Cyclic peptide ligand of targeted asialoglycoprotein receptor, pharmaceutically acceptable salt of cyclic peptide ligand and application and pharmaceutical composition of cyclic peptide ligand

Abstract

The invention relates to the technical field of liver targeting compounds, in particular to a cyclic peptide ligand of a targeting asialoglycoprotein receptor, pharmaceutically acceptable salt of the cyclic peptide ligand, application of the pharmaceutically acceptable salt and a pharmaceutical composition. The invention provides a cyclic peptide ligand of a targeted asialoglycoprotein receptor, a pharmaceutically acceptable salt of the cyclic peptide ligand, an application of the pharmaceutically acceptable salt and a pharmaceutical composition. The cyclic peptide ligand has better endocytosis activity.

Description

Technical Field

[0001] This invention relates to the field of liver-targeting compound technology, and more particularly to a cyclic peptide ligand targeting the desialylate glycoprotein receptor, its pharmaceutically acceptable salt, its application, and pharmaceutical compositions thereof. Background Technology

[0002] The liver is the largest metabolic organ in the human body, participating in the synthesis and metabolism of various chemical substances. Hepatic-targeted drug delivery is a particularly attractive strategy for treating metabolic diseases, cardiovascular diseases, and other liver conditions. The desialylate glycoprotein receptor (ASGPR), highly expressed specifically in the liver, is a well-defined lysosomal-targeting receptor responsible for clearing glycoproteins via clathrin-mediated endocytosis and lysosomal degradation. Its unique expression pattern, specific binding ligand N-acetylgalactosamine (GalNAc), and rapid recycling rate make ASGPR a liver-specific protein degrader and one of the most efficient ligand molecules in liver-targeted drug delivery technologies.

[0003] Tri-GalNAc glycoligands targeting the ASGPR receptor are among the most important lysosome-mediated endocytosis targeting molecules, playing a central role in lysosome-targeted chimeric (LYTAC) design and drug delivery. GalNAc-induced ASGPR receptors also provide an ideal pathway for targeted delivery of drug molecules or imaging reagents to liver tissue. Other studies have shown that LYTAC molecules designed based on ASGPR ligands are a hot topic in the research of extracellular protein targeted degraders, exhibiting unique biological activity and potential druggability. Currently, the Lytac drugs BHV1300 and BHV1400, designed based on tri-GalNac ligands, have both completed Phase I clinical trials. Although the clinical results are encouraging, there is still significant room for improvement in the degradation efficiency of target proteins, and many clinical needs remain unmet. Therefore, developing novel ASGPR ligand molecules with better endocytosis activity, improving the delivery efficiency of small nucleic acid drugs, and enhancing the degradation efficiency of pathological extracellular proteins by novel LYTAC drugs are currently critical technologies that urgently need to be addressed. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a cyclic peptide ligand targeting the desialylate glycoprotein receptor, a pharmaceutically acceptable salt thereof, its application, and pharmaceutical compositions thereof. The cyclic peptide ligand exhibits improved endocytic activity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a cyclic peptide ligand targeting the desialylate glycoprotein receptor and a pharmaceutically acceptable salt thereof, the cyclic peptide ligand having the structure shown in Formula I: Formula I; In Equation I, -S-R0-S- is -SS- or ; R1, R2, R3, R4, R5, R6, R7, and R8 are independently glycine, L-arginine, L-alanine, L-leucine, L-phenylalanine, L-proline, L-aspartic acid, L-lysine, L-glutamine, L-asparagine, L-histidine, L-glutamic acid, L-isoleucine, L-valine, L-methionine, L-tryptophan, L-serine, L-threonine, L-tyrosine, or R-cysteine; a, b, c, d, e, f, g, and h are independent integers between 0 and 3; R 13 -NH2, -OH, -NHR 14 or -OR 14 The -NHR 14 and -OR 14 Chinese R 14 Independently, it is -(CH2). y CH3 or -(CH2) y COOH; the -(CH2)yCH3 or -(CH2) y In COOH, y is an independent integer between 6 and 20; L1 is –(NH–(CH2) n –CO– or –(NH–(CH2CH2O) m –CO) p –, wherein the broken bond at the -NH- position in L1 is connected to the carbonyl group in Formula I, and the broken bond at the -CO- position in L1 is connected to the imino group in Formula I; in L1, n is an integer between 1 and 8, m is an integer between 1 and 8, and p is an integer between 0 and 3; L2 is -(CH2) z -, where z is an integer between 0 and 12; L3 is -CO-(CH2) q -NH-, -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- or -CO-(CH2CH2O) t -NH-CO-(CH2) q -NH-, wherein the broken bond at the -CO- position in L3 is connected to the imino group in Formula I; the broken bond at the -NH- position in L3 is connected to X in Formula I; the -CO-(CH2) q -NH-, -CO-(CH2) q-NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q In -NH-, q is an independent integer between 6 and 18; -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q In -NH-, t is an independent integer between 1 and 8, and -(CO-(CH2CH2O) t -NH) w In the -, w is a positive integer from 1 to 6; X is an isotope chelating agent, oligonucleotide, extracellular protein binder, or fluorescent dye.

[0006] Preferably, the isotope chelating agent includes , or .

[0007] The cyclic peptide ligand and its pharmaceutically acceptable salt described in this invention can target ASGPR via GalNAc-modified cyclic peptide compounds, thereby endocytosis into the lysosomal pathway. When X is an extracellular protein binder, the protein degrader can form a complex between the extracellular protein target and ASGPR, entering the lysosome and achieving targeted protein degradation. Furthermore, the cyclic peptide ligand and its pharmaceutically acceptable salt described in this invention exhibit comparable or significantly enhanced endocytosis and protein degradation activity compared to the targeting agent L96 used in already marketed RNAi drugs. Since the endocytic activity of such molecules is directly related to their drug delivery and extracellular protein degradation effects, the GalNAc cyclic peptide ligand in this invention can provide a new and superior technology for drug delivery of small nucleic acids and the development of novel LYTAC drugs for degrading pathological extracellular proteins, and has broad application prospects. Attached Figure Description

[0008] Figure 1 The LC-MS spectrum of IPM-2-2S-biotin described in Example 1; Figure 2 The LC-MS spectrum of IPM-1-2S-3PEG3-biotin described in Example 3; Figure 3 The LC-MS spectrum of IPM-2-2Br-biotin described in Example 5; Figure 4 The LC-MS spectrum of IPM-4-2S-biotin described in Example 8; Figure 5 The LC-MS spectrum of IPM-4-2Br-biotin described in Example 9; Figure 6 The LC-MS spectrum of IPM-5-2S-biotin described in Example 10; Figure 7 The LC-MS spectrum of IPM-5-2Br-biotin described in Example 11; Figure 8 The LC-MS spectrum of IPM-6-2S-biotin described in Example 12; Figure 9 The LC-MS spectrum of IPM-6-2Br-biotin described in Example 13; Figure 10 The LC-MS spectrum of IPM-7-2S-biotin described in Example 14; Figure 11 The LC-MS spectrum of IPM-8-2Br-biotin described in Example 17; Figure 12 The LC-MS spectrum of DOTA-IPM-1-2S-3PEG3 described in Example 24; Figure 13 The results of the cytotoxicity of IPM-4-2Br-biotin at different concentrations on 293T cells described in Example 9; Figure 14 The results of CD24 degradation by the product (IPM-1-4S-CD24-LYTAC) described in Example 22; Figure 15 PET imaging of the product (DOTA-IPM-1-2S) described in Example 23 in healthy mice. Detailed Implementation

