Polypeptide-drug conjugate, and preparation method therefor and use thereof

Abstract

The present invention relates to a SORTILIN-specific polypeptide-drug conjugate and a preparation method therefor and a use thereof. The SORTILIN-specific polypeptide-drug conjugate has high inhibitory activity against cell lines such as breast cancer cell lines 4T1 and MDA-MB-231, and ovarian cancer cell lines SK-OV-3, A2780-cisR, and Caov3. Meanwhile, the SORTILIN-specific polypeptide-drug conjugate has a long plasma half-life and has good pharmacokinetic and pharmacodynamic properties.

Description

A polypeptide drug conjugate, its preparation method and application

[0001] This disclosure claims priority to Chinese Patent Application No. 202411008570.5, filed on July 25, 2024, entitled "A polypeptide drug conjugate and its preparation method and application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, and in particular to SORTILIN-specific polypeptide-drug conjugates, their preparation methods, and applications. Background Technology

[0003] Cancer remains a leading cause of death in both developing and developed countries, and conventional chemotherapy remains the primary treatment for many cancers. Chemotherapy drugs, such as doxorubicin, paclitaxel, gemcitabine, and camptothecin, are highly effective cytotoxic agents that exert their effects through various mechanisms. However, the toxicity of these chemotherapeutic drugs is nonspecific and can also kill normal cells and tissues, leading to dosage limitations and reduced efficacy. Different strategies are employed in cancer treatment to improve the therapeutic effect of chemotherapeutic drugs and reduce their toxic side effects. One popular strategy is to use targeted ligands to deliver chemotherapeutic drugs (or cytotoxic payloads / drugs) to the cancer site and then release them, reducing damage to non-cancerous cells and tissues. The ligands used actively target specific cell surface receptors or biomarkers at the tumor site, allowing higher concentrations of the drug to reach the tumor. Targeted ligands, such as antibodies or peptides, can covalently bind to the surface of drugs or drug delivery systems for site-specific delivery. Antibodies or peptides are covalently coupled to drugs via linkers to create antibody-drug conjugates (ADCs) or peptide-drug conjugates (PDCs). Both ADCs and PDCs are promising treatment modalities that are rapidly emerging for cancer treatment. Due to their large molecular weight, ADCs typically deliver no more than 2% of their dose to tumor tissues near blood vessels via the bloodstream. In contrast, PDCs have smaller molecular weights and stronger tissue penetration capabilities, delivering more chemotherapy drugs to tumor tissues far from blood vessels. Furthermore, PDCs exhibit better batch-to-batch uniformity, making them more suitable for large-scale production.

[0004] SORTILIN is a key clearance receptor and the first member of the vacuole sorting 10 protein domain (Vps10p) family. Functional characterization suggests that SORTILIN plays a dual role in endocytosis and receptor transport, allowing its ligands to be sorted from the cell surface to specific subcellular compartments and transporting pro-neurotrophs such as the neuropeptides neurotensin (NT), proNGF, and proBDNF. Given its role in ligand internalization and cellular transport, it is considered one of the cell's own shuttle systems. SORTILIN is involved in cancer cell proliferation, migration, and invasion. Compared to healthy ovarian tissue, SORTILIN is particularly overexpressed in ovarian cancer and is associated with an aggressive cancer phenotype. Its expression is also elevated in the tumor microenvironment of several other human cancers, including breast cancer, prostate cancer, colon cancer, endometrial cancer, pancreatic cancer, skin cancer, and pituitary cancer.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] To address the aforementioned problems, this invention provides a polypeptide-drug conjugate of formula (I) or its stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives, and pharmaceutically acceptable salts. This polypeptide-drug conjugate exhibits excellent inhibitory activity against breast cancer cell lines 4T1 and MDA-MB-231, as well as ovarian cancer cell lines SK-OV-3, A2780-cisR, and Caov3.

[0008] Solution for solving the problem

[0009] To address the aforementioned technical problems, the present invention provides a polypeptide-drug conjugate of formula (I) or its stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives, and pharmaceutically acceptable salts thereof.

[0010] in:

[0011] A is a cytotoxic agent;

[0012] L is a connector between E and A. The wavy line in formula (I) indicates that L is covalently attached to the side chain residues or terminal groups of E.

[0013] a is an integer from 0 to 10;

[0014] E represents a SORTILIN-specific peptide ligand, wherein the peptide ligand comprises any one of the following amino acid sequences (i)-(iv):

[0015] (i) The amino acid sequence shown in formula (EI):

[0016] Equation (EI) is R1-X1-P1-X2-P2-X3-P3-X4-P4-X5-P5-P6-B1.

[0017] in:

[0018] R1 is a modifying group of the N-terminal amino group or is absent;

[0019] X1, X2, X3, X4, and X5 are independently selected from natural amino acids, non-natural amino acids, chemical modifications of natural or non-natural amino acids, or combinations thereof.

[0020] P2 is selected from Ala, Gly, Ile, Val, Leu, Pro, Nle, Sar, tBuA, Dpr, A2Bu, Dbu, Abu, Aib, OctG, PipAla, PirrAla, BnG, Cha, Cpa, C4al, C5al, Thea, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNl e, any one of 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNle, hCha and 4-PryAla;

[0021] P3 is selected from any one of Asn, Gln, Trp, Arg, Lys, His, Cit, Orn, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, Cha, C4al, C5al, Thi, Tza, Mso, Aha, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeArg, hCha, hArg, and 4-PryAla;

[0022] P4 is selected from Lys, Arg, His, Gln, Pro, Dab, Cit, Orn, Thi, Tza, Aha, Aoc, HyPro, hArg, 3AmiPhe, 4AmiPhe, Dimk, AcLys, MeLys, MeArg, Pip, Pzp, IPegDab and SPegDab;

[0023] P5 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, Thi, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe, and MePhe.

[0024] P6 is selected from Ala, Gly, Val, Ile, Leu, Pro, Met, Nle, 4-AmPyrr1, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C Any one of 4al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha and Mso;

[0025] B1 is selected from any one of Val, Ile, Leu, Nle, Aha, MeIle, MeLeu, MeVal, and MeNle;

[0026] (ii) The amino acid sequence shown in formula (E-II):

[0027] R2-A1-P7-X6-P8-P9-X7-A2-X8-P 10 -B2 formula (E-II),

[0028] in:

[0029] R2 is a modifying group of the N-terminal amino group or is absent;

[0030] X6, X7, and X8 are independently selected from non-existent or selected from natural amino acids, non-natural amino acids, chemical modifications of natural or non-natural amino acids, or combinations thereof.

