Peptide-oligonucleotide conjugate for inhibiting central nervous system target gene expression and use thereof

Abstract

The present invention relates to a peptide-oligonucleotide conjugate having the structure of formula (I) or (II) below, or a pharmaceutically acceptable salt or stereoisomer thereof, and use thereof. The peptide-oligonucleotide conjugate of the present invention can be used to inhibit the expression of one or more target genes in central nervous system tissue cells, with favorable in vivo efficacy, long duration of action, and high safety.

Description

Polypeptide oligonucleotide conjugate for inhibiting central nervous system target gene expression and its application

[0001] The present invention claims priority to the Chinese patent application with application number 2024103688430, filed with the Patent Office of China on March 28, 2024, and entitled “Polypeptide oligonucleotide conjugates for inhibiting the expression of target genes in the central nervous system and their applications”, and the Chinese patent application with application number 2024113531318, filed with the Patent Office of China on September 26, 2024, and entitled “Polypeptide oligonucleotide conjugates for inhibiting the expression of target genes in the central nervous system and their applications”. The entire contents of the two patent applications are incorporated into the present invention by reference. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide oligonucleotide conjugate for delivering oligonucleic acid drugs for inhibiting the expression of target genes in the central nervous system and its application. Background Art

[0003] Based on the development of peptide science, researchers have found that peptides can maintain their integrity in the blood circulation, and also show specificity and selectivity for targeting specific receptors, with good permeability and drug loading capacity. Therefore, peptide-drug conjugates (PDCs) have great prospects for biomedical applications. Currently, PDCs not only focus on antibody drugs, small molecule drugs, etc., but also use specific chemical reactions such as click chemistry to couple peptides with nucleic acid drugs (such as small interfering RNA, siRNA). Due to the targeting of peptides, targeted delivery of siRNA can be achieved, which greatly expands the application prospects of extrahepatic delivery of nucleic acid drugs (Rizvi SFA et al,.ACS Pharmacol Transl Sci.2024;7(2):309-334).

[0004] Compared to traditional small molecule drugs, small interfering RNA (siRNA) drugs utilize endogenous cellular systems to specifically degrade targeted target genes, offering promising clinical therapeutic prospects. Mechanistically, exogenously introduced double-stranded siRNAs are recognized by the cellular endoribonuclease Dicer and cleaved into a single-stranded guide strand and its complementary strand by the RNA-induced silencing complex (RISC). The guide stranded siRNA then binds to the Argonate 2 protein (AGO2) and directs it to the target RNA, where AGO2 mediates its degradation. In addition to degrading RNAs in the cytoplasm, siRNAs can also promote chromosome remodeling and histone modification in the nucleus, leading to transcriptional silencing (Matzke et al. Nature reviews. Genetics vol. 6, 1 (2005): 24-35.). The delivery of siRNA drugs is an important guarantee for RNAi therapy. The most mature siRNA intrahepatic delivery system currently uses N-oligosaccharide acetylgalactosamine (GalNAc) to specifically bind to the asialoglycoprotein receptor (ASGPR) specifically expressed by hepatocytes, thereby achieving active delivery to liver cells. It is an important breakthrough in the field of RNA therapy (Zhang L et al,. Front Pharmacol. 2022; 13: 1090237.). In addition to liver tissue, researchers have found that adenovirus targeting mouse brain containing BACE1 siRNA shows the potential to improve AD pathology (Zhou Y et al,. Sci Adv. 2020; 6 (41): eabc7031.). However, effective, safe and comprehensive siRNA extrahepatic delivery systems still face huge challenges, especially siRNA delivery to brain tissue where the blood-brain barrier exists.

[0005] Regarding the development of RNAi therapeutic drugs targeting the central nervous system, researchers have established a system of chemically modified siRNAs linked to fatty alcohol 2'-O-hexadecyl (C16) to target the Aβ peptide derived from amyloid precursor protein (APP) that induces AD. Intraventricular injection of C16-siRNA targeting APP into Alzheimer's disease (AD) mice can reduce amyloid β deposition, inflammation, and behavioral deficits. Similarly, after C16-siRNA was administered to the central nervous system of non-human primates, extensive siRNA distribution and RNAi activity were achieved, with target gene silencing greater than 75% for at least 3 months. This provides an important technical platform for extrahepatic targeted therapy of RNAi drugs (Brown KM et al,. Nat Biotechnol. 2022; 40(10): 1500-1508.). Using peptides to specifically bind to target cell surface receptor proteins to achieve transcellular transport of drugs, thereby crossing the blood-brain barrier and delivering them to target cells in central tissues is also an important idea for drug delivery to the central nervous system. Among them, low-density lipoprotein receptor-related protein (LRP1) is widely expressed in neurons and brain endothelial cells. It is a multifunctional endocytic receptor that participates in many biological processes, including the transport of metabolites (Aβ peptides) across the blood-brain barrier. Therefore, it is a very attractive target for drug delivery to the central nervous system (Ulery PG et al,. J Biol Chem. 2000; 275(10): 7410-7415.). LRP1 has multiple ligands, reflecting its multiple physiological functions. In recent years, researchers have developed a transport peptide called Angiopep-2 (ANG2), which can be used to transport ANG-coupled drugs to brain tissue by utilizing its interaction with the LRP1 receptor. Among them, ANG2-mediated paclitaxel delivery is the first example, and the related drug An1005 has completed Phase II clinical trials (Régina A et al,. Br J Pharmacol. 2008; 155(2): 185-197.). Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a polypeptide oligonucleotide conjugate based on a novel LRP-1 targeting peptide, which can be used to inhibit the expression of one or more target genes in central nervous system tissue cells, and has the advantages of good in vivo effect and weak non-target activity.

[0007] The present invention includes the following technical solutions.

[0008] In one aspect, the present invention provides a polypeptide oligonucleotide conjugate having a structure represented by the following formula (I) or (II), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0009] in, It is an oligonucleic acid drug;

[0010] P is a polypeptide ligand for the LRP-1 receptor, selected from an unmodified or modified polypeptide;

[0011] LE is a group used to link to an oligonucleic acid drug, which contains a hydroxyl group or a protected hydroxyl group;

[0012] Each LA is independently selected from the following structures:

[0013] LB is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: alkylene (preferably C1-C 10 Alkylene, more preferably C1-C6 alkylene), cycloalkyl (preferably C5-C 10 Cycloalkyl, more preferably C5-C7 cycloalkyl), heterocyclyl (preferably 5-10 membered heterocyclyl, more preferably 5-7 membered heterocyclyl), -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-;

[0014] LC is a connecting unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: alkylene (preferably C1-C 10 Alkylene, more preferably C1-C6 alkylene), cycloalkyl (preferably C5-C 10 Cycloalkyl, more preferably C5-C7 cycloalkyl), heterocyclyl (preferably 5-10 membered heterocyclyl, more preferably 5-7 membered heterocyclyl), -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-;

[0015] R1 is selected from: H, C1-C 10 Alkyl (preferably selected from: H, C1-C6 alkyl, more preferably selected from: H, C1-C3 alkyl);

[0016] R2 is selected from: C1-C 10 Alkyl (preferably selected from: H, C1-C6 alkyl, more preferably selected from: H, C1-C3 alkyl);

[0017] Each n is independently selected from: 1, 2, 3;

[0018] a and b are each independently 0 or 1, and at least one of them is 1;

[0019] m is selected from: an integer between 0 and 12;

[0020] Each n1 is independently selected from the group consisting of: 0, 1, 2, and 3.

[0021] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGCRGKRNNFKTEEY (SEQ ID NO: 1, referred to herein as Ang1), TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 2, referred to herein as Ang2), TWPKHFDKHTFYSILKLGKH (SEQ ID NO: 3, referred to herein as L57), TFFYGGKRGKRNNFKTEEY (SEQ ID NO: 5) or TFFYGGSRGZRNNFKTEEY (SEQ ID NO: 4), wherein Z represents L-norleucine.

[0022] In some embodiments, the polypeptide ligand has one or more of the following structural changes:

[0023] (1) L-amino acid residues in the amino acid sequence are replaced by D-amino acid residues;

[0024] (2) amino acid residues in the amino acid sequence are replaced by methylated amino acid residues;

[0025] (3) the presence or absence of acylation modification of the N-terminal amino group in the amino acid sequence;

[0026] (4) the presence or absence of amidation modification of the C-terminal carboxyl group in the amino acid sequence;

[0027] (4) Any aromatic amino acid residue in the amino acid sequence can be replaced by a non-aromatic amino acid residue;

[0028] (5) Any two amino acid sequences are linked by a linker, wherein the N-terminus of one amino acid sequence is linked to the C-terminus of the other amino acid sequence by a linker;

[0029] The linking group is preferably:

[0030] In some embodiments, the LA in P-LA is linked to the N-terminus of the polypeptide ligand.

[0031] In some embodiments, the LA in P-LA is linked to the C-terminus of the polypeptide ligand.

[0032] In some embodiments, the LA in P-LA is linked to the seventh amino acid residue from the N-terminus of the polypeptide ligand.

[0033] In some embodiments, the LA in P-LA is linked to the tenth amino acid residue from the N-terminus of the polypeptide ligand.

[0034] In some embodiments, the LA in P-LA is linked to the 15th amino acid residue from the N-terminus of the polypeptide ligand.

[0035] In some embodiments, the LA in P-LA is linked to an amino acid residue containing a side chain amino group in the polypeptide ligand.

[0036] In some embodiments, the LA in P-LA is linked to a lysine residue in the polypeptide ligand.

[0037] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGKRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid (i.e., lysine) residue from the N-terminus of the polypeptide ligand.

[0038] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGKRNNFKTEEY, and the LA in P-LA is linked to the 15th amino acid (i.e., lysine) residue from the N-terminus of the polypeptide ligand.

[0039] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGKRGKRNNFKTEEY, and the LA in P-LA is linked to the 7th, 10th, or 15th amino acid (i.e., lysine) residue in the polypeptide ligand from the N-terminus.

[0040] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGCRGKRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid (i.e., lysine) residue from the N-terminus of the polypeptide ligand.

[0041] In some embodiments, the LA in P-LA is linked to a norleucine residue in the polypeptide ligand.

[0042] In some embodiments, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGZRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid (norleucine) residue from the N-terminus of the polypeptide ligand.

[0043] In some embodiments, LA in P-LA is linked to an amino acid residue containing a side chain carboxyl group in the polypeptide ligand.

[0044] In some embodiments, the LA in P-LA is linked to a glutamic acid residue or an aspartic acid residue in the polypeptide ligand.

[0045] In some embodiments, LE is selected from the following structures:

[0046] Each R is independently selected from: H or a hydroxyl protecting group;

[0047] Each n2 is independently selected from an integer between 0-10.

[0048] In some embodiments, LE is selected from:

[0049] In some embodiments, LA is selected from the following structures:

[0050] In some embodiments, LB in formula (I) is selected from the following structures:

[0051] wherein each i, j, h, q, s, and e are independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0052] c is selected from: 0, 1;

[0053] X is selected from the group consisting of: O, S, CH2;

[0054] Y is absent or selected from: C=O, CH2.

[0055] In some embodiments, LB in formula (I) is selected from the following structures:

[0056] Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4;

[0057] c is selected from: 0, 1;

[0058] k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0059] X is selected from: CH2, O.

[0060] In some embodiments, LB in formula (I) is selected from the following structures:

[0061] In some embodiments, LB in formula (II) has the structure shown below:

[0062] Wherein, X is selected from: O, S, CH2;

[0063] L is selected from the following structures:

[0064] Each i, j, h, q, s, f, and e are independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0065] c is selected from: 0, 1;

[0066] X is selected from the group consisting of: O, S, CH2;

[0067] Y is absent or selected from: C=O, CH2.

[0068] In some embodiments, LB in formula (II) has the structure shown below:

[0069] X is selected from: O, CH2.

[0070] In some embodiments, L is selected from the following structures:

[0071] Each i and e are independently selected from: 0, 1, 2, 3, 4;

[0072] k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0073] X is selected from: CH2, O.

[0074] In some embodiments, L is selected from the following structures:

[0075] In some embodiments, LC in formula (II) has the structure shown below:

[0076] wherein each i, j, h, and e are independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0077] c is selected from: 0, 1;

[0078] X is selected from the group consisting of: O, S, CH2;

[0079] Y is absent or selected from: C=O, CH2.

[0080] In some embodiments, LC in formula (II) is selected from the following structures:

[0081] Each i and j are independently selected from: 0, 1, 2, 3, 4;

[0082] c is selected from: 0, 1;

[0083] X is selected from: CH2, O.

[0084] In some embodiments, LC in formula (II) is selected from the following structures:

[0085] In some embodiments, the polypeptide oligonucleotide conjugate is selected from the following compounds:

[0086] Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4;

[0087] c is selected from: 0, 1;

[0088] k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0089] X is selected from: CH2, O;

[0090] Each n1 is independently selected from the group consisting of: 0, 1, 2, and 3.

[0091] In some embodiments, the oligonucleic acid drug is selected from the group consisting of small interfering RNA (siRNA), antisense oligonucleotide (ASO) and peptide nucleic acid (PNA).

[0092] In some embodiments, the oligonucleic acid drug is a small interfering RNA (siRNA), and the LE is linked to the phosphate group or phosphorothioate group at the 3' and / or 5' end of the sense strand of the siRNA.

[0093] The polypeptide-oligonucleotide conjugate of the present invention is prepared by the following method: a click chemistry reaction is performed between an alkyne-containing linker-nucleic acid sense chain molecule and an azide-modified polypeptide to obtain a polypeptide-sense chain conjugate, and the polypeptide-sense chain conjugate is then annealed with a corresponding nucleic acid antisense chain to obtain the polypeptide-oligonucleotide conjugate; the polypeptide is a polypeptide ligand for the LRP-1 receptor;

[0094] The alkyne-containing linker-nucleic acid sense chain molecule has a structure as shown in the following formula (III) or formula (IV):

[0095] in, It is the positive chain in oligonucleic acid drugs;

[0096] LA, LB, LC, LE, n, a, and b are as described above;

[0097] R3 is selected from: ethynyl,

[0098] In some embodiments, the alkyne-containing linker-nucleic acid sense strand molecule is selected from the following structures:

[0099] wherein each i, j, and e are independently selected from: 0, 1, 2, 3, and 4;

[0100] c selection: 0, 1;

[0101] k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0102] X is selected from: CH2, O.