[0009] This invention provides a cyclic peptide ligand targeting the desialylate glycoprotein receptor and a pharmaceutically acceptable salt thereof, the cyclic peptide ligand having the structure shown in Formula I: Formula I; In Equation I, -S-R0-S- is -SS- or ; R1, R2, R3, R4, R5, R6, R7, and R8 are independently glycine, L-arginine, L-alanine, L-leucine, L-phenylalanine, L-proline, L-aspartic acid, L-lysine, L-glutamine, L-asparagine, L-histidine, L-glutamic acid, L-isoleucine, L-valine, L-methionine, L-tryptophan, L-serine, L-threonine, L-tyrosine, or R-cysteine; a, b, c, d, e, f, g, and h are independent integers between 0 and 3; R 13 -NH2, -OH, -NHR 14 or -OR 14 The -NHR 14 and -OR 14 Chinese R 14 Independently, it is -(CH2). y CH3 or -(CH2) y COOH; the -(CH2)yCH3 or -(CH2) y In COOH, y is an independent integer between 6 and 20; L1 is –(NH–(CH2) n –CO– or –(NH–(CH2CH2O) m –CO) p –, wherein the broken bond at the -NH- position in L1 is connected to the carbonyl group in Formula I, and the broken bond at the -CO- position in L1 is connected to the imino group in Formula I; in L1, n is an integer between 1 and 8, m is an integer between 1 and 8, and p is an integer between 0 and 3; L2 is -(CH2) z -, where z is an integer between 0 and 12; L3 is -CO-(CH2) q -NH-, -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- or -CO-(CH2CH2O) t -NH-CO-(CH2) q -NH-, wherein the broken bond at the -CO- position in L3 is connected to the imino group in Formula I; the broken bond at the -NH- position in L3 is connected to X in Formula I; the -CO-(CH2) q -NH-, -CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O)t -NH-CO-(CH2) q In -NH-, q is an independent integer between 6 and 18; -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q In -NH-, t is an independent integer between 1 and 8, and -(CO-(CH2CH2O) t -NH) w In the -, w is a positive integer from 1 to 6; X is an isotope chelating agent, oligonucleotide, extracellular protein binder, or fluorescent dye.

[0010] In this invention, the values ​​of a, b, c, d, e, f, g, and h are preferably 0, 1, 2, or 3.

[0011] In this invention, n in L1 is preferably 1, 2, 3, 4, 5, 6, 7 or 8; m in L1 is preferably 1, 2, 3, 4, 5, 6, 7 or 8; and p in L1 is preferably 0, 1, 2 or 3.

[0012] In this invention, z in L2 is preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0013] In this invention, the -CO-(CH2) q -NH-, -CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q The q in -NH- is preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; the -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q The t in -NH- is preferably 1, 2, 3, 4, 5, 6, 7, or 8, and the -(CO-(CH2CH2O) t -NH)w The preferred values ​​for w in - are 1, 2, 3, 4, 5, or 6.

[0014] In this invention, -(CH2)yCH3 or -(CH2) y The preferred values ​​for y in COOH are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0015] In this invention, when X is an isotope chelating agent, the isotope chelating agent preferably includes... , or .

[0016] In this invention, when X is an oligonucleotide, the oligonucleotide is collectively referred to as an RNAi agent (or RNAi drug), preferably including antisense oligonucleotides (ASO), short hairpin RNA (shRNA), micro-interfering RNA (miRNA), small interfering RNA (siRNA), or DNA / RNA hybrid molecules composed of a sense strand and DNA.

[0017] In this invention, each nucleotide of the sense and antisense strands of the oligonucleotide portion of the RNAi agent is modified. The modified nucleotides are preferably 2'-deoxy-thymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleic acids (LNA), open-ring nucleic acids (UNA), bridged nucleic acids (BNA), ethylene glycol nucleic acids (GNA), aspartate nucleic acids (TNA), configuration-restricted nucleotides, restricted ethyl nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, baseless nucleotides, inverted baseless nucleotides, inverted nucleotides, morpholino nucleotides, aminophosphates, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, or nucleotides containing 5'-phosphate mimics. In this invention, the preferred modification methods for the modified nucleotides are STC modification technology (replacing all 2'-OH with 2'-F and 2'-Ome, Alnylam, USA), ESC modification technology (increasing the 2'-Ome content and adding two thiophosphates at the 5' end of the Passenger, Alnylam, USA), and ESC+ (adding ethylene glycol nucleic acid to the siRNA fragment, Alnylam, USA).

[0018] In this invention, the oligonucleotide portion of the RNAi agent preferably includes a nucleotide overhang at one end of the duplex RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and antisense strand are completely base-paired at the molecule's ends, and no unpaired nucleotides extend beyond the double-stranded region. In this invention, the oligonucleotide portion of the RNAi agent preferably includes a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In this invention, the oligonucleotide portion of the RNAi agent preferably includes a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand; in this invention, the oligonucleotide portion of the RNAi agent preferably includes (i) a sense strand of 19 nucleotides in length and (ii) an antisense strand of 21 nucleotides in length, the two strands forming a double-stranded region of equal length to the sense strand. In this invention, the oligonucleotide portion of the RNAi agent preferably comprises (i) a sense strand of 21 nucleotides in length and (ii) an antisense strand of 23 nucleotides in length, the two strands forming a double-stranded region with a length equal to that of the sense strand.