[0031] A1 and A2 are each independently selected from Cys, Sec, and Pen;

[0032] P7 is selected from any one of the following: non-existent, Ser, Thr, Tyr, Trp, Cys, Thea, His, Sec, Pen, hSer, hCys, AlloT, sBzl, tBzl, and yBzl.

[0033] P8 is selected from any one of Asn, Glu, Cys, Sec, Pro, Arg, Lys, His, Cit, Orn, Dab, Aha, Thea, Dimk, Isorn, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, hCys, hArg, AcLys, MeArg, MeLys, IPegDab, and SPegDab;

[0034] P9 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe, MePhe, Thi, Pro, Arg, and Leu;

[0035] P 10 Selected from Ala, Gly, Val, Ile, Leu, Pro, Met, Nle, 4-AmPyrr1, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C4 Any of al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha and Mso;

[0036] B2 is selected from any one of Val, Ile, Leu, Trp, Nle, Aha, MeIle, MeLeu, MeVal, and MeNle;

[0037] (iii) An amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in Formula (EI), and retaining the activity of the amino acid sequence shown in Formula (EI);

[0038] (iv) An amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in Formula (E-II), and retaining the activity of the amino acid sequence shown in Formula (E-II).

[0039] The effects of the invention

[0040] The SORTILIN-specific peptide-drug conjugate provided by this invention exhibits excellent inhibitory activity against breast cancer cell lines 4T1 and MDA-MB-231, as well as ovarian cancer cell lines SK-OV-3, A2780-cisR, and Caov3. Furthermore, this SORTILIN-specific peptide-drug conjugate possesses a long plasma half-life and exhibits favorable pharmacokinetic and pharmacodynamic characteristics. Attached Figure Description

[0041] Figure 1 is the HPLC chromatogram of conjugate 1.

[0042] Figure 2 shows the MS diagram of coupling 1.

[0043] Figure 3 shows the HPLC chromatogram of conjugate 2.

[0044] Figure 4 shows the MS image of coupling compound 2.

[0045] Figure 5 shows the HPLC chromatogram of conjugate 3.

[0046] Figure 6 shows the MS image of coupling compound 3.

[0047] Figure 7 shows the HPLC chromatogram of conjugate 4.

[0048] Figure 8 shows the MS image of coupling compound 4.

[0049] Figure 9 shows the HPLC chromatogram of conjugate 5.

[0050] Figure 10 shows the MS image of coupling compound 5.

[0051] Figure 11 is the HPLC chromatogram of conjugate 6.

[0052] Figure 12 shows the MS diagram of coupling compound 6.

[0053] Figure 13 is the HPLC chromatogram of conjugate 7.

[0054] Figure 14 is the MS image of coupling compound 7.

[0055] Figure 15 shows the HPLC chromatogram of conjugate 8.

[0056] Figure 16 is the MS diagram of coupling 8.

[0057] Figure 17 is the HPLC chromatogram of conjugate 9.

[0058] Figure 18 shows the MS image of coupling compound 9.

[0059] Figure 19 shows the HPLC chromatogram of conjugate 10.

[0060] Figure 20 shows the MS diagram of coupling compound 10.

[0061] Figure 21 is the HPLC chromatogram of conjugate 11.

[0062] Figure 22 is the MS diagram of coupling 11.

[0063] Figure 23 is the HPLC chromatogram of conjugate 12.

[0064] Figure 24 is the MS image of coupling compound 12.

[0065] Figure 25 shows the HPLC chromatogram of conjugate 13.

[0066] Figure 26 is the MS image of coupling 13.

[0067] Figure 27 is the HPLC chromatogram of conjugate 14.

[0068] Figure 28 shows the MS image of coupling compound 14.

[0069] Figure 29 shows the HPLC chromatogram of conjugate 15.

[0070] Figure 30 shows the MS diagram of coupling compound 15.

[0071] Figure 31 is the HPLC chromatogram of conjugate 16.

[0072] Figure 32 is the MS image of coupling 16.

[0073] Figure 33 is the HPLC chromatogram of conjugate 17.

[0074] Figure 34 is an MS image of coupling compound 17.

[0075] Figure 35 shows the HPLC chromatogram of conjugate 18.

[0076] Figure 36 is the MS image of coupling 18.

[0077] Figure 37 is the HPLC chromatogram of conjugate 19.

[0078] Figure 38 shows the MS image of coupling 19.

[0079] Figure 39 shows the HPLC chromatogram of conjugate 20.

[0080] Figure 40 is an MS image of coupling compound 20.

[0081] Figure 41 is the HPLC chromatogram of conjugate 21.

[0082] Figure 42 is an MS image of coupling 21.

[0083] Figure 43 is the HPLC chromatogram of conjugate 22.

[0084] Figure 44 is the MS image of coupling 22.

[0085] Figure 45 shows the HPLC chromatogram of conjugate 23.

[0086] Figure 46 is the MS image of coupling 23.

[0087] Figure 47 is the HPLC chromatogram of conjugate 24.

[0088] Figure 48 shows the MS image of coupling 24.

[0089] Figure 49 shows the HPLC chromatogram of conjugate 25.

[0090] Figure 50 shows the MS image of coupling compound 25.

[0091] Figure 51 is the HPLC chromatogram of conjugate 26.

[0092] Figure 52 is the MS image of coupling 26.

[0093] Figure 53 is the HPLC chromatogram of conjugate 27.

[0094] Figure 54 shows the MS image of coupling compound 27.

[0095] Figure 55 is the HPLC chromatogram of conjugate 28.

[0096] Figure 56 is the MS image of coupling 28.

[0097] Figure 57 is the HPLC chromatogram of conjugate 29.

[0098] Figure 58 shows the MS image of coupling 29.

[0099] Figure 59 shows the HPLC chromatogram of conjugate 30.

[0100] Figure 60 is an MS image of coupling compound 30.

[0101] Figure 61 is the HPLC chromatogram of conjugate 31.

[0102] Figure 62 is an MS diagram of coupling 31.

[0103] Figure 63 is the HPLC chromatogram of conjugate 32.

