechanisms. Due to its high expression in renal tubular cells and extensive ligand binding capacity, LRP2 becomes an ideal candidate target for delivering siRNA drugs to the kidney. More importantly, SGLT2 and URAT1 are also expressed in the same renal tubular cells, making it possible to use LRP2-targeted peptides to deliver siRNA to precisely target these targets, thereby reducing the expression of target genes and treating related diseases.

[0011] The use of LRP2-targeting peptides to deliver siRNA drugs has several advantages. First, through the specific binding of the targeting peptide, efficient drug delivery can be achieved, significantly increasing the concentration of the drug in target cells. Second, siRNA drugs can fundamentally treat diseases by silencing specific genes, avoiding the side effects of traditional small molecule drugs. In addition, siRNA drugs have a longer duration of action, which can reduce the frequency of administration and improve patient compliance. In the treatment of kidney disease, the use of LRP2-targeting delivery of siRNA drugs can achieve precise targeting of key targets such as SGLT2 and URAT1, effectively reducing blood glucose and uric acid levels, and improving patient outcomes.

[0012] Therefore, a highly specific LRP2-targeting peptide-conjugated siRNA delivery system is designed and invented to improve the delivery efficiency and targeting of siRNA to kidney cells, and has better selectivity than existing small molecule drugs or antibody drugs, making it safer and more effective. SUMMARY

[0013] Based on this, the purpose of the present application is to provide a polypeptide delivery system based on targeting LRP2, which uses LRP2-targeting peptides to specifically deliver siRNA drugs to the kidney. This not only increases the effective concentration of siRNA drugs in the kidney and reduces the side effects of treatment, but also overcomes the limitations of existing small molecule drugs in terms of administration frequency and long-term effectiveness, with high in vivo delivery efficiency, high specificity, low toxicity, and reduced administration frequency.

[0014] To achieve the above-mentioned purposes of the application, the present application includes the following technical solutions.

[0015] In a first aspect, the present application provides a polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, comprising:

[0016] (1) at least one polypeptide capable of specifically binding to LRP2 receptor; and

[0017] (2) one or more oligonucleotide drugs for regulating the expression of kidney target genes.

[0018] In some embodiments, the polypeptide oligonucleotide conjugate comprises:

[0019] (1) at least one polypeptide capable of specifically binding to LRP2 receptor, the N-terminal to C-terminal amino acid sequence of which is represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 40;

[0020] or an amino acid sequence obtained by substitution, replacement, deletion, addition, insertion, and / or modification of the amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 40; and

[0021] (2) one or more oligonucleotide drugs for regulating the expression of a kidney target gene.

[0022] In some embodiments, the number of amino acid residues substituted, replaced, deleted, added, inserted, and / or modified in the amino acid sequence is 1 or more and 10 or less.

[0023] In some embodiments, the number of amino acid residues substituted, replaced, deleted, added, inserted, and / or modified in the amino acid sequence is 1 or more and 5 or less.

[0024] In some embodiments, the unmodified amino acid sequence of the polypeptide N- to C-terminus is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41;

[0025] or a substitution, a replacement, a deletion, an addition, an insertion and / or a modification of 1 or more and less than 5 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.

[0026] In some embodiments, the unmodified amino acid sequence of the polypeptide from N-terminus to C-terminus is

[0027] DDAEDRHDSGYEVHCQKLCFRRRR (SEQ ID NO: 42),

[0028] DDAEDRHDSGYEVHCQKLCFKRRRR (SEQ ID NO: 43),

[0029] DDAEDRHDSGYEVHCQKLCFRRRRRR (SEQ ID NO: 44),

[0030] DDAEDRHDSGYEVHCQKLCFKRRRRRR (SEQ ID NO: 45),

[0031] KDDAEDRHDSGYEVHCQKLCF (SEQ ID NO: 46),

[0032] KDDAEDRHDSGYEVHCQKLCFRRRR (SEQ ID NO: 47),

[0033] DDAEDRHDSGYCVHHQKLCF (SEQ ID NO: 48), or

[0034] DDAEDRHDSGCEVHHQKCVF (SEQ ID NO: 49);

[0035] or an amino acid sequence obtained by substitution, replacement, deletion, addition, insertion and / or modification of 1 or more and less than 5 amino acid residues in the amino acid sequence represented by SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49.

[0036] In some embodiments, the modification includes one or more of (i-1) - (i-9):

[0037] (i-1) a cyclization modification;

[0038] (i-2) modification of the side chain amino group of an amino acid residue with a long-chain fatty acid or long-chain fatty diacid having a carbon chain length of 12-22;

[0039] (i-3) modification of the side chain amino group of an amino acid residue with p-iodophenylbutyric acid;

[0040] (i-4) modification of the side chain amino group of an amino acid residue with 2-(2-(2-(4-(4-(4-iodophenyl)butyramido)ethoxy)ethoxy)acetic acid;

[0041] (i-5) N-terminal amino acylation modification in the amino acid sequence;

[0042] (i-6) C-terminal carboxy amide modification in the amino acid sequence;

[0043] (i-7) methylation modification of an amino acid residue;

[0044] (i-8) alkynyl modification of an amino acid residue;

[0045] (i-9) azido modification of an amino acid residue.

[0046] In some embodiments, the type of cyclization modification includes one or more of (a) - (e):

[0047] (a) cyclization by alkylation reaction of the side chain mercapto groups of two cysteine residues;

[0048] (b) cyclization by disulfide bond formation through the side chain mercapto groups of two cysteine residues;

[0049] (c) cyclization by amide bond formation through condensation reaction of the side chain amino group of a lysine residue with the side chain carboxyl group of a glutamic acid residue;

[0050] (d) insertion of an alkyne and azido-modified amino acid residue in the amino acid sequence or introduction of an alkyne and azido modification in the amino acid sequence and cyclization by click chemistry reaction of alkyne and azido;

[0051] (e) cyclization by olefin metathesis reaction.

[0052] In some of the embodiments, the alkylating agent that reacts with the side chain mercapto group of a cysteine residue includes one or more of 1,4-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene, 1,2-bis(bromomethyl)benzene, 1,3-dibromoacetone, 1,3-dibromopropane, diiodoethane, 4,4'-bis(bromomethyl)-1,1'-biphenyl, 1,3,5-tris(bromomethyl)benzene, 1,8-bis(bromomethyl)naphthalene, and 1,4-bis(bromomethyl)naphthalene.

[0053] In some of the embodiments, the number of amino acid residues in each loop in the cyclized polypeptide is 4, 5, 6, 7, or 8.

[0054] In some of the embodiments, the substitutions include one or more of (a-1) - (a-11):

[0055] (a-1) L-amino acid residues in the amino acid sequence are replaced by D-amino acid residues;

[0056] (a-2) D-amino acid residues in the amino acid sequence are replaced by L-amino acid residues;

[0057] (a-3) amino acid residues in the amino acid sequence are replaced by methylated amino acid residues;

[0058] (a-4) aromatic amino acids in the amino acid sequence are replaced by acidic amino acid residues;

[0059] (a-5) amino acid residues in the amino acid sequence are replaced by 2-(2-(2- aminoethoxy)ethoxy)acetic acid residues;

[0060] (a-6) amino acid residues in the amino acid sequence are replaced by (S)-2-amino- hept-6-ynoic acid residues;

[0061] (a-7) amino acid residues in the amino acid sequence are replaced by (S)-2-amino- 4-pentynoic acid residues;

[0062] (a-8) the amino acid residue in the amino acid sequence is replaced by a 2- amino isobutyric acid residue;

[0063] (a-9) the amino acid residue in the amino acid sequence is replaced by an L- tert-leucine residue;

[0064] (a-10) the amino acid residue in the amino acid sequence is replaced by an R-2- amino-2-methyl-6-heptenoic acid residue;

[0065] (a-11) any two amino acid residues separated by 4, 5, 6, 7 or 8 amino acid residues are simultaneously replaced by a cysteine residue.

[0066] In some of the embodiments, the replacement comprises: 1-3 amino acids in the amino acid sequence are replaced by different amino acids of the same property, i.e. mutual replacement between non-polar amino acids, mutual replacement between polar amino acids, mutual replacement between acidic amino acids, mutual replacement between basic amino acids. Among them, the non-polar amino acids are: Gly, Ala, Val, Leu, Ile, Met, Trp, Phe, Pro; the polar amino acids are: Ser, Thr, Cys, Tyr, Asn, Gln; the basic amino acids are: Lys, Arg, His; the acidic amino acids are: Asp, Glu.

[0067] In some of the embodiments, the replacement comprises: 1 amino acid in the amino acid sequence is replaced by a non-amino acid structure as follows:

[0068] In some of the embodiments, the addition insertion comprises one or more of (b-1) - (b-3):

[0069] (b-1) a new amino acid, N-methylglycine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, (S)-2-amino-hept-6-ynoic acid, (S)-2-amino-4-pentynoic acid, 2-aminoisobutyric acid, L-tert-leucine and / or R-2-amino-2-methyl-6-heptenoic acid is inserted in the amino acid sequence;

[0070] (b-2) lysine, arginine, glutamic acid, aspartic acid, N-methylglycine, 2-(2-(2- aminoethoxy)ethoxy)acetic acid and / or the following group is added at the N-terminus or C-terminus of the amino acid sequence:

[0071] (b-3) a polypeptide residue composed of 1, 2 or 3 amino acids selected from the group consisting of lysine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid and N- methylglycine is added at the N-terminus or C-terminus of the amino acid sequence.

[0072] In some embodiments, the amino acid sequence of the polypeptide from N- to C-terminus is:

[0073] DAEFRHDSGYEVHHQK-NH2;

[0074] DAEFRHDSGYEVHHQKLVFFAE-NH2;

[0075] KLVFFAEDVGSNKGAIIGLMVGGVV-NH2;

[0076] Ac-dVdHdHdQdKdLdPdFdFdAdEdDdVGdSdNdK-NH2;

[0077] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA-NH2;

[0078] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-OH;

[0079] FRHDSGYEVHHQKLVFFAE-NH2;

[0080] DSGYEVHHQKLVFFAE-NH2;

[0081] YEVHHQKLVFFAE-NH2;

[0082] Ac-dDdAdEdFdRdHdDdSGdYdEdVdHdHdQdKdLdVdFdFdAdE-NH2;

[0083] Ac-dEdAdFdFdVdLdKdQdHdHdVdEdYGdSdDdHdRdFdEdAdD-NH2;

[0084] DAEFRHDSGYEVHHQKLVFFAEDV-NH2;

[0085] GAIIGLMVGGVV-[AEEA]-KSK-NH2;

[0086] SGYEVHHQKLVF-NH2;

[0087] SGYEVHHQKdLdVF-NH2;

[0088] DSGYEVHHQKLVF-NH2;

[0089] DSGYEVHHQK-L(N-Me)-V(N-Me)-F-NH2;

[0090] DSGYEVHHQK-L(N-Me)-dV-F-NH2;

[0091] DSGYEVHHQK-dL-V(N-Me)-F-NH2;

[0092] DSGYEVHH-Q(N-Me)-K-L(N-Me)-V(N-Me)-F-NH2;

[0093] DSGYEVH-dH-Q(N-Me)-K-L(N-Me)-V(N-Me)-F-NH2;

[0094] DSGYEVH-dH-QK-L(N-Me)-V(N-Me)-F-NH2;

[0095] FRHDSGYEVHHQKLVF-NH2;

[0096] DAEFRHDSGYEVHHQKLVF-NH2;

[0097] DSGYEVHHQKdLdVF-NH2;

[0098] DSGYEVHCQKLCF-NH2;

[0099] DDDDSGYEVHCQKLCF-NH2;

[0100] DSGYEVHCQKLCFGFWFG-NH2;

[0101] GFWFGDSGYEVHCQKLCF-NH2;

[0102] DAEFRHDSGYEVHCQKLCF-NH2;

[0103] DDEDREDSGYEVHCQKLCF-NH2;

[0104] dDDAEdDRHDSGYEVHCQKLCF-NH2;

[0105] D-K(Pal)-EFRHDSGYEVHCQKLCF-NH2;

[0106] DAEFRHDSGYEVHCQKLCF-K(Pal)-NH2;

[0107] DAECRHDCGYEVHHQKLVFFAE-NH2;

[0108] DAEFRHDSGYEVHHQCLVFCAE-NH2;

[0109] DAEFRHDSGYEVHCQKLCFFAE-NH2;

[0110] DDDAEFRHDSGYEVHCQKLCFFAE-NH2;

[0111] dDdDDAEFRHDSGYEVHCQKLCFFAE-NH2;

[0112] dDDAEdDRHDSGYEVHCQKLCFdDAE-NH2;

[0113] DAEFRHDSGYEVCHQKCVFFAE-NH2;

[0114] DAEFRHDSGYEVCHQKCVFFAE-NH2;

[0115] DAEFRHDSGYEVHHCKLVCFAE-NH2;

[0116] DAEFRHDSGYEVHX2QKLX3FFAE-NH2;

[0117] DAEFRHDSGYEVHKQKLEFFAE-NH2;

[0118] DAEX1RHDX3GYEVHCQKLCFFAE-NH2;

[0119] RRRdDDAEdDRHDSGYEVHCQKLCF-NH2;

[0120] RRRRdDDAEdDRHDSGYEVHCQKLCF-NH2;

[0121] dDDAEdDRHDSGYEVHCQKLCF-K(X4)-NH2;

[0122] RRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2;

[0123] RRRRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2;

[0124] dDDAEdDRHDSGYEVHCQKLCFRRRR-[AEEA]-K(IBA)-NH2;

[0125] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2;

[0126] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-OH;

[0127] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(X5)-NH2;

[0128] K(X5)dDDAEdDRHDSGYEVHCQKLCF-NH2;

[0129] dDDAEdDRHDSGYEVHCQKLCFRRRR-NH2;

[0130] KdDDAEdDRHDSGYEVHCQKLCFRRRR-NH2;

[0131] dDDAEdDRHDSGYCVHHQKLCF-NH2;

[0132] dDDAEdDRHDSGCEVHHQKCVF-NH2;

[0133] dDDAEdDRHDSGYCVdHdHQKLCF-NH2;

[0134] dDDAEdDRHDSGYdCVdHdHQKLdCF-NH2;

[0135] dDDAEdDRHDSGYEVHdCQKLdCF-NH2;

[0136] dDDAEdDRHDSGYEVdHCQKLCdF-NH2;

[0137] dDDAEdDRHDSGYEVdHCQKLCF-NH2;

[0138] dDDAEdDRHDS[Aib]YEVHCQKLCF-NH2;

[0139] dDDAEdDRHDS[Aib]YEVdHCQKLCF-NH2;

[0140] dDDAEdDRHDS[Aib]YEdVdHCQKLCF-NH2;

[0141] dDDAEdDRHDSGYE[Aib]HCQKLCF-NH2;

[0142] dDDAEdDRHDS[Tle]YEVHCQKLCF-NH2;

[0143] dDDAEdDRHDSGYE[Tle]HCQKLCF-NH2;

[0144] dDDAEdDRHDSGYEVHX6QKLX6F-NH2; or

[0145] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-X7;

[0146] wherein d represents a D-amino acid, N-Me represents an N-methylated amino acid, 6-Azidohexanoic is 6-azidohexanoic acid, Pal is palmitic acid, IBA is p-iodophenylbutyric acid, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butyramido)ethoxy)ethoxy)acetic acid, AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid, X1 is L-propargylglycine, X2 is (S)-2-amino-hept-6-ynoic acid, X3 is LysN3, Aib is 2-aminoisobutyric acid, Tle is L-tert-leucine, X6 is R-2-amino-2-methyl-6-heptenoic acid, and X5 has the following structure:

[0147] or

[0148] X7 has the following structure:

[0149] In some embodiments, the polypeptide is a polypeptide that is cyclized by modifying the above polypeptides.