[0103] In some embodiments, the alkyne-containing linker-nucleic acid molecule sense strand is selected from the following structures:

[0104] In some embodiments, the azide-modified polypeptide is selected from the following structures:

[0105] In a second aspect, the present invention provides applications of the polypeptide-oligonucleotide conjugate, including the following:

[0106] The polypeptide oligonucleotide conjugate of the present invention or its pharmaceutically acceptable salt or stereoisomer is used as an active ingredient in the preparation of a drug for inhibiting the expression of a target gene in the central nervous system.

[0107] In some embodiments, the central nervous system target gene is selected from one or more of APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and NLRP3.

[0108] The polypeptide oligonucleotide conjugate of the present invention or its pharmaceutically acceptable salt or stereoisomer is used as an active ingredient in the preparation of a drug for treating diseases related to the central nervous system (CNS).

[0109] In some embodiments, the target of the disease related to the central nervous system (CNS) is selected from APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and NLRP3.

[0110] In some embodiments, the disease associated with the central nervous system is Alzheimer's disease, cachexia, cerebral amyloid angiopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, spinocerebellar disorders, prion diseases and Lafora disease.

[0111] In a third aspect, the present invention provides a drug for inhibiting the expression of target genes in the central nervous system, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the polypeptide oligonucleotide conjugate described in the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0112] In a fourth aspect, the present invention provides a drug for treating diseases related to the central nervous system, which is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient comprises the polypeptide oligonucleotide conjugate described in the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0113] In a fifth aspect, the present invention provides a method for inhibiting the expression of a target gene in the central nervous system, comprising: administering a safe and effective amount of the polypeptide oligonucleotide conjugate of the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof; or administering a safe and effective amount of the drug of the present invention for inhibiting the expression of a target gene in the central nervous system.

[0114] In the sixth aspect, the present invention provides a method for treating diseases related to the central nervous system, comprising: administering a safe and effective amount of the polypeptide oligonucleotide conjugate of the present invention or a pharmaceutically acceptable salt or a stereoisomer thereof; or administering a safe and effective amount of a drug for treating diseases related to the central nervous system.

[0115] The present invention couples the polypeptide ligand of the LRP-1 receptor with an oligonucleic acid drug via a linker group to obtain a polypeptide oligonucleotide conjugate based on a novel LRP-1 targeting peptide. The polypeptide oligonucleotide conjugate can be used to deliver nucleic acid drugs such as small interfering RNA (siRNA) for the treatment of central nervous system diseases, and is used to inhibit the expression of one or more target genes in central nervous system tissue cells. It has the advantages of good in vivo effect, long duration of action and good safety. DETAILED DESCRIPTION

[0116] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0117] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0118] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0120] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0121] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, including both branched and straight-chain groups, having a specified number of carbon atoms. For example, the definition of "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl. "Alkylene" is the corresponding "alkyl" group minus one hydrogen atom.

[0122] The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon group whose ring atoms are composed of carbon atoms. Bicyclic or polycyclic rings include spirocyclic, fused rings and bridged rings. For example, "cycloalkyl" includes but is not limited to the following groups: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wait.

[0123] As used herein, the term "heterocycloalkyl" or "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic cyclic substituent (including spirocyclic, bridged, fused, and parallel rings), wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon, and the bicyclic or polycyclic rings include spirocyclic, fused, and bridged rings. For example: morpholinyl, piperidinyl, tetrahydropyrrolyl, pyrrolidinyl, dihydro Imidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, etc., and N-oxides thereof. The attachment of the heterocyclic substituent can be achieved through a carbon atom or through a heteroatom.

[0124] The drugs or methods for inhibiting the expression of target genes in the central nervous system, as well as the drugs or methods for treating diseases related to the central nervous system, provided by the present invention, comprise (administered to patients or subjects) an active ingredient within a safe and effective amount (i.e., the polypeptide oligonucleotide conjugate of the present invention or a pharmaceutically acceptable salt or stereoisomer thereof), and a pharmaceutically acceptable excipient. When administering the drug, a safe and effective amount of the active ingredient is administered to a mammal (such as a human) in need of treatment, wherein the dosage at the time of administration is a pharmaceutically effective dosage. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0125] Here, "safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. "Pharmaceutically acceptable excipients" refers to: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components of the composition can be mixed with the active ingredient of the present invention (polypeptide oligonucleotide conjugate or its pharmaceutically acceptable salt or stereoisomer) and with each other without significantly reducing the efficacy of the active ingredient.

[0126] Pharmaceutically acceptable excipients include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, dissolution buffers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, and fragrances.

[0127] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0128] The names, abbreviations, and structures of nucleotide monomers used in nucleic acid sequence representation are as follows:

[0129] The specific structure is as follows:

[0130] According to existing technologies, those skilled in the art know that the pharmaceutically acceptable salt of the siRNA may be a sodium salt or a potassium salt. For example, a sodium salt of the siRNA is produced during purification.

[0131] The present invention is further described in detail below with reference to specific embodiments.

[0132] Example 1. Synthesis of polypeptide

[0133] Peptide synthesis was performed on a PurePep Chorus 4-channel peptide synthesizer at a 0.2 mmol scale according to conventional solid-phase peptide synthesis methods, such as those described in Adam G et al., Standard practices for Fmoc-based solid-phase peptide synthesis in the Nowick laboratory. The conditions of the main process are as follows: (1) Resin swelling: Fmoc-Tyr-Wang Resin and Rink-Amide-MBHA-Resin were placed in DMF and swelled at room temperature for 30 minutes; (2) Deprotection: The resin was treated with a DMF solution containing 20% ​​piperidine at 50°C for 2 minutes; (3) Amino acid condensation: 5eq (1eq of resin) Fmoc amino acid, HCTU as a condensing agent (5eq), DIPEA (5eq) as a base, and the reaction was carried out at 50°C for 10 minutes; (4) N-terminal acetylation: 5% N-acetylimidazole was used for reaction at room temperature for 1 hour; (5) TFA cleavage of the resin: After the synthesis was completed, the resin was treated with a cleavage reagent containing TFA (TFA / Tips / H2O=95:2.5:2.5; TFA / Tips / EDT / H2O=92.5:2.5:2.5:2.5) for more than 2 hours, the resin was filtered and discarded, and the filtrate was collected. (6) Precipitation and purification: Add pre-cooled anhydrous ether to the filtrate and centrifuge to obtain a white precipitate. Wash the precipitate three times with anhydrous ether and dry under reduced pressure at room temperature to obtain a crude polypeptide product as an off-white powder. Dissolve the crude polypeptide in acetonitrile / water and purify it by high performance liquid chromatography.

[0134] For peptides with 6-azidohexanoyl modified lysine side chains, the synthesis process and conditions are essentially the same as those described above, with the following differences: (1) Fmoc-Lys(Mtt)-OH is used instead of conventional Fmoc-Lys(Boc)-OH amino acid raw material; (2) After solid-phase synthesis, the Mtt protecting group on the amino group of the lysine side chain is selectively removed using a DCM solution containing 2% TFA, and the peptide is then reacted with 6-azidohexanoic acid according to the conventional amino acid condensation conditions described above before the peptide is cleaved. In addition, for peptides with both 2-(2-(2-aminoethoxy)ethoxy)acetyl and 6-azidohexanoyl modified lysine side chains, the Mtt protecting group on the amino group of the lysine side chain is first selectively removed using a DCM solution containing 2% TFA, and the peptide is then reacted with 2-(2-(2-aminoethoxy)ethoxy)acetic acid according to the conventional amino acid condensation and Fmoc removal conditions described above, and then reacted with 6-azidohexanoic acid before the peptide is cleaved.

[0135] Example 2. Synthesis of polypeptide Ang2 (K10N3)

[0136] Ang2(K10Mtt)-Wang Resin was synthesized on a 0.2 mmol scale using the conditions described in Example 1 on a PurePep Chorus 4-channel peptide synthesizer. 20 mL of DCM and 0.4 mL of TFA were added to Ang2(K10Mtt)-Wang Resin. After shaking at room temperature for 15 minutes, the resin was washed three times with DCM to obtain Ang2(K10NH2)-Wang Resin. Ang2(K10NH2)-Wang Resin was placed in 15 mL of DMF, and 6-azidohexanoic acid (157 mg, 1 mmol), HCTU (414 mg, 1 mmol), and DIEA (129 mg, 1 mmol) were added sequentially. The mixture was shaken at room temperature for 1 hour, washed with DMF and DCM, and cleaved with TFA at room temperature for 2 hours. The mixture was precipitated with diethyl ether and purified by high-performance liquid chromatography to obtain the target peptide Ang2(K10N3) (150 mg, white powdery solid) (theoretical molecular weight: 2482.70, measured molecular weight: 1242.30 [M+2H]). 2+ ).

[0137] Example 3. Synthesis of polypeptide Ang2-Ang2-AZK

[0138] Ang2-Wang resin was synthesized on a PurePep Chorus 4-channel peptide synthesizer at a 0.2 mmol scale according to the conditions described in Example 1. Ang2-Wang resin was mixed with 1.5 equivalents of 3-maleimidopropionic acid hydroxysuccinimide ester (BMPS) and reacted for 1 h to obtain maleimido-Ang2-Wang resin. Maleimido-Ang2 was then cleaved with TFA (TFA / Tips / H₂O = 95:2.5:2.5), precipitated with ether, and purified by HPLC. 1 eq of maleimido-Ang2 and 1.1 eq of AZK-Ang-Cys were dissolved in a DMF / H₂O = 1:1 mixture and reacted at room temperature for 2 h. After completion of the reaction, the product was evaporated under reduced pressure. The residue was dissolved in acetonitrile / water and purified by HPLC to obtain 25 mg of the target peptide Ang2-Ang2-AZK (theoretical molecular weight: 5010.46, measured: 1002.8 [M+5H]). 5+ ).

[0139] According to the method of Example 1-3, the azide-modified polypeptides shown in Table 1-1 were synthesized, and their theoretical molecular weights and measured molecular weights are shown in Table 1-1 below.

[0140] Table 1-1

[0141] The corresponding unmodified amino acid sequences of the azide-modified polypeptides in Table 1-1 are shown in Table 1-2.

[0142] Table 1-2 Unmodified amino acid sequences

[0143] Example 4 Synthesis of Compound B08

[0144] 1. Synthesis of Compound B08-1

[0145] Compound [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (10 g, 26.0 mmol), DIEA (6.7 g, 52.0 mmol), and HBTU (10.8 g, 28.6 mmol) were dissolved in DMF (100 ml) and stirred at room temperature for 5 min. Compound 17 (11.4 g, 27.3 mmol) was then dissolved in DMF (20 mL) and added to the reaction solution. The reaction system was stirred at room temperature for 2 h. The reaction mixture was added to saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 100:1, 2% TEA) to obtain 17.4 g of a colorless oil with a yield of 85.2%. LCMS: 786.9, found: 808.9 [M+Na] + .

[0146] 2. Synthesis of Compound B08-2

[0147] B08-1 (8.0 g, 10.2 mmol) was dissolved in EA (76 ml), and diethylamine (12.6 g) was added. The reaction system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM / MeOH = 20:1, 0.5% TEA → 10:1, 0.1% TEA) to obtain 3.8 g of the product as a white foamy solid, with a yield of 66.2%. LCMS: 564.7, found 565.8 [M+H] + .

[0148] 3. Synthesis of Compound B08-3

[0149] B08-2 (2.0 g, 3.5 mmol) was dissolved in ACN (35 ml), and ethyl trifluoroacetate (1.3 g, 8.8 mmol) was added. The reaction system was stirred at room temperature overnight. The reaction solution was concentrated and separated by column chromatography (DCM / MeOH = 30:1, 0.1% TEA) to obtain 1.87 g of the product as a white foamy solid, with a yield of 79.9%. LCMS: 660.7, found 682.7 [M+Na] + .

[0150] 4. Synthesis of Compound B08

[0151] B08-3 (1.5 g, 2.3 mmol) and DIEA (0.9 g, 6.8 mmol) were dissolved in anhydrous DCM (10 ml). Cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.6 g, 2.7 mmol) was added, and the reaction system was stirred at room temperature for 1 h. The reaction mixture was concentrated at 30°C and separated by column chromatography (PE, 2% TEA → PE / EA = 4:1, 2% TEA) to give 0.8 g of the product as an off-white foamy solid in a 38.8% yield. LCMS: 860.9, found 861.9 [M+H] + .

[0152] Example 5 Synthesis of Compound B12

[0153] 1. Synthesis of Compound B12-1

[0154] Compound 6-heptynoic acid (0.35 g, 2.77 mmol), DIEA (0.90 g, 6.94 mmol), and HATU (1.16 g, 3.05 mmol) were dissolved in DMF (15 ml) and stirred at room temperature for 5 min. Compound B08-2 (1.64 g, 2.91 mmol) was then dissolved in DMF (15 mL) and added to the reaction solution. The reaction system was stirred at room temperature for 2 h. The reaction mixture was added to saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 100:1, 2% TEA) to obtain 0.5 g of a colorless oil, with a yield of 26.8%. LCMS: 672.8 found: 695.0 [M+Na] + .

[0155] 2. Synthesis of Compound B12

[0156] Compound B12-1 (0.5 g, 0.74 mmol) and DIEA (0.3 g, 2.25 mmol) were dissolved in anhydrous DCM (10 ml). Cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.21 g, 0.89 mmol) was added, and the reaction system was stirred at room temperature for 1 h. The reaction mixture was concentrated at 30°C and separated by column chromatography (PE, 2% TEA → PE / EA = 4:1, 2% TEA) to give 0.39 g of the product as an off-white foamy solid, with a yield of 60.1%. LCMS: 873.0, found 874 [M+H] + .

[0157] Example 6 Synthesis of dipeptide linker BPL-1

[0158] Step 1. Preparation of N6-(tert-Butoxycarbonyl)-N2-(6-((tert-Butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (Compound A3): A mixture 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 at room temperature. 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 then added and the mixture was allowed to react for 1 hour. Spot plate (DCM:MeOH=30:1, R f =0.3) and LCMS monitoring, the reaction was complete, and dichloromethane was added to dilute the reaction solution for extraction. The organic layer was washed twice with saturated sodium bicarbonate and then twice with saturated ammonium chloride, and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol) to obtain the product N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (8.5 g, yield: 93.8%). MS (ES) + ):m / z=474(M+H) + .