[0104] Figure 64 is the MS image of coupling 32.

[0105] Figure 65 is the HPLC chromatogram of conjugate 33.

[0106] Figure 66 is the MS image of coupling 33.

[0107] Figure 67 is the HPLC chromatogram of conjugate 34.

[0108] Figure 68 is the MS image of coupling 34.

[0109] Figure 69 shows the HPLC chromatogram of conjugate 35.

[0110] Figure 70 shows the MS image of coupling compound 35.

[0111] Figure 71 is the HPLC chromatogram of conjugate 36.

[0112] Figure 72 is the MS image of coupling 36.

[0113] Figure 73 is the HPLC chromatogram of conjugate 37.

[0114] Figure 74 is the MS image of coupling compound 37.

[0115] Figure 75 shows the HPLC chromatogram of conjugate 38.

[0116] Figure 76 shows the MS image of coupling compound 38.

[0117] Figure 77 is the HPLC chromatogram of conjugate 39.

[0118] Figure 78 shows the MS image of coupling compound 39.

[0119] Figure 79 shows the HPLC chromatogram of conjugate 40.

[0120] Figure 80 is an MS image of coupling compound 40.

[0121] Figure 81 is the HPLC chromatogram of conjugate 41.

[0122] Figure 82 is an MS image of coupling 41.

[0123] Figure 83 is the HPLC chromatogram of conjugate 42.

[0124] Figure 84 is the MS image of coupling compound 42.

[0125] Figure 85 is the HPLC chromatogram of conjugate 43.

[0126] Figure 86 is the MS image of coupling compound 43.

[0127] Figure 87 is the HPLC chromatogram of conjugate 44.

[0128] Figure 88 is the MS image of coupling 44.

[0129] Figure 89 shows the HPLC chromatogram of conjugate 45.

[0130] Figure 90 shows the MS image of coupling compound 45.

[0131] Figure 91 is the HPLC chromatogram of conjugate 46.

[0132] Figure 92 shows the MS image of coupling compound 46.

[0133] Figure 93 is the HPLC chromatogram of conjugate 47.

[0134] Figure 94 shows the MS image of coupling compound 47.

[0135] Figure 95 shows the pharmacokinetic curve of TH1902 in mice.

[0136] Figure 96 shows the pharmacokinetic curve of conjugate 6 in mice.

[0137] Figure 97 shows the pharmacodynamics of conjugates 30, 31, and 43 in transplanted tumor models.

[0138] Figure 98 shows the pharmacodynamics of conjugate 45 in a transplanted tumor model. Detailed Implementation

[0139] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0140] Terminology Explanation:

[0141] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0142] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable connection point. The substituents are preferably independently selected independently from one or more substituents chosen from the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0143] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group, which is a residue derived from a parent alkane by removing two hydrogen atoms from the same carbon atom or two different carbon atoms. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) carbon atoms, more preferably alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), etc. The alkylene group can be substituted or unsubstituted, and when substituted, it can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents selected from alkenyl, alkynyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclic alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, and oxo.

[0144] The term "alkenyl" refers to an alkyl compound containing at least one carbon-carbon double bond in its molecule, wherein the definition of alkyl is as described above. Alkenyl groups can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl substituents.

[0145] The term "alkynyl" refers to an alkyl compound containing at least one carbon-carbon triple bond in its molecule, wherein the definition of alkyl is as described above. The alkynyl group can be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl substituents.

[0146] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7, and 8) carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0147] The term "spirocycloalkyl" refers to a 5- to 20-membered polycyclic group that shares a single carbon atom (called a spiro atom) between its rings, and may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Spirocycloalkyl groups are classified as monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, it is a 3 / 5-membered, 3 / 6-membered, 4 / 4-membered, 4 / 5-membered, 4 / 6-membered, 5 / 5-membered, or 5 / 6-membered monospirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0148] The term "fused cycloalkyl" refers to a 5- to 20-membered polycyclic aromatic hydrocarbon group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 3 / 4-membered, 3 / 5-membered, 3 / 6-membered, 4 / 4-membered, 4 / 5-membered, 4 / 6-membered, 5 / 4-membered, 5 / 5-membered, 5 / 6-membered, 6 / 3-membered, 6 / 4-membered, 6 / 5-membered, and 6 / 6-membered bicyclic alkyl groups. Non-limiting examples of fused cycloalkyl groups include:

[0149] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms, and may contain one or more double bonds. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0150] The cycloalkyl ring comprises a cycloalkyl group (including monocyclic, spirocyclic, fused, and bridged rings) fused to an aryl, heteroaryl, or heterocyclic alkyl ring as described above, wherein the ring attached to the parent structure is a cycloalkyl group. Non-limiting examples include... etc.; preferred

[0151] The cycloalkyl group can be substituted or unsubstituted, and when substituted, it can be substituted at any usable connection point. The substituent is preferably selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0152] The term "alkoxy" refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl and cycloalkyl are defined as described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, they are preferably one or more of the following groups, independently selected from D atoms, halogens, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclic oxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups.

[0153] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic non-aromatic cyclic substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), but excluding the -OO-, -OS-, or -SS- ring moiety, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) ring atoms, wherein 1 to 4 (e.g., 1, 2, 3, and 4) are heteroatoms; more preferably, it comprises 3 to 8 ring atoms (e.g., 3, 4, 5, 6, 7, and 8), wherein 1 to 3 (e.g., 1, 2, and 3) are heteroatoms; even more preferably, it comprises 3 to 6 ring atoms, wherein 1 to 3 are heteroatoms; most preferably, it comprises 5 or 6 ring atoms, wherein 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0154] The term "spiroheterocyclic group" refers to a 5- to 20-membered non-aromatic polycyclic heterocyclic group in which one or more ring atoms share a single atom (called a spiro atom) between the rings, wherein the one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming a sulfoxide or sulfone), and the remaining ring atoms are carbon. It may contain one or more double bonds. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered (e.g., 7, 8, 9, or 10-membered). Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups according to the number of shared spiro atoms between the rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 3 / 5-membered, 3 / 6-membered, 4 / 4-membered, 4 / 5-membered, 4 / 6-membered, 5 / 5-membered, or 5 / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic groups include:

[0155] The term "fused heterocyclic group" refers to a 5- to 20-membered non-aromatic polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 3-membered / 4-membered, 3-membered / 5-membered, 3-membered / 6-membered, 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 4-membered, 5-membered / 5-membered, 5-membered / 6-membered, 6-membered / 3-membered, 6-membered / 4-membered, 6-membered / 5-membered, and 6-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0156] The term "bridged heterocyclic group" refers to a 5- to 14-membered, non-aromatic polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, wherein the sulfur may optionally be oxidized (i.e., forming sulfoxide or sulfone), and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0157] The heterocyclic ring comprises a heterocyclic group (including monocyclic, spirocyclic, fused heterocyclic, and bridged heterocyclic rings) fused to an aryl, heteroaryl, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0158] wait.