[0150] In some embodiments, the polypeptide has an amino acid sequence from N-terminus to C-terminus and is cyclized in the following manner:

[0151] DSGYEVHHQKLVF-NH2, E and K side chains form an amide bond to cyclize;

[0152] DSGYEVHHQKdLdVF-NH2, E and K side chains form an amide bond to cyclize;

[0153] DSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize;

[0154] DDDDSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize;

[0155] DSGYEVHCQKLCFGFWFG-NH2, Cys reacts with m-dibromobenzyl to cyclize;

[0156] GFWFGDSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize;

[0157] DSGYEVHCQKLCF-NH2, disulfide bond formation to cyclize;

[0158] DAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0159] DDEDREDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0160] dDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0161] D-K(Pal)-EFRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0162] DAEFRHDSGYEVHCQKLCF-K(Pal)-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0163] DAEFRHDSCYEVHCQKLCF-NH2, Cys reacted with 1,3,5-tris(bromomethyl)benzene to cyclize;

[0164] dDDAEdDRHDSGYEVHCQKLCF-NH2, disulfide bond formation to cyclize;

[0165] DAECRHDCGYEVHHQKLVFFAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0166] DAEFRHDSGYEVHHQCLVFCAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0167] DAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0168] DDDAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0169] dDdDDAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0170] dDDAEdDRHDSGYEVHCQKLCFdDAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0171] DAEFRHDSGYEVCHQKCVFFAE-NH2, Cys reacted with m-dibromobenzyl to cyclize;

[0172] DAEFRHDSGYEVHHCKLVCFAE-NH2, Cys reacted with m-dibenzylbromide to form a ring;

[0173] DAEFRHDSGYEVHX2QKLX3FFAE-NH2, X2 is (S)-2-amino-hept-6-ynoic acid, X3 is LysN3, X2 and X3 form a ring by click chemistry reaction;

[0174] DAEFRHDSGYEVHKQKLEFFAE-NH2, the side chain of the 14th amino acid residue (K) and the 18th amino acid residue (E) form an amide bond to form a ring;

[0175] DAEX1RHDX3GYEVHCQKLCFFAE-NH2, X1 is L-propargylglycine, X3 is LysN3, X1 and X3 form a ring by click chemistry reaction and Cys reacts with m-dibenzylbromide to form a ring;

[0176] RRRdDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0177] RRRRdDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0178] dDDAEdDRHDSGYEVHCQKLCF-K(X4)-NH2, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butyramide)ethoxy)ethoxy)acetic acid; Cys reacts with m-dibenzylbromide to form a ring;

[0179] RRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0180] RRRRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0181] dDDAEdDRHDSGYEVHCQKLCFRRRR-[AEEA]-K(IBA)-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0182] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacts with m-dibenzylbromide to form a ring;

[0183] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-OH, Cys reacts with m-dibenzylbromide to form a ring;

[0184] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(X 5-3 )-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0185] K(X 5-3 )dDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0186] dDdDDAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with p-dibromobenzyl to form a ring;

[0187] dDdDDAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with 1,8-bis(bromomethyl)naphthalene to form a ring;

[0188] dDDAEdDRHDSGYCVHHQKLCF-NH2, Cys reacted with 4,4'-dibromomethylbiphenyl to form a ring;

[0189] dDDAEdDRHDSGCEVHHQKCVF-NH2, Cys reacted with 4,4'-dibromomethylbiphenyl to form a ring;

[0190] dDDAEdDRHDSGYCVdHdHQKLCF-NH2, Cys reacted with 4,4'-dibromomethylbiphenyl to form a ring;

[0191] dDDAEdDRHDSGYdCVdHdHQKLdCF-NH2, Cys reacted with 4,4'-dibromomethylbiphenyl to form a ring;

[0192] dDDAEdDRHDSGYEVHdCQKLdCF-NH2, Cys reacted with 4,4'-dibromomethylbiphenyl to form a ring;

[0193] dDDAEdDRHDSGYEVHdCQKLdCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0194] dDDAEdDRHDSGYEVdHCQKLCdF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0195] dDDAEdDRHDSGYEVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0196] dDDAEdDRHDS[Aib]YEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0197] dDDAEdDRHDS[Aib]YEVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0198] dDDAEdDRHDS[Aib]YEdVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0199] dDDAEdDRHDSGYE[Aib]HCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0200] dDDAEdDRHDS[Tle]YEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring;

[0201] dDDAEdDRHDSGYE[Tle]HCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; or

[0202] dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-X7, Cys reacted with m-dibromobenzyl to form a ring.

[0203] In some embodiments, the polypeptide is selected from the following structures:

[0204] or a polypeptide obtained by substitution, replacement, deletion, addition, insertion and / or modification of 1 or more and less than 5 amino acid residues in the polypeptide;

[0205] wherein IBA is p-iodophenylbutyric acid, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butyramido)ethoxy)ethoxy)acetic acid, X5 has the following structure: or X7 has the following structure:

[0206] In some embodiments, the polypeptide is covalently linked to the oligonucleotide drug through a linker group.

[0207] In some embodiments, the linker group has the following structure: or LE is a group linked to the oligonucleotide drug, which contains or does not contain a hydroxyl group or a protected hydroxyl group;

[0208] LA is linked to the polypeptide and is selected from the following structures:

[0209] LB is a linker unit connected by one or more of the following groups without violating the principle of chemical bonding: alkylene, cycloalkyl, heterocyclyl, heteroaryl, aryl, -C(=0)NR1-, -C(=0)0-, -C(=0)S-, -C(=0)-, -C(=S)-, -NR1-, -0-, -S-;

[0210] R1is selected from the group consisting of: H, C1-C 10 alkyl;

[0211] R2is selected from the group consisting of: C1-C 10 alkyl;

[0212] m is selected from the group consisting of: an integer between 0-12;

[0213] each n1and n2is independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6.

[0214] In some embodiments, LE is selected from C1-C3 alkylene or the following structure:

[0215] each R4is independently selected from the group consisting of: H or a hydroxyl protecting group;

[0216] each n2is independently selected from the group consisting of: an integer between 0-10.

[0217] In some embodiments, LE is selected from:

[0218] In some embodiments, LE is connected to the 5' or / and 3' end of the sense strand of RNA in the oligonucleotide drug.

[0219] In some embodiments, LE is connected to the phosphate group or the phosphorothioate group of the 5' or / and 3' end of the sense strand of RNA in the oligonucleotide drug by a phosphodiester bond or a phosphorothioate bond.

[0220] In some embodiments, LA is selected from the following structure:

[0221] In some embodiments, LA is connected to the N-terminus of the polypeptide.

[0222] In some embodiments, LA is connected to the C-terminus of the polypeptide.

[0223] In some embodiments, LA is connected to an amino acid residue containing a side chain amino group in the polypeptide, preferably a lysine residue.

[0224] In some embodiments, the LA is attached to an amino acid residue in the polypeptide that contains a side chain carboxyl group.

[0225] In some embodiments, the LA is attached to an amino acid residue in the polypeptide that contains a side chain thiol group.

[0226] In some embodiments, the LA is attached to a side chain amino group of a non-amino acid type structure contained in the polypeptide.

[0227] In some embodiments, the LB is selected from a C1-C8 alkylene or the following structures:

[0228] wherein each i, j, h, q, s, y is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0229] X is selected from -O-, -S-, -CH2-;

[0230] Y is absent or selected from -C(=O)-, -CH2-.

[0231] In some embodiments, the LB is selected from the following structures:

[0232] each i, j is independently selected from 0, 1, 2, 3, 4;

[0233] k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0234] y is selected from 0, 1, 2, 3, 4;

[0235] X is selected from -CH2-, -O-.

[0236] In some embodiments, the LB is selected from the following structures:

[0237] In some embodiments, the linking group is selected from the following structures:

[0238] n2 is selected from 0, 1, 2, 3, 4, 5, 6;

[0239] R3 is selected from H or acetyl.

[0240] In some embodiments, the oligonucleotide drug is selected from the group consisting of small interfering RNA (siRNA), antisense oligonucleotide (ASO), small activating RNA (saRNA) and peptide nucleic acid (PNA).

[0241] In some embodiments, the oligonucleotide drug is selected from the group consisting of small interfering RNA (siRNA), and the polypeptide is linked to the 3' and / or 5' end of the sense strand of the small interfering RNA via a linker group.

[0242] In a second aspect, the present application provides a polypeptide capable of binding to LRP2 receptor.

[0243] In a third aspect, the present application provides uses of the polypeptide or polypeptide oligonucleotide conjugate or pharmaceutically acceptable salt thereof or stereoisomer thereof, including the following technical solutions.

[0244] Use of the polypeptide capable of binding to LRP2 receptor in the preparation of a delivery system for delivering an oligonucleotide drug to kidney cells in vivo.

[0245] Use of the polypeptide oligonucleotide conjugate or pharmaceutically acceptable salt thereof or stereoisomer thereof as an active ingredient in the preparation of a medicament for inhibiting the expression of a target gene in the kidney.

[0246] In some embodiments, the target gene in the kidney is selected from the group consisting of URAT1, SGLT-2, HIF-PH, ACE, TGF-β, Endothelin-1, TGF-b, NF-κB, PDE5, NOX1, Egln1, ALDH2.

[0247] Use of the polypeptide oligonucleotide conjugate or pharmaceutically acceptable salt thereof or stereoisomer thereof as an active ingredient in the preparation of a medicament for preventing or treating a disease mediated or related to URAT1, SGLT-2, HIF-PH, ACE, TGF-β, Endothelin-1, TGF-b, NF-κB, PDE5, Egln1, ALDH2 or NOX1 genes.

[0248] Use of the polypeptide oligonucleotide conjugate or pharmaceutically acceptable salt thereof or stereoisomer thereof as an active ingredient in the preparation of a medicament for preventing or treating a disease related to the kidney.

[0249] In some embodiments, the kidney-related disease is selected from the group consisting of gout and hyperuricemia, diabetes and diabetic nephropathy, IgA nephropathy (IgAN), chronic kidney disease (CKD), polycystic kidney disease (PKD), acute kidney injury (AKI), lupus nephritis, Alport syndrome, membranous nephropathy, focal segmental glomerulosclerosis (FSGS), renal amyloidosis, hemolytic-uremic syndrome, nodular interstitial nephritis, renal tumors, anemia, and hypertension.

[0250] In a fourth aspect, the present application provides a medicament for treating and / or preventing a kidney-related disease, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the polypeptide oligonucleotide conjugate of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

[0251] In a fifth aspect, the present application provides a method for treating and / or preventing a kidney-related disease, which comprises: administering a safe and effective amount of the polypeptide oligonucleotide conjugate of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; or administering a safe and effective amount of the medicament for treating and / or preventing a kidney-related disease of the present application.

[0252] In some embodiments, the kidney-related disease is selected from the group consisting of gout and hyperuricemia, diabetes and diabetic nephropathy, IgA nephropathy, chronic kidney disease, polycystic kidney disease, acute kidney injury, lupus nephritis, Alport syndrome, membranous nephropathy, focal segmental glomerulosclerosis, renal amyloidosis, hemolytic-uremic syndrome, nodular interstitial nephritis, renal tumors, anemia, and hypertension.

[0253] The present application synthesizes a series of polypeptides which can specifically bind to LRP2 receptor and target the delivery of nucleic acid drugs such as small interfering RNA (siRNA) to the kidney. The LRP2-targeting peptide synthesized by the present application is bonded and coupled with siRNA to obtain a polypeptide oligonucleotide conjugate based on LRP2-targeting peptide, which can be used to deliver nucleic acid drugs such as small interfering RNA (siRNA) to the kidney for inhibiting the expression of one or more kidney target genes. It can use LRP2-targeting peptide to specifically deliver siRNA drugs to the kidney, not only can improve the effective concentration of siRNA drugs in the kidney, reduce the side effects of treatment, but also can overcome the limitations of existing small molecule drugs in terms of drug frequency and long-term effect, has the advantages of high in vivo delivery efficiency, high specificity, good in vivo treatment effect, low toxic side effects and reducing drug frequency, etc., and opens up a new way for the treatment of kidney diseases. BRIEF DESCRIPTION OF DRAWINGS

[0254] Figure 1 is the result of Western Blot (WB) analysis of the inhibitory effect of the polypeptide oligonucleotide conjugate on the expression of target protein in the kidney tissue of a hyperuricemic mouse. DETAILED DESCRIPTION

[0255] For the purposes of promoting an understanding of the principles of the application, the application will now be described in more detail. The application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0256] The experimental methods in the following examples, unless otherwise specified, are generally performed according to conventional conditions, for example, as described in the fourth edition of Molecular Cloning: A Laboratory Manual, edited by Green and Sambrook, published in 2013, or as suggested by the manufacturer. The various common chemical reagents used in the examples are commercially available products.

[0257] Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0258] In addition, as used in this application, the term "or" is the inclusive "or" and not the

[0259] The names, abbreviations, and structures of the nucleotide monomers used in the nucleic acid sequence listing are as follows:

[0260] The specific structure is as follows:

[0261] The application will now be further described in connection with the following specific examples.

[0262] Example 1

[0263] 1.1 Synthesis of linear polypeptides

[0264] Peptide synthesis on a PurePep Chorus 4 channel peptide synthesizer was performed on a 0.2 mmol scale according to the standard solid-phase peptide synthesis method, for example, according to the protocol described in Adam G et al., Standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory. The conditions for the main processes are as follows: (1) Resin swelling: Fmoc-Val-Wang Resin, Rink-Amide-MBHA-Resin was placed in DMF and swelled at room temperature for 30 min; (2) Deprotection: the resin was treated with a 20% piperidine solution in DMF at 50°C for 2 min; (3) Amino acid condensation: 5 eq (resin is 1 eq) of Fmoc-amino acid, HCTU as condensing agent (5 eq), DIPEA (5 eq) as base, reaction at 50°C for 10 min; (4) N-terminal acetylation: 5% N-acetylimidazole, reaction at room temperature for 1 h; (5) Resin cleavage: after completion of synthesis, the resin was treated with cleavage reagent containing TFA (TFA / Tips / H2O = 95:2.5:2.5) for more than 2 hours, the resin was filtered and discarded, and the filtrate was collected. (6) Precipitation and purification: pre-cooled anhydrous ether was added to the filtrate, and white precipitate was obtained by centrifugation. The precipitate was washed with anhydrous ether for 3 times, and white powder-like crude peptide product was obtained by drying at room temperature under reduced pressure. The crude peptide was dissolved in acetonitrile / water system and purified by high performance liquid chromatography.

[0265] 1.2 Cyclization of polypeptides

[0266] 1.2.1 Cyclization reaction based on cysteine and m-dibromobenzyl, p-dibromobenzyl or tris-dibromobenzyl The experimental method is as follows: linear polypeptide synthesis and purification were performed according to the conventional method of 1.1, and the purified linear polypeptide (1 mM) was dissolved in a mixed solvent of acetonitrile and sodium borate buffer (0.1 M, pH 9.5) (acetonitrile: sodium borate buffer = 1:1 (v / v)), and bromobenzyl cyclization reagent (i.e. m-dibromobenzyl, p-dibromobenzyl or tris-dibromobenzyl) (1.3 eq.) was added under stirring, and stirred at room temperature for 30 min, and the reaction solution was purified by high performance liquid chromatography to obtain the target cyclic peptide.

[0267] 1.2.2 Cyclization based on Lys and Glu side chain amide bond The experimental method is as follows: linear polypeptide synthesis is carried out according to the general method of 1.1, and Fmoc-Lys(Alloc)-OH and Fmoc-Glu(OAll)-OH amino acid monomers are used at the cyclization position. After completing the solid-phase synthesis, 0.2 mmol of the peptide resin is placed in 10 mL of dichloromethane, and Pd(PPh3)4 (10 eq.) and phenylsilane (20 eq.) are added under N2 protection, and the reaction is oscillated at room temperature for 30 min (repeated three times) to remove the Alloc and OAll protecting groups of the corresponding amino acid residues side chain. Subsequently, the peptide resin is placed in 10 mL of DMF, and HATU (2 eq.) and DIEA (4 eq.) are added, and the reaction is carried out at room temperature for 3 h to condense the side chain to form an amide group. The resin is cleaved and purified according to the method of 1.1 to obtain the target cyclic peptide.

[0268] 1.2.3 Cyclization based on copper-catalyzed click chemistry reaction The experimental method is as follows: linear polypeptide synthesis is carried out according to the general method of 1.1, and Fmoc-LysN3-OH and Fmoc-(S)-2-amino-hept-6-yne acid (or Fmoc-L- propargylglycine) are used at the cyclization position. After completing the synthesis of the peptide sequence containing LysN3 and the side chain alkyne moiety, 0.2 mmol of the peptide resin is placed in 10 mL of a mixed solvent of DMSO and H2O (DMSO:H2O = 9:1 (v / v)), and cuprous bromide (I) dimethyl sulfide complex (2.5 eq.) and DIEA (5 eq.) are added under N2 protection, and the reaction is oscillated at room temperature overnight to complete the click chemistry reaction. Subsequently, the complete polypeptide is synthesized, the resin is cleaved, and purification is carried out according to the method of 1.1 to obtain the target cyclic peptide.

[0269] 1.2.4 Cyclization based on disulfide bond The experimental method is as follows: linear polypeptide synthesis and purification are carried out according to the general method of 1.1, and the purified linear polypeptide (1 mM) is dissolved in a mixed solvent of acetonitrile and aqueous ammonium bicarbonate solution (0.1 M) (acetonitrile:aqueous ammonium bicarbonate solution = 1:1 (v / v)), and stirred at room temperature for 60 min. The reaction solution is purified by high performance liquid chromatography to obtain the target cyclic peptide.

[0270] The theoretical molecular weight and measured molecular weight of the polypeptide products synthesized by the present application are shown in Table 1-1 and Table 2-1.

[0271] Table 1-1: Linear polypeptide name, polypeptide sequence structure and mass spectrometry characterization

[0272] Table 2-1: Cyclic peptide name, polypeptide sequence structure and mass spectrometry characterization

[0273] wherein d represents a D-amino acid, N-Me represents an N-methylated amino acid, Sar is sarcosine (N-methylglycine), 6-Azidohexanoic refers to 6-azidohexanoic acid, Pal represents palmitic acid, IBA refers to p-iodophenylbutyric acid, X4 refers to 2-(2-(2-(4-(4-(4-iodophenyl)butylamido)ethoxy)ethoxy)acetic acid, AEEA refers to 2-(2-(2-aminoethoxy)ethoxy)acetic acid, (AEEA)2 refers to two 2-(2-(2-aminoethoxy)ethoxy)acetic acids, (AEEA)3 refers to three 2-(2-(2-aminoethoxy)ethoxy)acetic acids, and so on, and X7 has the following structure:

[0274] The modified polypeptides (cyclic peptides) in Table 1-1 and Table 2-1, their corresponding unmodified amino acid sequences are shown in Table 1-2 and 2-2, the amino acids in the sequences listed in Table 1-2 and Table 2-2 are amino acids in the natural configuration without modification.