[0159] Step 2. Preparation of (6-aminohexanoyl)-L-lysine methyl ester (Compound A4): N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexanoyl)-L-lysine methyl ester (1.0 g, 2.11 mmol, 1.0 equiv) and a solution of hydrogen chloride in dioxane (10 mL, 2 mol / L) were mixed and reacted for 1.5 hours at room temperature. The reaction was complete as monitored by LCMS. The reaction solution was concentrated under reduced pressure, water (30 mL) was added, and sodium hydroxide solution was added to adjust the pH to approximately 7-8. The aqueous phase was concentrated under reduced pressure using an oil pump. The resulting solid was washed with a solution of dichloromethane / methanol (10 / 1) with stirring. The filtrate was filtered and concentrated under reduced pressure to afford (6-aminohexanoyl)-L-lysine methyl ester (450 mg, yield: 77.9%) as a pale yellow oil. MS (ES) + ):m / z=274(M+H) + .

[0160] Step 3. Preparation of N6-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine methyl ester (Compound A5): Under nitrogen protection, (6-aminohexanoyl)-L-lysine methyl ester (450 mg) was added. , 1.65 mmol, 1.0 equiv), ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (960 mg, 3.29 mmol, 2.0 equiv) and N,N-diisopropylethylamine (468 mg, 3.63 mmol, 2.2 equiv) in N,N-dimethylformamide (5 ml) were stirred at room temperature for 1 hour. Spot plate (DCM:MeOH=10:1, R f =0.4) and LCMS monitoring, the reaction was complete, and after cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was diluted with water (20 ml) and extracted with dichloromethane / methanol = 10 / 1 (30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 30 / 1) to obtain a white solid N6-((((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R, 8S, 9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine methyl ester (280 mg, yield: 27.1%). MS (ES + ):m / z=626(M+H) + .

[0161] Step 4. Preparation of N6-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine (Compound A6): To a mixture of tetrahydrofuran, methanol, and water (6 ml) was added (280 mg, 0.448 mmol, 1.0 equiv) of (1R, 8S, 9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine methyl ester (280 mg, 0.448 mmol, 1.0 equiv). A lithium hydroxide (13 mg, 0.538 mmol, 1.2 equiv) aqueous solution (1 ml) was then added, and the mixture was stirred at room temperature for 1 hour. Following completion of the reaction as monitored by LCMS, the reaction solution was concentrated under reduced pressure, water (30 ml) was added, and the pH was adjusted to approximately 3-4 by adding 1 mol / L dilute hydrochloric acid. The mixture was diluted with water (20 ml), and extracted with dichloromethane (30 ml). The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure to give N6-((((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine (220 mg, yield: 80.2%) as a white solid. MS (ES) + ):m / z=612(M+H) + .

[0162] Step 5. Preparation of 2,5-dioxopyrrolidin-1-yl N6-((1R,8S,9s)-bicyclo[6.1.0]non-4-yl-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)hexanoyl)-L-lysine salt (BPL-1): N6-(((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)-N2- (6-(1R,8S,9r)-bicyclo[6.1.0]non-4-en-9-ylmethoxy)carbonyl)amino)hexanoyl)-L-lysine (220 mg, 0.36 mmol, 1.0 eq), N-hydroxysuccinimide (49.7 mg, 0.43 mmol, 1.2 eq), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (82.5 mg, 0.432 mmol, 2.2 eq) were added to dichloromethane, and the mixture was stirred at room temperature for 1.5 hours. LCMS monitoring confirmed the completion of the reaction, and the reaction solution was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate and then twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. A white solid 2,5-dioxopyrrolidin-1-yl (N6-((1R,8S,9s)-bicyclo[6.1.0]non-4-yl-9-yl)methoxy)carbonyl)-N2-(6-(1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)hexanoyl)-L-lysine salt (200 mg, yield: 78.7%) was obtained and used directly in the next reaction without purification. MS (ES) + ):m / z=709(M+H) + .

[0163] Example 7 Synthesis of dipeptide linker BPL-2

[0164] Step 1. Preparation of (S)-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester (Compound B1): (6-aminohexanoyl)-L-lysine methyl ester (3.0 g, 10.9 mmol, 1.0 eq), 2,2-dimethyl-4-oxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oic acid methyl ester were added at room temperature. A mixture of 3,8,11-trioxo-5-azatridecane-13-oic acid (6.35 g, 24.2 mmol, 2.2 eq.) and N,N-diisopropylethylamine (5.7 ml, 32.9 mmol, 3.0 eq.) in dichloromethane was stirred uniformly, and then 1-hydroxybenzotriazole (3.7 g, 27.5 mmol, 2.5 eq.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.24 g, 27.5 mmol, 2.5 eq.) were added and reacted for 1 hour. The plate was stirred (DCM:MeOH=20:1, R f =0.3) and LCMS monitoring, the reaction was complete, and dichloromethane was added to dilute the reaction solution and extract. The organic layer was washed twice with saturated sodium bicarbonate and then washed 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-triazatricosane-23-oic acid methyl ester (3.9 g, yield: 46.8%), MS (ES) + ):m / z=764(M+H) + .

[0165] Step 2. Preparation of methyl-N2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (Compound B2): Methyl-22-(2,2-dimethyl-4,13-dioxo-3,8-11-trioxa-5,14-diazaoctadec-18-yl)-2,2-dimethyl-4,3,20-trioxa-3,8,11-trioxa-5,14,21-triazatricosan-23-oate (3.9 g, 5.11 mmol, 1.0 eq.) and hydrogen chloride were added at room temperature. The mixture was stirred with a dioxane solution (40 mL, 2 mol / L) for 1.5 hours. The reaction was complete under LCMS monitoring. The reaction solution was concentrated under reduced pressure, and water (30 mL) was added. Sodium hydroxide solution was added to adjust the pH to approximately 7-8. The aqueous phase was concentrated under reduced pressure using an oil pump. The resulting solid was washed with a solution of dichloromethane / methanol = 10 / 1, stirred, and filtered. The filtrate was concentrated under reduced pressure to afford methyl N2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (2.9 g, yield: 85.8%) as a pale yellow oil. MS (ES) + ):m / z=564(M+H) + .

[0166] Step 3. Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid methyl ester (Compound B3): Methyl N2-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)acetylamino)hexanoyl)-N6-(2-(2-(2-aminoethoxy)ethoxy)acetyl)-L-lysine ester (440 mg, A mixture of dichloromethane (0.691 mmol, 1.0 equivalent), 6-heptynoic acid (178 mg, 1.41 mmol, 2.04 equivalent), N,N-diisopropylethylamine (263 μl, 1.52 mmol, 2.2 equivalent) was stirred evenly, and then 1-hydroxybenzotriazole (205 mg, 1.52 mmol, 2.2 equivalent) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (290 mg, 1.52 mmol, 2.2 equivalent) were added and reacted for 1 hour. Spot plate (DCM:MeOH=20:1, R f=0.2) and LCMS monitoring, the reaction was complete, and dichloromethane was added to dilute the reaction solution and extract. The organic layer was washed twice with saturated sodium bicarbonate and then washed 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-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid methyl ester (310 mg, yield: 57.6%), MS (ES) + ):m / z=780(M+H) + .

[0167] Step 4. Preparation of (S)-2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25ynoic acid (Compound B4): Methyl 1-hydroxy-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoate (310 mg, 0.398 mmol, 1.0 eq) was added to a mixture of tetrahydrofuran, methanol, and water (6 mL). A solution of lithium hydroxide (20.1 mg, 0.478 mmol, 1.2 eq) in water (1 mL) was then added and stirred at room temperature for 1 hour. The reaction was complete as monitored by LCMS. The reaction mixture was concentrated under reduced pressure, water (25 mL) was added, and the pH was adjusted to approximately 3-4 with 1 mol / L dilute hydrochloric acid. The mixture was diluted with water (20 mL) and extracted with dichloromethane (25 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure to give a white solid (S) 2-(6,15-dioxo-8,11-dioxo-5,14-diazaicos-20-yn-1-yl)-4,11,20-trioxo-13,16-dioxo-3,10,19-triazahexadec-25-ynoic acid (290 mg, yield: 95.4%). 1H NMR (500MHz, CDCl3) δ6.90 (s, 2H), 6.83 (d, J = 8.3Hz, 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=4.8Hz,10H),3.41-3.35(m,10H ),2.45-2.36(m,4H),2.32(t,J=6.8Hz,2H),2.24(t,J=6.9Hz,2H),2.15(dt,J=7. 1,6.2Hz,7H),1.89(dd,J=15.2,8.1Hz,4H),1.72-1.64(m,4H),1.52-1.46(m,4H). MS(ES + ):m / z=766(M+H) + .

[0168] Step 5. Preparation of BPL-2: Compound B4 (290 mg, 0.38 mmol) was dissolved in 4 mL of dichloromethane, and N-hydroxysuccinimide (52 mg, 0.46 mmol, 1.2 eq.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88 mg, 0.46 mmol, 1.2 eq.) were added in sequence. The mixture was stirred at room temperature for 1.5 hours. The reaction was completed under LCMS monitoring. Dichloromethane was added to dilute the reaction solution and extracted. The organic layer was washed twice with saturated sodium bicarbonate and then washed twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. BPL-2 (310 mg, yield: 94.5%) was obtained as a white viscous solid and used directly in the next step without purification. MS (ES) + ):m / z=864(M+H) + .

[0169] Example 8 Synthesis of dipeptide linker BPL-3

[0170] Step 1. Preparation of (S)-1-((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yl-9-yl)-21-(1-(1R, 8S, 9s)-bicyclo[6.1.0]hept-4-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3,12,19-trioxy-2,7,10-trioxa-4,13,20-triazadocosa-22-oic acid methyl ester (Compound C1): Under nitrogen protection, methyl N2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)acetamido) A solution of ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (928 mg, 3.19 mmol, 2.0 equiv) and N,N-diisopropylethylamine (450 mg, 3.49 mmol, 2.2 equiv) in N,N-dimethylformamide (10 ml) was stirred at room temperature for 1 hour. A plate (DCM:MeOH=10:1, R f =0.3) and LCMS monitoring for complete reaction. After cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was diluted with water (30 ml) and extracted with dichloromethane / methanol = 10 / 1 (30 ml). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 30 / 1) to give (S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yl-9-yl)-21-(1-(1R,8S,9s)-bicyclo[6.1.0]hept-4-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3,12,19-trioxy-2,7,110-trioxa-4,13,20-triazadocosan-22-oic acid methyl ester (580 mg, yield: 39.8%) as a white solid. MS (ES) + ):m / z=916(M+H) + .

[0171] Step 2. Preparation of (S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-butan-9-yl)-21-(1-(1R,8S,9)-bicyclo[1.1.0]non-4-butan-9-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3,12,19-trioxa-2,7,110-trioxa-4,13,20-triazadocosan-22-oic acid (Compound C2): (S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yl-9-yl)-21 -(1-(1R,8S,9s)-bicyclo[6.1.0]hept-4-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3.12,19-trioxy-2,7,110-trioxa-4,13,20-triazadocosa-22-oic acid methyl ester (300 mg, 0.327 mmol, 1.0 eq) was added to a mixed solvent of tetrahydrofuran, methanol and water (7.5 ml), and then lithium hydroxide (9.5 mg, 0.40 mmol, 1.2 eq) aqueous solution (0.5 ml) was added, and the mixture was stirred at room temperature for 1 hour. The reaction was completed under LCMS monitoring. The reaction solution was concentrated under reduced pressure, and water (25 mL) was added. 1 mol / L dilute hydrochloric acid solution was added to adjust the pH to about 3-4, and the mixture was diluted with water (25 mL). Dichloromethane (30 mL) was added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a white solid (S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-butan-9-yl)-21-(1-(1R,8S,9)-bicyclo[1.1.0]non-4-butan-9-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3,12,19-trioxa-2,7,110-trioxa-4,13,20-triazadocosan-22-oic acid (260 mg, yield: 88.4%). MS (ES) + ):m / z=902(M+H) + .

[0172] Step 3. Preparation of 2,5-dioxopyrrolidin-1-yl-(S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-21-(1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3,12-dioxo-2,7,10-trioxo-4,13-diazaheptadecan-17-yl)-3,12,19-trioxo-2,7,10-trioxo-4,13,20-triazadocosan-22-oate (BPL-3): (S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-butan-9-yl)-21-(1-(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3,12-dioxo-2,7,10-trioxo-4,13-diazaheptadecan-17-yl)-3,12,19-trioxo-2,7,10-trioxo-4,13,20-triazadocosan-22-oate 8S,9-(8S,9-bicyclo[1.1.0]non-4-but-9-yl)-3,12-dioxo-2,7,10-trioxa-4,13-diazaheptadecan-17-yl)-3,12,19-trioxa-2,7,110-trioxa-4,13,20-triazadocosan-22-oic acid (260 mg, 0.288 mmol, 1.0 eq.), N-hydroxysuccinimide (40 mg, 0.35 mmol, 1.2 eq.), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (121 mg, 0.634 mmol, 2.2 eq.) were dissolved in dichloromethane, and the mixture was stirred at room temperature for 1.5 hours. The reaction was complete as monitored by LCMS, and the reaction solution was diluted with dichloromethane and extracted. The organic layer was washed twice with saturated sodium bicarbonate and then twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. A white solid 2,5-dioxopyrrolidin-1-yl-(S)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-21-(1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3,12-dioxo-2,7,10-trioxo-4,13-diazaheptadecan-17-yl)-3,12,19-trioxo-2,7,10-trioxo-4,13,20-triazadocosanoic-22-oic acid ester (200 mg, yield: 69.7%) was obtained and used directly in the next step without purification. MS (ES) + ):m / z=999(M+H) + .

[0173] Example 9 Synthesis of dipeptide linker BPL-4

[0174] 1. Synthesis of Compound D2: D1 (10 g, 52.2 mmol, 1.0 eq) was dissolved in 1,4-dioxane (100 mL). Acrylonitrile (9.7 g, 183.7 mmol, 3.5 eq) and 3 mL of an aqueous potassium hydroxide solution (292 mg, 5.22 mmol, 0.1 eq) were added with stirring. The system was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with water (60 mL), and extracted with dichloromethane (100 mL). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to a yellow oil. The residue was purified by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 8:1) to obtain D2 (15.2 g, 97.2% yield) as a clear oil. MS (ES) + ):m / z=326(M+H) + .

[0175] 2. Synthesis of compound D3: D2 (5 g, 16.8 mmol, 1.0 eq) was dissolved in 10 M hydrochloric acid / ethanol (50 ml), heated in an oil bath to 79°C, and stirred overnight. TLC (PE:EA=1:1, R f =0.2) and LCMS monitoring showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and water (50 ml) was added to dilute the reaction system. Saturated sodium carbonate solution was added to adjust the pH to 9, and the mixture was extracted with dichloromethane. The organic layer was washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude product D3 (3.49 g, yield: 71.3%). MS (ES + ):m / z=292(M+H) + .