[0159] The heterocyclic group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0160] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (fused polycyclic) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring comprises an aryl ring fused to a heteroaryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring attached to the parent structure is an aryl ring, and non-limiting examples include:

[0161] The aryl group can be substituted or unsubstituted, and when substituted, it can be substituted at any usable connection point. The substituent is preferably selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0162] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 (e.g., 1, 2, 3, and 4) heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered (e.g., 5, 6, 7, 8, 9, or 10-membered), more preferably 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, etc. The heteroaryl ring comprises a heteroaryl group fused to an aryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0163] The heteroaryl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected independently from one or more substituents selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0164] The aforementioned cycloalkyl, heterocyclic, aryl, and heteroaryl groups include residues derived from removing one hydrogen atom from a parent ring atom, or residues derived from removing two hydrogen atoms from the same or two different ring atoms of the parent, namely "divalent cycloalkyl", "divalent heterocyclic", "aryl", and "heteroaryl".

[0165] The term “cycloalkyloxy” refers to cycloalkyl-O-, where the cycloalkyl group is as defined above.

[0166] The term “heterocyclic oxy group” refers to the heterocyclic group -O-, where the heterocyclic group is as defined above.

[0167] The term "alkylthio" refers to alkyl-S-, where the alkyl group is as defined above.

[0168] The term "haloalkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0169] The term "haloalkoxy" refers to an alkoxy group that is substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0170] The term "hydroxyalkyl" refers to an alkyl group that is replaced by one or more hydroxyl groups, wherein the alkyl group is as defined above.

[0171] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0172] The term "hydroxyl group" refers to -OH.

[0173] The term "thiol" refers to -SH.

[0174] The term "amino" refers to -NH2.

[0175] The term "cyano" refers to -CN.

[0176] The term "nitro" refers to -NO2.

[0177] The term "oxo" or "oxo" refers to "=O".

[0178] The term "carbonyl" refers to C=O.

[0179] The term "carboxyl group" refers to -C(O)OH.

[0180] The compounds disclosed herein contain their isotopic derivatives. The term "isotopic derivative" refers to a compound whose structure differs only in the presence of one or more isotopically enriched atoms. For example, compounds having the structure disclosed herein, using "deuterium" or "tritium" instead of hydrogen, or using... 18 F-fluorine labeling ( 18 F isotopes) can be used instead of fluorine, or... 11 C-, 13 C-, or 14 C-enriched carbon ( 11C-, 13 C-, or 14 C-carbon labeling; 11 C-, 13 C-, or 14 Compounds in which carbon atoms are replaced by C-isotopes are within the scope of this disclosure. Such compounds can be used as analytical tools or probes in, for example, biological assays, or as in vivo diagnostic imaging tracers for diseases, or as tracers for pharmacodynamic, pharmacokinetic, or receptor studies. The various deuterated forms of compounds disclosed herein refer to compounds in which each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds by referring to relevant literature. Commercially available deuteration starting materials can be used in the preparation of deuterated forms of compounds, or they can be synthesized using conventional techniques with deuteration reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane. Deuterated compounds generally retain activity comparable to undeuterated compounds, and better metabolic stability can be achieved when deuterated at certain specific sites, resulting in certain therapeutic advantages.

[0181] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0182] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions without much effort (through experimentation or theory). For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0183] The terms "SORT1 protein" and "Sortilin" are used interchangeably, both referring to a neuronal type 1 membrane glycoprotein encoded by the SORT1 gene (GeneID: 6272), which belongs to the vacuolar protein sorting 10 protein (Vps10) receptor family.

[0184] Unless otherwise specified, all amino acids are used in the L-configuration.

[0185] Table I shows some common amino acid names and their three-letter and single-letter abbreviations:

[0186] Table I

[0187] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, such as an α-carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function similarly to naturally occurring amino acids.

[0188] The term "non-naturally occurring amino acid" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid but is not incorporated into a growing polypeptide chain via a translation complex. "Non-naturally occurring amino acids" also includes, but is not limited to, amino acids obtained by modifying (e.g., post-translational modification) naturally encoded amino acids (including, but not limited to, 20 common amino acids) but which are not themselves naturally incorporated into a growing polypeptide chain via a translation complex. A non-limiting list of examples of non-naturally occurring amino acids that can be inserted into or replace wild-type residues in a polypeptide sequence includes β-amino acids, high-molecular-weight amino acids, cyclic amino acids, and amino acids with derivatized side chains.

[0189] In this invention, when referring to peptides, "amino acid" and "amino acid residue" have the same meaning, referring to the fact that when amino acids are linked by chemical bonds, some of their groups are lost due to participation in the formation of the linking bonds, and the remaining amino acid portion is called an amino acid residue.

[0190] The terms “homology” and “identity” indicate sequence similarity to wild-type amino acid sequences or wild-type nucleic acid sequences, and homology comparisons can be performed visually or using commercially available comparison programs. Using commercially available computer programs, homology between two or more sequences can be expressed as a percentage (%), and homology (%) between adjacent sequences can be calculated.

[0191] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0192] "Carrier" refers to a system that does not cause significant stimulation to the organism and does not eliminate the biological activity and properties of the given compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the target organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

[0193] "Excipient" refers to an agent that is not itself a therapeutic agent but is used as a diluent, excipient, binder, and / or medium to be added to a pharmaceutical composition to improve its disposal or storage properties or to allow or promote the formation of a unit dosage form of the compound or pharmaceutical composition for administration. As is known to those skilled in the art, pharmaceutical excipients can provide a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, and croscarmellose (e.g., sodium croscarmellose); (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter. (9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) Diols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; and (22) Other non-toxic compatible substances used in pharmaceutical preparations.