[0019] In this invention, the RNAi agent refers to a reagent containing an RNA molecule that, upon introduction into a cell, can downregulate the expression of a target gene by interfering with the matrix via RNA interference, including modified or unmodified oligonucleotides linked to a target moiety (e.g., L96 or the target compound involved in this invention). RNAi (RNA interference) is the process by which nucleic acid molecules induce the cleavage and degradation of target RNA molecules (such as mRNA molecules) in a sequence-specific manner (e.g., via the RNA-induced silencing complex (RISC) pathway). RNAi agents herein include ASO, miRNA, siRNA, shRNA, and related DNA / RNA hybrid molecules consisting of a sense strand and DNA, sometimes collectively referred to herein as double-stranded RNA (dsRNA), which comprises two antiparallel consecutive nucleotide chains that are sufficiently complementary to each other to hybridize and form a double-stranded region. "Hybridization" refers to the pairing of complementary polynucleotides, typically through hydrogen bonds between complementary bases in the two polynucleotides (e.g., Watson-Crick bonds, Wobble bonds, Hoogsteen bonds, or reverse Hoogsteen bonds). "Double-stranded region" refers to a region in two complementary or substantially complementary polynucleotides that hybridize to form a base pair, thereby forming a double strand between the two polynucleotide chains; "siRNA" refers to a nucleic acid that forms a double-stranded RNA. When siRNA and a target gene are present in the same cell, the double-stranded RNA has the ability to reduce or inhibit the expression of the target gene. siRNA is typically about 15 to 30 base pairs in length, most commonly about 19 to 25 base pairs, for example, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs in length. Antisense oligonucleotides (ASOs) are single-stranded DNA or RNA complementary to a selected sequence. In the case of antisense DNA, they can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, this hybrid can be degraded by nuclease H1 (RNAase H1). A typical example of an ASO is a "gapmer," which has an internal "gap segment" flanked by two external "wing segments." The gap segment consists of multiple nucleotides that support nuclease H1 cleavage, and each wing segment consists of one or more nucleotides that are chemically different from those within the gap segment. For example, the 5' and 3' wings of a gapmer consist of nucleotides modified with 2'-oxy-methoxyethyl (2'-MOE), the gap segment consists of deoxyribonucleotides, and optionally, the linkages between all nucleotides are phosphate thioester bonds.

[0020] In this invention, when X is an extracellular protein binder, the extracellular protein binder preferably includes a membrane protein binder, a secretory protein binder, or a nucleic acid drug; the membrane protein binder is preferably a PD-L1 antibody or a small molecule and peptide inhibitor, preferably atezolizumab, durvalumab, or tagolizumab; a phosphatidylinositol proteoglycan-3 (GPC3) antibody or a small molecule and peptide inhibitor, preferably codrituzumab; a carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) antibody, preferably besilesomab or sacituzumab; a CD3 monoclonal antibody, preferably catutxomab; an epithelial cell adhesion molecule antibody; an epidermal growth factor (EGFR) antibody, preferably cetuximab; or a vascular endothelial growth factor antibody. (VEGF) antibody, preferably bevacizumab; epidermal growth factor receptor-2 (Her2) antibody, preferably trastuzumab; the binding agent of the secreted protein is preferably an IgE antibody, preferably omalizumab; the IgG ligand is preferably a disulfide cyclic peptide DCAWHLGELVWCT (seq1) or PPAWHLGELVW (seq2); the IgA antibody is preferably a disulfide cyclic peptide HMVCLSYRGRPVCFSL (seq3); VDNKFNKETIQASQEIRLLPNLNGRQKLAFIHSLLDDPSQSANLLAEAKKLNDAQAPKVPS-(Thr-Alpha-GalNAc)-PP-(Thr-Alpha-GalNAc)-P-(Ser-Alpha-GalNAc)-P-(Ser- Alpha-GalNAc)-(Thr-Alpha-GalNAc)-PP-(Thr-Alpha-GalNAc)-PSPS (NephrologyDialysis Transplantation, 2012, 30, 1315-1321), Gly-Gly-PS-(Thr-Alpha-GalNAc)-PP (Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 2018 37:6, 252-256); the TNF-α antibody is preferably adalimumab; the PCSK9 antibody is preferably tafolecimab, recaticimab, or alirocumab, more preferably evolocumab.The IL-6 antibody is tocilizumab; preferably, alirocumab; the nucleic acid drug is preferably Givlaari, inclisiran, Oxlumo, leqvio, or Amvuttra, more preferably Givlaari, inclisiran, or Oxlumo.

[0021] In this invention, when X is a fluorescent dye, the fluorescent dye preferably includes 5FAM, FITC, Cy3, Cy5, Cy5.5 or Cy7.

[0022] In this invention, the cyclic peptide ligand preferably has the structure shown in any one of Formula I-1 to Formula I-4: Formula I-1 Formula I-2 Formula I-3 or Formula I-4; In Formulas I-1 to I-4, X is an isotope chelating agent, oligonucleotide, or extracellular protein binder.

[0023] In this invention, the cyclic peptide ligand is preferably: Formula 1 (IPM-1-2S-biotin) Formula 2 (IPM-1-2Br-biotin) Formula 3 Formula 4 (IPM-1-2S-3PEG3-biotin), Formula 5 (IPM-2-2S-biotin) Formula 6 (IPM-2-2Br-biotin) Formula 7 (IPM-3-2S-biotin) Formula 8 (IPM-3-2Br-biotin) Formula 9 (IPM-4-2S-biotin) Formula 10 (IPM-4-2Br-biotin) Formula 11 (IPM-5-2S-biotin) Formula 12 (IPM-5-2Br-biotin) Formula 13 (IPM-6-2S-biotin) Formula 14 (IPM-6-2Br-biotin) Formula 15 (IPM-7-2S-biotin) Formula 16 (IPM-7-2Br-biotin) Formula 17 (IPM-8-2S-biotin) Formula 18 (IPM-8-2Br-biotin) Formula 19 (IPM-9-2S-biotin) Formula 20 (IPM-9-2Br-biotin) Formula 21 (IPM-10-2S-biotin) Formula 22 (IPM-10-2Br-biotin) Formula 23 (IPM-1-4S-CD24-LYTAC) or Formula 24 (DOTA-IPM-1-2S).

[0024] In this invention, the method for preparing the cyclic peptide ligand preferably includes the following steps: A condensation reaction is performed between a compound having the structure shown in formula a and a compound having the structure shown in formula b to obtain a compound having the structure shown in formula c. After removing the Fmoc protection from the compound with the structure shown in formula c, a condensation reaction is carried out with the Fmoc-protected linker to obtain the compound with the structure shown in formula d. The compound having the structure shown in formula d was deprotected from Fmoc protection and then condensed with biotin to obtain the compound having the structure shown in formula e. The compound having the structure shown in formula e was cut from the resin and simultaneously subjected to a Trt protecting group removal reaction to obtain the compound having the structure shown in formula f. The compound having the structure shown in formula f was subjected to a cyclization reaction in the presence of elemental iodine to obtain the compound having the structure shown in formula g. The compound having the structure shown in formula g was subjected to an Ac protecting group removal reaction under MeONa basic conditions to obtain the structure shown in formula I.

[0025] Formula a, Formula b, Equation c, Formula d, Equation e, Formula f, Formula g.

[0026] The present invention also provides the application of the cyclic peptide ligands and their pharmaceutically acceptable salts described in the above-described technical solutions in the preparation of RNAi drugs or in LYTAC protein degrading agents for degrading extracellular proteins.