[0176] 3. Synthesis of Compound D4: D3 (3.2 g, 10.99 mmol, 1.0 eq) was dissolved in dichloromethane (25 ml) at room temperature, followed by the addition of mono-tert-butyl dodecyl diacid ester (3.77 g, 13.2 mmol, 1.2 eq), DIPEA (3.8 ml, 21.98 mmol, 2.0 eq), HOBt (2.23 g, 16.49 mmol, 1.5 eq) and EDCI (3.15 g, 16.49 mmol, 1.5 eq) and the reaction was allowed to proceed at room temperature for 1 hour. TLC (PE:EA = 2:1, R f =0.5) and LCMS monitoring of the reaction completion, dichloromethane was added to dilute the reaction solution. The organic layer was washed twice with saturated sodium bicarbonate and twice with saturated ammonium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to obtain D4 (4.8 g, yield: 78%) as a light yellow oil. MS (ES + ):m / z=560(M+H) + .

[0177] 4. Synthesis of Compound D5: D4 (2.57 g, 4.59 mmol, 1.0 eq) was dissolved in a mixed solvent of THF, MeOH, and H2O (THF / MeOH / H2O = 1:1:1, 25 mL). A solution of lithium hydroxide (264 mg, 11.02 mmol, 2.4 eq) in water (5 mL) was then added, and the system was stirred at room temperature for 1 hour. The reaction was complete as monitored by LCMS. The reaction solution was concentrated under reduced pressure, diluted with water (30 mL), and the pH was adjusted to 3-4 with 1 mol / L dilute hydrochloric acid. The mixture was extracted with dichloromethane (30 mL). The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield a crude yellow oil. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to yield D5 (2.0 g, yield: 86.5%). MS (ES) + ):m / z=504(M+H) + .

[0178] 5. Synthesis of Compound D6: D5 (700 mg, 1.39 mmol, 1.0 eq), N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)hept-6-ynamide (783 mg, 3.06 mmol, 2.2 eq), DIPEA (846 μl, 4.87 mmol, 3.5 eq), and HOBt (469 mg, 3.48 mmol, 2.5 eq) were dissolved in dichloromethane (8 ml) at room temperature. EDCI (666 mg, 3.48 mmol, 2.5 eq) was then added and the reaction was stirred at room temperature for 1.5 hours. TLC (DCM:MeOH = 15:1, R f =0.3) and LCMS monitoring of the reaction completion. Dichloromethane was added to dilute the reaction solution. The organic layer was washed twice with saturated sodium bicarbonate solution and then twice with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 15 / 1) to obtain D6 (1.15 g, yield: 84.5%) as a colorless, transparent oil. MS (ES + ):m / z=980(M+H) + .

[0179] 6. Synthesis of compound D7: D6 (1.15 g, 1.17 mmol, 1.0 eq) was dissolved in a 20% TFA solution in DCM (12 ml) at room temperature, and the system was reacted at room temperature for 2 hours. TLC (DCM:MeOH=10:1, R f=0.3) and LCMS monitored the completion of the reaction. The reaction solution was concentrated under reduced pressure, diluted with water (30 ml), and adjusted to pH 9 by adding sodium carbonate solution. The mixture was then extracted with dichloromethane, and the organic phase was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase HPLC to give D7 (620 mg, yield: 57.4%) as a white oil. 1 H NMR (500MHz, CDCl3) δ6.70 (s, 2H), 6.59 (d, J = 8.0Hz, 1H), 6.32 (s, 2H), 4.12 (s, 1H), 3.73 -3.60(m,4H),3.59-3.52(m,9H),3.50(dd,J=9.0,4.8Hz,9H),3.43-3.27(m,10H),2.42- 2.35(m,4H),2.26(t,J=7.0Hz,2H),2.15(dd,J=15.3,7.5Hz,10H),1.89(s,2H),1.69(dt ,J=15.3,7.6Hz,4H),1.51(ddd,J=22.2,16.1,6.3Hz,8H),1.20(d,J=15.9Hz,12H),MS(ES + ):m / z=924(M+H) + .

[0180] 7. Synthesis of compound BPL-4: D7 (620 mg, 0.67 mmol) was dissolved in DCM (20 mL), and NHS (93 mg, 0.81 mmol, 1.2 eq.) and EDCI (154 mg, 0.81 mmol, 1.2 eq.) were added with stirring. The reaction was allowed to react at room temperature for 2 hours. The reaction was complete after monitoring by LC-MS. The reaction solution was diluted with 20 mL of DCM, and the organic phase was washed twice with saturated NH4Cl solution. The reaction was concentrated under reduced pressure to obtain a white solid BPL-4, which was used directly in the next step without purification. MS (ES + ):m / z=1021(M+H) + .

[0181] Example 10 Synthesis of solid phase carrier Q20-CPG

[0182] Step 1: Preparation of methyl 1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatriacontane-13-hydroxy)piperidine-4-carboxylic acid (Compound Q20-2)

[0183] 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 piperidine 4-carboxylate (2.72 g, 18.99 mmol, 1.0 eq), N,N-diisopropylethylamine (4.90 g, 37.98 mmol, 2.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.66 g, 22.78 mmol, 1.2 eq) in dichloromethane (50 ml) was stirred at room temperature for 30 minutes. The solvent was concentrated under reduced pressure, and 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-azatriacontane-13-hydroxy)piperidine-4-carboxylic acid as a yellow oil (12.7 g, crude product). LCMS (ESI): m / z = 389 [M+H] + .

[0184] Step 2: Preparation of methyl 1-(2-(2-(2-aminoethoxy)acetyl)piperidine-4-carboxylate hydrochloride (Compound Q20-3):

[0185] A solution of methyl 1-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatriacontane-13-hydroxy)piperidine-4-carboxylic acid (crude) in hydrogen chloride-dioxane (40 mL) was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was used directly in the next step without purification (crude product). LCMS (ESI): m / z = 289 [M+H] + .

[0186] Step 3: Preparation of methyl 1-(2-(2-(6-heptylamino)ethoxy)acetyl)piperidine-4-carboxylate (Compound Q20-4)

[0187] A mixture of methyl 1-(2-(2-(2-aminoethoxy)acetyl)piperidine-4-carboxylate hydrochloride (crude), 6-heptynoic acid (2.35 g, 18.69 mmol), N,N-diisopropylethylamine (7.23 g, 56.06 mmol) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.52 g, 22.42 mmol) 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 methyl 1-(2-(2-(6-heptylamido)ethoxy)acetyl)piperidine-4-carboxylate (8.19 g, crude) as a yellow oil. LCMS (ESI): m / z = 397 [M+H] + .

[0188] Step 4: Preparation of 1-(2-(2-(6-heptylamino)ethoxy)acetyl)piperidine-4-carboxylic acid (Compound Q20-5)

[0189] A mixture of methyl 1-(2-(2-(6-heptylamino)ethoxy)acetyl)piperidine-4-carboxylate (8.19 g, 20.6 mmol, 1.0 eq) and 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 hour. The solvent was concentrated under reduced pressure, 1 M hydrochloric acid solution was added to adjust the pH of the residue to 3, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated to give 1-(2-(2-(6-heptylamino)ethoxy)acetyl)piperidine-4-carboxylic acid (6.0 g, crude) as a yellow oil. LCMS (ESI): m / z=383[M+H] + .

[0190] Step 5: Preparation 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-ynamido)ethyl)acetyl)piperidine-4-carboxamide (Compound Q20-6)

[0191] A mixture of 1-(2-(2-(6-heptamido)ethoxy)acetyl)piperidine-4-carboxylic acid (1.1 g, 2.87 mmol, 1.0 eq), 2-(2-(2-aminoethoxy)ethoxy-1-(2S,4R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)ethan-1-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-azabenzotriazole)-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 hour. The mixture was separated and purified using RP-HPLC (column: UniSil 10-120C18, 30×250 mm) to give 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-ynamido)ethyl)acetyl)piperidine-4-carboxamide) as a white solid product (320 mg, yield: 12%). LCMS (ESI): m / z=627 [(M-303)+H] + .

[0192] Step 6: Preparation of 4-(((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(2-(2-(2-(2-(hept-6-ynylcarboxamido)ethoxy)ethyl)acetyl)piperidine-4-carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (Compound Q20-7)

[0193] 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-ynamido)ethyl)acetyl)piperidine-4-carboxamide (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 (4.1 mg, 0.034 mmol, 0 A mixture of 4-[((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(2-(2-(2-(hept-6-ynylcarboxamido)ethoxy)ethyl)acetyl)piperidine-4-carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid was stirred at room temperature overnight. The mixture was washed three times with 10% sodium bicarbonate solution and extracted with dichloromethane. The organic phase was washed again 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-ynylcarboxamido)ethoxy)ethyl)acetyl)piperidine-4-carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid as a yellow solid (380 mg, crude). 1 H NMR(500MHz,MeOD)δ7.42-7.12(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=14.8,9.4Hz,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=17.2,12.8,5.6Hz,5H),1.81-1.60(m,5H),1.51(dt,J=14.4,7.0Hz,3H). LCMS (ESI): m / z=727[(M-303)+H] + .

[0194] Step 7: Preparation of Q20-CPG

[0195] To a solution of 4-(((3R,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(2-(2-(-2-(2-(hept-6-yncarboxamido)ethoxy)ethyl)acetyl)piperidine-4-carboxamidoethoxyacetyl)pyrrolidin-3-yl)oxy)-4-oxobutanoic acid (380 mg, 0.37 mmol, 1.0 eq) in N,N-dimethylformamide (10 ml) were added 2-(7-azabenzotriazole)-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. Then, solid support CPG-NH2 (1.9 g, substitution ratio 1.19 g / mmol) was added to the reaction mixture and stirred at room temperature overnight. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane in sequence and dried under vacuum for 1 hour. The residue was placed in a pyridine / acetic anhydride (7.5 ml / 2.5 ml) mixed solution and stirred at room temperature for 3 hours. The mixture was filtered, and the filter cake was washed with dichloromethane, acetonitrile, and dichloromethane in sequence, and dried under vacuum for 1 hour to obtain a light yellow solid support Q20-CPG (2.08 g).

[0196] Example 11 Synthesis of solid phase carrier Q22-CPG

[0197] Step 1. A mixture of B4 (5.0 g, 6.53 mmol, 1.0 eq.), Q22-1 (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 hours. The reaction system was diluted with ethyl acetate, washed twice with water, saturated Na2CO3 solution, and NH4Cl solution, and then washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give 6.14 g of crude Q22-2. MS (ES) + ):m / z=1079.46(M+H) + .

[0198] Step 2. Dissolve the crude product Q22-2 (6.14 g) in 5 mL of dichloromethane, add 5 mL of trifluoroacetic acid with stirring, and stir at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure, dilute the residue with dichloromethane, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate to obtain 5.53 g of crude product Q22-3. MS (ES) + ):m / z=1023.25(M+H)+

[0199] Step 3. A mixture of crude Q22-3 (5.53 g), B08-2 (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 hours. The reaction was diluted with ethyl acetate, washed twice with water, twice with saturated NaCO solution, and then with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by RP-HPLC to afford the desired product Q22-4 as a white oil (5.1 g, 49.7% yield over three steps). 1 H NMR(500MHz,DMSO-d6)δ7.90-7.77(m,5H),7.61(t,J=5.9Hz,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=14.0Hz,1H),4.19(td,J=8.6,5.1Hz,2H),4.11(t,J=13.4Hz,2H),4.06 (s,1H),3.85(d,J=1.7Hz,4H),3.73(s,7H),3.62-3.46(m,17H),3.46-3.35(m,9 H),3.19(p,J=7.5,6.6Hz,9H),3.10-3.01(m,5H),2.90(d,J=12.7Hz,1H),2.72 (d,J=2.6Hz,2H),2.54(s,1H),2.43-2.30(m,1H),2.18-2.04(m,11H),2.01(dd, J=12.5,5.9Hz,1H),1.84(ddd,J=12.9,8.3,4.4Hz,1H),1.64(s,2H),1.56(p,J =7.5Hz, 5H), 1.43 (ddq, J = 24.8, 17.3, 10.1, 8.8Hz, 14H), 1.22 (q, J = 7.5Hz, 4H). MS(ES + ):m / z=1267.30[(M-303)+H] + .

[0200] Step 4. Q22-4 (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 sequentially with stirring. The reaction mixture was allowed to react overnight at room temperature. The reaction mixture was diluted with dichloromethane and washed three times with 10% sodium bicarbonate solution and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate and concentrated to yield a brown oil. 1H NMR(500MHz,CDCl3)δ7.57(dt,J=20.0,5.0Hz,1H),7.35(d,J=10.0Hz,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=5.0Hz,4H),3.79-3.78(m,7H),3.67-3.60(m,16H),3.57(q,J=5.0Hz,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=10.0,5.0Hz,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=10.0,5.0Hz,1H),2.26-2.17(m,10H),2.11(td,J=10.0,5.0Hz,1H),1.98(t,J=5.0Hz,2H),1.84(dt,J=10.0,5.0Hz,2H),1.75(dt,J=15.0,5.0Hz,6H),1.65(dt,J=15.0,5.0Hz,4H),1.55(dq,J=15.0,5.0Hz,9H),1.39-1.33(m,4H).13C NMR(126MHz,CDCl3)δ174.77,174.60,173.25,173.11,173.02,172.93,172.20,169.98,169.87,168.55,168.04,167.58,167.51,158.66,158.47,144.85,135.86,129.95,128.00,127.84,126.84,113.27,113.13,86.06,84.18,84.16,73.19,70.96,70.92,70.61,70.54,70.12,70.04,70.01,70.00,68.69,55.22,52.78,52.19,45.02,39.44,39.26,39.07,38.69,38.57,36.24,35.90,35.86,32.87,32.48,31.22,30.57,29.32,29.14,28.72,27.97,26.51,25.30,25.25,24.79,22.73,18.20,9.05. MS(ES. + ):m / z=1367.35[(M-303)+H] + .