[0275] Table 1-2: Linear polypeptide name and unmodified amino acid sequence

[0276] Table 2-2: Cyclic peptide name and unmodified amino acid sequence (from N-terminus to C-terminus)

[0277] Some structural examples are as follows:

[0278] Example 2 Synthesis of polypeptide-fluorophore conjugates

[0279] 2.1 Synthesis of A1-22-FITC

[0280] A1-22-AEEA-Resin was synthesized according to the method of Example 1. 0.05 mM (280 mg) of A1-22-AEEA-Resin was placed in DMF, DIEA (9.7 mg, 0.075 mM, 1.5 eq.) and FITC (21 mg, 0.055 mM, 1.1 eq.) were added sequentially, and the system was allowed to react at room temperature for 1 hour. After filtration, the resulting polypeptide fluorophore resin A1-22-AEEA-FITC-Resin was washed with DMF (3 x 15 mL), shrunken with methanol, cleaved with TFA (4 mL) for 2 hours, filtered to remove the resin, and the filtrate was precipitated in cold ether (45 mL), centrifuged, and the supernatant was discarded. The precipitate was dissolved in acetonitrile / water containing 0.1% formic acid (1 mL), and separated by RP-HPLC to obtain 15 mg of the target compound A1-22-FITC (99.14% purity, 9.4% yield, MS [M+3H] 3+ : 1066.45).

[0281] 2.2 Synthesis of A1-22-TAMRA

[0282] Polypeptide A1-22 (16.6 mg, 5.3 μM, 1.0 eq.) was dissolved in DMF / H2O (1 mL, DMF / H2O = 1:1), followed by the sequential addition of a DMF solution (0.2 mL) of 5-TAMRA alkyne (2 mg, 6.4 μM, 1.2 eq.), a DMF solution (0.3 mL) of CuSO4.5H2O (27 mg, 0.11 mM, 20 eq.) and THPTA (23 mg, 0.053 mM, 10 eq.), and solid Na2CO3 (41 mg, 0.21 mM, 40 eq.). After the system was allowed to react at room temperature for 1 hour, 100 eq. of ammonia water and 100 eq. of EDTA were added to quench the reaction. Separation by RP-HPLC yielded 7 mg of the target compound A1-22-TAMRA (97.9% purity, 36.8% yield, [M+5H] 5+ : 715.7).

[0283] A series of TAMRA fluorophore derivatives of polypeptides were synthesized according to the method of Example 2.

[0284] Table 3: Polypeptide TAMRA fluorophore derivative name, theoretical molecular weight, and measured molecular weight

[0285] Example 3 Synthesis of polypeptide cytotoxic agent conjugates

[0286] Dissolve polypeptide A1-22-Cys (5 mg, 1.7 μM, 1.0 eq.) in DMF / H20 (0.5 mL, DMF / H20 = 1 : 1), then add cytotoxic agent MC-Val-Cit-PAB-MMAE (2.3 mg, 1.7 μM, 1.0 eq.), and allow the system to react at room temperature for 1 hour. Isolate 1.5 mg of target compound A1-22-Cys-MMAE (99.7% purity, 20.5% yield, MS [M+4H] 4+ : 1057.9).

[0287] Example 4 Synthesis of linker-solid support

[0288] 4.1 Synthesis of Q20-CPG

[0289] Step 1: Preparation of methyl 1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5- azatricosan-13-hydroxy)piperidine-4-carboxylate (Compound Q20-2)

[0290] A mixture of 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-oic acid (5.0 g, 18.99 mmol, 1.0 eq.), methyl 4-carboxypiperidinecarboxylate (2.72 g, 18.99 mmol, 1.0 eq.), N,N-diisopropylethylamine (4.90 g, 37.98 mmol, 2.0 eq.) and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.47 g, 22.28 mmol, 1.2 eq.) in dichloromethane (50 mL) was stirred at room temperature for 30 min. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give methyl 1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatricosan-13-hydroxy)piperidine-4-carboxylate as a yellow oil (12.7 g, crude). LCMS (ESI): m / z = 389 [M+H] + .

[0291] Step 2: Preparation of methyl 1-(2-(2-(2-aminoethoxy)acetyl)piperidine-4-carboxylate hydrochloride (Compound Q20-3):

[0292] A solution of methyl 1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatricosan-13- hydroxy)piperidine-4-carboxylate (crude) in hydrogen chloride-dioxane (40 mL) was stirred at room temperature for 1 h. The solvent was removed under vacuum. The residue was used in the next step without purification (crude). LCMS (ESI): m / z = 289 [M+H] + .

[0293] Step 3: Preparation of methyl 1-(2-(2-(6-heptanamido)ethoxy)acetyl)piperidine-4- carboxylate (Compound Q20-4)

[0294] A mixture of methyl 1-(2-(2-(2-aminoethoxy)acetyl)piperidine-4-carboxylate hydrochloride (crude), 6-heptynoic acid (2.35 g, 18.69 mmol, 1.0 eq), N,N- diisopropylethylamine (7.23 g, 56.06 mmol, 3.0 eq) and 2-(7-azabenzotriazol- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.52 g, 22.42 mmol, 1.2 eq) in dichloromethane (50 mL) was stirred at room temperature for 30 min. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate to dichloromethane / methanol = 20 / 1) to give the product methyl 1-(2-(2-(6-heptanamido)ethoxy)acetyl)piperidine-4-carboxylate (8.19 g, crude) as a yellow oil. LCMS (ESI): m / z = 397 [M+H] + .

[0295] Step 4: Preparation of 1-(2-(2-(6-heptanamido)ethoxy)acetyl)piperidine-4-carboxylic acid (Compound Q20-5)

[0296] A mixture of methyl 1-(2-(2-(6-heptanamido)ethoxy)acetyl)piperidine-4-carboxylate (8.19 g, 18.69 mmol, 1.0 eq), sodium hydroxide (1.24 g, 30.94 mmol, 1.5 eq) in tetrahydrofuran / methanol / water (20 mL / 10 mL / 10 mL) was stirred at room temperature for 1 h. The solvent was concentrated under reduced pressure, the residue was adjusted to pH = 3 and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the product 1-(2-(2-(6-heptanamido)ethoxy)acetyl)piperidine-4-carboxylic acid (6.0 g, crude) as a yellow oil. LCMS (ESI): m / z = 383 [M+H] + .

[0297] Step 5: Preparation of N-(2-(2-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 4-hydroxypyrrolidin-l-yl)-2-oxoethoxy)ethoxyethyl)-l-(2-(2-(2-(hept-6-ynoylamido) ethyl)acetyl)piperidine-4-carboxamide (Compound Q20-6)

[0298] A mixture of l-(2-(2-(6-heptanoylamido)ethoxy)acetyl)piperidine-4-carboxylic acid (1.1 g, 2.87 mmol, 1.0 eq), 2-(2-(2-aminoethoxy)ethoxy-l-(2S,4R)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-l-yl)ethan-l-one (1.22 g, 2.15 mmol, 0.75 eq), N,N-diisopropylethylamine (1.11 g, 8.62 mmol, 3.0 eq) and 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.31 g, 3.45 mmol, 1.2 eq) in dichloromethane (10 mL) was stirred at room temperature for 1 h. The mixture was separated and purified using HPLC (column: UniSil 10-120 C18, 30 x 250 mm) to give the product N-(2-(2-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxypyrrolidin-l-yl)-2-oxoethoxy)ethoxyethyl)-l-(2-(2-(2-(hept-6-ynoylamido) ethyl)acetyl)piperidine-4-carboxamide (320 mg, yield: 16%) as a white solid. LCMS (ESI): m / z = 627 [(M-303)+H] + .

[0299] Step 6: Preparation of 4-(((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-l-(2-(2-(-2-(2-(hept-6-ynoylamido)ethoxy)ethyl)acetyl)piperidine-4-carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (Compound Q20-7)

[0300] A mixture of N-(2-(2-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4- hydroxypyrrolidin-1-yl)-2-oxoethoxy)ethoxyethyl)-1-(2-(2-(2-(hept-6-ynoylamido)ethyl)acetyl)piperidin-4-yl)formamide (320 mg, 0.34 mmol, 1.0 equiv), succinic anhydride (207 mg, 2.07 mmol, 6.0 equiv), triethylamine (418 mg, 4.14 mmol, 12.0 equiv), and 4-dimethylaminopyridine (5 mg, 0.034 mmol, 0.1 equiv) in dichloromethane (5 mL) was stirred at room temperature overnight. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated to give 4-(((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(2-(2-(2-(hept-6-ynoylamido)ethoxy)ethyl)acetyl)piperidin-4-yl)formamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid as a yellow solid (380 mg, crude). 1 H NMR (500 MHz, MeOD) δ 7.42-7.12 (m, 9H), 6.93-6.71 (m, 4H), 5.55-5.28 (m, 1H), 4.40 (dd, J = 44.2, 7.2 Hz, 2H), 4.32-4.16 (m, 3H), 3.91-3.82 (m, 2H), 3.77 (s, 6H), 3.72-3.60 (m, 8H), 3.54 (td, J = 13.4, 6.2 Hz, 6H), 3.35 (dd, J = 14.8, 9.4 Hz, 4H), 3.18-3.05 (m, 6H), 3.02-2.82 (m, 1H), 2.64-2.41 (m, 6H), 2.39-2.24 (m, 1H), 2.17 (dtd, J = 17.2, 12.8, 5.6 Hz, 5H), 1.81-1.60 (m, 5H), 1.51 (dt, J = 14.4, 7.0 Hz, 3H). LCMS (ESI): m / z = 727 [(M-303)+H] + .

[0301] Step 7: Preparation of Q20-CPG

[0302] To a solution of 4-(((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)- 1 -(2-(2-(-2-(2-(hept-6-ynoylamido)ethoxy)ethyl)acetyl)piperidin-4-yl)carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (380 mg, 0.37 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL) was added 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (169 mg, 0.44 mmol, 1.2 eq), 1-hydroxybenzotriazole (70 mg, 0.52 mmol, 1.4 eq) and N,N-diisopropylethylamine (191 mg, 1.48 mmol, 4.0 eq) and the mixture was stirred at room temperature for 5 minutes. To the reaction mixture was then added CPG-NH2 (1.9 g) and stirred at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour. The residue was added to pyridine / acetic anhydride (7.5 mL / 2.5 mL) and stirred at room temperature for 3 hours. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 hour to give the product Q20-CPG as a light yellow solid (2.08 g).

[0303] 4.2 Preparation of Q22-CPG

[0304] Step 1. Preparation of N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (Compound A3): A solution of N6-(tert-butoxycarbonyl)-L-lysine methyl ester (5.0 g, 19.2 mmol, 1.0 eq), tert-butoxycarbonyl-6-aminohexanoic acid (4.89 g, 21.1 mmol, 1.1 eq) and N,N-diisopropylethylamine (5.46 g, 42.3 mmol, 2.2 eq) in dichloromethane was stirred well at room temperature, then 1-hydroxybenzotriazole (5.71 g, 42.3 mmol, 2.2 eq) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.08 g, 42.3 mmol, 2.2 eq) were added and the reaction was allowed to proceed for 1 hour. The reaction was monitored by TLC (DCM:MeOH = 30:1, Rf = 0.3) and LCMS and when the reaction was complete, the reaction was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate solution, then twice with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (eluent: dichloromethane:methanol) to give the product N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (8.5 g, yield: 93.8 %), MS (ESI) m / z 538.3 (M+H)+. f + ​):m / z=474(M+H) + .

[0305] Step 2. Preparation of (6-aminohexanoyl)-L-lysine methyl ester (compound A4): Compound A3 (1.0 g, 2.11 mmol, 1.0 equivalent) and a 10 mL solution of dioxane in hydrogen chloride (2 mol / L) were mixed and reacted for 1.5 h at room temperature. The reaction was completed under LCMS monitoring. The mixture was concentrated under reduced pressure, and water (30 mL) was added. The pH was adjusted to approximately 7-8 by adding sodium hydroxide solution. The aqueous phase was concentrated under reduced pressure using an oil pump. The solid was washed with a 10 / 10 solution of dichloromethane and methanol, filtered, and the filtrate was concentrated under reduced pressure to obtain a pale yellow oily (6-aminohexanoyl)-L-lysine methyl ester (450 mg, yield: 77.9%). MS (ES) + ):m / z=274(M+H) + .

[0306] Step 3. Preparation of (S)-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadecane-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatridecane-23-olate (compound A5): At room temperature, compound A4 (3.0 g, 10.9 mmol, 1.0 equivalent), 2,2-dimethyl-4-oxo-3,8 A solution of 1,1-trioxo-5-azatridecane-13-acid (6.35 g, 24.2 mmol, 2.2 equivalences) and N,N-diisopropylethylamine (5.7 mL, 32.9 mmol, 3.0 equivalences) in dichloromethane was stirred until homogeneous. Then, 1-hydroxybenzotriazole (3.7 g, 27.5 mmol, 2.5 equivalences) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.24 g, 27.5 mmol, 2.5 equivalences) were added, and the mixture was reacted for 1 hour. TLC was performed (DCM:MeOH = 20:1, R...). f =0.3) and under LCMS monitoring, the reaction was completed, and the reaction solution was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate, then twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol) to obtain the product (S)22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadecane-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatoctadecane-23-oic acid methyl ester (3.9 g, yield: 46.8%). MS (ES) + ):m / z=764(M+H) + .

[0307] Step 4. Preparation of methyl-N2-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (compound A6): Compound A5 (3.9 g, 5.11 mmol, 1.0 equivalent) was mixed with a 40 mL solution of dioxane (2 mol / L) of hydrogen chloride at room temperature and reacted for 1.5 h. The reaction was completed under LCMS monitoring, and the reaction solution was concentrated under reduced pressure. Add water (30 mL), adjust the pH to approximately 7-8 with sodium hydroxide solution, concentrate the aqueous phase under reduced pressure using an oil pump, and wash the solid with a 10 / 10 dichloromethane / methanol solution. Filter and concentrate the filtrate under reduced pressure to obtain a pale yellow oily methyl N2-(6-(2-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (2.9 g, yield: 85.8%). MS (ES) + ):m / z=564(M+H) + .

[0308] Step 5. Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaeicosico-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadecano-25-ynyl methyl ester (compound A7): At room temperature, compound A6 (440 mg, 0.691 mmol, 1.0 equivalent), 6-heptanynic acid (178 mg) and methyl 2-(6,15-dioxo-8,11-dioxo-5,14-diazaeicosico-20-yn-1-yl) were prepared. A solution of N,N-diisopropylethylamine (263 μL, 1.52 mmol, 2.2 equivalences) in dichloromethane was stirred until homogeneous. Then, 1-hydroxybenzotriazole (205 mg, 1.52 mmol, 2.2 equivalences) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (290 mg, 1.52 mmol, 2.2 equivalences) were added and reacted for 1 hour. TLC was performed (DCM:MeOH = 20:1, R...). f =0.2) and under LCMS monitoring, the reaction was completed, and the reaction solution was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate, then twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol) to obtain the product (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaeicosico-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadecano-25-ynyl methyl ester (310 mg, yield: 57.6%). MS (ES) +): m / z = 780 (M+H) + .

[0309] Step 6. Preparation of (S)-2-(6, 15-dioxo-8, 11-dioxo-5, 14-diazahenicos-20-yn-1-yl)- 4, 11, 20-trio xo-13, 16-dioxo-3, 10, 19-triazahexacos-25-ynoic acid (Compound A8): Compound A7 (310 mg, 0.398 mmol, 1.0 eq) was added into a mixture solvent of tetrahydrofuran, methanol and water (6 mL), then lithium hydroxide (20.1 mg, 0.478 mmol, 1.2 eq) aqueous solution (1 mL) was added, and stirred at room temperature for 1 hour. The reaction was monitored by LCMS and was complete. The reaction solution was concentrated under reduced pressure, water (25 mL) was added, 1 mol / L dilute hydrochloric acid solution was added to adjust the pH value to about 3-4, diluted with water (20 mL), and dichloromethane (25 mL) was added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to obtain white solid (S)-2-(6, 15-dioxo-8, 11-dioxo-5, 14-diazahenicos-20-yn-1-yl)-4, 11, 20-trio xo-13, 16-dioxo-3, 10, 19-triazahexacos-25-ynoic acid (290 mg, yield: 95.4%). 1 H NMR (500 MHz, CDC13) δ 6.90 (s, 2H), 6.83 (d, J = 8.3 Hz, 1H), 6.55 (s, 2H), 4.15-4.10 (m, 1H), 3.69-3.62 (m, 4H), 3.55 (s, 8H), 3.50 (t, J = 4.8 Hz, 10H), 3.41-3.35 (m, 10H), 2.45-2.36 (m, 4H), 2.32 (t, J = 6.8 Hz, 2H), 2.24 (t, J = 6.9 Hz, 2H), 2.15 (dt, J = 7.1, 6.2 Hz, 7H), 1.89 (dd, J = 15.2, 8.1 Hz, 4H), 1.72-1.64 (m, 4H), 1.52-1.46 (m, 4H). MS (ES + ): m / z = 766 (M+H) + .