[0027] In this invention, the RNAi drug preferably includes the LYTAC protein degrader.

[0028] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a cyclic peptide ligand and a pharmaceutically acceptable salt thereof; The cyclic peptide ligand and its pharmaceutically acceptable salt are the cyclic peptide ligand and its pharmaceutically acceptable salt described in the above technical solution.

[0029] In this invention, the RNAi drug is preferably used to treat primates, humans or other mammals, more preferably humans, horses, cattle, pigs, sheep, poultry, dogs or cats.

[0030] In this invention, pharmaceutically acceptable means compounds, materials, compositions, and / or dosage forms that are within the range of correct medical judgment, suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0031] In this invention, a pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition, or carrier that participates in carrying or transporting the cyclic peptide ligands described in this invention from one organ or site of the body to another organ or site of the body, such as liquid or solid fillers, diluents, excipients, electret or solvent encapsulation materials known in the art that can serve as carriers; each carrier must be "acceptable" in the sense of being compatible with the other components of the composition and harmless to the patient.

[0032] In this invention, the pharmaceutically acceptable salt refers to a salt prepared by reacting the cyclic peptide ligand of the invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the cyclic peptide ligand of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the cyclic peptide ligand of the present invention contains a relatively basic functional group, it is preferable to obtain an acid addition salt by contacting the neutral form of such a compound with a sufficient amount of pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acids preferably include inorganic acids and / or organic acids, wherein the inorganic acids preferably include one or more of hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, and sulfuric acid; and the organic acids preferably include one or more of acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentic acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)) and amino acids (e.g., glutamic acid, arginine). When the cyclic peptide ligands of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into basic addition salts or acid addition salts.

[0033] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Preparation of Fmoc-GalNAc-1: ; Preparation process: Compound Fmoc-Lys(Boc)-OH (10.0 g, 21.4 mmol, 1.0 eq) was dissolved in DMF (200 mL), and K2CO3 (8.8 g, 64.2 mmol, 3.0 eq) was added. Benzyl bromide (4.5 mL, 32.0 mmol) was added under ice bath conditions. After 10 min, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 2 h. The reaction progress was monitored by TLC until the reaction was complete. 1 M hydrochloric acid aqueous solution was added to the reaction solution, and the product was extracted using DCM. The obtained organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator to obtain the crude product of compound 1, which was directly used in the next reaction. Compound 1 (~20 mmol) was dissolved in 50 mL of a 1:1 mixture of TFA (trifluoroacetic acid) and DCM, and reacted at room temperature for 2 h. DCM and TFA were removed by rotary evaporation to obtain the crude product of compound 2. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain compound 2 (white solid, 8.0 g, yield 81.6%). Compound 2 was dissolved using DCM, and then N-acetylgalactosamine ligand (10.7 g, 24.4 mmol), HATU (10.1 g, 24.4 mmol, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and DIPEA (9.1 mL, 52.4 mmol, diisopropylethylamine) were added. After reacting at room temperature for 2 h, 1 mol / L hydrochloric acid aqueous solution was added to the reaction solution to adjust the pH to 6. The product was extracted using DCM, the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and compound 3 (white powder solid, 13.3 g, yield 85.7%) was purified by column chromatography using silica gel (DCM / MeOH system). 13.3 g (15.0 mmol) of compound 3 was dissolved in 400 mL of methanol, and 3.0 g of palladium on carbon catalyst (palladium mass percentage 10%) was added. The mixture was then purged with a hydrogen balloon to create a hydrogen atmosphere, and reacted at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, palladium on carbon was removed with diatomaceous earth, and the organic solution was concentrated by rotary evaporation. The crude product was purified by column chromatography with silica gel (DCM / MeOH system) to obtain Fmoc-GalNAc-1 (white solid, 12.0 g, yield 93.3%). Preparation of Fmoc-GalNAc-2: ; The preparation of compound 2 is the same as that of compound 2 in the preparation of Fmoc-GalNAc-1, and will not be repeated here. Compound 2 was dissolved using DCM, and then 4-[(tert-butylcarbonyl)amino]butyric acid (4.9 g, 24.4 mmol), HATU (10.1 g, 24.4 mmol, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and DIPEA (9.1 mL, 52.4 mmol, diisopropylethylamine) were added. After reacting at room temperature for 2 h, 1 mol / L hydrochloric acid aqueous solution was added to the reaction solution to adjust the pH to 6. The product was extracted using DCM, the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator to obtain the crude product of compound 4, which was directly used in the next reaction. The crude product of compound 4 (~20 mmol) was dissolved in 50 mL of a 1:1 mixture of TFA (trifluoroacetic acid) and DCM, and reacted at room temperature for 2 h. DCM and TFA were removed by rotary evaporation to obtain the crude product of compound 5. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain compound 5 (white solid, 9.2 g, yield 85.0%). Compound 5 was dissolved using DCM, and then N-acetylgalactosamine ligand (10.4 g, 23.8 mmol), HATU (9.9 g, 23.8 mmol, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and DIPEA (8.9 mL, 51.2 mmol, diisopropylethylamine) were added. After reacting at room temperature for 2 h, 1 mol / L hydrochloric acid aqueous solution was added to the reaction solution to adjust the pH to 6. The product was extracted using DCM, the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain compound 6 (white powder solid, 13.6 g, yield 82.4%). 13.6 g (14.0 mmol) of compound 6 was dissolved in 400 mL of methanol, and 3.2 g of palladium catalyst on carbon (palladium mass percentage 10%) was added. The mixture was then purged with a hydrogen balloon to create a hydrogen atmosphere, and reacted at room temperature for 2 h. The reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, palladium on carbon was removed with diatomaceous earth, and the organic solution was concentrated by rotary evaporation. The crude product was purified by column chromatography with silica gel (DCM / MeOH system) to obtain Fmoc-GalNAc-2 (white solid, 10.9 g, yield 88.6%).