[0201] The above oil (5.26g) was dissolved in DMF, and DIEA (1.63g, 12.6mmol), HATU (1.44g, 3.78mmol) and HOBt (510mg, 3.78mmol) were added with stirring. The mixture was stirred at room temperature for 5 minutes, and 5.3g of controlled pore glass support (CPG, substitution rate 1.2mmol / g) was added to the reaction mixture. The system was stirred at room temperature overnight, filtered, and the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in sequence, and dried under vacuum for 1 hour. The above dried solid support was placed in pyridine / acetic anhydride (20mL / 7mL), shaken at room temperature for 3 hours, filtered, and the filter cake was washed with dichloromethane, acetonitrile and dichloromethane in sequence, and dried under vacuum for 1 hour to obtain a light yellow solid support Q22-CPG (10.2g).

[0202] Example 12 Synthesis of Solid Phase Support DT-Linker CPG

[0203] Step 1. Synthesis of compound DT-Linker-1

[0204] Compound 3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)propanoic acid (4.0 g, 11.3 mmol), DIEA (3.6 g, 27.9 mmol), and HATU (4.3 g, 11.3 mmol) were dissolved in DMF (40 mL) and stirred at room temperature for 5 min. Compound 17 (3.93 g, 9.37 mmol) was dissolved in 10 mL of DMF and added to the above solution, and the system was stirred at room temperature for 1 h. The reaction solution was diluted with saturated NaHCO3 and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 50:1, 0.5% TEA) to obtain 6.5 g of a colorless oil, with a yield of 76.4%. LCMS: 756, Found 779 [M+23] + .

[0205] Step 2. Synthesis of compound DT-Linker-2

[0206] DT-Linker-1 (6.5 g, 8.60 mmol) was dissolved in ethyl acetate (76 mL), and diethylamine (12.6 g) was added. The system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was separated by column chromatography (DCM / MeOH = 20:1, 0.5% TEA → 10:1, 0.1% TEA) to obtain 3.1 g of a white foamy solid, with a yield of 67.5%. LCMS: 534, found 535 [M+H] + . 1 HNMR(500MHz,Chloroform-d)δ7.36(d,J=6.3Hz,2H),7.33-7.13(m,7H),6.81(dt,J=10.8,6.3Hz,4H),4.57-4.24(m,2H),4.17-3.91(m,1H),3.78 (t,J=5.4Hz,8H),3.63(h,J=5.9,5.0Hz,1H),3.54-3.33(m,3H),3.17(dt ,J=21.6,5.2Hz,1H),2.88-2.64(m,3H),2.47(s,3H),2.29-1.96(m,3H).

[0207] Step 3. Synthesis of compound DT-Linker-3

[0208] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (2.8 g, 6.59 mmol), DIEA (1.4 g, 10.8 mmol), and HATU (2.7 g, 7.10 mmol) were dissolved in DMF (20 mL) and stirred at room temperature for 5 min. DT-Linker-2 (2.9 g, 5.43 mmol) was dissolved in DMF (9 mL) and added to the above solution, and the system was stirred at room temperature for 2 h. The reaction solution was diluted with saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 50:1, 0.5% TEA) to obtain 4.48 g of an off-white solid, with a yield of 87.7%. LCMS: 941, found 640 [M-DMTr+H] + .

[0209] Step 4. Synthesis of compound DT-Linker-4

[0210] DT-Linker-3 (4.48 g, 4.76 mmol) was dissolved in ethyl acetate (70 mL), and diethylamine (6.94 g) was added. The system was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and separated by column chromatography (DCM / MeOH = 50:1, 0.5% TEA) to obtain 3.46 g of an orange foamy solid. Yield: 101.1%. LCMS: 719, found 720 [M+H] + .

[0211] Step 5. Synthesis of compound DT-Linker-5

[0212] 6-Azidohexanoic acid (906 mg, 5.77 mmol), DIEA (1.9 g, 14.7 mmol), and HATU (2.4 g, 6.31 mmol) were dissolved in DMF (20 mL) and stirred at room temperature for 5 min. DT-Linker-4 (3.46 g, 4.81 mmol) was dissolved in 15 mL of DMF and added to the solution. The system was stirred at room temperature for 1 h. The reaction solution was diluted with saturated NaHCO₃ and extracted with DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography (DCM / MeOH = 50:1, 0.5% TEA) to obtain 2.8 g of a yellow oil in a 68.0% yield. LCMS: 858, found 881 [M+Na] + .

[0213] Step 6. Synthesis of compound DT-Linker-6

[0214] DT-Linker-5 (2.8 g, 3.26 mmol) was dissolved in a mixture of THF (17 mL), MeOH (8.5 mL), and H₂O (8.5 mL). LiOH·H₂O (1.38 g, 32.6 mmol) was added with stirring, and the system was stirred at room temperature overnight. AcOH (9 eq.) was added to neutralize the reaction, concentrated, and extracted with DCM. The organic solution was separated, dried, concentrated, and separated by column chromatography (DCM / MeOH = 30:1, 0.1% TEA → 5:1, 0.1% TEA) to afford 850 mg of an off-white oil in a 32.7% yield. LCMS: 802, found 801 [MH] - .

[0215] Step 7: Synthesis of compound DT-Linker-7:

[0216] A mixture of compound DT-Linker-6 (2.4 g, 2.99 mmol, 1.0 eq.), DIEA (965 mg, 7.48 mmol, 2.5 eq.), N-(2-(2-aminoethoxy)ethyl)-2,2,2-trifluoroacetamide hydrochloride (601 mg, 2.99 mmol, 1.0 eq.), and HCTU (1.85 g, 4.49 mmol, 1.5 eq.) in N,N-dimethylformamide (20 mL) was stirred at room temperature for one hour. The reaction 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: dichloromethane / methanol = 100 / 1 to 20 / 1) to obtain the yellow oily product DT-Linker-7 (1.75 g, yield: 59.3%). LCMS (ESI): m / z = 985 [M+H] + .

[0217] Step 8: Preparation of the Synthesis of Compound DT-Linker-8

[0218] A mixture of DT-Linker-7 (1.75 g, 1.78 mmol, 1.0 eq.), succinic anhydride (534 mg, 5.34 mmol, 3.0 eq.), 4-dimethylaminopyridine (22 mg, 0.18 mmol, 0.1 eq.), and triethylamine (1.17 g, 11.55 mmol, 6.5 eq.) in dichloromethane (20 mL) was stirred at room temperature overnight. The reaction mixture 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 afford DT-Linker-8 (1.72 g, yield: 89.6%) as a dark purple oil. LCMS (ESI): m / z = 1085 [M+H] + .

[0219] Step 9: Synthesis of solid support DT-Linker-CPG

[0220] A mixture of DT-Linker-8 (1.25 g, 1.15 mmol, 1.0 eq.), HATU (525 mg, 1.38 mmol, 1.2 eq.), 1-hydroxybenzotriazole (218 mg, 1.61 mmol, 1.4 eq.), and DIEA (594 mg, 4.61 mmol, 4.0 eq.) in dimethylformamide (60 mL) was stirred at room temperature for 5 minutes. A blank solid support, CPG (6.25 g, substitution ratio: 1.19 g / mmol), was added to the reaction solution, and the mixture was stirred overnight on a shaker at room temperature. The mixture was filtered, and the filter cake was washed three times with dichloromethane, acetonitrile, and dichloromethane, followed by vacuum drying for one hour. The filter cake was then placed in a mixture of pyridine (45 mL) and acetic anhydride (15 mL) and shaken on a shaker for two hours. The mixture was filtered again, and the filter cake was washed three times with dichloromethane, acetonitrile, and dichloromethane in sequence. The filter cake was dried under vacuum for one hour to obtain the product DT-Linker-CPG (6.43 g) as a yellow solid.

[0221] Example 13 Synthesis and Annealing of siRNA

[0222] Oligonucleotides were synthesized in 10-40 μmol batches using a solid-phase oligonucleotide synthesis method on an Edgex MerMade-12-channel nucleic acid synthesizer. An aminolysis reagent was added to the synthesized oligonucleotide, and the oligonucleotide was incubated at 45-80°C to separate the oligonucleotide from the solid phase support and free the oligonucleotide. The crude oligonucleotide was then precipitated with ethanol, and the supernatant was discarded by high-speed centrifugation. This process was repeated twice to obtain the crude oligonucleotide, and the precipitate was resuspended in DEPC water. The crude oligonucleotide was purified by ion-pairing HPLC, and the collected product was dried to a powder in a vacuum centrifuge. The purified product was dissolved in DEPC water and analyzed by time-of-flight liquid chromatography-mass spectrometry (TOF-LC-MS).

[0223] After obtaining the purified peptide-sense strand conjugate, the nucleotide concentration was determined by dissolving it in DEPC-treated water. The volumes required for equimolar amounts of the sense and antisense strands were calculated. The equimolar amounts of the peptide, sense, and antisense strands were then mixed and heated at 75°C for 5 minutes, followed by annealing with natural cooling to room temperature to produce peptide-siRNA duplexes. After cooling to room temperature, the annealed product was diluted with 10 volumes of DEPC-treated water and transferred to a Millipore 15 mL ultrafiltration centrifuge tube (molecular cutoff 3 kDa) for ultrafiltration. This step removes residual chemicals (such as n-hexylamine) from the peptide and nucleic acid preparation process. The ultrafiltration tube was centrifuged at 4000 RPM for approximately 40 minutes. Once the product volume remained at approximately 1 mL, the product was collected and washed with 3 mL of DEPC-treated water. The ultrafiltration product was sterilized by filtration through a 0.22 μm filter membrane. The collected product was then dried to a powder in a vacuum centrifuge. After dissolution with the appropriate solvent, it could be used for subsequent in vitro and in vivo experiments.

[0224] Example 14 Synthesis of polypeptide oligonucleotide conjugate Ang2(K10N3)-12006.2-2

[0225] 1. Using conventional nucleotide monomers and the special monomer B04, a standard solid-phase oligonucleotide procedure was performed according to the method of Example 13 to synthesize and purify the oligonucleotide sense strand 12006SM2-B04 (33 mg, 4.56 μmol) containing a B04 modification at the 3' end (molecular weight: 7230.8, measured: 7230.8).

[0226] 2. Synthesis of siRNA-BCN: 12006SM2-B04 (33 mg, 4.56 μmol) was dissolved in 30 mL of phosphate buffer (0.1 M, pH 7.4), and BCN-NHS (93 mg, 320 μmol) was dissolved in 15 mL of DMF. The BCN-NHS solution in DMF and the 12006SM2-B04 solution in phosphate buffer were mixed and shaken at 40°C for 30 min. After the reaction was complete as monitored by LC-MS, 4.5 mL of 3 M NaCl solution and 150 mL of pre-cooled ethanol were added to the reaction solution, which was thoroughly shaken and centrifuged for 3 min (4°C, 15,000 rpm). The supernatant was discarded, and the precipitate was dissolved in approximately 3 mL of DEPC water. The precipitate was purified by HPLC and lyophilized to obtain a white powder solid 12006SM2-B04-BCN (23.4 mg, 3.16 μmol, yield: 69.3%) (molecular weight: 7407.02, measured: 7408.14).

[0227] 3. 12006SM2-B04-BCN (5 mg, 0.85 μmol) was dissolved in 2.5 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2(K10N3) (2.5 mg, 1.0 μmol) was dissolved in 1 mL of DMF. The phosphate buffer of 12006SM2-B04-BCN and the DMF solution of Ang2(K10N3) were mixed and shaken for 2 hours under N2 protection. After the reaction was complete as monitored by LC-MS, 0.3 mL of 3 M NaCl solution and 10 mL of pre-cooled ethanol were added to the reaction solution. After thorough shaking, the solution was centrifuged for 3 min (4 ° C, 15000 rpm), the supernatant was discarded, and about 2 mL of The precipitate was dissolved in DEPC water, purified by HPLC, and freeze-dried to obtain a white powder solid Ang2(K10N3)-B04-12006SM2 (3.5 mg, 0.35 μmol, yield: 41%) (molecular weight: 9889.73, measured: 9890.58).

[0228] 4. Mix Ang2(K10N3)-B04-12006SM2 and 12006AM2, and anneal according to the method in Example 13 to obtain Ang2(K10N3)-12006.2-2.

[0229] Example 15 Synthesis of polypeptide oligonucleotide conjugate Ang2(K10N3)-12006.1-1

[0230] 1. Using conventional nucleotide monomers and the special monomer B12, a standard solid-phase oligonucleotide procedure was performed according to the method in Example 13 to synthesize and purify the oligonucleotide sense strand 12006SM1-B12 (molecular weight: 7388.78, measured: 7387.59) containing a terminal alkyne group at the 3' end.

[0231] 2. 12006SM1-B12 (10 mg, 1.35 μmol) was dissolved in 2.5 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2(K10N3) (5 mg, 2.0 μmol) was dissolved in 1 mL of DMF. The phosphate buffer of 12006SM1-B12 and the DMF solution of Ang2(K10N3) were mixed. Under N2 protection, CuSO4 pentahydrate (6.8 mg, 27 μmol), tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA, 5.9 mg, 13.5 μmol), and L-ascorbic acid sodium salt (NaVc, 10.3 mg, 54 μmol) were added. The reaction was carried out at 40°C for 1 hour. After the reaction was completed by LC-MS monitoring, 0.3 mL of 3M NaCl solution, 10 mL of pre-cooled ethanol, shake thoroughly and then centrifuge for 3 minutes (4°C, 15000 rpm), discard the supernatant, add about 2 mL of DEPC water to dissolve the precipitate, purify by HPLC, and lyophilize to obtain a white powder solid Ang2(K10N3)-B12-12006SM1 (4.6 mg, 0.47 μmol, yield: 35%) (molecular weight: 9868.88, measured: 9869.42).

[0232] 3. Mix Ang2(K10N3)-B12-12006SM1 and 12006AM1, and anneal according to the method in Example 13 to obtain Ang2(K10N3)-12006.1-1.

[0233] Example 16 Synthesis of BiAng2(K10N3)-12006.1-1

[0234] 1. Using conventional nucleotide monomers and the special monomer B08, a standard solid-phase oligonucleotide procedure was performed according to the method in Example 13 to synthesize and purify the oligonucleotide sense strand 12006SM1-B08 (molecular weight: 7279.63, measured: 7279.64) containing an amino group at the 3' end.

[0235] 2. 12006SM1-B08 (160 mg, 22 μmol) was dissolved in 20 mL of phosphate buffer (0.1 M, pH 7.4), and BPL-2 (569 mg, 660 μmol) was dissolved in 10 mL of DMF. The DMF solution of BPL-2 and the phosphate buffer of 12006SM1-B08 were mixed and shaken at 40°C for 60 min. After completion of the reaction as monitored by LC-MS, 3 mL of 3 M NaCl solution and 90 mL of pre-chilled ethanol were added to the reaction solution. The mixture was shaken thoroughly and centrifuged for 3 min (4°C, 15,000 rpm). The supernatant was discarded, and the precipitate was dissolved in approximately 5 mL of DEPC water. The precipitate was purified by HPLC and lyophilized to obtain 12006SM1-B08-BPL-2 (120 mg, 14.9 μmol, yield: 68%) as a white powder (molecular weight: 8028.56, measured: 8027.78).