[0194] For the purposes of pharmaceuticals or pharmacologically active agents, the term "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. The appropriate effective amount in a given case can be determined by a person skilled in the art based on routine testing.

[0195] "Solvate" refers to the physical combination of a compound of this disclosure with one or more, preferably one to three, solvent molecules, whether organic or inorganic. This physical combination includes hydrogen bonds. In some cases, for example, when one or more, preferably one to three, solvent molecules are incorporated into the lattice of a crystalline solid, the solvate will be separated. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0196] "Prodrugs" refer to compounds that can be converted in the body to produce active drug compounds under physiological conditions, such as through hydrolysis in the blood.

[0197] "Pharmaceutical acceptable" means that these compounds, materials, compositions, and / or dosage forms are suitable for contact with patient tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use.

[0198] As used herein, the singular forms of “a,” “an,” and “the” include plural references, and vice versa, unless the context clearly indicates otherwise.

[0199] "Pharmaceutically acceptable salt" refers to a salt of the compounds disclosed herein that is safe and effective in mammalian use and possesses the intended biological activity. The salt can be prepared separately during the final isolation and purification of the compound, or by reacting a suitable group with a suitable base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases as well as organic bases. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids as well as organic acids.

[0200] The term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may include one or more chiral centers and / or double bonds, and thus exist as a stereoisomer, such as a double-bonded isomer (i.e., a geometric E / Z isomer) or a diastereomer (e.g., an enantiomer (i.e., (+) or (-)) or a cis / trans isomer). This disclosure covers any and all isomers of the compounds described herein, including stereoisomeric pure forms (e.g., geometrically pure, enantiomeric pure, or diastereomeric pure) and mixtures of enantiomers and stereoisomers, such as racemates. Mixtures of enantiomers and stereoisomers of compounds, and the ways in which they are resolved into their constituent enantiomers or stereoisomers, are well known.

[0201] On the one hand, the present invention provides a polypeptide-drug conjugate of formula (I) or its stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives, and pharmaceutically acceptable salts thereof.

[0202] in:

[0203] A is a cytotoxic agent;

[0204] L is a connector between E and A. The wavy line in formula (I) indicates that L is covalently attached to the side chain residues or terminal groups of E.

[0205] a is an integer from 0 to 10;

[0206] E represents a SORTILIN-specific peptide ligand, wherein the peptide ligand comprises any one of the following amino acid sequences (i)-(iv):

[0207] (i) The amino acid sequence shown in formula (EI):

[0208] R1-X1-P1-X2-P2-X3-P3-X4-P4-X5-P5-P6-B1 (EI)

[0209] in:

[0210] R1 is a modifying group of the N-terminal amino group or is absent;

[0211] X1, X2, X3, X4, and X5 are independently selected from natural amino acids, non-natural amino acids, chemical modifications of natural or non-natural amino acids, or combinations thereof.

[0212] P2 is selected from Ala, Gly, Ile, Val, Leu, Pro, Nle, Sar, tBuA, Dpr, A2Bu, Dbu, Abu, Aib, OctG, PipAla, PirrAla, BnG, Cha, Cpa, C4al, C5al, Thea, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNl e, any one of 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNle, hCha and 4-PryAla;

[0213] P3 is selected from any one of Asn, Gln, Trp, Arg, Lys, His, Cit, Orn, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, Cha, C4al, C5al, Thi, Tza, Mso, Aha, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeArg, hCha, hArg, and 4-PryAla;

[0214] P4 is selected from Lys, Arg, His, Gln, Pro, Dab, Cit, Orn, Thi, Tza, Aha, Aoc, HyPro, hArg, 3AmiPhe, 4AmiPhe, Dimk, AcLys, MeLys, MeArg, Pip, Pzp, IPegDab and SPegDab;

[0215] P5 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, Thi, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe, and MePhe.

[0216] P6 is selected from Ala, Gly, Val, Ile, Leu, Pro, Met, Nle, 4-AmPyrr1, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C Any one of 4al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha and Mso;

[0217] B1 is selected from any one of Val, Ile, Leu, Nle, Aha, MeIle, MeLeu, MeVal, and MeNle;

[0218] (ii) The amino acid sequence shown in formula (E-II):

[0219] R2-A1-P7-X6-P8-P9-X7-A2-X8-P 10 -B2 formula (E-II),

[0220] in:

[0221] R2 is a modifying group of the N-terminal amino group or is absent;

[0222] X6, X7, and X8 are independently selected from non-existent or selected from natural amino acids, non-natural amino acids, chemical modifications of natural or non-natural amino acids, or combinations thereof.

[0223] A1 and A2 are each independently selected from Cys, Sec, and Pen;

[0224] P7 is selected from any one of the following: non-existent, Ser, Thr, Tyr, Trp, Cys, Thea, His, Sec, Pen, hSer, hCys, AlloT, sBzl, tBzl, and yBzl.

[0225] P8 is selected from any one of Asn, Glu, Cys, Sec, Pro, Arg, Lys, His, Cit, Orn, Dab, Aha, Thea, Dimk, Isorn, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, hCys, hArg, AcLys, MeArg, MeLys, IPegDab, and SPegDab;

[0226] P9 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe, MePhe, Thi, Pro, Arg, and Leu;

[0227] P 10Selected from Ala, Gly, Val, Ile, Leu, Pro, Met, Nle, 4-AmPyrr1, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C4 Any of al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha and Mso;

[0228] B2 is selected from any one of Val, Ile, Leu, Trp, Nle, Aha, MeIle, MeLeu, MeVal, and MeNle;

[0229] (iii) An amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in Formula (EI), and retaining the activity of the amino acid sequence shown in Formula (EI);

[0230] (iv) An amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in Formula (E-II), and retaining the activity of the amino acid sequence shown in Formula (E-II).

[0231] In some implementations, P1 is Asn, Glu, Arg, Lys, Cit, Orn, or Dab.

[0232] In some implementations, P1 is Cit, Orn, or Arg.

[0233] In some implementations, P1 is Arg.

[0234] In some implementations, P2 is Ala, Gly, Ile, Val, Leu, Pro, Nle, Dbu, Aha, or Dpr.

[0235] In some implementations, P2 is Aha, Aib, Nle, Ile, Val, or Leu.

[0236] In some implementations, P2 is Val.