[0035] Example 1 IPM-1-2S-biotin: The preparation process is as follows: 120 mg of CTC resin (loading capacity 1.319 mmol / g) was placed in a peptide synthesis tube, and 4 mL of DMF was added to swell the resin for 30 min, followed by washing. 3.0 molar equivalents of Fmoc-Ala-OH and 6.0 molar equivalents of DIPEA (diisopropylethylamine) were dissolved in 4 mL of DCM. The resulting reaction solution was added to the peptide synthesis tube, and the reaction was allowed to proceed for 2 h to obtain intermediate compound 7. Step a: Add 4 mL of a 1:4 mixture of piperidine and DMF to intermediate compound 7. After reacting for 10 min, wash the resin three times each with alternating amounts of DCM and DMF. Step b: Dissolve 3.0 molar equivalents of Fmoc-Cys(Trt)-OH, 3.0 molar equivalents of HOBt (1-hydroxybenzotriazole), and 3.0 molar equivalents of DIC (N,N'-diisopropylcarbodiimide) in 4 mL of DCM. Shake for 10 min to activate the amino acids. Add the resulting reaction solution to the above-mentioned peptide synthesis tube and react for 2 h to obtain intermediate compound 8. Detect the reaction progress using the ninhydrin colorimetric method. By repeatedly performing steps a and b until all the required amino acids are coupled to the resin, compound 9 is obtained, wherein the condensation conditions of Fmoc-GalNAc-1 are the same as those of Fmoc-Ala-OH described above. Finally, the Fmoc protecting group at the N-terminus of the peptide in compound 9 is removed by a DMF solution of piperidine at a volume ratio of 1:4. The peptide is then lysed from the resin using 5 mL of a mixture of TFA, TIS, and H2O at a volume ratio of 95:2.5:2.5. After 2 h, the lysate is collected and TFA and DCM are removed by rotary evaporation. The lysate is then added dropwise to ice-cold diethyl ether, and the crude acetyl-protected peptide product (compound 10) is collected by centrifugation. The crude product of compound 10 was dried at room temperature until it became powder. It was dissolved in water and methanol in a volume ratio of 1:1. A methanol solution containing 0.1 mol / L iodine was added dropwise until the solution turned yellow. After stirring for 30 min, an aqueous solution containing 0.1 mol / L sodium thiosulfate was added dropwise to quench the reaction until the yellow color of the solution disappeared. Insoluble substances were removed using a filter. The filtrate was purified by HPLC and lyophilized to obtain compound 11. The crude acetyl-protected cyclic peptide was dissolved in methanol, and then 10 molar equivalents of sodium methoxide were added to remove the acetyl groups for 30 min. The reaction progress was monitored by LC-MS until the reaction was completed. The pH was adjusted to 7 by adding 1 M hydrochloric acid aqueous solution, and excess solvent was removed by rotary evaporation. The remaining substance was dissolved in a mixed solvent of water and methanol, and insoluble substances were removed by filter. The filtrate was purified by HPLC and lyophilized to obtain compound 12 (white powder product, denoted as 1-2S-NH2). 4 mL of a 1:4 DMF solution of piperidine was added to the peptide synthesis tube containing compound 9. After reacting for 10 min, the resin was washed three times each with alternating DCM and DMF. Then, 3.0 molar equivalents of Biotin-COOH, 6.0 eq of HOBt, and 6.0 eq of DIC were dissolved in 4 mL of DMF and shaken for 10 min to activate the carboxylic acid. After activation, the reaction solution was added to the peptide synthesis tube and reacted for 4 h to obtain intermediate compound 13. The reaction progress was detected using the ninhydrin colorimetric method. Add 4 mL of a 1:4 DMF solution of piperidine and react for 10 min. Wash the resin three times each with alternating DCM and DMF. Dissolve 3.0 molar equivalents of Biotin-COOH, 3.0 molar equivalents of HOBt, and 3.0 molar equivalents of DIC in 4 mL of DMF and shake for 10 min to activate the carboxylic acid. After activation, add the resulting reaction solution to a peptide synthesis tube and react for 2 h to obtain intermediate compound 14. The reaction progress is detected using the ninhydrin colorimetric method. Use 5 mL of a 95:2.5:2.5 mixture of TFA, TIS, and H2O to lyse the peptide from the resin. After 2 h, collect the lysate and remove TFA and DCM using a rotary evaporator. Then, add the lysate dropwise to ice-cold diethyl ether, centrifuge, and collect the acetyl-protected crude peptide product (compound 15). The crude product of compound 15 was dried at room temperature until it became powder. It was dissolved in water and methanol in a volume ratio of 1:1. A methanol solution containing 0.1 mol / L iodine was added dropwise until the solution turned yellow. After stirring for 30 min, an aqueous solution containing 0.1 mol / L sodium thiosulfate was added dropwise to quench the reaction until the yellow color of the solution disappeared. Insoluble substances were removed using a filter. The filtrate was purified by HPLC and lyophilized to obtain compound 16. The crude acetyl-protected cyclic peptide was dissolved in methanol, and 10 molar equivalents of sodium methoxide were added to remove the acetyl groups for 30 min. The reaction progress was monitored by LC-MS until the reaction was completed. 1 M hydrochloric acid aqueous solution was added to adjust the pH to 7, and excess solvent was removed by rotary evaporation. The remaining substances were dissolved in a mixed solvent of water and methanol, and insoluble substances were removed by filter. The filtrate was purified by HPLC and lyophilized to obtain IPM-1-2S-biotin. The detection results of the IPM-1-2S-biotin are as follows: LC-MS (ESI) calculated for C 91 H 156 N 16 O 32 S3(2082.51), 1042.26 [M+2H] 2+ , found 1041.78;940.72[M-203.08(GalNAc)+2H] 2+ , found 940.54.

[0036] Example 2 IPM-1-2Br-biotin: The preparation method is the same as in Example 1, except for the cyclization method: after obtaining the white powder compound 15, it was dissolved in a 1:1 volume ratio mixture of acetonitrile and ethanol, and 1.5 equivalents of 1,3,5-tribromomethylbenzene and 6 equivalents of DIPEA (N,N-diisopropylethylamine) were added. The mixture was stirred for 2 hours, and insoluble substances were removed using a filter. The resulting filtrate was purified by HPLC and lyophilized to obtain compound 15-2Br. ; The crude acetyl-protected cyclic peptide was dissolved in methanol, and 10 molar equivalents of sodium methoxide were added to remove the acetyl groups for 30 min. The reaction progress was monitored by LC-MS until the reaction was completed. 1 M hydrochloric acid aqueous solution was added to adjust the pH to 7, and excess solvent was removed by rotary evaporation. The remaining substances were dissolved in a mixed solvent of water and methanol, and insoluble substances were removed by filter. The filtrate was purified by HPLC and lyophilized to obtain IPM-1-2Br-biotin. The detection results of IPM-1-2Br-biotin are as follows: LC-MS (ESI) calculated for C 99 H 164 N 16 O 32 S3(2186.66), 1094.33 [M+2H] 2+ , found 1093.95.

[0037] Example 3 IPM-1-2S-3PEG3-biotin: The preparation method is the same as in Example 1, except that Fmoc-12-aminododecanoic acid in Example 1 is replaced with three Fmoc-NH-PEG3-CH2CH2COOH.

[0038] The detection results of IPM-1-2S-3PEG3-biotin are as follows: LC-MS (ESI) calculated for C 106 H 184 N 18 O 43 S3(2494.90), 1248.45 [M+2H] 2+ , found 1247.60; 854.62 [M+3Na] 3+ , found854.46 [M+3Na] 3+ .