[0236] 3. 12006SM1-B08-BPL-2 (20 mg, 2.5 μmol) was dissolved in 10 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2(K10N3) (30 mg, 12 μmol) was dissolved in 10 mL of DMF. The DMF solution of Ang2(K10N3) and the phosphate buffer of 12006SM1-B08-BPL-2 were mixed. Under N2 protection, CuSO4 pentahydrate (12.5 mg, 50 μmol), tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA, 10.9 mg, 25 μmol), and L-ascorbic acid sodium salt (NaVc, 19.1 mg, 100 μmol) were added. The reaction was carried out at 40°C for 2 hours. After the reaction was completed by LC-MS monitoring, 0.3 mL of 3M NaCl solution, 90 mL of pre-cooled ethanol, shake thoroughly and then centrifuge for 3 min (4°C, 15000 rpm), discard the supernatant, add about 2 mL of DEPC water to dissolve the precipitate, purify by HPLC, and lyophilize to obtain a white powder solid BiAng2(K10N3)-B08-12006SM1 (9.4 mg, 0.72 μmol, yield: 29%) (molecular weight: 12993.25, measured: 12992.92).

[0237] 4. Mix BiAng2(K10N3)-B08-12006SM1 and 12006AM1, and anneal according to the method in Example 13 to obtain BiAng2(K10N3)-12006.1-1.

[0238] Example 17 Synthesis of BiAng2-12006.2-2

[0239] 1. Dissolve 12006SM2-B04 (9 mg, 1.24 μmol) in 4 mL of 0.1 M phosphate buffer (pH 7.4) and BPL-3 (61 mg, 70 μmol) in 4 mL of DMF. Mix the BPL-3 / 12006SM2-B04 ...

[0240] 2. 12006SM2-B04-BPL-3 (3.9 mg, 0.48 μmol) was dissolved in 2.5 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2-AZK (4.7 mg, 1.91 μmol) was dissolved in 1.5 mL of DMF. The phosphate buffer of 12006SM2-B04-BPL-3 and the DMF solution of Ang2-AZK were mixed and shaken for 2 hours under N2 protection. After the reaction was complete as monitored by LC-MS, 0.7 mL of 3 M NaCl solution and 20 mL of pre-cooled ethanol were added to the reaction solution. After thorough shaking, the mixture was centrifuged for 3 min (4 ° C, 15000 rpm), the supernatant was discarded, and about 2 mL of The precipitate was dissolved in DEPC water, purified by HPLC, and freeze-dried to obtain a white powder solid BiAng2-B04-12006SM2 (3.1 mg, 0.24 μmol, yield: 47.9%) (molecular weight: 13027.26, measured: 13027.09).

[0241] 3. Mix BiAng2-B04-12006SM2 and 12006AM2, and anneal according to the method in Example 13 to obtain BiAng2-12006.2-2.

[0242] Example 18 Synthesis of BiAng2-2-12006.2-2

[0243] 1. Synthesis of siRNA-BCN: 12006SM2-B04 (9 mg, 1.24 μmol) was dissolved in 5 mL of phosphate buffer (0.1 M, pH 7.4), and BPL-1 (61 mg, 70 μmol) was dissolved in 5 mL of DMF. The DMF solution of BPL-1 and the phosphate buffer of 12006SM2-B04 were mixed and shaken at room temperature for 60 min. After completion of the reaction as monitored by LC-MS, 1 mL of 3 M NaCl solution and 30 mL of pre-chilled ethanol were added to the reaction mixture. The mixture was shaken thoroughly and centrifuged for 3 min (4°C, 15,000 rpm). The supernatant was discarded, and the precipitate was dissolved in approximately 3 mL of DEPC water. The mixture was purified by HPLC and lyophilized to obtain 12006SM2-B04-BPL-1 (6.8 mg, 0.87 μmol, yield: 70.2%) as a white powder (molecular weight: 7826.58, measured: 7826.49).

[0244] 2. Click chemistry reaction without Cu(I) catalysis: 12006SM2-B04-BPL-1 (6.8 mg, 0.87 μmol) was dissolved in 3.5 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2-AZK (8.5 mg, 3.5 μmol) was dissolved in 2 mL of DMF. The phosphate buffer of 12006SM2-B04-BPL-1 and the DMF solution of Ang2-AZK were mixed and reacted at room temperature under N2 protection for 2 hours. After the reaction was completed as monitored by LC-MS, 0.5 mL of 3 M NaCl solution and 15 mL of pre-cooled ethanol were added to the reaction solution. After thorough shaking, the solution was centrifuged for 3 minutes (4°C, 15000 rpm). The supernatant was discarded and about 2 mL of ethanol was added. The precipitate was dissolved in DEPC water, purified by HPLC, and freeze-dried to obtain a white powder solid BiAng2-2-B04-12006SM2 (3.8 mg, 0.31 μmol, yield: 35.6%) (molecular weight: 12735.94, measured: 12736.97).

[0245] 3. Mix BiAng2-2-B04-12006SM2 and 12006AM2, and anneal according to the method in Example 13 to obtain BiAng2-2-12006.2-2.

[0246] Example 19 Synthesis of BiAng2(K10N3)-Q22-12006.3-5

[0247] 1. Synthesis of 12006SM5-Q22: Using the solid support Q22-CPG, a standard solid-phase oligonucleotide procedure was performed according to the method of Example 13 to obtain the sense oligonucleotide 12006SM5-Q22 (molecular weight: 8276.07, measured: 8275.66) modified with a diacetylene group at the 3' end (molecular weight: 8276.07).

[0248] 2. Synthesis of BiAng2(K10N3)-Q22-12006SM5: 12006SM5-Q22 (25.5 mg, 3.08 μM) was dissolved in 3 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2(K10N3) (30.6 mg, 12.3 μmol) was dissolved in 3 mL of DMF. The phosphate buffer of 12006SM5-Q22 and the DMF solution of Ang2(K10N3) were mixed. Under N2 protection, CuSO4 pentahydrate (23.1 mg, 92.4 μmol), tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA, 20.1 mg, 46.2 μmol) and L-ascorbic acid sodium salt (NaVc, 35.3 mg, 184.8 μmol) were added. The reaction was carried out at 40°C for 1 hour. After the reaction was completed as monitored by LC-MS, 0.6 mL of 3M NaCl solution, 20 mL of pre-cooled ethanol, shake thoroughly and then centrifuge for 3 min (4°C, 15000 rpm), discard the supernatant, add about 2 mL of DEPC water to dissolve the precipitate, purify by HPLC, and lyophilize to obtain a white solid powder BiAng2(K10N3)-Q22-12006SM5 (18.3 mg, 1.38 μmol, yield: 44.8%) (molecular weight: 13241.48, measured: 13241.31).

[0249] 4. Mix BiAng2(K10N3)-Q22-12006SM5 and 12006AM3, and anneal according to the method in Example 13 to obtain BiAng2(K10N3)-Q22-12006.3-5.

[0250] Example 20 Synthesis of the dual-target compound BiAng2(K10N3)-12006-8022

[0251] 1. Synthesis of 12006SM2-DT: Using the solid-phase support DT-Linker CPG, standard solid-phase oligonucleotide synthesis and purification were performed according to the method in Example 13 to obtain the oligonucleotide sense chain 12006SM2-DT (molecular weight: 7588.29, measured: 7587.44), which was modified with both an azide and a primary amine reactive group at the 3' end.

[0252] 2. Synthesis of 8022SM2-B12: The oligonucleotide sense strand 8022SM2-B12 (molecular weight: 7445.06, measured: 7444.33) modified with a single alkynyl group at the 3' end was synthesized according to the method described in Example 15.

[0253] 3. Synthesis of 12006SM2-8022SM2-DT: 12006SM2-DT (20 mg, 2.64 μM, 1 eq.) and 8022SM2-B12 (20.6 mg, 2.77 μM, 1.05 eq.) were dissolved in 2 mL of phosphate buffer (0.1 M, pH 7.4). Under N2 protection, a DMF solution (2 mL) of CuSO4 pentahydrate (13.2 mg, 52.8 μmol, 20 eq.) and tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA, 11.5 mg, 26.4 μmol, 10 eq.) and sodium L-ascorbic acid (NaVc, 20.2 mg, 105.6 μmol, 40 eq.) were added. The reaction was continued at 40°C for 1 hour. After the reaction was completed as monitored by LC-MS, 0.4 mL of 3 M NaCl solution, 15 mL of pre-cooled ethanol, thoroughly shaken and centrifuged for 3 min (4°C, 15000 rpm), the supernatant discarded, and about 2 mL of DEPC water was added to dissolve the precipitate. Ultrafiltration was performed to obtain 40 mg of 12006SM2-8022SM2-DT crude product (molecular weight: 15033.38, measured: 15033.49).

[0254] 4. The above crude 12006SM2-8022SM2-DT was dissolved in 5 mL of phosphate buffer (0.1 M, pH 7.4), and then a DMF solution (5 mL) of BPL-2 (92 mg, 105.6 μM) was added. The reaction was carried out at 40°C for 1 hour. After the reaction was completed by LC-MS monitoring, ultrafiltration was performed to obtain 32 mg of crude 12006SM2-8022SM2-DT-BPL-2 (molecular weight: 15779.31, measured: 15781.37).

[0255] 5. The above 12006SM2-8022SM2-DT-BPL-2 crude product (10 mg) was dissolved in 5 mL of phosphate buffer (0.1 M, pH 7.4), and Ang2(K10N3) (7.8 mg, 3.14 μmol) was dissolved in 3 mL of DMF. The above liquids were mixed, and under N2 protection, a DMF solution (2 mL) of CuSO4 pentahydrate (3.2 mg, 12.6 μmol) and tris-(3-hydroxypropyltriazolylmethyl)amine (THPTA, 2.7 mg, 6.3 μmol) and L-ascorbic acid sodium salt (NaVc, 4.8 mg, 25.2 μmol) were added. The reaction was carried out at 40 ° C for 1 hour. After the reaction was completed by LC-MS monitoring, 1 mL of 3M NaCl solution and 30 mL of pre-cooled ethanol were added to the reaction solution. After sufficient shaking, the mixture was centrifuged for 3 min (4 ° C, 15000 rpm), the supernatant was discarded, and 2 mL of 4% ethanol was added. The precipitate was dissolved in DEPC water, purified by HPLC, and freeze-dried to obtain a white powder solid BiAng2(K10N3)-12006SM2-8022SM2 (5.2 mg, 0.27 μmol) (molecular weight: 20744.72, measured: 20745.9).

[0256] 6. Mix BiAng2(K10N3)-12006SM2-8022SM2, 12006AM2 and 8022AM4, and anneal according to the method in Example 13 to obtain BiAng2(K10N3)-12006-8022.

[0257] The structures and sequences of the polypeptide oligonucleotide conjugates and C16 modified nucleotides synthesized by the above method are shown in Tables 2-1 and 2-2 below.

[0258] Table 2-1

[0259] The unmodified sequences corresponding to the modified nucleotide sequences in Table 2-1 are shown in Table 2-2 below.

[0260] Table 2-2 Unmodified nucleotide sequences

[0261] The specific structure of the polypeptide oligonucleotide conjugate is as follows:

[0262] The molecular weight and purity of the positive chain of the polypeptide oligonucleotide conjugate and C16-modified nucleotide synthesized by the present invention are shown in Table 3 below.

[0263] Table 3

[0264] Example 21 Effect of the peptide attachment site on the in vivo activity of peptide-siRNA by intrathecal injection in wild-type mice

[0265] 1. Drug Preparation

[0266] After freeze-drying to obtain APP-targeting polypeptide-siRNA double-stranded powder (polypeptide oligonucleotide conjugate), artificial cerebrospinal fluid (CSF) was used as a solvent in a sterile environment to prepare a polypeptide-siRNA drug with a stock concentration of 20 mg / mL.

[0267] 2. Experimental Grouping and Medication

[0268] Four-week-old female C57 wild-type mice of similar weight were selected as experimental subjects and randomly divided into groups of six mice per group. After anesthesia, the mice were injected intrathecally with the peptide-siRNA drug at a dose of 10 mg / kg (approximately 0.25 mg per mouse). On the day of drug injection, the mice were observed after they emerged from anesthesia, and their weights were recorded daily.

[0269] 3. Sample collection and testing

[0270] On the 10th day of administration, the mice were killed and samples were collected from the cerebral cortex, brainstem, hypothalamus, hippocampus, and cervical spinal cord. The samples were stored in RNA Later to prevent RNA degradation. RNA was then extracted from each brain tissue using TRI REAGENT (MRC, Catalog No.: TR118), and the extracted RNA was reverse transcribed into cDNA using PrimeScript RT regent Kit (Takara, Catalog No.: RR047A). The prepared QPCR system was added to a 96-well PCR plate, sealed with a plate film, and QPCR was performed on the StepOnePlus real-time PCR system (applied biosystems) to detect the expression level of mAPP (mouse APP). The content of the antisense chain targeting mAPP in brain tissue was detected by liquid chromatography-mass spectrometry.

[0271] On day 10 of drug administration, mice were sacrificed and samples were collected for analysis of mAPP expression and content. The results are shown in Table 4. At a dose of 10 mg / kg, the in vivo activity of APP siRNAs delivered by different peptides varied. C16(7-U)-12006.2-2 (with a hexadecyl fatty chain modification at position 7 of the sense strand) served as a positive control, while 12006.2-2 was a bare-strand sequence without peptide or C16 conjugation. Multiple peptide-siRNA sequences demonstrated significant in vivo degradation of the target gene in mice.

[0272] Table 4. Effects of different attachment sites on Ang2 on the activity of peptide-siRNA in mice

[0273] The results in Table 4 show that Ang2-12006.2-2 (Ang2 peptide linked to siRNA at its N-terminus) exhibited strong activity in various brain tissues, particularly in the cerebral cortex, where it exhibited greater activity than the positive control, C16(7-U)-12006.2-2. In contrast, the naked 12006.2-2 peptide exhibited significant activity only in the cervical spinal cord. This suggests that the Ang2 peptide can deliver mAPP-targeting siRNA to mouse brain tissue cells, achieving therapeutic efficacy, with superior delivery compared to C16. Further testing of the effect of the Ang2 linker on efficacy revealed that peptide-siRNA linked to mAPP siRNA at amino acid position 10 (Ang2(K10AZK)-12006.2-2) exhibited the highest activity. However, mAPP siRNA linked to the C-terminus of Ang2 (Reverse-Ang2-12006.2-2) exhibited poorer mAPP siRNA delivery than Ang2(K10AZK)-12006.2-2. This result shows that the way of connecting the 10th amino acid of Ang2 can more effectively deliver mAPP siRNA to the brain tissue cells of mice, so that the siRNA targeting mAPP can exert better efficacy.