[0237] In some implementations, P3 is Asn, Gln, Arg, Lys, or His.

[0238] In some implementations, P3 is Asn or Gln.

[0239] In some implementations, P3 is Gln.

[0240] In some implementations, P4 is Lys, Arg, His, Dab, Dbu, or Aha.

[0241] In some implementations, P4 is Lys, Dab, Dbu, or Aha.

[0242] In some implementations, P4 is Lys.

[0243] In some implementations, P5 is Phe, Tyr, Trp, His, Thi, or Phg.

[0244] In some implementations, P5 is Phe, Tyr, Trp, and His.

[0245] In some implementations, P5 is Tyr.

[0246] In some implementations, P6 is Ala, Gly, Val, Ile, Leu, Pro, Nle, Abu, Aha, or Aib.

[0247] In some implementations, P6 is Abu, Aib, Val, or Ile.

[0248] In some implementations, P6 is Ile.

[0249] In some implementations, B1 is Val, Ile, Leu, Nle, Aha, MeIle, MeLeu, MeVal, or MeNle.

[0250] In some implementations, B1 is Leu, Aha, or Nle.

[0251] In some implementations, B1 is Leu.

[0252] In some implementations, X1 is Ala, Gly, Ile, Leu, Pro, Phe, Val, Tyr, Trp, Aib, Nle, Pip, Pzp, Sar, tBuG, or a combination thereof.

[0253] In some implementations, X2 is Aha, Dab, tBuG, Ala, Gly, Ile, Lys, Cys, Sar, Leu, Val, or a combination thereof.

[0254] In some implementations, X3 is Cit, Orn, Cys, Arg, or a combination thereof.

[0255] In some implementations, X4 is Mso, Nle, Cys, Leu, Met, Phe, Tyr, Trp, or a combination thereof.

[0256] In some implementations, X5 is Pip, Pzp, Pro, or a combination thereof.

[0257] In some embodiments, X1 consists of 1 to 3 amino acid residues.

[0258] In some embodiments, X1 is two amino acid residues.

[0259] In some implementations, X1 is Pip-Phe, Pip-Tyr, Pip-Leu, Pip-Val, Pzp-Phe, Pzp-Tyr, Pzp-Leu, Pzp-Val, Sar-Ile, Sar-Leu, Sar-Val, tBuG-Ile, tBuG-Leu, tBuG-Val, Ala-Phe, Gly-Ile, Gly-Leu, Gly-Val, Ile-Val, Leu-Phe, Pro-Aib, Pro-Ala, Pro-Phe, Pro-Leu, Pro-Val, Pro-Trp, Pro-Tyr, or Val-Phe.

[0260] In some implementations, X1 is Ala-Phe, Gly-Val, Leu-Phe, Ile-Val, Pro-Tyr, Val-Phe, Pro-Phe, or Pro-Ala.

[0261] In some embodiments, X2 consists of 2 to 5 amino acid residues.

[0262] In some embodiments, X2 is four amino acid residues.

[0263] In some implementations, X2 is Aha-Ile-Gly-Ala, Dab-Ile-Gly-Ala, tBuG-Lys-Ala-Sar, Ala-Aha-Leu-Ile, Ala-Aha-Ala-Gly, Ala-Dab-Ala-Gly, Ala-Dab-Leu-Ile, Ala-Lys-Leu-Ile, Ala-Lys-Ala-Sar, Ala-Lys-Ala-Gly, Ala-Lys-Val-Leu, Gly-Aha-Ala-Gly, Gly-Dab-Ala-Gly, Gly-Lys-Ala-Gly, Gly-Lys-Val-Leu, Ile-Aha-Ala-Gly, I le-Aha-Leu-Ile, Ile-Dab-Ala-Gly, Ile-Dab-Ala-Sar, Ile-Dab-Gly-Ala, Ile-Dab-Leu-Ile, Ile-Cys-Gly-Ala, Ile-Cys-Ala-Gly, Ile-Cys-Sar-Ala, Ile-Cys-A la-tBuG, Ile-Cys-Leu-Ile, Ile-Lys-Leu-Ile, Ile-Lys-Sar-Ala, Ile-Lys-Ala-Gly, Ile-Lys-Ala-Sar, Ile-Lys-Gly-Ala, Lys-Ile-Gly-Ala or Lys-Ile-tBuG-Ala.

[0264] In some implementations, X2 is Ala-Lys-Leu-Ile, Ala-Lys-Ala-Gly, Ala-Lys-Val-Leu, Gly-Lys-Ala-Gly, Gly-Lys-Val-Leu, Ile-Lys-Gly-Ala, Ile-Cys-Gly-Ala, or Ile-Lys-Ala-Gly.

[0265] In some embodiments, X3 is 1 to 2 amino acid residues.

[0266] In some embodiments, X3 is a single amino acid residue.

[0267] In some implementations, X3 is Cit, Orn, Cys, or Arg.

[0268] In some implementations, X3 is Arg.

[0269] In some embodiments, X4 consists of 1 to 3 amino acid residues.

[0270] In some embodiments, X4 is two amino acid residues.

[0271] In some implementations, X4 is Mso-Phe, Mso-Tyr, Mso-Trp, Nle-Phe, Nle-Tyr, Nle-Trp, Cys-Phe, Cys-Tyr, Cys-Trp, Leu-Phe, Leu-Tyr, Leu-Trp, Met-Phe, Me-Tyr, or Met-Trp.

[0272] In some implementations, X4 is Met-Phe, Met-Tyr, or Met-Trp.

[0273] In some embodiments, X5 is 1 to 2 amino acid residues.

[0274] In some embodiments, X5 is a single amino acid residue.

[0275] In some implementations, X5 is Pip, Pzp, or Pro.

[0276] In some implementations, X5 is Pro.

[0277] In some embodiments, the peptide ligand comprises the amino acid sequence shown in formula (EI-1):

[0278] R1-X 1-1 -X 1-2 -P1-X 2-1 -X 2-2 -X 2-3 -X 2-4 -P2-X3-P3-X 4-1 -X 4-2 -P4-X5-P5-P6-B1 (EI-1),

[0279] Wherein, R1, P1, P2, P3, P4, P5, P6 and B1 are as defined in any of the preceding items;

[0280] X 1-1 X 1-2 X 2-1 X 2-2 X 2-3 X 2-4 X3, X 4-1 X 4-2 X5 is independently selected from natural amino acids, non-natural amino acids, and chemical modifications of natural or non-natural amino acids.