[0310] Step 7. Preparation of compound A10: A solution of A8 (5.0 g, 6.53 mmol, 1.0 eq.), A9 (2.26 g, 6.86 mmol, 1.05 eq.), DIEA (1.68 g, 13.06 mmol, 2.0 eq.) and HATU (3.72 g, 9.8 mmol, 1.5 eq.) in DMF (10 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with water, saturated Na2CO3 and NH4Cl solution twice, then washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give A10 crude 6.14 g. MS (ES + ): m / z = 1079.46 (M+H) + .

[0311] Step 8. Preparation of compound A11: A10 crude (6.14 g) was dissolved in 5 mL of dichloromethane, 5 mL of trifluoroacetic acid was added with stirring, the system was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, the residue was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give A11 crude 5.53 g. MS (ES + ): m / z = 1023.25 (M+H) + .

[0312] Step 9. Preparation of compound A13: A solution of A11 crude (5.53 g), A12 (3.2 g, 5.67 mmol), DIEA (1.4 g, 10.8 mmol) and HATU (3.07 g, 8.1 mmol) in DMF (10 mL) was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with water, saturated Na2CO3 solution twice, then washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by RP-HPLC to give the target product A13 (5.1 g, three-step reaction yield: 49.7%) as a white oil. 1HNMR (500 MHz, DMSO-d6) δ 7.90-7.77 (m, 5H), 7.61 (t, J = 5.9 Hz, 2H), 7.35-7.26 (m, 4H), 7.21-7.17 (m, 4H), 6.91-6.84 (m, 4H), 4.97 (s, 1H), 4.42-4.36 (m, 1H), 4.27 (d, J = 14.0 Hz, 1H), 4.19 (td, J = 8.6, 5.1 Hz, 2H), 4.11 (t, J = 13.4 Hz, 2H), 4.06 (s, 1H), 3.85 (d, J = 1.7 Hz, 4H), 3.73 (s, 7H), 3.62-3.46 (m, 17H), 3.46-3.35 (m, 9H), 3.19 (p, J = 7.5, 6.6 Hz, 9H), 3.10-3.01 (m, 5H), 2.90 (d, J = 12.7 Hz, 1H), 2.72 (d, J = 2.6 Hz, 2H), 2.54 (s, 1H), 2.43-2.30 (m, 1H), 2.18-2.04 (m, 11H), 2.01 (dd, J = 12.5, 5.9 Hz, 1H), 1.84 (ddd, J = 12.9, 8.3, 4.4 Hz, 1H), 1.64 (s, 2H), 1.56 (p, J = 7.5 Hz, 5H), 1.43 (ddq, J = 24.8, 17.3, 10.1, 8.8 Hz, 14H), 1.22 (q, J = 7.5 Hz, 4H). MS (ES + ): m / z = 1267.30 [(M-303)+H] + .

[0313] Step 10. Preparation of Q22-CPG: A13 (5.1 g, 3.25 mmol, 1 eq.) was dissolved in dichloromethane (20 mL), succinic anhydride (1.95 g, 19.5 mmol, 6.0 eq), triethylamine (3.94 g, 39 mmol, 12.0 eq.) and 4-dimethylaminopyridine (39 mg, 0.325 mmol, 0.1 eq.) were added successively with stirring. The system was allowed to react at room temperature overnight, the reaction system was diluted with dichloromethane, washed with 10% sodium bicarbonate solution three times, and saturated sodium chloride solution once. The organic phase was dried over anhydrous sodium sulfate and concentrated to give a brown oil. 1H NMR (500 MHz, CDC13 ) δ 7.57 (dt, J = 20.0, 5.0 Hz, 1H), 7.35 (d, J = 10.0 Hz, 2H), 7.29 - 7.19 (m, 6H), 7.22 - 7.17 (m, 3H), 7.08 - 6.90 (m, 4H), 6.69 - 6.59 (m, 1H), 6.50 - 6.39 (m, 1H), 5.46 - 5.38 (m, 1H), 4.58 - 4.43 (m, 2H), 4.33 - 4.01 (m, 4H), 3.98 (d, J = 5.0 Hz, 4H), 3.79 - 3.78 (m, 7H), 3.67 - 3.60 (m, 16H), 3.57 (q, J = 5.0 Hz, 8H), 3.52 - 3.49 (m, 2H), 3.48 - 3.43 (m, 5H), 3.43 - 3.38 (m, 2H), 3.38 - 3.31 (m, 1H), 3.27 (dq, J = 10.0, 5.0 Hz, 4H), 3.16 - 3.11 (m, 1H), 3.06 (s, 1H), 2.58 - 2.50 (m, 5H), 2.46 - 2.36 (m, 1H), 2.31 (dd, J = 10.0, 5.0 Hz, 1H), 2.26 - 2.17 (m, 10H), 2.11 (td, J = 10.0, 5.0 Hz, 1H), 1.98 (t, J = 5.0 Hz, 2H), 1.84 (dt, J = 10.0, 5.0 Hz, 2H), 1.75 (dt, J = 15.0, 5.0 Hz, 6H), 1.65 (dt, J = 15.0, 5.0 Hz, 4H), 1.55 (dq, J = 15.0, 5.0 Hz, 9H), 1.39 - 1.33 (m, 4H). 13 C NMR (126 MHz, CDC13 ) δ

[0314] 174.77, 174.60, 173.25, 173.11, 173.02, 172.93, 172.20, 169.98, 169.87, 168.55, 168.04, 167.58, 167.51, 158.66, 158.47, 144.85, 135.86, 129.95, 128.00, 127.84, 126.84, 113.27, 113.13, 86.06, 84.18, 84.16, 73.19, 70.96, 70.92, 70.61, 70.54, 70.12, 70.04, 70.01, 70.00, 68.69, 55.22, 52.78, 52.19, 45.02, 39.44, 39.26, 39.07, 38.69, 38.57, 36.24, 35.90, 35.86, 32.87, 32.48, 31.22, 30.57, 29.32, 29.14, 28.72, 27.97, 26.51, 25.30, 25.25, 24.79, 22.73, 18.20, 9.05. MS (ES + ): m / z = 1367.35 [(M-303)+H] + .

[0315] The above oil (5.26 g) was dissolved in DMF, DIEA (1.63 g, 12.6 mmol), HATU (1.44 g, 3.78 mmol) and HOBt (510 mg, 3.78 mmol) were added with stirring, the mixture was stirred at room temperature for 5 minutes, 5.3 g of controlled-pore glass support (CPG, substitution rate 1.2 mmol / g) was added to the reaction mixture. The system was stirred at room temperature overnight, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour. The above dried solid phase support was placed in pyridine / acetic anhydride (20 mL / 7 mL) and shaken at room temperature for 3 hours, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour to obtain a light yellow solid phase support Q22-CPG (10.2 g).

[0316] 4.3 Preparation of Q23-CPG

[0317] Step 1: Preparation of compound Q23-3

[0318] A solution of Q23-1 (9.8 g, 19.38 mmol, 1.0 eq), Q23-2 (4.74 g, 19.38 mmol, 1.0 eq), N,N-diisopropylethylamine (7.5 g, 58.14 mmol, 3.0 eq) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.84 g, 23.26 mmol, 1.2 eq) in dichloromethane (150 mL) was stirred at room temperature for one hour. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 5 / 1 to 2 / 1) to give Q23-3 as a yellow oil (12.2 g, yield: 86.04%). LCMS (ESI): m / z = 732 [M+H] + .

[0319] Step 2: Preparation of compound Q23-4:

[0320] A solution of Q23-3 (12 g, 16.39 mmol, 1.0 eq) in dichloromethane (50 mL) was added to trifluoroacetic acid (50 mL) at 0 °C. The mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used in the next step without purification (crude). LCMS (ESI): m / z = 564 [M+H] + .

[0321] Step 3: Preparation of compound Q23-5

[0322] A solution of Q23-4 (crude), tert-butyl (2-aminoethyl)carbamate (8.35 g, 52.2 mmol, 3.2 eq), N,N-diisopropylethylamine (16.83 g, 130.5 mmol, 8.0 eq) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (20.46 g, 53.8 mmol, 3.3 eq) in dichloromethane (150 mL) was stirred at room temperature for one hour. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 200 / 1 to 20 / 1) to give the product Q23-5 as a yellow oil (10.21 g, yield: 63.14%). LCMS (ESI): m / z = 991 [M+H] + .

[0323] Step 4: Preparation of compound Q23-6:

[0324] A solution of Q23-5 (10.2 g, 10.29 mmol, 1.0 eq) in hydrogen chloride-dioxane (40 mL) and methanol (20 mL) was stirred at room temperature for 1 h. The solvent was removed under vacuum. The residue was used in the next step without purification (crude). LCMS (ESI): m / z = 691 [M+H] + .

[0325] Step 5: Preparation of compound Q23-7

[0326] Q23-6 (crude) was added slowly dropwise into a mixture of hept-6-ynoic acid (4.15 g, 32.94 mmol, 3.2 eq), N,N-diisopropylethylamine (6.64 g, 51.46 mmol, 5.0 eq) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (12.91 g, 33.97 mmol, 3.3 eq) in N,N-dimethylformamide (50 mL). The mixture was stirred at room temperature for 1 h. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1) to give the product Q23-7 (8.25 g, yield: 78.95%) as a yellow solid. LCMS (ESI): m / z = 1015 [M+H] + .

[0327] Step 6: Preparation of compound Q23-8

[0328] A mixture of Q23-7 (4.2 g, 4.14 mmol, 1.0 eq), lithium hydroxide (209 mg, 4.97 mmol, 1.2 eq) in tetrahydrofuran / methanol / water (10 mL / 10 mL / 10 mL) was stirred at room temperature for 2.5 h. The solvent was concentrated under reduced pressure, the residue was adjusted to pH = 4 and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to give the product Q23-8 (4.1 g, crude) as a white solid. LCMS (ESI): m / z = 1001 [M+H] + .

[0329] Step 7: Preparation of compound Q23-10

[0330] A solution of Q23-8 (4.1 g, 4.1 mmol, 1.0 eq), Q23-9 (1.72 g, 4.1 mmol, 1.0 eq), N,N-diisopropylethylamine (2.11 g, 16.38 mmol, 4.0 eq) and benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (1.63 g, 4.3 mmol, 1.05 eq) in N,N-dimethylformamide (50 mL) was stirred at room temperature for 1 h. The mixture was separated and purified by HPLC (column: UniSil 10-120 C18, 30 x 250 mm) to give the product Q23-10 (4.17 g, yield: 72.77%) as a yellow solid. LCMS (ESI): m / z = 1100 [(M-303)+H] + .

[0331] Step 8: Preparation of compound Q23-11

[0332] A solution of Q23-10 (2.1 g, 1.5 mmol, 1.0 eq), succinic anhydride (900 mg, 9.0 mmol, 6.0 eq), triethylamine (1.82 g, 18.0 mmol, 12.0 eq) and 4-dimethylaminopyridine (19 mg, 0.15 mmol, 0.1 eq) in dichloromethane (10 mL) was stirred at room temperature for 2 h. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated to give Q23-11 as a yellow solid (2.2 g, crude). LCMS (ESI): m / z = 1200 [(M-303)+H] + .

[0333] Step 9: Preparation of compound Q23-CPG

[0334] To a solution of Q23-11 (1.1 g, 0.73 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL) was added 2-(7-azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (335 mg, 0.88 mmol, 1.2 eq), 1 -hydroxybenzotriazole (139 mg, 1.03 mmol, 1.4 eq) and N,N-diisopropylethylamine (379 mg, 2.93 mmol, 4.0 eq), the mixture was stirred at room temperature for 5 min. Then CPG-NH2 (5.0 g) was added to the reaction mixture, stirred at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h. The residue was added with pyridine / acetic anhydride (7.5 mL / 2.5 mL), stirred at room temperature for 3 h. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h to give the product Q23-CPG as a light yellow solid. (5.12 g, loading: 76.87 umol / g).

[0335] 4.4 Preparation of Q25-CPG

[0336] Step 1: Preparation of compound Q25-2

[0337] A solution of Q23-1 (4.2 g, 8.31 mmol, 1.0 eq), (1r,4r)-4-(methoxycarbonyl)cyclohexane-1 - carboxylic acid (1.55 g, 8.31 mmol, 1.0 eq), N,N-diisopropylethylamine (3.21 g, 24.92 mmol, 3.0 eq) and 2-(7-azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.79 g, 9.97 mmol, 1.2 eq) in dichloromethane (50 mL) was stirred at room temperature for 1 h. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 5 / 1 to 2 / 1) to give Q25-2 as a yellow solid (12.2 g, yield: 73.32%). LCMS (ESI): m / z = 674 [M+H] + .

[0338] Step 2: Preparation of compound Q25-3:

[0339] A solution of Q25-2 (4.05 g, 6.0 mmol, 1.0 eq) in dichloromethane (15 mL) was added with trifluoroacetic acid (15 mL) at 0 °C. The mixture was stirred at room temperature for 1 h. The solvent was removed under vacuum. The residue was used in the next step without purification (crude). LCMS (ESI): m / z = 506 [M+H]+ .

[0340] Step 3: Preparation of compound Q25-4

[0341] A solution of compound Q25-3 (crude), tert-butyl (2-aminoethyl)carbamate (3.08 g, 19.23 mmol, 3.2 eq), N,N-diisopropylethylamine (3.88 g, 30.04 mmol, 5.0 eq) and 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.31 g, 19.23 mmol, 3.2 eq) in dichloromethane (30 mL) was stirred at room temperature for one hour. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 200 / 1 to 20 / 1) to give the product Q25-4 (5.05 g, yield: 90.02%) as a yellow solid. LCMS (ESI): m / z = 933 [M+H] + .

[0342] Step 4: Preparation of compound Q25-5

[0343] A solution of Q25-4 (5.05 g, 5.41 mmol, 1.0 eq) in hydrogen chloride-dioxane (30 mL) and methanol (15 mL) was stirred at room temperature for one hour. The solvent was removed under vacuum. The residue was used in the next step without purification (crude). LCMS (ESI): m / z = 633 [M+H] + .

[0344] Step 5: Preparation of compound Q25-6

[0345] Q25-5 (crude) was slowly added dropwise into a mixture of hept-6-ynoic acid (2.18 g, 17.32 mmol, 3.2 eq), N,N-diisopropylethylamine (3.49 g, 27.06 mmol, 5.0 eq) and 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.58 g, 17.32 mmol, 3.2 eq) in N,N-dimethylformamide (30 mL). The mixture was stirred at room temperature for one hour. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1) to give the product Q25-6 (3.91 g, yield: 75.48%) as a yellow solid. LCMS (ESI): m / z = 957 [M+H] + .

[0346] Step 6: Preparation of compound Q25-7

[0347] A mixture of Q25-6 (3.5 g, 3.66 mmol, 1.0 eq), lithium hydroxide (184 mg, 4.39 mmol, 1.2 eq) in tetrahydrofuran / methanol / water (10 mL / 10 mL / 10 mL) was stirred at room temperature for 2.5 hours. The solvent was concentrated under reduced pressure, the residue was adjusted to pH = 7 and lyophilized to give the yellow solid product Q25-7 (3.67 g, crude). LCMS (ESI): m / z = 943 [M+H] + .

[0348] Step 7: Preparation of compound Q25-9

[0349] A solution of Q25-7 (3.67 g, crude), Q25-8 (2.2 g, 3.66 mmol, 1.0 eq), N,N- diisopropylethylamine (1.89 g, 14.63 mmol, 4.0 eq) and benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (1.46 g, 3.84 mmol, 1.05 eq) in N,N-dimethylformamide (30 mL) was stirred at room temperature for 1 hour. The mixture was separated and purified by HPLC (column: UniSil 10-120 C18, 30 x 250 mm) to give the yellow solid product Q25-9 (3.5 g, yield: 62.17%). LCMS (ESI): m / z = 1239 [(M-303)+H] + .

[0350] Step 8: Preparation of compound Q25-10

[0351] A solution of Q25-9 (1.2 g, 0.78 mmol, 1.0 eq), succinic anhydride (468 mg, 4.68 mmol, 6.0 eq), triethylamine (944 mg, 9.35 mmol, 12.0 eq) and 4-dimethylaminopyridine (10 mg, 0.078 mmol, 0.1 eq) in dichloromethane (6 mL) was stirred at room temperature for 2 hours. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated to give Q25-10 as a yellow solid (1.3 g, crude). LCMS (ESI): m / z = 1339 [(M-303)+H] + .

[0352] Step 9: Preparation of compound Q25-CPG

[0353] To a solution of Q25-10 (1.3 g, 0.79 mmol, 1.0 eq) in N,N-dimethylformamide (11 mL) was added 2-(7-azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (421 mg, 1.11 mmol, 1.4 eq), 1 -hydroxybenzotriazole (160 mg, 1.19 mmol, 1.5 eq) and N,N-diisopropylethylamine (409 mg, 3.17 mmol, 4.0 eq), the mixture was stirred at room temperature for 5 min. Then CPG-NH2 (5.5 g) was added to the reaction mixture, stirred at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h. The residue was added with pyridine / acetic anhydride (7.5 mL / 2.5 mL), stirred at room temperature for 3 h. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h to give the product Q25-CPG as a light yellow solid. (6.34 g, loading: 69.21 umol / g).