[0274] Example 22: Effect of Linking Single or Double Peptides on the In Vivo Activity of Peptide-siRNA in Wild-Type Mice by Intrathecal Injection

[0275] This example tested the effects of Ang2 conjugation, tandem dipeptides, and parallel dipeptides on the in vivo activity of peptide-siRNA. Four-week-old female C57 wild-type mice were administered intrathecally according to the method of Example 21. The mice were sacrificed on day 11, and mAPP expression was measured in various brain tissues. The results are shown in Table 5.

[0276] Table 5. Effects of different forms of single and double peptides on the activity of peptide-siRNA in mice

[0277] The results in Table 5 show that each peptide-siRNA exhibited good activity in the brainstem and cervical spinal cord. In the cerebral cortex, compared with mAPP siRNA linked to the N-terminus of Ang2 and mAPP siRNA linked to the C-terminus of L57, linking mAPP siRNA, tandem dipeptides (Ang2-Ang2), or parallel dipeptides (BiAng2) to lysine 10 of Ang2 (Ang2(K10N3)) significantly improved the activity of the peptide-siRNA, with the BiAng2-12006.2-2 group showing the best activity. This indicates that linking a dipeptide to siRNA can improve its delivery efficiency, and further demonstrates that the site of attachment to the peptide has a significant effect on the activity of the peptide-siRNA. It was also found that the activity of BiAng2-2-12006.2-2 was poorer than that of other peptide-siRNAs, indicating that the structure of the linker portion also has a significant effect on the activity of the peptide-siRNA.

[0278] Example 23: Testing the Effect of the Linking Site of the Dipeptide on the In Vivo Activity of Peptide-siRNA by Injection into the Cisterna magna in Wild-Type Mice

[0279] This example tested the effects of different dipeptide linkages on peptide-siRNA activity. Aside from the different administration methods, the experimental procedures were the same as in Example 21. In this example, drug was administered via cisterna magna injection, with each mouse receiving a dose of 0.25 mg. After injection, the mice were sacrificed on day 17, and various brain tissues were collected to measure mAPP expression. The results are shown in Table 6.

[0280] Table 6. Effects of different linkage modes of dipeptides on the activity of peptide-siRNA in mice

[0281] The results in Table 6 show that the parallel Ang2 showed good drug delivery effects in various brain tissues, among which the siRNA linked to the 10th lysine of Ang2 (BiAng2(K10N3)-12006.1-1) had higher activity than the siRNA linked from the N-terminus.

[0282] Example 24: Effect of single or double peptide conjugation on the in vivo activity of polypeptide-siRNA in wild-type rats by cisterna magna injection

[0283] This example tested the in vivo activity of the polypeptide-siRNA of the present invention in rats. Aside from the different administration methods, the experimental methods were the same as in Example 21. In this example, the drug was injected into the rats via cisterna magna injection, with each rat receiving a 1 mg dose. Rats were sacrificed on days 21 and 42, and different brain tissues were collected to measure rAPP (rat APP) expression. The results are shown in Tables 7 and 8.

[0284] Table 7. Effects of different forms of Ang2 on the activity of peptide-siRNA in rats for 21 days

[0285] The results in Table 7 indicate that at 21 days, the activity of the single-peptide Ang2(K10N3)-12006.1-1 was significantly higher than that of the dual-peptide BiAng2-12006.1-1 linked to the N-terminus of Ang2, further demonstrating that siRNA linked to lysine 10 of Ang2 has higher activity. Furthermore, the activity of the dual-Ang2(K10N3) peptide-siRNA (BiAng2(K10N3)-12006.1-1) was further enhanced compared to the single-Ang2(K10N3) peptide-siRNA (Ang2(K10N3)-12006.1-1), further demonstrating that linking a dual-peptide to siRNA can enhance its delivery efficiency.

[0286] Table 8. Effects of different forms of Ang2 on the activity of peptide-siRNA in rats for 42 days

[0287] The results in Table 8 show that BiAng2(K10N3)-12006.1-1 and BiAng2-12006.1-1 maintained extremely high target inhibitory activity at 42 days, demonstrating that siRNA delivered using Ang2 peptides can achieve very long-lasting efficacy. Furthermore, the dual-Ang2(K10N3) peptide-siRNA (BiAng2(K10N3)-12006.1-1) exhibited significantly better activity retention at 42 days than the dual-peptide BiAng2-12006.1-1.

[0288] Example 25 Testing the in vivo activity of peptide-siRNA in wild-type mice by intrathecal injection

[0289] This example compares the in vivo activity of the polypeptide-siRNA (BiAng2(K10N3)-12006.1-1) of the present invention with that of the hexadecyl chain-modified oligonucleotide C16(6-G)-12006.1-1 in mice (C16(6-G)-12006.1-1 has a hexadecyl fatty chain modification on the 6th nucleotide residue of the sense chain. Hexadecyl modification of the 6th nucleotide residue is an effective strategy to improve the efficiency of nucleic acid delivery). Naked-chain siRNA not coupled to a polypeptide or C16 was used as a control. In this example, the drug was injected into mice by intrathecal injection. The injection dose for each mouse was 0.25 mg. Other experimental methods were the same as in Example 21. The mice were sacrificed on the 14th and 28th days, and different brain tissues were taken to detect the expression of mAPP. The results are shown in Table 9

[0290] Table 9. Comparison of the activity of peptide-siRNA and C16-siRNA in mice

[0291] The results in Table 9 show that at 14 days, the activity of the dual-peptide BiAng2(K10N3)-12006.1-1 was comparable to that of C16(6-G)-12006.1-1 delivered by C16, and at 28 days, BiAng2(K10N3)-12006.1-1 was even higher. This suggests that siRNA delivered by the peptide Ang2 is more long-lasting and specific than siRNA delivered by C16.

[0292] Example 26 Testing the in vivo activity of low-dose peptide-siRNA by injecting it into the cisterna magna in wild-type rats

[0293] This example compares the in vivo activity of the polypeptide-siRNA (BiAng2(K10N3)-12006.1-1) of the present invention with that of the hexadecyl chain-modified oligonucleotide C16(6-G)-12006.1-1 in rats (C16(6-G)-12006.1-1 has a hexadecyl fatty chain modification on the 6th nucleotide residue of the sense chain. Hexadecyl modification of the 6th nucleotide residue is an effective strategy to improve the efficiency of nucleic acid delivery). Naked-chain siRNA not coupled to a polypeptide or C16 was used as a control. In this example, the drug was injected into rats by cisterna magna injection. The injection dose for each rat was reduced to 0.3 mg. Other experimental methods were the same as in Example 21. The rats were sacrificed on the 14th day and different brain tissues were taken to detect the expression level of rAPP. The results are shown in Table 10.

[0294] Table 10. Comparison of the activity of low-dose peptide-siRNA and C16-siRNA in rats for 14 days

[0295] The results in Table 10 show that under low-dose conditions, the activity of BiAng2(K10N3)-12006.1-1 in the form of a dipeptide was significantly improved at 14 days compared to C16(6-G)-12006.1-1 delivered by C16. This indicates that the siRNA delivered by the polypeptide Ang2 has more significant specificity and efficiency than the siRNA delivered by C16, and low-dose injection can also reduce toxic side effects. Therefore, the polypeptide Ang2 of the present invention is an ideal carrier for delivering siRNA to the central nervous system.

[0296] Example 27 Testing the in vivo activity of symmetric or asymmetric peptide-siRNA by injection into the cisterna magna of wild-type rats

[0297] This example compares the in vivo activity of the polypeptide-siRNA of the present invention (BiAng2(K10N3)-12006.1-1) with two polypeptide-siRNAs containing a symmetrical dipeptide linker structure (Bi-sym-Ang2(K10N3)-12006.1-1 and Bi-sym-Ang2(K10Peg2N3)-12006.1-1) in rats. In this example, the drug was injected into the rats via the cisterna magna injection, with each rat receiving an injection dose of 0.3 mg. Other experimental methods were the same as in Example 21. The rats were sacrificed on day 26, and different brain tissues were collected to detect rAPP expression. The results are shown in Table 11.

[0298] Table 11. Effects of different dipeptides on the 26-day activity of APP-targeted peptide-siRNA in rats

[0299] The results in Table 11 show that BiAng2(K10N3)-12006.1-1 and Bi-sym-Ang2(K10N3)-12006.1-1 have comparable activities, indicating that the symmetry of the bi-peptide linker structure has no significant effect on activity. Bi-sym-Ang2(K10N3)-12006.1-1 has superior activity to Bi-sym-Ang2(K10Peg2N3)-12006.1-1, indicating that the length of the linker between the two peptides has a significant effect on activity.

[0300] Example 28: Testing the in vivo activity of peptide-siRNA with different conjugation patterns in wild-type mice by injection into the cisterna magna

[0301] In this example, the activity of BiANG2(S7KN3)-12006.3-1 (Ang2 peptide with serine 7 replaced with lysine, then bivalently linked to siRNA targeting APP) and BiANG2(K10N3)-12006.3-1 were compared. In this example, the drug was injected into mice via cisterna magna injection, with the dose reduced to 0.1 mg per mouse. Other experimental procedures were the same as in Example 21. On day 28, the mice were sacrificed, and different brain tissues were collected to measure mAPP expression. The results are shown in Table 12.

[0302] Table 12. Effects of different peptide forms on the 28-day activity of APP-targeted peptide-siRNA in mice

[0303] The results in Table 12 show that compared with the amino acid at position 7, the lysine at position 10 in the Ang2 polypeptide has a better ability to deliver siRNA when used as the siRNA attachment site.

[0304] Example 29: Testing the in vivo activity of different peptide-siRNA forms by injecting them into the cisterna magna in wild-type rats

[0305] This example compares the siRNA delivery capabilities of the LRP-1 ligands Ang1 and Ang2. The corresponding peptide-siRNA conjugates, BiAng2(K10N3)-12006.1-1 and BiAng1(K10N3)-12006.1-1, both utilize lysine 10 as the siRNA attachment site and possess a bi-peptide structure. In this example, rats were injected into the cisterna magna with a dose of 0.3 mg per rat. Other experimental procedures were the same as in Example 21. Rats were sacrificed on day 28, and rAPP expression was measured in various brain tissues. The results are shown in Table 13.

[0306] Table 13. Effects of different peptide forms on the 28-day activity of APP-targeted peptide-siRNA in rats

[0307] The results in Table 13 show that after 28 days of administration, the delivery efficiency of Ang2 polypeptide was slightly better than that of Ang1.

[0308] Example 30: Testing the in vivo activity of peptide-siRNA containing different linkers by injection into the cisterna magna of wild-type mice

[0309] This example compared the in vivo activity of a peptide-siRNA (BiAng2(K10N3)-12006.1-1) from the present invention with that of a peptide-siRNA (BiAng2(K10N3)-Q22-12006.3-5) using a Q22 linker in mice. In this example, the drugs were injected into the mice via cisterna magna injection, with the dose reduced to 0.1 mg per mouse. Other experimental procedures were the same as in Example 21. On day 14, the mice were sacrificed, and different brain tissues were collected to measure mAPP expression. The results are shown in Table 14.

[0310] Table 14. Comparison of the effects of different linkers on the 14-day activity of APP-targeted peptide-siRNA in mice

[0311] The results in Table 14 show that the activity of BiAng2(K10N3)-Q22-12006.3-5 was higher than that of BiAng2(K10N3)-12006.3-5 at 14 days, indicating that the structure of the linker part also has a significant effect on the activity of polypeptide-siRNA.

[0312] Example 31 Comparison of the effects of different injection methods on the in vivo activity of polypeptide-siRNA in wild-type mice

[0313] This example compares the in vivo activity of the polypeptide-siRNA (BiAng2(K10N3)-12006.3-5) of the present invention with that of the hexadecyl chain-modified oligonucleotide C16(6-G)-12006.3-5 in mice (C16(6-G)-12006.3-5 has a hexadecyl fatty chain modification at the 6th nucleotide residue of the sense strand. Hexadecyl modification of the 6th nucleotide residue is an effective strategy for improving the efficiency of nucleic acid delivery). This example uses different injection methods, namely intrathecal injection (IT) and cisterna magna injection (ICM), to inject the drug into mice. The injection dose for each mouse is 0.25 mg. Other experimental methods are the same as in Example 21. On the 7th day, the mice were sacrificed and different brain tissues were taken to detect the expression level of mAPP. The results are shown in Table 15.

[0314] Table 15. Comparison of the effects of different injection methods on the 7-day activity of APP-targeted peptide-siRNA in mice

[0315] The results in Table 15 show that, at the same drug dose, ICM injection resulted in higher efficacy than IT injection. At 7 days, the activity of the bi-peptide BiAng2(K10N3)-12006.3-5 in various mouse brain regions was significantly enhanced compared to C16(6-G)-12006.3-5 delivered via C16. This demonstrates that siRNA delivery using this peptide exhibits superior delivery efficiency compared to C16.

[0316] Example 32: Injection of SNCA-targeted polypeptide-siRNA into wild-type mice to test its in vivo activity

[0317] In this example, AD1747580 (WO2023192977A2), a known siRNA targeting α-synuclein (SNCA) for the treatment of Parkinson's disease, was linked to the dual-ANG2 polypeptide of the present invention. The activity of this peptide-siRNA was tested in mice. This example used cisterna magna injection to deliver the peptide-siRNA to mice, with a dose of 0.25 mg per mouse. Other experimental procedures were the same as in Example 21. On day 14, the mice were sacrificed, and different brain tissues were collected to measure mSNCA (mouse SNCA) expression. The results are shown in Table 16.

[0318] Table 16. In vivo activity of peptide-siRNA targeting SNCA

[0319] The results in Table 16 show that at a dose of 0.25 mg, BiAng2(K10N3)-Q22-AD1747580 exhibited significant inhibitory effects on target genes in various brain regions, indicating that the polypeptide of the present invention can knock down different central nervous system targets.