[0039] Example 4 IPM-2-2S-biotin: The preparation method is the same as in Example 1, except that the 120 mg CTC resin in Example 1 is replaced with 120 mg Rink Amide MBHA resin (sample loading amount is 0.631 mmol / g), and Fmoc-Ala-OH is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0040] The detection results of IPM-2-2S-biotin (LC-MS spectrum as shown) Figure 1 As shown below, LC-MS (ESI) calculated for C 97 H 171 N 23 O 31 S3(2251.75), 1126.88 [M+2H] 2+ , found 1126.13;751.58[M+3H] 3+Found 751.35; 563.94 [M+4H] 4+ , found 563.87.

[0041] Example 5 IPM-2-2Br-biotin: The preparation method is the same as in Example 2, except that the 120 mg CTC resin in Example 2 is replaced with 120 mg Rink Amide MBHA resin (sample loading amount is 0.631 mmol / g), and Fmoc-Ala-OH is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0042] The detection results of IPM-2-2Br-biotin (LC-MS spectrum as shown) Figure 2 As shown below, LC-MS (ESI) calculated for C 105 H 179 N 23 O 31 S3(2355.90), 786.30 [M+3H] 3+ Found 785.94; 589.48[M+4H] 4+ , found 589.82.

[0043] Example 6 IPM-3-2S-biotin: The preparation method is the same as in Example 1, except that the 120 mg CTC resin in Example 1 is replaced with 120 mg Rink Amide MBHA resin (sample loading amount is 0.631 mmol / g), and Fmoc-Ala-OH is replaced with Fmoc-Pro-OH.

[0044] The detection results of the IPM-3-2S-biotin are as follows: LC-MS (ESI) calculated for C 95 H 161 N 17 O 31 S3(2133.60), 1067.80 [M+2H] 2+ , found 1067.04;712.20 [M+3H] 3+ , found711.97.

[0045] Example 7 IPM-3-2Br-biotin: The preparation method is the same as in Example 2, except that the 120 mg CTC resin in Example 2 is replaced with 120 mg Rink Amide MBHA resin (sample loading amount is 0.631 mmol / g), and Fmoc-Ala-OH is replaced with Fmoc-Pro-OH.

[0046] The detection results of IPM-3-2Br-biotin are as follows: LC-MS (ESI) calculated for C 103 H 169 N 17 O 31 S3(2237.75), 1119.88 [M+2H] 2+ , found 1119.06;746.92 [M+3H] 3+ , found746.83.

[0047] Example 8 IPM-4-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Arg(Pbf)-OH.

[0048] The detection results of IPM-4-2S-biotin (LC-MS spectrum as shown) Figure 3 As shown below, LC-MS (ESI) calculated for C 109 H 194 N 30 O 34 S3(2565.11), 1283.56[M+2H] 2+ Found 1283.40; 856.04 [M+3H] 3+ Found 855.89; 642.28[M+4H] 4+ , found 642.20; 788.34[M-203.08(GalNAc)+3H] 3+ , found 788.19.

[0049] Example 9 IPM-4-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Arg(Pbf)-OH.

[0050] The detection results of IPM-4-2Br-biotin (LC-MS spectrum as shown) Figure 4 As shown below, LC-MS (ESI) calculated for C 117 H 202 N 30 O 34 S3(2669.26), 890.75 [M+3H] 3+ Found 890.40; 668.34 [M+4H] 4+ , found 668.15; 534.85 [M+5H] 5+ , found 534.78.

[0051] Example 10 IPM-5-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0052] The detection results of IPM-5-2S-biotin (LC-MS spectrum as shown) Figure 5 As shown below, LC-MS (ESI) calculated for C 107 H 184 N 24 O 34 S3(2446.96), 816.65 [M+3H] 3+ Found 816.42; 612.74 [M+4H] 4+ , found 612.62.

[0053] Example 11 IPM-5-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0054] The detection results of IPM-5-2Br-biotin (LC-MS spectrum as shown) Figure 6 As shown below, LC-MS (ESI) calculated for C 115 H 192 N 24 O 34 S3(2551.11), 851.37 [M+3H] 3+ Found 850.96; 638.78 [M+4H] 4+, found 638.68; 783.68[M-203.08+3H] 3+ , found 783.23.

[0055] Example 12 IPM-6-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0056] The detection results of IPM-6-2S-biotin (LC-MS spectrum as shown) Figure 7 As shown below, LC-MS (ESI) calculated for C 119 H 208 N 32 O 36 S3(2759.34), 920.78 [M+3H] 3+ Found 920.65; 690.84 [M+4H] 4+ , found 690.71;852.09[M-203.08+3H] 3+ , found 852.89.

[0057] Example 13 IPM-6-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Pro-OH and Fmoc-Arg(Pbf)-OH.

[0058] The detection results of IPM-6-2Br-biotin (LC-MS spectrum as shown) Figure 8 As shown below, LC-MS (ESI) calculated for C 127 H 216 N 32 O 36 S3(2863.49), 955.49 [M+3H] 3+ , found 954.98;716.87 [M+4H] 4+ , found 716.57; 887.80 [M-203.08+ 3H] 3+ , found 887.24; 666.10 [M-203.08+4H] 4+ , found 665.77.

[0059] Example 14 IPM-7-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Phe-OH and Fmoc-Arg(Pbf)-OH.

[0060] The detection results of IPM-7-2S-biotin (LC-MS spectrum as shown) Figure 9 As shown below, LC-MS (ESI) calculated for C 115 H 188 N 24 O 34 S3(2547.08), 850.03 [M+3H] 3+ , found 849.96; 637.77 [M+4H] 4+ , found 637.66.

[0061] Example 15 IPM-7-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Phe-OH and Fmoc-Arg(Pbf)-OH.

[0062] The detection results of IPM-7-2Br-biotin are as follows: LC-MS (ESI) calculated for C 123 H 196 N 24 O 34 S3(2651.23), 1326.62 [M+2H] 2+ , found 1325.68;884.74 [M+3H] 3+ , found884.67.

[0063] Example 16 IPM-8-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Arg(Pbf)-OH, and Fmoc-GalNAc 1 is replaced with Fmoc-GalNAc 2.

[0064] The detection results of the IPM-8-2S-biotin are as follows: LC-MS (ESI) calculated for C 121 H215 N 33 O 37 S3(2820.43), 1411.22 [M+2H] 2+ , found 1410.25;941.14 [M+3H] 3+ , found941.07.

[0065] Example 17 IPM-8-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Arg(Pbf)-OH, and Fmoc-GalNAc-1 is replaced with Fmoc-GalNAc-2.

[0066] The detection results of IPM-8-2Br-biotin are as follows: LC-MS (ESI) calculated for C 129 H 223 N 33 O 37 S3(2924.58), 1463.29 [M+2H] 2+ , found 1463.21;975.86 [M+3H] 3+ , found975.85.

[0067] Example 18 IPM-9-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-His(Trt)-OH.