[0354] 4.5 Preparation of Q16-CPG

[0355] Step 1 : Preparation of Q16-2

[0356] A solution of 2-(2-(2-aminoethoxy)ethoxy-1 -(2S,4R)-2-(bis(4- methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1 -yl)ethan-1 -one (2.5 g, 4.4 mmol, 1.0 eq), hept-6-ynoic acid (0.55 g, 4.4 mmol, 1.0 eq), N,N-diisopropylethylamine (1.7 g, 13.2 mmol, 3.0 eq) and 2-(7-azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.0 g, 5.3 mmol, 1.2 eq) in dichloromethane (20 mL) was stirred at room temperature for 1 h. The solvent was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography to give the product Q16-2 (2.7 g, yield: 91 %) as a yellow solid. LCMS (ESI): m / z = 369 [(M-303)+H] + .

[0357] Step 2: Preparation of Q16-CPG

[0358] A solution of Q16-2 (2.7 g, 4.0 mmol, 1.0 eq), succinic anhydride (2.4 g, 24 mmol, 6.0 eq), triethylamine (4.85 g, 48 mmol, 12.0 eq) and 4-dimethylaminopyridine (48.8 mg, 0.4 mmol, 0.1 eq) in dichloromethane (6 mL) was stirred at room temperature for 2 h. The mixture was washed with 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was washed with 10% sodium bicarbonate solution and acetonitrile. The organic phase was concentrated and dried under reduced pressure to give a solid.

[0359] To a solution of the above solid in N,N-dimethylformamide (20 mL) was added 2-(7-azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.12 g, 5.6 mmol, 1.4 eq), 1 -hydroxybenzotriazole (810 mg, 6.0 mmol, 1.5 eq) and N,N-diisopropylethylamine (2.06 g, 16 mmol, 4.0 eq) and the mixture was stirred at room temperature for 5 min. CPG-NH2 (27.8 g) was then added to the reaction mixture and stirred at room temperature overnight. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h. The residue was added to pyridine / acetic anhydride (30 mL / 10 mL) and stirred at room temperature for 3 h. The mixture was filtered, the filter cake was washed with dichloromethane, acetonitrile, dichloromethane and dried under vacuum for 1 h to give a light yellow solid product Q16-CPG. (31.2 g, loading: 78.4 umol / g).

[0360] 4.6 Synthesis of Q27-CPG

[0361] Step 1. Synthesis of Q27-2

[0362] Q27-1 (9.04 g, 19.17 mmol, 1.0 eq) was dissolved in acetonitrile (181 mL), N,N,N',N'-tetramethyluronium hexafluorophosphate (19.37 g, 69.01 mmol, 3.6 eq) and N-methylimidazole (16.53 g, 201.29 mmol, 10.5 eq) were added successively, stirred for 10 min, then Int-1 (9.89 g, 69.01 mmol, 3.6 eq) was added, stirred at room temperature for 30 min. The reaction solution was rotary evaporated, then the residue was dissolved in 200 mL EA, the organic phase was washed successively with saturated NaHC03, saturated NH4C1 and 1 M HC1 solution, the organic phase was collected, dried with anhydrous Na2S04, filtered, rotary evaporated, finally 13.5 g of crude product was obtained. LCMS (ESI): m / z = 847 [(M+H] + The product was used directly in the next step without further purification.

[0363] Step 2. Synthesis of Q27-3

[0364] Q27-2 (13.5 g, 15.95 mmol, 1.0 eq) was dissolved in hexafluoroisopropanol (65 mL), methanesulfonic acid (15.3 g, 159.50 mmol, 10.0 eq) was added, and the reaction was placed at 50 °C for 2 h. The reaction was spun dry, dissolved in 100 mL DCM, diluted with 100 mL water, and the pH of the reaction was adjusted to 8-9 with saturated Na2CO3 solution. Extraction was performed with DCM (150 mL x 3), the organic phases were combined, dried, and concentrated to give 11.3 g of crude product. LCMS (ESI): m / z = 713 [(M+H] + The product was used directly in the next step without further purification.

[0365] Step 3. Synthesis of Q27-4

[0366] Int-2 (14.8 g, 79.3 mmol, 5.0 eq) was dissolved in acetonitrile (270 mL), N,N,N',N'-tetramethylchloroformamidium hexafluorophosphate (26.7 g, 95.16 mmol, 6.0 eq) and N-methylimidazole (26.0 g, 317.2 mmol, 20 eq) were added sequentially, stirred for 10 min, and then Q27-3 (11.3 g, 15.86 mmol, 1.0 eq, dissolved in acetonitrile) was added. The reaction was left overnight. The reaction was spun dry, dissolved in 300 mL EA, washed sequentially with 1 M HC1 solution and saturated Na2CO3 solution (300 mL each), the organic phase was collected, dried with anhydrous Na2SO4, filtered, spun dry, and finally 40.0 g of crude product was obtained. Column chromatography on silica gel gave 11.8 g of a light yellow foamy solid. LCMS (ESI): m / z = 882 [(M+H] + (Eltuant solvent gradient: 0.5%, 1%, 2%, 3.3%, 5% MeOH / DCM).

[0367] Step 4. Synthesis of Q27-5

[0368] Q27-4 (11.8 g, 13.4 mmol, 1.0 eq) was dissolved in THF (40 mL), MeOH (40 mL), H2O (40 mL), and LiOH*H2O (1.7 g, 40.20 mmol, 3.0 eq) were added sequentially, and the reaction was stirred at room temperature for 60 min. The THF and MeOH were spun dry from the reaction, the pH of the reaction was adjusted to 3-4 with 1 M HC1 solution, and extraction was performed with EA (150 mL x 3). The organic phases were combined, dried, and concentrated to give 9.8 g of crude product. LCMS (ESI): m / z = 867 [(M+H] +The product was used in the next step without further purification.

[0369] Step 5. Synthesis of Q27-6

[0370] Q27-5 (9.8 g, 11.06 mmol, 1.0 eq) was dissolved in acetonitrile (200 mL), followed by the addition of N,N,N',N'-tetramethylchloroformamidium hexafluorophosphate (9.3 g, 33.18 mmol, 3.0 eq) and N-methylimidazole (9.1 g, 110.60 mmol, 10 eq), stirred for 10 min, then Int-3 (9.4 g, 16.59 mmol, 1.5 eq) was added, stirred at room temperature for 2 h. The reaction was concentrated to dryness, the residue was purified by silica gel column chromatography (elution solvent gradient: 0.5%, 1%, 2%, 3.3%, 5% MeOH / DCM, all elution solutions were added with 1% Et3N) to give 5 g of product, then purified by HPLC prep to give 1.50 g of white foamy solid as pure product. LCMS (ESI): m / z = 1111 [(M-303)+H] + .

[0371] Step 6. Synthesis of Q27-7

[0372] Q27-6 (1.50 g, 1.06 mmol, 1.0 eq) was dissolved in anhydrous dichloromethane (7.5 mL), followed by the addition of triethylamine (0.64 g, 6.36 mmol, 6.0 eq), 4-dimethylaminopyridine (0.0130 g, 0.106 mmol, 0.1 eq) and succinic anhydride (0.32 g, 3.18 mmol, 3.0 eq), stirred at room temperature for 20 h under nitrogen protection. 7.5 mL dichloromethane, 7.5 mL water were added, stirred at room temperature for 5 min, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give 1.59 g of yellow foamy solid, yield 93.0%. LCMS (ESI): m / z = 1211 [(M-303)+H] + .

[0373] Step 7. Synthesis of Q27-CPG

[0374] Q27-7 (1.59 g, 0.98 mmol, 1.0 eq) was dissolved in anhydrous DMF (80 mL), HBTU (0.45 g, 1.18 mmol, 1.2 eq) and DIEA (0.51 g, 3.92 mmol, 4.0 eq) were added successively, the solution was shaken at room temperature for 10 min until it was clear, then the solid support CPG (8.00 g, 1.27 mmol, 1.3 eq) was added, and shaken at room temperature for 18 h. Filtration, washing with acetonitrile, dichloromethane, the resulting solid was added to a mixed solution of anhydrous pyridine (60 mL) and acetic anhydride (20 mL), shaken at room temperature for 1 h, filtered, washed with acetonitrile, dichloromethane, the resulting solid was dried at room temperature for 2 h, and finally Q27-CPG solid 8.56 g was obtained with a loading of 68.47 pmol / g.

[0375] 4.7 Preparation of Q26-CPG

[0376] Step 1. Preparation of Q26-1 : A solution of A11 crude (2.5 g), Int-1 (1.48 g, 2.45 mmol), DIEA (0.94 g, 7.36 mmol) and HATU (1.4 g, 3.68 mmol) in DMF (7 mL) was stirred at room temperature for 2 h. The reaction was diluted with ethyl acetate and washed with water, saturated Na2CO3 solution twice, and saturated brine once. The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by RP-HPLC to give the target product 1.77 g as a white oil. MS (ES + ): m / z = 1305.04 [(M-303) + H] + .

[0377] Step 2. Preparation of Q26-CPG: Q26-1 (1.77 g, 1.1 mmol, 1 eq.) was dissolved in dichloromethane (10 mL), succinic anhydride (6.6 g, 6.6 mmol, 6.0 eq), triethylamine (1.33 g, 13,2 mmol, 12.0 eq.) and 4-dimethylaminopyridine (13.4 mg, 0.11 mmol, 0.1 eq.) were added successively with stirring. The system was reacted at room temperature overnight, the reaction was diluted with dichloromethane, washed with 10% sodium bicarbonate solution three times, and saturated sodium chloride solution once. The organic phase was dried over anhydrous sodium sulfate and concentrated to give a brown oil. MS (ES + ): m / z = 1405.12 [(M-303) + H] + .

[0378] The oily substance (1.8 g) was dissolved in DMF, DIEA (0.82 g, 6.32 mmol), HATU (0.61 g, 1.58 mmol) and HOBt (213 mg, 1.58 mmol) were added under stirring, the mixture was stirred at room temperature for 5 minutes, 1.3 g of controlled-pore glass carrier (CPG, substitution rate 1.2 mmol / g) was added to the reaction mixture. The system was stirred at room temperature overnight, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour. The dried solid phase carrier was placed in pyridine / acetic anhydride (10 mL / 3.5 mL) and shaken at room temperature for 3 hours, filtered, the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in turn, and dried under vacuum for 1 hour to obtain a light yellow solid phase carrier Q26-CPG (2.1 g).

[0379] Example 5 Synthesis and annealing of siRNA

[0380] On the 48-channel nucleic acid synthesizer of Jiangsu Lingkun Biotechnology Co., Ltd., 0.2-1 μmol of oligonucleotide synthesis was carried out using a solid-phase oligonucleotide synthesis protocol. For siRNA sense strands SS-Q16 (R=O or S), SS-Q20 (R=O or S), SS-Q22 (R=O or S), SS-Q23 (R=O or S), SS-Q25 (R=O or S), SS-Q26 (R=O or S) and SS-Q27 with alkyne modification at the 3'-end, they were synthesized by using special controlled-pore glass (CPG) carrier as shown in the following reaction formula. According to the existing technology, the pharmaceutically acceptable salt of the siRNA can be a sodium salt or a potassium salt, for example, a sodium salt of siRNA is produced during purification.

[0381] After the completion of solid-phase synthesis, an aminolysis reagent was added to the oligonucleotide solid phase carrier, and incubated at 45-80°C, the oligonucleotide was separated from the solid phase carrier to free the oligonucleotide. Then the crude oligonucleotide was precipitated with ethanol, the supernatant was discarded by high-speed centrifugation, and the process was repeated twice to obtain the crude oligonucleotide, and the precipitate was resuspended in DEPC water. The crude oligonucleotide was purified by ion pair HPLC method, and the collected product was dried in a vacuum centrifugal dryer to powder. The product obtained after purification was dissolved in DEPC water, and analyzed by TOF LC-MS. The names, modified and unmodified sequences, and corresponding theoretical and actual molecular weights of the siRNA sense strands, antisense strands and sense strands containing alkyne modification synthesized in the present application are summarized in Tables 4-1 and 4-2 below.

[0382] Table 4-1: Mass spectrometric characterization of siRNA sense strands, alkyne-sense strands and antisense strands.

[0383] Table 4-2: Unmodified nucleic acid sequences of sense strand, alkynyl-sense strand and antisense strand of siRNA.

[0384] Example 6 Synthesis of polypeptide oligonucleotide conjugates

[0385] The present application utilizes click chemistry to efficiently synthesize polypeptide oligonucleotide conjugates. In which, the 3' end of siRNA sense strand is modified with one, two or three alkynyl groups, while the polypeptide is modified with azido group. The general conditions and procedures for the reaction between mono-alkynyl-nucleic acid and azido-polypeptide are as follows: mono-alkynyl-nucleic acid (sense strand-Q16, sense strand-Q20, sense strand-Q22, sense strand-Q23 or sense strand-Q25) is dissolved in phosphate buffer (0.1 M, pH 7.4) with a concentration of 5 mg / mL, then N-methyl pyrrolidone solution of azido-polypeptide (2.5 eq.) is added, followed by the addition of copper sulfate pentahydrate (30 eq.) and tris(3-hydroxypropyltriazolylmethyl)amine (15 eq.) in N-methyl pyrrolidone and sodium L-ascorbate (60 eq.) under nitrogen protection. The volume ratio of phosphate buffer and N-methyl pyrrolidone in the system is between 1:1 and 2:1. After the system is reacted at 40°C for 1 hour under nitrogen protection, 200 eq. of ammonia and 200 eq. of EDTA are added to quench the reaction, followed by the addition of 1 / 10 times the volume of saturated NaCl solution and 3 times the volume of cold ethanol to precipitate the product, high-speed centrifugation for 3 minutes (12000 rpm, 4°C), discard the supernatant, dissolve the precipitate in ddH2O, and purify by RP-HPLC. The experimental procedure for the click chemistry coupling reaction between di-alkynyl-nucleic acid and azido-polypeptide is similar to that between mono-alkynyl-nucleic acid and azido-polypeptide, with the exception of the following reagent equivalents: the equivalents of polypeptide, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolylmethyl)amine and sodium L-ascorbate are 5, 60, 40 and 120 equivalents, respectively. The experimental procedure for the click chemistry coupling reaction between tri-alkynyl-nucleic acid and azido-polypeptide is similar to that between mono-alkynyl-nucleic acid and azido-polypeptide, with the exception of the following reagent equivalents: the equivalents of polypeptide, copper sulfate pentahydrate, tris(3-hydroxypropyltriazolylmethyl)amine and sodium L-ascorbate are 10, 60, 40 and 120 equivalents, respectively.

[0386] After obtaining the purified polypeptide-sense strand conjugate, the nucleotide concentration was determined by dissolving the sample in DEPC water, and the volume of polypeptide-sense strand and antisense strand required for equimolar amount was calculated. The polypeptide-siRNA double strand was prepared by mixing equimolar amount of polypeptide-sense strand and antisense strand, heating at 75°C for 5 minutes, and then naturally cooling to room temperature. After the annealing product was cooled to room temperature, it was diluted with 10 times volume of DEPC water, and then transferred to a Millipore 15 mL ultrafiltration centrifuge tube (molecular weight cut-off 3 KDa) for ultrafiltration, which aimed to remove the residual chemical reagents (such as n-hexylamine) in the preparation process of polypeptide and nucleic acid. The ultrafiltration centrifuge tube was centrifuged at 4000 rpm for about 40 min, and when the volume of the product was reduced to about 1 mL, the product in the ultrafiltration tube was collected and the residual product in the tube was washed with 3 mL of DEPC water. The ultrafiltration product was filtered through a 0.22 μm filter to remove bacteria, and then the collected product was dried to powder in a vacuum centrifugal dryer. After being dissolved in the corresponding solvent, the product can be used for subsequent in vitro and in vivo experiments.

[0387] 6.1 Synthesis of A1-22-Q20-m24017.1-1

[0388] m24017SM1-Q20 (10 mg, 1.33 μM, 1 eq.) was dissolved in 2 mL of phosphate buffer (0.1 M, pH 7.4), and then a solution of A1-22 (10.2 mg, 3.3 μM, 2.5 eq.) in N-methylpyrrolidone (1.5 mL) was added. Under nitrogen protection, a solution of copper sulfate pentahydrate (10 mg, 40 μM, 30 eq.) and tris(3-hydroxypropyltriazolylmethyl)amine (8.7 mg, 20 μM, 15 eq.) in N-methylpyrrolidone (0.5 mL) and sodium L-ascorbate (15 mg, 80 μM, 60 eq.) were added. After the system was reacted at 40°C for 1 hour under nitrogen protection, 36.5 μl of ammonia solution (28% in water) and 90 mg of EDTA-2Na were added, and the mixture was shaken for 10 minutes. Then, 0.4 mL of saturated NaCl solution and 13 mL of cold ethanol were added, and the mixture was centrifuged at high speed for 3 minutes (12000 rpm, 4°C). The supernatant was discarded, and the precipitate was dissolved in 2 mL of ddH2O. The polypeptide-siRNA sense strand conjugate A1-22-Q20-m24017SM1 was obtained by RP-HPLC purification (7.4 mg, yield: 52.3%).