[0320] Example 33: Testing the in vivo activity of a peptide-siRNA containing two genes simultaneously targeted by injection into the cisterna magna of wild-type mice

[0321] This example evaluates the dual-target peptide-siRNA conjugate BiAng2(K10N3)-12006-8022 in mice. This conjugate utilizes the bivalent Ang2(K10N3) structure to simultaneously deliver siRNA targeting the amyloid precursor protein APP gene and siRNA targeting the melanocortin receptor 4 (MC4R). BiAng2(K10N3)-12006.2-2 is a single-target peptide-siRNA control targeting APP. In this example, the drug was injected into the mice via the medullary cisterna magna injection, with an injection dose of 15 nmol per mouse. Other experimental methods were the same as in Example 21. On day 21, the mice were sacrificed and different brain tissues were taken to detect the expression levels of mAPP and mMC4R (mouse MC4R). The results are shown in Table 17.

[0322] Table 17. Activity results of peptide-siRNA targeting both APP and MC4R in mice for 21 days

[0323] The results in Table 17 indicate that both the single-target peptide-siRNA targeting mAPP (BiAng2(K10N3)-12006.2-2) and the dual-peptide dual-target peptide-siRNA targeting both mAPP and mMC4R (BiAng2(K10N3)-12006-8022) had significant inhibitory effects on mAPP at 21 days. Furthermore, the dual-peptide dual-target peptide-siRNA targeting both mAPP and mMC4R (BiAng2(K10N3)-12006-8022) also had an inhibitory effect on mMC4R, demonstrating that the peptide-siRNA of the present invention can be used for dual-gene siRNA delivery.

[0324] Example 34 Safety Testing of Peptide-siRNA in Wild-Type Mice

[0325] The results of the above examples suggest that the dipeptide BiAng2(K10N3)-Q22-12006.3-5 exhibits extremely high inhibitory activity against the target gene APP in various brain tissues of both mice and adults. In this example, 0.7 mg of BiAng2(K10N3)-Q22-12006.3-5 peptide-siRNA was injected into the brains of wild-type C57 / Bl6 mice via cisterna magna injection. Each treatment consisted of six mice, with mice receiving artificial cerebrospinal fluid injection serving as a negative control. 24 and 48 hours after cisterna magna injection, the mice's functional observational composite (FOB) scores were assessed according to seven different criteria to evaluate the safety of the peptide-siRNA. The criteria for FOB were (1) the mouse was intelligent, alert, and responsive; (2) the mouse stood or arched its back without stimulation; (3) the mouse showed any movement without stimulation; (4) the mouse exhibited forward movement after being lifted; (5) the mouse showed any movement after being lifted; (6) the mouse responded to a tail pinch; and (7) the mouse breathed evenly. For each of the seven criteria, the mouse was given a score of 0 if it met the criteria and 1 if it did not. After evaluating all seven criteria, the scores for each mouse were summed and averaged within each treatment group. The results are presented in Table 18.

[0326] Table 18. FOB scores of mice after injection of peptide-siRNA

[0327] The results in Table 18 show that after high-dose peptide-siRNA was injected into the mouse brain via the cisterna magna, mild functional abnormalities appeared at 0.5 hours. Over time, the mouse behavior returned to normal, and after 24 hours, the mouse FOB score was 0. This indicates that the peptide-siRNA was well tolerated in mice.

[0328] Table 19. Blood biochemical test results of mice after injection of peptide-siRNA

[0329] The results in Table 19 show that 15 days after drug injection, the blood of the mice was collected for biochemical testing to detect liver damage indicators (ALT, AST, ALP) and kidney damage indicators (CREA and UREA). The results showed that BiAng2(K10N3)-Q22-12006.3-5 did not cause liver and kidney damage, indicating that the polypeptide-siRNA in the present invention has good safety.

[0330] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A polypeptide oligonucleotide conjugate having a structure represented by the following formula (I) or (II) or a pharmaceutically acceptable salt or stereoisomer thereof: in, It is an oligonucleic acid drug; P is a polypeptide ligand for the LRP-1 receptor, selected from an unmodified or modified polypeptide; LE is a group used to link to an oligonucleic acid drug, which contains a hydroxyl group or a protected hydroxyl group; Each LA is independently selected from the following structures: LB is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: alkylene, cycloalkyl, heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-; LC is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: alkylene, cycloalkyl, heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-; R1 is selected from: H, C1-C 10 alkyl; R2 is selected from: C1-C 10 alkyl; Each n is independently selected from: 1, 2, 3; a and b are each independently 0 or 1, and at least one of them is 1; m is selected from: an integer between 0 and 12; Each n1 is independently selected from the group consisting of: 0, 1, 2, and 3.

2. The polypeptide-oligonucleotide conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The unmodified amino acid sequence of the polypeptide ligand is TFFYGGCRGKRNNFKTEEY, TFFYGGSRGKRNNFKTEEY, TWPKHFDKHTFYSILKLGKH, TFFYGGKRGKRNNFKTEEY or TFFYGGSRGZRNNFKTEEY, wherein Z represents L-norleucine.

3. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 2, characterized in that: The polypeptide ligand has one or more of the following structural changes: (1) L-amino acid residues in the amino acid sequence are replaced by D-amino acid residues; (2) amino acid residues in the amino acid sequence are replaced by methylated amino acid residues; (3) the presence or absence of acylation modification of the N-terminal amino group in the amino acid sequence; (4) the presence or absence of amidation modification of the C-terminal carboxyl group in the amino acid sequence; (4) Any aromatic amino acid residue in the amino acid sequence can be replaced by a non-aromatic amino acid residue; (5) Any two amino acid sequences are linked by a linker, wherein the N-terminus of one amino acid sequence is linked to the C-terminus of the other amino acid sequence by a linker; The linking group is preferably:

4. The polypeptide-oligonucleotide conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The LA in P-LA is linked to the N-terminus of the polypeptide ligand; Alternatively, LA in P-LA is linked to the 7th, 10th or 15th amino acid residue of the polypeptide ligand from the N-terminus, preferably to the 10th amino acid residue.

5. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 1, characterized in that: The LA in P-LA is linked to the C-terminus of the polypeptide ligand.

6. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 1, characterized in that: The LA in P-LA is linked to an amino acid residue containing a side chain amino group in the polypeptide ligand.

7. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 1, characterized in that: The LA in P-LA is linked to a lysine residue or a norleucine residue in the polypeptide ligand.

8. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 1, characterized in that: The unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGKRNNFKTEEY, and the LA in P-LA is connected to the 10th or 15th amino acid residue in the polypeptide ligand from the N-terminus; Alternatively, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGKRGKRNNFKTEEY, and LA in P-LA is linked to the 7th, 10th, or 15th amino acid residue in the polypeptide ligand from the N-terminus; Alternatively, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGCRGKRNNFKTEEY, and the LA in P-LA is linked to the 10th amino acid residue from the N-terminus of the polypeptide ligand; Alternatively, the unmodified amino acid sequence of the polypeptide ligand is TFFYGGSRGZRNNFKTEEY, and the LA in P-LA is connected to the 10th amino acid residue from the N-terminus of the polypeptide ligand.

9. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 1, characterized in that: The LA in P-LA is linked to the amino acid residue containing a side chain carboxyl group in the polypeptide ligand.

10. The polypeptide-oligonucleotide conjugate according to claim 9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The LA in P-LA is linked to a glutamic acid residue or an aspartic acid residue in the polypeptide ligand.

11. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LE is selected from the following structures: Each R is independently selected from: H or a hydroxyl protecting group; Each n2 is independently selected from an integer between 0-10.

12. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 11, characterized in that: LE is selected from:

13. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LA is selected from the following structures:

14. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The LB in formula (I) is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C 10 Alkylene, C5-C 10 Cycloalkyl, 5-10 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C6 alkyl; Preferably, LB in formula (I) is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C6 alkylene, C5-C7 cycloalkyl, 5-7 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C3 alkyl; Preferably, LB in formula (I) is selected from the following structures: wherein each i, j, h, q, s, and e are independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; c is selected from: 0, 1; X is selected from the group consisting of: O, S, CH2; Y is absent or selected from: C=O, CH2.

15. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or a stereoisomer thereof according to claim 14, characterized in that: LB in formula (I) is selected from the following structures: Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4; c is selected from: 0, 1; k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; X is selected from: CH2, O.

16. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 15, characterized in that: LB in formula (I) is selected from the following structures:

17. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LB in formula (II) is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C 10 Alkylene, C5-C 10 Cycloalkyl, 5-10 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C6 alkyl; Preferably, LB in formula (II) is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C6 alkylene, C5-C7 cycloalkyl, 5-7 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C3 alkyl; Preferably, LB in formula (II) has the structure shown below: Wherein, X is selected from: O, S, CH2; L is selected from the following structures: Each i, j, h, q, s, f, and e are independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; c is selected from: 0, 1; X is selected from the group consisting of: O, S, CH2; Y is absent or selected from: C=O, CH2.

18. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 17, characterized in that: LB in formula (II) has the structure shown below: X is selected from: O, CH2.

19. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 17, characterized in that: L is selected from the following structures: Each i and e are independently selected from: 0, 1, 2, 3, 4; k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; X is selected from: CH2, O.

20. The polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 19, characterized in that: L is selected from the following structures:

21. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LC in formula (II) is a connecting unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C 10 Alkylene, C5-C 10 Cycloalkyl, 5-10 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C6 alkyl; Preferably, LC in formula (II) is a linking unit formed by connecting one or more of the following groups without violating the principle of chemical bonding: C1-C6 alkylene, C5-C7 cycloalkyl, 5-7 membered heterocyclyl, -C(=O)NR1-, -C(=O)O-, -C(=O)S-, -C(=O)-, -C(=S)-, -NR1-, -O-, -S-, wherein R1 is selected from: H, C1-C3 alkyl; Preferably, LC in formula (II) has the structure shown below: wherein each i, j, h, and e are independently selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; c is selected from: 0, 1; X is selected from the group consisting of: O, S, CH2; Y is absent or selected from: C=O, CH2.

22. The polypeptide-oligonucleotide conjugate according to claim 21, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LC in formula (II) is selected from the following structures: Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4; c is selected from: 0, 1; X is selected from: CH2, O.

23. The polypeptide oligonucleotide conjugate according to claim 22, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: LC in formula (II) is selected from the following structures:

24. The polypeptide-oligonucleotide conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The polypeptide oligonucleotide conjugate is selected from the following compounds: Each i, j, and e are independently selected from: 0, 1, 2, 3, and 4; c is selected from: 0, 1; k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; X is selected from: CH2, O; Each n1 is independently selected from the group consisting of: 0, 1, 2, and 3.

25. The polypeptide-oligonucleotide conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The polypeptide oligonucleotide conjugate is selected from the following compounds:

26. The polypeptide-oligonucleotide conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The polypeptide oligonucleotide conjugate is selected from the following compounds:

27. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The oligonucleic acid drug is selected from the group consisting of small interfering RNA, antisense oligonucleotide, small activating RNA and peptide nucleic acid.

28. The polypeptide-oligonucleotide conjugate according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The oligonucleic acid drug is a small interfering RNA, and the LE is connected to the phosphate group or the thiophosphate group at the 3' and / or 5' end of the sense strand of the small interfering RNA.

29. A polypeptide-oligonucleotide conjugate or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The polypeptide-oligonucleotide conjugate is prepared by the following method: a linker-nucleic acid sense chain molecule containing an alkyne group is subjected to a click chemistry reaction with an azide-modified polypeptide to obtain a polypeptide-sense chain conjugate, and the polypeptide-sense chain conjugate is then annealed with a corresponding nucleic acid antisense chain to obtain the polypeptide-oligonucleotide conjugate; the polypeptide is a polypeptide ligand for the LRP-1 receptor; The alkyne-containing linker-nucleic acid sense chain molecule has a structure as shown in the following formula (III) or formula (IV): in, It is the positive chain in oligonucleic acid drugs; LA, LB, LC, LE, n, a, b are as described in any one of claims 1-26; R3 is selected from: ethynyl, 30. The polypeptide-oligonucleotide conjugate according to claim 29, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The alkyne-containing linker-nucleic acid molecule sense chain is selected from the following structures: wherein each i, j, and e are independently selected from: 0, 1, 2, 3, and 4; k is selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; c selection: 0, 1; X is selected from: CH2, O.

31. The polypeptide oligonucleotide conjugate according to claim 30, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The alkyne-containing linker-nucleic acid molecule sense chain is selected from the following structures:

32. The polypeptide-oligonucleotide conjugate according to claim 29, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The azide-modified polypeptide is selected from the following structures:

33. The polypeptide oligonucleotide conjugate according to claim 32, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The azide-modified polypeptide is selected from the following structures:

34. The polypeptide-oligonucleotide conjugate according to claim 29, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: The azide-modified polypeptide is selected from the following structures:

35. Use of the polypeptide oligonucleotide conjugate according to any one of claims 1 to 34, or a pharmaceutically acceptable salt or stereoisomer thereof, as an active ingredient in the preparation of a drug for inhibiting the expression of a target gene in the central nervous system.

36. The use according to claim 35, characterized in that The central nervous system target gene is selected from one or more of APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and NLRP3.

37. Use of the polypeptide-oligonucleotide conjugate according to any one of claims 1 to 34, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof as an active ingredient in the preparation of a medicament for treating diseases related to the central nervous system.

38. The use according to claim 37, characterized in that The diseases associated with the central nervous system are Alzheimer's disease, cachexia, cerebral amyloid angiopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, spinocerebellar disorders, prion diseases and Lafora disease.

39. A drug for treating diseases related to the central nervous system, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient comprises the polypeptide oligonucleotide conjugate according to any one of claims 1 to 34, or a pharmaceutically acceptable salt or a stereoisomer thereof.

40. A method for inhibiting the expression of a target gene in the central nervous system, characterized in that: include: Administering a safe and effective amount of the polypeptide oligonucleotide conjugate according to any one of claims 1 to 34, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; Or administer a safe and effective amount of the drug according to claim 39.

41. The method according to claim 40, wherein The central nervous system target gene is selected from one or more of APP, MC4R, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK and NLRP3.

42. A method for treating diseases related to the central nervous system, characterized in that: include: Administering a safe and effective amount of the polypeptide oligonucleotide conjugate according to any one of claims 1 to 34, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof; Or administer a safe and effective amount of the drug according to claim 39.

43. The method according to claim 42, characterized in that The diseases associated with the central nervous system are Alzheimer's disease, cachexia, cerebral amyloid angiopathy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia, spinocerebellar disorders, prion diseases and Lafora disease.