[0068] The detection results of the IPM-9-2S-biotin are as follows: LC-MS (ESI) calculated for C 109 H 174 N 26 O 34 S3(2488.92), 1245.46 [M+2H] 2+ , found 1244.59; 830.64 [M+3H] 3+ , found830.51.

[0069] Example 19 IPM-9-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-His(Trt)-OH.

[0070] The detection results of IPM-9-2Br-biotin are as follows: LC-MS (ESI) calculated for C 117 H 182 N 26 O 34 S3(2593.07), 1297.54 [M+2H] 2+ , found 1296.62;865.36 [M+3H] 3+ , found865.23.

[0071] Example 20 IPM-10-2S-biotin: The preparation method is the same as in Example 1, except that Fmoc-Ala-OH in Example 1 is replaced with Fmoc-Asp(OtBu)-OH.

[0072] The detection results of IPM-10-2S-biotin are as follows: LC-MS (ESI) calculated for C 101 H 166 N 18 O 42 S3(2400.70), 1201.35 [M+2H] 2+ , found 1200.53;801.23 [M+3H] 3+ , found800.98.

[0073] Example 21 IPM-10-2Br-biotin: The preparation method is the same as in Example 2, except that Fmoc-Ala-OH in Example 2 is replaced with Fmoc-Asp(OtBu)-OH.

[0074] The detection results of IPM-10-2Br-biotin are as follows: LC-MS (ESI) calculated for C 109 H 174 N 18 O 42 S3(2504.85), 1253.42 [M+2H] 2+ , found 1252.56;835.28 [M+3H] 3+ , found835.17.

[0075] Example 22 IPM-1-4S-CD24-LYTAC: The preparation method is the same as in Example 1, except that Biotin-COOH in Example 1 is replaced with Ac-Cys(Trt)-COOH to obtain intermediate compound 17, which is then reacted with peptide compound 18 targeting CD24. The reaction is carried out in an acetonitrile aqueous solution at pH 8 for 1 hour to obtain IPM-1-4S-CD24-LYTAC. The detection result of the IPM-1-4S-CD24-LYTAC is as follows: LC-MS (ESI) calculated for C 170 H 283 N 39 O 56 S3(3865.53), 967.38 [M+4H] 4+ , found 967.13;774.11 [M+5H] 5+ , found773.86.

[0076] Example 23 DOTA-IPM-1-2S: The preparation method is the same as in Example 1, except that Biotin-COOH in Example 1 is replaced with DOTA-3tBu-COOH. The detection results of DOTA-IPM-1-2S are as follows: LC-MS (ESI) calculated for C 97 H 168 N 18 O 37 S2(2346.77), 1122.31 [M+2H] 2+ , found 1121.87;783.26 [M+3H] 3+ , found 783.07.

[0077] Example 24 DOTA-IPM-1-2S-3PEG3: The preparation method was the same as in Example 1. Compound 12 obtained in Example 1 was dissolved in DMF, and DIPEA and NHS-3(PEG)3-DOTA were added. After reacting at room temperature for 2 hours, the mixture was purified. The detection result of DOTA-IPM-1-2S-3PEG3 was as follows: LC-MS (ESI) calculated for C112 H 196 N 20 O 48 S2(2655.01), 1328.51 [M+2H] 2+ , found1328.49;886.00 [M+3H] 3+ , found 885.82; 664.75 [M+4H] 4+ , found 664.66.

[0078] Test case 1. Assay for the endocytic activity of cyclic peptide ligands: Test procedure: Human hepatocytes (HepG2) were cultured in Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher / Gibco) low-glucose medium containing 10% fetal bovine serum until 80% confluence. Then, cells were seeded at 4.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1 / 2 cells per well in 96-well culture plates and cultured at 37°C and 5% CO2 for 24 hours. The test sample (1 μM) was pre-incubated with Streptavidin-650 fluorescent label for 30 min, and the resulting mixture was then added to the cell culture medium. The cells were cultured at 37°C and 5% CO2 for another 8 hours. After culture, the cells were washed three times with phosphate-buffered saline (PBS) to remove unbound label. The fluorescence intensity of the cells was measured using a Spectra Maxi3x microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Statistical analysis of the fluorescence intensity data was performed using GraphpadPism 9.5 software. Table 1 shows the fold increase in the endocytosis effect of the products described in Examples 1, 3, 4-5, and 8-19 and L-96-biotin on Streptavidin-650 in stem cells (with L-96-biotin set to 1). Table 1. Magnification of the products described in Examples 1, 3, 4-5 and 8-19 and L-96-biotin to Streptavidin-650 endocytosis in stem cells.