[0389] The polypeptide-siRNA sense strand conjugate A1-22-Q20-m24017SM1 was annealed with the antisense strand m24017AM1 to obtain the polypeptide oligonucleotide conjugate A1-22-Q20-m24017.1-1.

[0390] Referring to the above method, the structures of the polypeptide oligonucleotide conjugates synthesized in this invention, the mass spectrometry characterization data of the polypeptide-positive chain conjugates, and their modified and unmodified nucleic acid sequences are summarized in Tables 5-1, 5-2, and 6 below.

[0391] Table 5-1: Names, structural types, and sequences of polypeptide oligonucleotide conjugates

[0392] Table 5-2: Names and unmodified nucleotide sequences of polypeptide oligonucleotide conjugates

[0393] The structural formula of the polypeptide oligonucleotide conjugate is as follows:

[0394] Table 6: Mass spectrometry characterization data of peptide-sense chain conjugates

[0395] Example 7: In vivo distribution of polypeptide oligonucleotide conjugates by tail vein injection in wild-type mice.

[0396] 7.1. Drug Preparation

[0397] After obtaining the polypeptide-siRNA double-stranded powder (polypeptide oligonucleotide conjugate) by freeze-drying, a polypeptide oligonucleotide conjugate solution with a mother liquor concentration of 0.5 mg / mL was prepared in a sterile environment using physiological saline (Saline) as a solvent.

[0398] 2. Experimental grouping and drug administration

[0399] 5-6 weeks old male C57 wild type mice with similar body weight were selected as experimental subjects and randomly divided into groups, 4 mice in each group. After the mice were anesthetized, the polypeptide oligonucleotide conjugate solution was injected into the mice through the tail vein at a dose of 5 mg / kg. On the day of drug injection, the state was observed and daily weight records were made.

[0400] 3. Sampling detection

[0401] On the 10th day of administration, the mice were sacrificed and sampled. The sampling site was bilateral kidneys, and the content of the antisense strand of the polypeptide oligonucleotide conjugate in the kidney tissue was detected by liquid chromatography-mass spectrometry.

[0402] 4. Quantification of polypeptide oligonucleotide conjugate in kidney tissue

[0403] The kidney tissue sample was weighed and an appropriate amount of lysis solution was added to make the final volume of lysis solution added to the tissue mass ratio 10:1 (mL: g). Then magnetic beads were added for grinding. After grinding, it was placed at 37°C for 3h to obtain kidney homogenate. Take 50 μL of kidney homogenate, add 100 μL of ultrapure water (containing 2.5 μg / mL internal standard), mix well, then sample to the activated solid phase extraction plate, perform solid phase extraction, and after blowing dry on the nitrogen blowing instrument, the residue is redissolved with ultrapure water. After centrifugation at 3500 rpm and 4°C for 10 minutes, LC-HRMS analysis was performed. The results are shown in Table 7.

[0404] Table 7: Content of antisense strand of polypeptide oligonucleotide conjugate in mouse kidney tissue

[0405] Table 7 shows that when the administration dose is 5 mg / kg, polypeptides of different structures have different delivery efficiencies, and siRNA delivered by polypeptides A1-40 and A1-22 has a higher kidney enrichment amount.

[0406] Further, the distribution of antisense strands of A1-40-Q16-12006.2-2 and A1-22-Q16-12006.2-2 in various tissues of mice was detected after 10 days of administration.

[0407] Table 8: Content of antisense strand in various tissues of mice

[0408] Note: ND indicates below the lower limit of quantification

[0409] Table 8 shows that siRNA delivered by A1-40 and A1-22 polypeptides has very specific kidney targeting, and the content of siRNA antisense strand in other tissues and organs such as heart, brain, etc. is below the lower limit of quantification, indicating good safety.

[0410] Example 8 Testing the binding efficiency of polypeptide A1-22 in vitro

[0411] 1. Drug preparation: The prepared powder of polypeptide fluorescent probe A1-22-TAMRA was dissolved in DEPC water as a solvent in a sterile environment to prepare a polypeptide solution with a mother liquor concentration of 0.1 mg / mL.

[0412] 2. In vitro experiments were performed in 293Ta cells using DMEM + 10% FBS + 1X penicillin streptomycin + 1X non-essential amino acid medium, and the cells were trypsinized when the cell plating area reached 80%, and the cell density was determined using a Scepter automatic cell counter (Millipore, #PHCC00000). The A1-22-TAMRA polypeptide was then diluted to concentrations of 1400 nM, 840 nM, and 280 nM, and 2.5 ug of APOJ was added to each milliliter of culture medium containing the A1-22-TAMRA polypeptide. After mixing, an equal amount of 293Ta cells was added to each well, and the 6-well plate was incubated at 37°C in a 5% CO2 incubator for 24 hours. The cells were collected, and the fluorescence signal intensity was detected by flow cytometry.

[0413] Table 9: Fluorescence signal intensity in 293Ta cells

[0414] The in vitro results showed that the binding of A1-22-TAMRA polypeptide in 293Ta cells was dose-dependent.

[0415] Example 9 Testing the binding efficiency of polypeptides A1-22, A4-22, D-A1-22 in vitro

[0416] According to the method of Example 8, the fluorescence signal intensity was detected by flow cytometry.

[0417] Table 10: Fluorescence signal intensity in 293Ta cells

[0418] The in vitro results showed that the binding efficiency of A1-22-TAMRA and A4-22-TAMRA polypeptides in 293Ta cells was similar, and the binding efficiency of D-A1-22-TAMRA was lower than that of A1-22-TAMRA and A4-22-TAMRA.

[0419] Example 10 Testing the binding efficiency of polypeptides A1-22, A1-22(C), A1-22(K16N3) in vitro

[0420] According to the method of Example 8, the fluorescence signal intensity was detected by flow cytometry.

[0421] Table 11: Fluorescence signal intensity in 293Ta cells

[0422] In vitro results show that the position of TAMRA group attachment has an effect on the binding efficiency of the polypeptide.

[0423] Example 11 Testing in vivo distribution of polypeptide oligonucleotide conjugates in wild type mice by tail vein injection

[0424] According to the method of Example 7, after administration to 5-6 week old male C57 wild type mice by tail vein injection, the mice were sacrificed on day 12 and kidney tissues were taken for detection of siRNA antisense strand content in the kidney tissues.

[0425] Table 12: Content of antisense strand of polypeptide oligonucleotide conjugates in kidney tissues of mice

[0426] As shown in Table 12, siRNA delivered by polypeptides has higher content in kidney tissues, and the delivery efficiency of the four polypeptides to the kidney is much higher than that of siRNA without delivery system. Among them, the delivery efficiency of polypeptides A1-22 and A4-22 is higher.

[0427] Example 12 Testing in vivo distribution of polypeptide oligonucleotide conjugates in wild type mice by tail vein injection

[0428] According to the method of Example 7, after administration to 5-6 week old male C57 wild type mice by tail vein injection, the mice were sacrificed on day 10 and kidney tissues were taken for detection of polypeptide oligonucleotide conjugate content in the kidney tissues of mice.

[0429] Table 13: Content of antisense strand of polypeptide oligonucleotide conjugates in kidney tissues of mice

[0430] As shown in Table 13, different configurations of A1-22 have different delivery efficiencies, and the delivery efficiency of siRNA by D-A1-22, a derivative of A1-22 in all-D configuration, and RI-A1-22, a derivative of A1-22 in reverse anti-D configuration, is much lower than that of A1-22 in natural configuration.

[0431] Example 13 Testing in vivo activity of polypeptide oligonucleotide conjugates in a hyperuricemic mouse model by tail vein injection

[0432] 13.1 Experimental materials

[0433] 5-6 weeks old male Kunming mice, potassium oxonate (PO): MCT dissolved and prepared into a suspension, with a preparation concentration of 30 mg / mL, hypoxanthine (HX): MCT dissolved and prepared into a suspension, with a preparation concentration of 30 mg / mL, allopurinol (ALL): MCT dissolved and prepared into a suspension, with a preparation concentration of 0.5 mg / mL, polypeptide-siRNA conjugate: dissolved in normal saline, with a preparation concentration of 1 mg / mL.

[0434] 13.2 Construction of hyperuricemia mouse model and administration mode

[0435] 5-6 weeks old male Kunming mice were randomly divided into 4 groups, 5 mice in each group. The groups were blank group, model group, allopurinol positive group, and polypeptide oligonucleotide conjugate group. Normal feed and pure water were provided for free drinking. The model group, allopurinol positive group, and polypeptide oligonucleotide conjugate group were intraperitoneally injected with potassium oxonate (300 mg / kg / day) for 18 consecutive days. The blank group was injected with 0.5% CMC-Na solvent (0.2 mL / 10 g). At the same time, the polypeptide oligonucleotide conjugate group was injected via tail vein on the first day (5 mg / kg). On the 18th day of the experiment, 1 hour after modeling, blood was collected in vivo to obtain serum for determination of inosine (CRE) and uric acid (UA) levels, and then the mice were sacrificed to obtain kidneys for detection of mURAT1 expression.

[0436] Table 14: Residual amount of mURAT1 in kidney tissue of mice

[0437] The results show that the mRNA level of mURAT1 in the A1-22-Q16-m24017.1-1 group of mice is reduced by 50% compared with the model mice.

[0438] Table 15: Inosine (CRE) and uric acid (UA) content in serum of mice

[0439] The results show that the uric acid level (UA-S) and the ratio of serum uric acid to serum creatinine (UA-S / CRE-S) of the A1-22-Q16-m24017.1-1 group of mice are significantly reduced compared with the model mice. This indicates that the polypeptide A1-22 can well deliver the siRNA targeting mURAT1 to the kidney, thereby improving its effect of reducing uric acid in mice.

[0440] Example 14 Testing of the targeting of polypeptide oligonucleotide conjugate in the kidney of wild-type mice by tail vein injection

[0441] According to the method of Example 7, the content of siRNA antisense strand in the kidney 3 hours after in vivo testing of the polypeptide oligonucleotide conjugate by tail vein injection

[0442] Table 16: Content of antisense strand of polypeptide oligonucleotide conjugate in kidney tissue of mice

[0443] Table 16 shows that siRNA delivered by A1-22 and A1-19 polypeptides has very strong kidney targeting.

[0444] Example 15: Testing of kidney targeting of polypeptide oligonucleotide conjugate by tail vein injection in wild type mice

[0445] According to the method of Example 7, the content of siRNA antisense strand in kidney after 30 min of polypeptide oligonucleotide conjugate by tail vein injection in vivo was tested

[0446] Table 17: Content of antisense strand of polypeptide oligonucleotide conjugate in kidney tissue of mice

[0447] The results show that after a single dose of polypeptide oligonucleotide conjugate injection, polypeptide delivered siRNA antisense strand shows high accumulation in the kidney of mice, with an average content of 64478.8 ng / g. This indicates that the polypeptide provided by the present application can quickly reach the kidney and has high delivery efficiency and targeting ability for siRNA.

[0448] Example 16: Testing of kidney targeting of polypeptide oligonucleotide conjugate by tail vein injection in wild type mice

[0449] According to the method of Example 7, the content of siRNA antisense strand in kidney after 1 h of polypeptide oligonucleotide conjugate by tail vein injection in vivo was tested

[0450] Table 18: Content of antisense strand of polypeptide oligonucleotide conjugate in kidney tissue of mice

[0451] The results show that after a single dose of polypeptide oligonucleotide conjugate injection, most of the polypeptide delivered siRNA antisense strand shows high accumulation in the kidney of mice, with an average content of 113070.5 ng / g. This indicates that the polypeptide provided by the present application has high delivery efficiency and can effectively deliver siRNA to the target organ.

[0452] Example 17: Testing of kidney targeting of polypeptide oligonucleotide conjugate by tail vein injection in wild type mice

[0453] According to the method of Example 7, the content of siRNA antisense strand in kidney after 7 days of polypeptide oligonucleotide conjugate by tail vein injection in vivo was tested

[0454] Table 19: Content of antisense strand of polypeptide oligonucleotide conjugate in mouse kidney tissue

[0455] The results show that after a single dose injection of polypeptide oligonucleotide conjugate for 7 days, the delivered siRNA antisense strand still shows a high accumulation in the mouse kidney, with an average content of 17313 ng / g, which is 24 times higher than that of the naked strand. This indicates that the polypeptide provided by the present application can significantly improve the targeting delivery efficiency of siRNA, and exhibits its potential as an siRNA delivery system.

[0456] Example 18: Evaluation of the knockdown efficiency of polypeptide oligonucleotide conjugate on the URAT1 gene in the kidney of wild-type mice

[0457] Experimental method: 5 mg / kg of polypeptide oligonucleotide conjugate was injected into the mouse body through the tail vein, and the mouse was sacrificed after 14 days. The kidney was removed and the renal tubular cells were isolated, and the RNA was extracted for qRT-PCR analysis.

[0458] Mouse primary renal tubular epithelial cell isolation method: At the end of the experiment, C57BL / 6 male mice were sacrificed by cervical dislocation and disinfected with 75% alcohol. The double kidneys were removed by cutting open along the back of the double kidney area and placed in a culture dish containing pre-cooled PBS (4°C) and repeatedly washed to remove blood residues. After removing the kidney capsule, continue to wash with pre-cooled PBS for 2-3 times. Then, the renal cortex and medulla were separated, and the renal cortex part was retained and placed in a culture dish, and the renal cortex was cut into small pieces with tissue scissors. After mixing the fragments with pre-cooled PBS, transfer to a centrifuge tube and centrifuge at 1500 rpm for 5 minutes to remove the supernatant. Then, 2 mL of 0.1% collagenase type II solution was added to the centrifuge tube, and the mixture was mixed by repeatedly blowing and placed in a 37°C shaking incubator for 30 minutes. After digestion, the digestion was terminated by adding medium containing 10% FBS. The digestion solution was filtered through a 70 μm filter, and the filtered liquid was mixed again by blowing and transferred to a centrifuge tube, which was centrifuged at 1500 rpm for 5 minutes to collect the cell precipitate. After washing once with pre-cooled PBS, the RNA was extracted using Trizol reagent according to the standard operating procedure.

[0459] Table 20: Remaining amount of mURAT1 in mouse kidney tissue

[0460] The results show that most of the polypeptide oligonucleotide conjugates can effectively degrade the target gene. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0461] Example 19: Effect of polypeptide oligonucleotide conjugate on the knockdown efficiency of mouse Egln1 gene

[0462] Experimental Methods: Several polypeptide oligonucleotide conjugates were randomly selected and injected into Kunming mice via tail vein at a dose of 5 mg / kg. The mice were sacrificed 7 days after administration. Subsequently, one kidney was removed and divided into four equal parts along the transverse and coronal planes. The proximal quarter of the kidney tissue was preserved in RNAlater to ensure RNA stability. Finally, RNA was extracted from the kidney tissue, and the effects of the polypeptide oligonucleotide conjugates on gene expression in mouse kidney tissue were evaluated by qRT-PCR.

[0463] Table 21: Residual amount of mEgln1 in mouse kidney tissue

[0464] The results showed that, 7 days after administration, the polypeptide oligonucleotide conjugate effectively degraded the target gene. This indicates that the polypeptide oligonucleotide conjugate provided by this invention can effectively inhibit the expression of the target gene.

[0465] Example 20: In vivo activity of polypeptide oligonucleotide conjugates was tested by tail vein injection in an adenine-induced chronic renal anemia model.

[0466] Adenine-induced chronic renal anemia model and administration method

[0467] Male C57BL / 6 mice aged 5-6 weeks were randomly divided into five groups of four each: a control group, a model group, a positive control group, and two groups of oligonucleotide conjugates (randomly selected). The control group mice were fed a normal diet, while the model group mice were fed a diet containing 0.2% adenine. After two weeks of receiving the 0.2% adenine diet, the positive control group (Roxadustat) was administered the drug (10 mg / kg) daily by gavage. Simultaneously, the oligonucleotide conjugate group mice received a single intravenous injection of 5 mg / kg of the conjugate via tail vein. The model and experimental groups continued to receive the 0.2% adenine diet for two weeks, after which they were switched to a normal diet. Thirty-five days after the oligonucleotide conjugate injection, all mice underwent submandibular blood sampling to collect anticoagulated whole blood samples for testing complete blood count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), and renal function indicators such as creatinine and blood urea nitrogen levels.

[0468] Table 22: Blood routine indicators in the adenine-induced chronic renal anemia model.

[0469] Table 23: Renal function indicators in a mouse model of adenine-induced chronic renal anemia.

[0470] The results show that a single dose injection of the polypeptide oligonucleotide conjugate can significantly improve anemia and kidney function indicators. In addition, the positive drug roxadustat can also improve anemia indicators, but requires continuous administration every day to maintain the effect. This result suggests that the polypeptide oligonucleotide conjugate has an advantage in improving anemia indicators with a single dose.