[0281] In some implementations, the X 1-1For Pip, Pzp, Sar, tBuG, Ala, Gly, Ile, Leu, Pro, or Val.

[0282] In some implementations, the X 1-1 For Ala, Gly, Ile, Leu, Pro, or Val.

[0283] In some implementations, the X 1-2 For Aib, Nle, Ala, Ile, Leu, Val, Tyr, Phe, or Trp.

[0284] In some implementations, the X 1-2 For Ala, Phe, Ile, Val, or Tyr.

[0285] In some implementations, the X 2-1 For Aha, Dab, tBuG, Ala, Gly, Ile, or Lys.

[0286] In some implementations, the X 2-1 It can be either Gly or Ile.

[0287] In some implementations, the X 2-2 It can be Cys, Ile, Aha, Dab, or Lys.

[0288] In some implementations, the X 2-2 For Cys or Lys.

[0289] In some implementations, the X 2-3 For Sar, tBuG, Ala, Gly, Leu, or Val.

[0290] In some implementations, the X 2-3 For Gly, Leu, or Val.

[0291] In some implementations, the X 2-4 For Sar, tBuG, Ala, Gly, Ile, or Leu.

[0292] In some implementations, the X 2-4 It can be Ala, Gly, Ile, or Leu.

[0293] In some implementations, X3 is Cit, Orn, Cys, or Arg.

[0294] In some implementations, X3 is Cys or Arg.

[0295] In some implementations, the X 4-1For Mso, Nle, Cys, Leu, or Met.

[0296] In some implementations, the X 4-1 For Cys or Met.

[0297] In some implementations, the X 4-2 For Phe, Tyr, or Trp.

[0298] In some implementations, the X 4-2 For Phe or Tyr.

[0299] In some implementations, X5 is Pip, Pzp, or Pro.

[0300] In some implementations, X5 is Pro.

[0301] In some embodiments, the peptide ligand comprises the amino acid sequence shown in formula (EI-1-1):

[0302] R1-X 1-1 -X 1-2 -Arg-X 2-1 -X 2-2 -X 2-3 -X 2-4 -Val-X3-P3-X 4-1 -X 4-2 -Lys-X5-Tyr-P6-Leu formula (EI-1-1),

[0303] Among them, R1, X 1-1 X 1-2 X 2-1 X 2-2 X 2-3 X 2-4 X3, P3, X 4-1 X 4-2 X5 and P6 are as defined in any of the preceding items.

[0304] In some embodiments, the modifying group of the N-terminal amino group in R1 is selected from Ac, Cbz, Boc, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB, Bn, methoxycarbonyl, and ethoxycarbonyl.

[0305] In some embodiments, the N-terminal amino group in R1 is an Ac group.

[0306] In some implementations, A1 and A2 are each independently Cys or Pen.

[0307] In some implementations, A1 and A2 are each independently Cys.

[0308] In some implementations, P7 is Ser, Thr, Tyr, Trp, Cys, Thea, His, Sec, Pen, or is absent.

[0309] In some implementations, P7 is Ser, Thr, Tyr, Trp, Thea, or His.

[0310] In some implementations, P7 is Thr.

[0311] In some implementations, P8 is Asn, Glu, Cys, Pro, Arg, Lys, Cit, Orn, Dab, or Aha.

[0312] In some implementations, P8 is Arg, Cit, or Orn.

[0313] In some implementations, P8 is Arg.

[0314] In some implementations, P9 is Phe, Tyr, Trp, His, Phg, Tza, Thi, Pro, Arg, or Leu.

[0315] In some implementations, P9 is Phe, Tyr, Trp, or His.

[0316] In some implementations, P9 is Tyr.

[0317] In some implementations, the P 10 For Ala, Gly, Val, Ile, Leu, Pro, Nle, Abu, or Aib.

[0318] In some implementations, the P 10 It can be Ile, Abu, or Aib.

[0319] In some implementations, the P 10 For Ile.

[0320] In some implementations, B2 is Leu, Trp, Nle, or Aha.

[0321] In some implementations, B2 is Leu, Trp, or Nle.

[0322] In some implementations, B2 is Leu.

[0323] In some embodiments, X6 is Lys, Aha, Cit, Orn, Dab, Glu, Asn, Pro, Arg, Trp, Ser, Gly, Ile, Ala, or a combination thereof.

[0324] In some implementations, X7 is absent, Abu, Aib, Dbu, Dpr, Sar, tBuG, Ala, Gly, Ile, Leu, Pro, Arg, Val, or a combination thereof.

[0325] In some implementations, X8 is either absent or Tyr.

[0326] In some implementations, X8 is absent.

[0327] In some embodiments, X6 consists of 2 to 5 amino acid residues.

[0328] In some embodiments, X6 consists of 3 to 5 amino acid residues.

[0329] In some implementations, X6 is Aha-Ser-Aha, Aha-Ser-Cit, Aha-Ser-Orn, Aha-Ser-Lys, Aha-Ser-Asn, Cit-Ser-Aha, Cit-Ser-Lys, Dab-Ser-Cit, Dab-Ser-Dab, Dab-Ser-Orn, Dab-Ser-Lys, Dab-Ser-Asn, Dab-Ser-Arg, Orn-Ser-Aha, Orn-Ser-Lys, Glu-Ser-Glu, Glu-Ser-Ile, Gly-Ser-Lys, Gly-Ser-Ile, Lys-Ser-Gly , Lys-Ser-Ile, Lys-Lys-Ile, Lys-Ser-Lys, Lys-Ser-Asn, Lys-Ser-Arg, Lys-Ser-Aha, Lys-Ser-Cit, Lys-Ser-Dab, Lys-Ser-Orn, Asn-Ser-Aha, Asn-S er-Dab, Asn-Ser-Lys, Pro-Ser-Lys, Arg-Ser-Lys, Arg-Lys-Lys, Ser-Lys-Ile, Trp-Ser-Ile, Trp-Lys-Lys, Arg-Lys-Ala-Lys or Arg-Lys-Pro-Lys-Pro.

[0330] In some implementations, X6 is Gly-Ser-Lys, Lys-Ser-Lys, Arg-Lys-Lys, Trp-Lys-Lys, Pro-Ser-Lys, Arg-Lys-Ala-Lys, or Arg-Lys-Pro-Lys-Pro.