[0079] As shown in Table 1, since the endocytic activity of the cyclic peptide ligands described in this invention is directly related to their drug delivery and extracellular protein degradation effects, the cyclic peptide ligands described in this invention can provide new and better technologies for drug delivery of small nucleic acids and the development of drugs for the degradation of pathological extracellular proteins by novel LYTAC drugs. 2. Cytotoxicity test 293T cells were cultured in DMEM medium containing 10% FBS at a density of 8 × 10⁶ cells / year. 3 The cells were placed in an incubator with an atmosphere of 95% air and 5% CO2 at 37°C for 24 hours. Then, 200 μL of the analyte was added to each well at a final concentration ranging from 500 μM to 6.25 μM. After incubation for 24 hours, 20 μL of 5 mg / mL MTT solution was added, and the cells were incubated for another 4 hours at 37°C. The culture medium in each well was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to dissolve the blue-violet compound formazan. The OD was measured using a multi-mode microplate reader. 570 ; Figure 13 The results of the cytotoxicity of IPM-4-2Br-biotin on 293T cells at different concentrations as described in Example 9 were obtained from... Figure 13 It can be seen that the IPM-4-2Br-biotin described in Example 9 has a survival rate of over 70% for L929 cells at a concentration of 10 μM; 3. Degradation test of extracellular protein degrading agents on target extracellular proteins (taking CD24 as an example): HepG2 cells were seeded in 24-well plates at a density of 250,000 cells / well and incubated for 24 hours. ASGPR extracellular protein degrading agent (the product described in Example 22, compound concentration of 5 μM) was added to the culture medium and premixed for 30 min. After premixing, the culture medium was added to the cells and incubated for 8 h. After incubation, the culture medium was discarded, and the cells were washed twice with PBS. Fresh culture medium was added, and 10 μL of RIPA was added at 0, 1, 2, and 4 h respectively. The cells were digested on ice for 20 min, centrifuged at 1200 rpm, and the supernatant was added to loading buffer. The cells were boiled at 100 °C for 10 min and stored at -20 °C. Subsequently, protein gel electrophoresis, transfer to a membrane, antibody staining and imaging were performed to detect the amount of CD24 protein. Figure 14 The results of CD24 degradation by the product (IPM-1-4S-CD24-LYTAC) described in Example 22 are obtained from... Figure 14 It can be seen that the product (IPM-1-4S-CD24-LYTAC) described in Example 22 can significantly degrade the target protein CD24, with the most obvious effect at 4h and 8h; 4. [ 68 The product (DOTA-IPM-1-2S) described in Example 23, labeled with Ga, was used to visualize mouse livers. 68 The GaCl3 solution was prepared in hydrochloric acid using a germanium-gallium generator (ITM Medical Isotopes GmbH, Germany) (concentration 0.1 M, volume 4 mL). The solution was then... 68GaCl3 solution (148-195 MBq, 1 mL) was mixed with sodium acetate solution (0.5 M, 250 μL) to adjust the pH to 4.0-4.5. Then, 40 μg of the DOTA-IPM-1-2S peptide was mixed in a centrifuge tube. The resulting mixture was heated to 95°C and reacted at 300 rpm for 15 min with mechanical stirring. After the reaction was complete, deionized water was added to terminate the reaction. The mixture was then purified using a Sep-Pak C18 Light solid-phase extraction column (Water Systems, Inc., USA) to obtain […]. 68 Ga]Ga-DOTA-IPM-1-2S; Radioactive labeling rate and radiochemical purity were determined using a high-performance liquid chromatography system (Agilent LC-2050, USA). Chromatographic conditions: mobile phase B (acetonitrile solution containing 0.1% trifluoroacetic acid) was linearly increased from 5% to 95% over 20 min at a flow rate of 1 mL / min; mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid. PET / CT scans were performed using a Trans PET Discoverist 180 device (Suzhou Ruikang Technology Co., Ltd., China). Normal healthy mice were intravenously injected with 3.7-7.4 MBq of radiotracer and then scanned 30 min, 1 h, and 2 h after injection under 2% isoflurane anesthesia. Each scan lasted 14 min, including 4 min of CT scan and 10 min of PET scan. PET images were reconstructed after attenuation correction, and quantitative analysis of relevant regions was performed using Inveon Research Workplace analysis software (Siemens, Germany). Figure 15 PET imaging of the product (DOTA-IPM-1-2S) described in Example 23 in healthy mice, by... Figure 15 It is known that the product (DOTA-IPM-1-2S) described in Example 23 can specifically and rapidly reach the liver site within 15 minutes (%ID / g>30), while its uptake in the heart, lungs, kidneys and muscles is very low (%ID / g<5). Therefore, the GalNAc cyclic peptide ligand in this invention has very high liver specificity, which can provide new and better technologies for drug delivery of small nucleic acids and the development of drugs that degrade pathological extracellular proteins using novel LYTAC drugs. It can also provide a new PET probe tool for non-invasive diagnosis of liver health.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cyclic peptide ligand targeting the desialylate glycoprotein receptor and a pharmaceutically acceptable salt thereof, characterized in that, The cyclic peptide ligand has the structure shown in Formula I: Formula I; In Equation I, -S-R0-S- is -SS- or ; R1, R2, R3, R4, R5, R6, R7, and R8 are independently glycine, L-arginine, L-alanine, L-leucine, L-phenylalanine, L-proline, L-aspartic acid, L-lysine, L-glutamine, L-asparagine, L-histidine, L-glutamic acid, L-isoleucine, L-valine, L-methionine, L-tryptophan, L-serine, L-threonine, L-tyrosine, or R-cysteine; a, b, c, d, e, f, g, and h are independent integers between 0 and 3; R 13 -NH2, -OH, -NHR 14 or -OR 14 The -NHR 14 and -OR 14 Chinese R 14 Independently, it is -(CH2). y CH3 or -(CH2) y COOH; the -(CH2) y CH3 or -(CH2) y In COOH, y is an independent integer between 6 and 20; L1 is –(NH–(CH2) n –CO– or –(NH–(CH2CH2O) m –CO) p –, wherein the broken bond at the -NH- position in L1 is connected to the carbonyl group in Formula I, and the broken bond at the -CO- position in L1 is connected to the imino group in Formula I; in L1, n is an integer between 1 and 8, m is an integer between 1 and 8, and p is an integer between 0 and 3; L2 is -(CH2) z -, where z is an integer between 0 and 12; L3 is -CO-(CH2) q -NH-, -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- or -CO-(CH2CH2O) t -NH-CO-(CH2) q -NH-, wherein the broken bond at the -CO- position in L3 is connected to the imino group in Formula I; the broken bond at the -NH- position in L3 is connected to X in Formula I; the -CO-(CH2) q -NH-, -CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q In -NH-, q is an independent integer between 6 and 18; -(CO-(CH2CH2O) t -NH) w -、-CO-(CH2) q -NH-CO-(CH2CH2O) t -NH- and -CO-(CH2CH2O) t -NH-CO-(CH2) q In -NH-, t is an independent integer between 1 and 8, and -(CO-(CH2CH2O) t -NH) w In the -, w is a positive integer from 1 to 6; X is an isotope chelating agent, oligonucleotide, extracellular protein binder, or fluorescent dye.

2. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in claim 1, characterized in that, The isotope chelating agent includes , or .

3. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in claim 1, characterized in that, The oligonucleotides include ASO, shRNA, miRNA, siRNA, or DNA / RNA hybrid molecules composed of a sense strand and DNA.

4. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in claim 1, characterized in that, The extracellular protein binders include membrane protein binders, secretory protein binders, or nucleic acid drugs.

5. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in claim 1, characterized in that, The fluorescent dyes include 5FAM, FITC, Cy3, Cy5, Cy5.5, or Cy7.

6. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in any one of claims 1 to 5, characterized in that, The cyclic peptide ligand has the structure shown in any one of Formula I-1 to Formula I-4: Formula I-1 Formula I-2 Formula I-3 or Equation I-4; In Formulas I-1 to I-4, X is an isotope chelating agent, oligonucleotide, or extracellular protein binder.

7. The cyclic peptide ligand and its pharmaceutically acceptable salt as described in any one of claims 1 to 5, characterized in that, The cyclic peptide ligand is: Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 Formula 8 Formula 9 Formula 10 Formula 11 Formula 12 Formula 13 Formula 14 Formula 15 Formula 16 Formula 17 Formula 18 Formula 19 Formula 20 Formula 21 Equation 22 Formula 23 or Equation 24.

8. The use of the cyclic peptide ligand and its pharmaceutically acceptable salt as described in any one of claims 1 to 7 in the preparation of RNAi drugs or as a LYTAC protein degrading agent for degrading extracellular proteins.

9. The application as described in claim 8, characterized in that, The RNAi drug includes the LYTAC protein degrader.

10. A pharmaceutical composition, characterized in that, This includes pharmaceutically acceptable carriers and cyclic peptide ligands and their pharmaceutically acceptable salts; The cyclic peptide ligand and its pharmaceutically acceptable salt are the cyclic peptide ligand and its pharmaceutically acceptable salt as described in any one of claims 1 to 7.

Images