[0471] Example 21 Influence of polypeptide oligonucleotide conjugate on kidney Egln1 gene knockdown efficiency in wild-type mice

[0472] Experimental method: In this example, several polypeptide oligonucleotide conjugates were randomly selected and injected into Kunming mice at a dose of 5 mg / kg through the tail vein. The mice were sacrificed after 18 days of administration. Subsequently, one kidney was divided into four equal parts along the transverse and coronal planes, and the 1 / 4 kidney tissue near the heart end was taken and stored in RNAlater for RNA stabilization. Finally, RNA was extracted and qRT-PCR analysis was performed to evaluate the effect of polypeptide oligonucleotide conjugates on gene expression in mouse kidney tissue.

[0473] Table 24: Remaining amount of mEgln1 in mouse kidney tissue

[0474] The results show that after 18 days of administration, the polypeptide oligonucleotide conjugate can effectively degrade the target gene in the kidney tissue compared to the naked chain 25006-Q20.1-1. This finding indicates that the polypeptide oligonucleotide conjugate provided by the present application has good specificity and effectiveness in targeted gene inhibition, and can significantly reduce the expression level of the target gene.

[0475] Example 22 Influence of polypeptide oligonucleotide conjugate on URAT1 gene knockdown efficiency in mice

[0476] Experimental method: In this example, several polypeptide oligonucleotide conjugates were randomly selected and injected into Kunming mice at a dose of 5 mg / kg through the tail vein. The mice were sacrificed after 45 days of administration. Subsequently, one kidney was removed and divided into four equal parts along the transverse and coronal planes. The 1 / 4 kidney tissue near the heart end was stored in RNAlater to ensure RNA stability. Finally, RNA was extracted from the kidney tissue and qRT-PCR analysis was performed to evaluate the effect of polypeptide oligonucleotide conjugates on gene expression in mouse kidney tissue.

[0477] Table 25: Remaining amount of mURAT1 in mouse kidney tissue

[0478] The results show that the polypeptide oligonucleotide conjugate can effectively degrade the target gene after 45 days of administration. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0479] Example 23: Test the in vivo activity of the polypeptide oligonucleotide conjugate by tail vein injection in a hyperuricemia mouse model.

[0480] Construction of a hyperuricemia mouse model and administration method

[0481] 5-6 week old male Kunming mice were randomly divided into 4 groups. They were blank group, model group, benzbromarone positive drug group, and polypeptide oligonucleotide conjugate group. The blank group was given ordinary feed, pure water, and free diet. The model group, benzbromarone positive drug group, and polypeptide oligonucleotide conjugate group were given high uric acid feed (20% yeast extract + 5% oxonic acid potassium), pure water, and free diet. The polypeptide oligonucleotide conjugate group was injected (5 mg / kg) into the tail vein on the first day. The benzbromarone group was given gavage (10 mg / kg) every day. On the 77th day of the experiment, blood was collected in vivo to collect serum, and the uric acid (UA) level was determined using a uric acid kit from Nanjing Jiancheng. On the 80th day, the mice were sacrificed to collect the kidneys for detection of mURAT1 expression and protein expression.

[0482] Table 26: Serum uric acid index of different polypeptide oligonucleotide conjugates in hyperuricemia mice on the 77th day

[0483] The results show that the uric acid level (UA-S) of the BEBT-P89066 group of mice is significantly lower than that of the model mice. This indicates that the BEBT-P89066 polypeptide oligonucleotide conjugate has good activity.

[0484] Table 27: Activity detection results of polypeptide oligonucleotide conjugates in the kidneys of hyperuricemia mice on the 80th day

[0485] The results show that the polypeptide oligonucleotide conjugate can effectively degrade the target gene. This indicates that the BEBT-P89066 polypeptide oligonucleotide conjugate has good activity.

[0486] Figure 1 shows the inhibition of target protein expression by polypeptide oligonucleotide conjugates in the kidneys of hyperuricemia mice on the 80th day, as analyzed by Western Blot (WB). POC is the polypeptide oligonucleotide conjugate BEBT-P89066. The results show that the polypeptide oligonucleotide conjugate can effectively degrade the target gene. This indicates that the BEBT-P89066 polypeptide oligonucleotide conjugate has good activity.

[0487] Example 24: Effect of polypeptide oligonucleotide conjugate on SGLT2 gene knockdown efficiency in mice

[0488] Experimental method: In this example, two polypeptide oligonucleotide conjugates were randomly selected and injected into the tail vein of a diabetic model mouse at a dose of 20 mg / kg. After 34 days of administration, the mice were sacrificed. Subsequently, one kidney was removed, and the tissue at the junction of the cortex and medulla of the far end was preserved in RNAlater to ensure the stability of the RNA. Finally, the RNA in the kidney tissue was extracted, and the effect of the polypeptide oligonucleotide conjugate on the gene expression in the mouse kidney tissue was evaluated by qRT-PCR analysis.

[0489] Table 28: Remaining amount of mSGLT2 in mouse kidney tissue

[0490] The results show that after 34 days of administration, the polypeptide oligonucleotide conjugate can effectively degrade the target gene. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0491] Example 25 Effect of polypeptide oligonucleotide conjugate on knockdown efficiency of mouse ALDH2 gene

[0492] Experimental method: In this example, one polypeptide oligonucleotide conjugate Bi-CycloA1-19-9-Q22-ALDH2.2-1 was randomly selected and injected subcutaneously into C57BL / 6 mice at a dose of 5 mg / kg and 30 mg / kg. The mice were sacrificed on the 7th day of administration. Subsequently, the cortex region of the proximal end of the kidney was preserved in RNAlater to ensure the stability of the RNA. Finally, the RNA in the kidney tissue was extracted, and the effect of the polypeptide oligonucleotide conjugate on the gene expression in the mouse kidney tissue was evaluated by qRT-PCR analysis.

[0493] Table 29: Remaining amount of mALDH2 in mouse kidney tissue

[0494] The results show that after 7 days of administration, the polypeptide oligonucleotide conjugate can effectively degrade the target gene. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0495] Example 26 Effect of polypeptide oligonucleotide conjugate on knockdown efficiency of mouse URAT1 gene

[0496] Experimental method: In this example, a polypeptide oligonucleotide conjugate BEBT-P89074 was selected at random and injected subcutaneously into C57BL / 6 mice at a dose of 10 mg / kg and 20 mg / kg. The mice were sacrificed on day 10 of administration. Subsequently, the renal cortex region of the proximal end of one side of the kidney, "upper pole", was preserved in RNAlater to ensure the stability of RNA. Finally, RNA was extracted from the kidney tissue and analyzed by qRT-PCR to evaluate the effect of the polypeptide oligonucleotide conjugate on gene expression in the kidney tissue of mice.

[0497] Table 30: Remaining amount of mURAT1 in the kidney tissue of mice

[0498] The results show that the polypeptide oligonucleotide conjugate can effectively degrade the target gene on day 10 of administration. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0499] Example 27: Effect of polypeptide oligonucleotide conjugate on the knockdown efficiency of mouse URAT1 gene

[0500] Experimental method: In this example, a polypeptide oligonucleotide conjugate BEBT-P89074 was selected at random and injected subcutaneously into C57BL / 6 mice at a dose of 20 mg / kg. The mice were sacrificed on day 21 of administration. Subsequently, the renal cortex region of the proximal end of one side of the kidney, "upper pole", was preserved in RNAlater to ensure the stability of RNA. Finally, RNA was extracted from the kidney tissue and analyzed by qRT-PCR to evaluate the effect of the polypeptide oligonucleotide conjugate on gene expression in the kidney tissue of mice.

[0501] Table 31: Remaining amount of mURAT1 in the kidney tissue of mice

[0502] The results show that the polypeptide oligonucleotide conjugate can effectively degrade the target gene on day 21 of administration. This indicates that the polypeptide oligonucleotide conjugate provided by the present application can effectively inhibit the expression of the target gene.

[0503] Example 28: Effect of polypeptide oligonucleotide conjugate on the knockdown efficiency of mouse SGLT2 gene

[0504] Experimental method: several polypeptide oligonucleotide conjugates were randomly selected and injected subcutaneously into C57BL / 6 female mice at a dose of 5 mg / kg, and the mice were sacrificed on the 14th day of administration. Subsequently, the renal cortex region at the junction of the near-end cortical medulla of one side of the kidney was preserved in RNAlater to ensure the stability of RNA. Finally, RNA in the kidney tissue was extracted and analyzed by qRT-PCR to evaluate the effect of polypeptide oligonucleotide conjugates on gene expression in mouse kidney tissue.

[0505] Table 32: Remaining amount of mSGLT2 in mouse kidney tissue

[0506] Example 29: Effect of polypeptide oligonucleotide conjugates on the knockdown efficiency of mouse SGLT2 gene

[0507] Experimental method: several polypeptide oligonucleotide conjugates were randomly selected and injected subcutaneously into C57BL / 6 male mice at a dose of 5 mg / kg, and the mice were sacrificed on the 14th day of administration. Subsequently, the renal cortex region at the junction of the near-end cortical medulla of one side of the kidney was preserved in RNAlater to ensure the stability of RNA. Finally, RNA in the kidney tissue was extracted and analyzed by qRT-PCR to evaluate the effect of polypeptide oligonucleotide conjugates on gene expression in mouse kidney tissue.

[0508] Table 33: Remaining amount of mSGLT2 in mouse kidney tissue

[0509] The results show that polypeptide oligonucleotide conjugates can effectively degrade target genes after 14 days of administration.

[0510] Example 30: Effect of polypeptide oligonucleotide conjugates on the knockdown efficiency of SGLT2 gene in cynomolgus monkeys

[0511] Experimental method: a polypeptide oligonucleotide conjugate was randomly selected and injected subcutaneously into a cynomolgus monkey at a dose of 20 mg / kg, and renal tissue was punctured on the 14th day of administration and preserved in RNAlater to ensure the stability of RNA. Finally, RNA in the kidney tissue was extracted and analyzed by qRT-PCR to evaluate the effect of polypeptide oligonucleotide conjugates on gene expression in cynomolgus monkey kidney tissue.

[0512] Table 34: Remaining amount of SGLT2 in cynomolgus monkey kidney tissue

[0513] The results show that polypeptide oligonucleotide conjugates can effectively degrade target genes in cynomolgus monkeys after 14 days of administration. This indicates that the polypeptide oligonucleotide conjugates provided by the present application can effectively inhibit the expression of target genes.

[0514] Based on our experimental data, a number of nucleic acid conjugates were tested to assess their ability to modulate the expression level of a target gene. The results showed that the vast majority of conjugates exhibited a down-regulation effect, and this effect was not significantly correlated with the overall kidney uptake level.

[0515] The above-described embodiments only express several implementation manners of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A polypeptide oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that, It comprises: (1) at least one polypeptide capable of specifically binding to the LRP2 receptor; and (2) one or more oligonucleotide drugs for regulating expression of a kidney target gene.

2. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, wherein, It comprises: (1) at least one polypeptide capable of specifically binding to the LRP2 receptor, an unmodified amino acid sequence of the polypeptide from N terminus to C terminus is represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 40; or an amino acid sequence obtained by substitution, replacement, deletion, addition, insertion, and / or modification of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 40; and (2) one or more oligonucleotide drugs for regulating expression of a kidney target gene.

3. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 2, wherein, The number of the amino acid residues substituted, replaced, deleted, added, inserted, and / or modified in the amino acid sequence is 1 or more and 10 or less.

4. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 3, wherein The number of the amino acid residues substituted, replaced, deleted, added, inserted, and / or modified in the amino acid sequence is 1 or more and 5 or less.

5. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 2, wherein the unmodified amino acid sequence of the polypeptide N- to C-terminally is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41; or a substitution, a replacement, a deletion, an addition, an insertion and / or a modification of 1 or more and less than 5 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO:

41.

6. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, wherein the unmodified amino acid sequence of the polypeptide from N terminus to C terminus is represented by DDAEDRHDSGYEVHCQKLCFRRRR (SEQ ID NO: 42), DDAEDRHDSGYEVHCQKLCFKRRRR (SEQ ID NO: 43), DDAEDRHDSGYEVHCQKLCFRRRRRR (SEQ ID NO: 44), DDAEDRHDSGYEVHCQKLCFKRRRRRR (SEQ ID NO: 45), KDDAEDRHDSGYEVHCQKLCF (SEQ ID NO: 46), KDDAEDRHDSGYEVHCQKLCFRRRR (SEQ ID NO: 47), DDAEDRHDSGYCVHHQKLCF (SEQ ID NO: 48), or DDAEDRHDSGCEVHHQKCVF (SEQ ID NO: 49); or an amino acid sequence obtained by substitution, replacement, deletion, addition, insertion, and / or modification of 1 or more and less than 5 amino acid residues in the amino acid sequence represented by SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO:

49.

7. The polypeptide oligonucleotide conjugate of any one of claims 2-6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, The modification includes one or more of (i-1) to (i-9): (i-1) a cyclization modification; (i-2) modification of a side chain amino group of an amino acid residue with a long-chain fatty acid or a long-chain fatty diacid having a carbon chain length of 12 to 22; (i-3) modification of a side chain amino group of an amino acid residue with p-iodophenylbutyric acid; (i-4) modification of a side chain amino group of an amino acid residue with 2-(2-(2-(4-(4-(4-iodophenyl)butyramido)ethoxy)ethoxy)acetic acid; (i-5) N-terminal amino acylation modification in the amino acid sequence; (i-6) C-terminal carboxy amide modification in the amino acid sequence; (i-7) methylation modification of an amino acid residue; (i-8) alkyne modification of an amino acid residue; (i-9) azido modification of an amino acid residue.

8. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 7, wherein, The type of the cyclization modification includes one or more of (a) to (e): (a) cyclization by alkylation reaction of side chain mercapto groups of two cysteine residues; (b) cyclization by disulfide bond formation from side chain mercapto groups of two cysteine residues; (c) cyclization by amide bond formation from condensation reaction of a side chain amino group of a lysine residue and a side chain carboxyl group of a glutamic acid residue; (d) cyclization by click chemistry reaction of alkyne and azido groups introduced in the amino acid sequence or modification of amino acid residues with alkyne and azido groups in the amino acid sequence; (e) cyclization by olefin metathesis reaction; Preferably, the alkylation reagent that reacts with the side chain mercapto group of a cysteine residue includes one or more of 1,4-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene, 1,2-bis(bromomethyl)benzene, 1,3-dibromopropane, diiodoethane, 4,4'-bis(bromomethyl)-1,1'-biphenyl, 1,3,5-tris(bromomethyl)benzene, 1,8-bis(bromomethyl)naphthalene, and 1,4-bis(bromomethyl)naphthalene.

9. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 7, wherein In the cyclization-modified polypeptide, the number of amino acid residues in each ring is 4, 5, 6, 7, or 8.

10. The polypeptide oligonucleotide conjugate of any one of claims 2-6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, The substitution includes one or more of (a-1) to (a-11): (a-1) substitution of an L-amino acid residue in the amino acid sequence with a D-amino acid residue; (a-2) substitution of a D-amino acid residue in the amino acid sequence with an L-amino acid residue; (a-3) substitution of an amino acid residue in the amino acid sequence with a methylated amino acid residue; (a-4) substitution of an aromatic amino acid in the amino acid sequence with an acidic amino acid residue; (a-5) substitution of an amino acid residue in the amino acid sequence with a 2-(2-(2-aminoethoxy)ethoxy)acetic acid residue; (a-6) substitution of an acidic amino acid residue in the amino acid sequence with a 2-(2-(2-carboxyethoxy)ethoxy)acetic acid residue; (a-6) an amino acid residue in the amino acid sequence is replaced by an (S)-2-amino-hept-6-ynoic acid residue; (a-7) an amino acid residue in the amino acid sequence is replaced by an (S)-2-amino-4-pentynoic acid residue; (a-8) an amino acid residue in the amino acid sequence is replaced by a 2-amino isobutyric acid residue; (a-9) an amino acid residue in the amino acid sequence is replaced by an L-tertiary leucine residue; (a-10) an amino acid residue in the amino acid sequence is replaced by an R-2-amino-2-methyl-6-heptenoic acid residue; (a-11) any two amino acid residues separated by 4, 5, 6, 7 or 8 amino acid residues are simultaneously replaced by a cysteine residue.

11. The polypeptide oligonucleotide conjugate of any one of claims 2-6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, The addition insertions include one or more of (b-1) - (b-3): (b-1) inserting a new amino acid, N-methylglycine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, (S)-2-amino-hept-6-ynoic acid, (S)-2-amino-4-pentynoic acid, 2-amino isobutyric acid, L-tertiary leucine and / or R-2-amino-2-methyl-6-heptenoic acid into the amino acid sequence; (b-2) addition of lysine, arginine, glutamic acid, aspartic acid, N- methylglycine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid and / or the following groups at the N- or C-terminus of the amino acid sequence of the peptide: (b-3) adding a polypeptide residue consisting of 1, 2 or 3 amino acids selected from the group consisting of lysine, 2-(2-(2-aminoethoxy)ethoxy)acetic acid and N-methylglycine at the N-terminus or C-terminus of the amino acid sequence.

12. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 2, wherein, The amino acid sequence of the polypeptide from N-terminus to C-terminus is: DAEFRHDSGYEVHHQK-NH2; DAEFRHDSGYEVHHQKLVFFAE-NH2; KLVFFAEDVGSNKGAIIGLMVGGVV-NH2; Ac-dVdHdHdQdKdLdPdFdFdAdEdDdVGdSdNdK-NH2; DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA-NH2; DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-OH; FRHDSGYEVHHQKLVFFAE-NH2; DSGYEVHHQKLVFFAE-NH2; YEVHHQKLVFFAE-NH2; Ac-dDdAdEdFdRdHdDdSGdYdEdVdHdHdQdKdLdVdFdFdAdE-NH2; Ac-dEdAdFdFdVdLdKdQdHdHdVdEdYGdSdDdHdRdFdEdAdD-NH2; DAEFRHDSGYEVHHQKLVFFAEDV-NH2; GAIIGLMVGGVV-[AEEA]-KSK-NH2; SGYEVHHQKLVF-NH2; SGYEVHHQKdLdVF-NH2; DSGYEVHHQKLVF-NH2; DSGYEVHHQK-L(N-Me)-V(N-Me)-F-NH2; DSGYEVHHQK-L(N-Me)-dV-F-NH2; DSGYEVHHQK-dL-V(N-Me)-F-NH2; DSGYEVHH-Q(N-Me)-K-L(N-Me)-V(N-Me)-F-NH2; DSGYEVH-dH-Q(N-Me)-K-L(N-Me)-V(N-Me)-F-NH2; DSGYEVH-dH-QK-L(N-Me)-V(N-Me)-F-NH2; FRHDSGYEVHHQKLVF-NH2; DAEFRHDSGYEVHHQKLVF-NH2; DSGYEVHHQKdLdVF-NH2; DSGYEVHCQKLCF-NH2; DDDDSGYEVHCQKLCF-NH2; DSGYEVHCQKLCFGFWFG-NH2; GFWFGDSGYEVHCQKLCF-NH2; DAEFRHDSGYEVHCQKLCF-NH2; DDEDREDSGYEVHCQKLCF-NH2; dDDAEdDRHDSGYEVHCQKLCF-NH2; D-K(Pal)-EFRHDSGYEVHCQKLCF-NH2; DAEFRHDSGYEVHCQKLCF-K(Pal)-NH2; DAEFRHDSCYEVHCQKLCF-NH2; DAECRHDCGYEVHHQKLVFFAE-NH2; DAEFRHDSGYEVHHQCLVFCAE-NH2; DAEFRHDSGYEVHCQKLCFFAE-NH2; DDDAEFRHDSGYEVHCQKLCFFAE-NH2; dDdDDAEFRHDSGYEVHCQKLCFFAE-NH2; dDDAEdDRHDSGYEVHCQKLCFdDAE-NH2; DAEFRHDSGYEVCHQKCVFFAE-NH2; DAEFRHDSGYEVHHCKLVCFAE-NH2; DAEFRHDSGYEVHX2QKLX3FFAE-NH2; DAEFRHDSGYEVHKQKLEFFAE-NH2; DAEX1RHDX3GYEVHCQKLCFFAE-NH2; RRRdDDAEdDRHDSGYEVHCQKLCF-NH2; RRRRdDDAEdDRHDSGYEVHCQKLCF-NH2; dDDAEdDRHDSGYEVHCQKLCF-K(X4)-NH2; RRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2; RRRRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2; dDDAEdDRHDSGYEVHCQKLCFRRRR-[AEEA]-K(IBA)-NH2; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-OH; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(X5)-NH2; K(X5)dDDAEdDRHDSGYEVHCQKLCF-NH2; dDDAEdDRHDSGYEVHCQKLCFRRRR-NH2; KdDDAEdDRHDSGYEVHCQKLCFRRRR-NH2; dDDAEdDRHDSGYCVHHQKLCF-NH2; dDDAEdDRHDSGCEVHHQKCVF-NH2; dDDAEdDRHDSGYCVdHdHQKLCF-NH2; dDDAEdDRHDSGYdCVdHdHQKLdCF-NH2; dDDAEdDRHDSGYEVHdCQKLdCF-NH2; dDDAEdDRHDSGYEVdHCQKLCdF-NH2; dDDAEdDRHDSGYEVdHCQKLCF-NH2; dDDAEdDRHDS[Aib]YEVHCQKLCF-NH2; dDDAEdDRHDS[Aib]YEVdHCQKLCF-NH2; dDDAEdDRHDS[Aib]YEdVdHCQKLCF-NH2; dDDAEdDRHDSGYE[Aib]HCQKLCF-NH2; dDDAEdDRHDS[Tle]YEVHCQKLCF-NH2; dDDAEdDRHDSGYE[Tle]HCQKLCF-NH2; dDDAEdDRHDSGYEVHX6QKLX6F-NH2; or dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-X7; wherein d represents a D-amino acid, N-Me represents an N-methylated amino acid, 6- Azidohexanoic is 6-azidohexanoic acid, Pal is palmitic acid, IBA is p-iodophenylbutyric acid, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butylamido)ethoxy)ethoxy)acetic acid, AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid, X1 is L-propargylglycine, X2 is (S)-2-amino-hept-6-ynoic acid, X3 is LysN3, Aib is 2-aminoisobutyric acid, Tle is L-tert-leucine, X6 is R-2-amino-2-methyl-6-heptenoic acid, and X5 has the following structure: or X7 has the following structure:

13. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 12, wherein, the polypeptide is a cyclized modification of the polypeptide in claim 12.

14. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 13, wherein, the N-terminal to C-terminal amino acid sequence of the polypeptide and the way it is cyclized is: DSGYEVHHQKLVF-NH2, E and K side chains form an amide bond to cyclize; DSGYEVHHQKdLdVF-NH2, E and K side chains form an amide bond to cyclize; DSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize; DDDDSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize; DSGYEVHCQKLCFGFWFG-NH2, Cys reacts with m-dibromobenzyl to cyclize; GFWFGDSGYEVHCQKLCF-NH2, Cys reacts with m-dibromobenzyl to cyclize; DSGYEVHCQKLCF-NH2, forms a disulfide bond to cyclize; DAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; DDEDREDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; D-K(Pal)-EFRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVHCQKLCF-K(Pal)-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSCYEVHCQKLCF-NH2, Cys reacted with 1,3,5-tris(bromomethyl)benzene to form a ring; dDDAEdDRHDSGYEVHCQKLCF-NH2, disulfide bond formation to form a ring; DAECRHDCGYEVHHQKLVFFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVHHQCLVFCAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DDDAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDdDDAEFRHDSGYEVHCQKLCFFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCFdDAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVCHQKCVFFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVHHCKLVCFAE-NH2, Cys reacted with m-dibromobenzyl to form a ring; DAEFRHDSGYEVHX2QKLX3FFAE-NH2, X2 is (S)-2-amino-hept-6-ynoic acid, X3 is LysN3, X2 and X3 reacted by click chemistry to form a ring; DAEFRHDSGYEVHKQKLEFFAE-NH2, the side chain of the 14th amino acid residue and the 18th amino acid residue form an amide bond to form a ring; DAEX1RHDX3GYEVHCQKLCFFAE-NH2, X1 is L-propargylglycine, X3 is LysN3, X1 and X3 reacted by click chemistry to form a ring and Cys reacted with m-dibromobenzyl to form a ring; RRRdDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; RRRRdDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCF-K(X4)-NH2, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butanamide)ethoxy)ethoxy)acetic acid, Cys reacted with m-dibromobenzyl to form a ring; RRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacted with m-dibromobenzyl to form a ring; RRRRRRdDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCFRRRR-[AEEA]-K(IBA)-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(IBA)-OH, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVHCQKLCF-[AEEA]-K(X 5-3 -NH2, Cys is cyclized with m-benzhydryl bromide; K(X 5-3 dDDAEdDRHDSGYEVHCQKLCF-NH2, Cys reacted with m-benzhydryl bromide to form a ring; dDdDDAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with p-dibromobenzyl to form a ring; dDdDDAEFRHDSGYEVHCQKLCF-NH2, Cys reacted with 1,8-bis(bromomethyl)naphthalene to form a ring; dDDAEdDRHDSGYCVHHQKLCF-NH2, Cys reacted with 4,4'-dibromomethyl biphenyl to form a ring; dDDAEdDRHDSGCEVHHQKCVF-NH2, Cys reacted with 4,4'-dibromomethyl biphenyl to form a ring; dDDAEdDRHDSGYCVdHdHQKLCF-NH2, Cys reacted with 4,4'-dibromomethyl biphenyl to form a ring; dDDAEdDRHDSGYdCVdHdHQKLdCF-NH2, Cys reacted with 4,4'-dibromomethyl biphenyl to form a ring; dDDAEdDRHDSGYEVHdCQKLdCF-NH2, Cys reacted with 4,4'-dibromomethyl biphenyl to form a ring; dDDAEdDRHDSGYEVHdCQKLdCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVdHCQKLCdF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYEVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDS[Aib]YEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDS[Aib]YEVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDS[Aib]YEdVdHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYE[Aib]HCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDS[Tle]YEVHCQKLCF-NH2, Cys reacted with m-dibromobenzyl to form a ring; dDDAEdDRHDSGYE[Tle]HCQKLCF-NH2, Cys reacted with m-benzhydryl bromide to form a ring; or dDDAEdDRHDSGYE[Tle]HCQKLCF-[AEEA]-X7, Cys reacted with m-benzhydryl bromide to form a ring.

15. The polypeptide oligonucleotide conjugate of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, H—D-A-E-F-R-H-D-S-G-Y—E-V-H-H-Q-K—NH2; H—D-A-E-F-R-H-D-S-G-Y—E-V-H-H-Q-K-L-V-F-F—A-E—NH2; H—K-L-V-F-F-A-E-D-V-G—S-N-K-G-A-I-I-G-L-M—V-G-G-V-V—NH2; H—F-R-H-D-S-G-Y-E-V-H—H-Q-K-L-V-F-F-A-E—NH2; H—D-S-G-Y-E-V-H-H-Q-K—L-V-F-F-A-E—NH2; H—D-A-E-F-R-H-D-S-G-Y—E-V-H-H-Q-K-L-V-F-F—A-E-D-V—NH2; H—S-G-Y-E-V-H-H-Q-K-L—V-F-F—NH2; H—S-G-Y-E-V-H-H-Q-K-dL-dV-F—NH2; H—D-S-G-Y-E-V-H-H-Q-K-L(N-Me)-V(N-Me)-F—NH2; H—D-S-G-Y-E-V-H-H-Q-K-L(N-Me)-dV-F—NH2; H—D-S-G-Y-E-V-H-H-Q-K-dL-V(N-Me)-F—NH2; H—D-S-G-Y-E-V-H-H-Q(N-Me)-K-L(N-Me)-V(N-Me)-F—NH2; H—D-S-G-Y-E-V-H-dH-Q(N-Me)-K-L(N-Me)-V(N-Me)-F—NH2; H—D-S-G-Y-E-V-H-dH-dQ-K-L(N-Me)-V(N-Me)-F—NH2; H—F-R-H-D-S-G-Y-E-V-H—H-Q-K-L-V-F—NH2; H—D-A-E-F-R-H-D-S-G-Y—E-V-H-H-Q-K-L-V-F—NH2; or a polypeptide obtained by substitution, replacement, deletion, addition, insertion and / or modification of one or more and less than five amino acid residues in the polypeptide; wherein IBA is p-iodophenylbutyric acid, X4 is 2-(2-(2-(4-(4-(4-iodophenyl)butyramido)ethoxy)ethoxy)acetic acid, and X5 has the following structure: or X7 has the following structure:

16. The polypeptide oligonucleotide conjugate of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, the polypeptide is covalently linked to the oligonucleotide drug through a linker group.

17. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 16, wherein The linking group has the structure: or LE is a group linked to the oligonucleotide drug, which contains or does not contain a hydroxyl group or a protected hydroxyl group; LA is linked to the polypeptide and is selected from the group consisting of: LB is a linking unit formed by one or more of the following groups connected without violating the principle of chemical bonding: alkylene, cycloalkyl, heterocyclyl, heteroaryl, aryl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-; R1is selected from: H, C1-C 10 alkyl; R2 is selected from: C1-C 10 alkyl; m is selected from an integer between 0 and 12; each n1 and n2 is independently selected from 0, 1, 2, 3, 4, 5, 6.

18. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein, LE is selected from C1-C3 alkylene or the following structure: each R4 is independently selected from H or a hydroxyl protecting group; each n2 is independently selected from an integer between 0 and 10.

19. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 18, wherein, LE is selected from:

20. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein LE is linked to the 5' or / and 3' end of the sense strand of RNA in the oligonucleotide drug.

21. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 20, wherein, LE is linked to the phosphate group or the phosphorothioate group at the 5' or / and 3' end of the sense strand of RNA in the oligonucleotide drug by forming a phosphoric acid ester bond or a phosphorothioate bond.

22. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein LA is selected from the following structures:

23. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein LA is linked to the N-terminus of the polypeptide; Alternatively, LA is linked to the C-terminus of the polypeptide; Alternatively, LA is linked to an amino acid residue containing a side chain amino group in the polypeptide, preferably a lysine residue; Alternatively, LA is linked to an amino acid residue containing a side chain carboxyl group in the polypeptide; Alternatively, LA is linked to an amino acid residue containing a side chain sulfhydryl group in the polypeptide.

24. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein LB is selected from a C1-C8 alkylene group or the following structure: wherein each i, j, h, q, s, y, k is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; X is selected from -O-, -S-, -CH2-; Y is absent or selected from -C(=O)-, -CH2-.

25. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 24, wherein, LB is selected from the following structures: each i, j is independently selected from 0, 1, 2, 3, 4; k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; y is selected from 0, 1, 2, 3, 4; X is selected from -CH2-, -O-.

26. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 25, wherein LB is selected from the following structures:

27. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 17, wherein The linking group is selected from the following structures: n2 is selected from 0, 1, 2, 3, 4, 5, 6; R3 is selected from H or acetyl.

28. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 1, wherein The polypeptide oligonucleotide conjugate is selected from the following compounds: wherein AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid, Sar is sarcosine, d represents a D-amino acid, and N-Me represents an N-methylated amino acid; is preferably selected from:

29. The polypeptide oligonucleotide conjugate of any one of claims 1-28, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein, the oligonucleotide drug is selected from small interfering RNA, antisense oligonucleotide, small activating RNA and peptide nucleic acid.

30. The polypeptide oligonucleotide conjugate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof of claim 29, wherein, the oligonucleotide drug is selected from small interfering RNA, and the polypeptide is linked to the 3' and / or 5' end of the sense strand of small interfering RNA through a linker group.

31. The polypeptide capable of binding to LRP2 receptor according to any one of claims 1-15.

32. Use of a polypeptide capable of binding to the LRP2 receptor as described in any one of claims 1-15 in the manufacture of a delivery system for the in vivo delivery of an oligonucleotide drug to a kidney cell.

33. Use of a polypeptide oligonucleotide conjugate as described in any one of claims 1-30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as an active ingredient in the manufacture of a medicament for inhibiting the expression of a kidney target gene.

34. The use according to claim 33, characterized in that The kidney target gene is selected from the group consisting of URAT1, SGLT-2, HIF-PH, ACE, TGF-β, Endothelin-1, TGF-b, NF-κB, PDE5, NOX1, Egln1, ALDH2.

35. Use of a polypeptide oligonucleotide conjugate as described in any one of claims 1-30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as an active ingredient in the manufacture of a medicament for preventing or treating a URAT1, SGLT-2, HIF-PH, ACE, TGF-β, Endothelin-1, TGF-b, NF-κB, PDE5, Egln1, ALDH2, or NOX1 gene-mediated or associated disease.

36. Use of a polypeptide oligonucleotide conjugate as described in any one of claims 1-30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, as an active ingredient in the manufacture of a medicament for preventing or treating a kidney-related disease.

37. The use according to claim 36, wherein The kidney-related disease is gout and hyperuricemia, diabetes and diabetic nephropathy, IgA nephropathy, chronic kidney disease, polycystic kidney disease, acute kidney injury, lupus nephritis, Alport syndrome, membranous nephropathy, focal segmental glomerulosclerosis, renal amyloidosis, hemolytic uremic syndrome, nodular interstitial nephritis, kidney tumors, anemia, and hypertension.

38. A medicament for the treatment and / or prophylaxis of a disease relating to the kidney, characterized in that a compound as described in embodiment 1 is used. prepared from an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises a polypeptide oligonucleotide conjugate as described in any one of claims 1-30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

39. A method for treating and / or preventing a kidney-related disease, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-38, or a pharmaceutically acceptable salt thereof. comprising: administering a safe and effective amount of a polypeptide oligonucleotide conjugate as described in any one of claims 1-30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; or administering a safe and effective amount of a medicament as described in claim 38.

40. The method of claim 39, wherein, The kidney-related disease is gout and hyperuricemia, diabetes and diabetic nephropathy, IgA nephropathy, chronic kidney disease, polycystic kidney disease, acute kidney injury, lupus nephritis, Alport syndrome, membranous nephropathy, focal segmental glomerulosclerosis, renal amyloidosis, hemolytic uremic syndrome, nodular interstitial nephritis, kidney tumors, anemia, and hypertension.

Images