[0331] In some embodiments, X7 is absent or consists of 1 to 2 amino acid residues.

[0332] In some embodiments, X7 is a single amino acid residue.

[0333] In some implementations, X7 is Abu, Aib, Dbu, Dpr, Sar, tBuG, Ala, Gly, Ile, Leu, Pro, Arg, or Val.

[0334] In some implementations, X7 is Ile.

[0335] In some embodiments, the peptide ligand comprises the amino acid sequence shown in formula (E-II-1):

[0336] R2-A1-P7-X 6-1 -X 6-2 -X 6-3 -X 6-4 -X 6-5 -P8-P9-X7-A2-X8-P 10 -B2 formula (E-II-1),

[0337] Among them, R2, A1, P7, P8, P9, X7, A2, X8, P 10 B2 is as defined in any of the above items;

[0338] X 6-1 X 6-2 X 6-3 X 6-4 X 6-5 Independent chemical modifications selected from non-existent or natural amino acids, non-natural amino acids, or natural or non-natural amino acids.

[0339] In some implementations, the X 6-1 For Aha, Cit, Dab, Orn, Glu, Gly, Lys, Asn, Pro, Arg, Ser, or Trp.

[0340] In some implementations, the X 6-1 For Lys, Arg, or Trp.

[0341] In some implementations, the X 6-1For Lys or Arg.

[0342] In some implementations, the X 6-2 For Ser or Lys.

[0343] In some implementations, the X 6-2 For Ser.

[0344] In some implementations, the X 6-3 If it does not exist, it is Pro or Ala.

[0345] In some implementations, the X 6-3 For Pro or Ala.

[0346] In some implementations, the X 6-4 For Aha, Cit, Dab, Orn, Glu, Gly, Ile, Lys, Asn, or Arg.

[0347] In some implementations, the X 6-4 For Ile or Lys.

[0348] In some implementations, the X 6-4 For Lys.

[0349] In some implementations, the X 6-5 If it does not exist or is Pro.

[0350] In some embodiments, the peptide ligand comprises the amino acid sequence shown in formula (E-II-1-1):

[0351] R2-Cys-P7-X 6-1 -Ser-X 6-3 -X 6-4 -X 6-5 -P8-P9-X7-Cys-Ile-B2 formula (E-II-1-1),

[0352] Among them, R2, P7, X 6-1 X 6-3 X 6-4 X 6-5 P8, P9, X7, and B2 are as defined in any of the above items.

[0353] In some embodiments, the N-terminal amino group in R2 is selected from Ac, Cbz, Boc, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB, Bn, methoxycarbonyl, and ethoxycarbonyl.

[0354] In some embodiments, the N-terminal amino group in R2 is an Ac group.

[0355] In some embodiments, the formula (EI) peptide ligand is a linear peptide.

[0356] In some embodiments, the (E-II) peptide ligand is a cyclic peptide.

[0357] In some embodiments, the amino acids at positions A1 and A2 are covalently linked to form a cyclic peptide.

[0358] In some embodiments, a disulfide bond or diselenide bond is formed between the amino acid at position A1 and the amino acid at position A2 to form a cyclic peptide.

[0359] In this invention, when A1 and A2 are each independently selected from Cys or Pen, they can form disulfide bonds with any cysteine ​​in the polypeptide.

[0360] In this invention, when A1 and A2 are each independently selected from Sec, they form a cyclic peptide through a diselenide bond.

[0361] In some embodiments, the amino acid sequence of the peptide ligand is selected from any one of SEQ ID NO.1 to SEQ ID NO.234, and the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.234 are shown in Table II:

[0362] Table II

[0363] Furthermore, although described in this invention as a “peptide composed of a specific SEQ ID NO”, as long as the peptide has the same or corresponding activity as the peptide composed of the amino acid sequence of the corresponding SEQ ID NO, it does not exclude mutations that may occur by the addition of meaningless sequences upstream or downstream of the amino acid sequence of the corresponding SEQ ID NO, or naturally occurring mutations, or their silent mutations, and it is clearly within the scope of this invention even when sequence addition or mutation is present.

[0364] In some embodiments, the polypeptide-drug conjugate has a structure as shown in formula (II).

[0365] in:

[0366] E is as defined in any of the above items;

[0367] A' and A” are cytotoxic agents independently of each other;

[0368] Each wavy line in formula (II) indicates that L1 or L1' is covalently attached to a side chain residue or terminal group of E;

[0369] m and p are independent of each other and can be 0, 1, 2, 3, 4 or 5;

[0370] L1 and L1' are each independently selected from formula (L1-I):

[0371] in:

[0372] T 1 T 2 T 3 T 4 T 5 Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, -S(=NR t )-、-C(=NR t )-、-C(R t (=N)-、-P(R) t )-、-P(=O)(R t )-、C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups and natural or non-natural amino acids, wherein C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups, and native or non-native amino acids are each independently bounded by one or more R groups. t replace;

[0373] Each R t Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and OR. s -SR s -N(R) sa (R) sb -C(O)R s -CO2R s -C(O)C(O)R s -C(O)CH2C(O)R s -S(O)R s -S(O)2Rs C(O)N(R) sa (R) sb -SO2N(R) sa (R) sb -OC(O)R s -N(R)SO2R s C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are bound by one or more R groups. s replace;

[0374] Each R s Each R sa Each R sb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl;

[0375] b, c, d, e, and f are each independently 0, 1, or 2;

[0376] L2 and L2' are each independently a bond or selected from formula (L2-I):

[0377] in:

[0378] Each W is independently selected from -C(R) wa (R) wb )-、-N(R wx -C(=O)-、-C(=O)-N(R) wx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R wx)-, -O-, -S-, -S(=O)-, -SO2-, -P(R wx )-、-P(=O)(R wx )-、-N(R wx -SO2-, -SO2-N(R) wx -, -C(=S)-, -C(=NR) wx -, -N = N-, -C(=N2)-、C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups and natural or non-natural amino acids, wherein C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups, and native or non-native amino acids are each independently bounded by one or more R groups. wx replace;

[0379] Y is -(OCH2CH2) yn -O yp -;

[0380] Each Z is independently selected from -C(R) za (R) zb )-、-N(R zx -C(=O)-、-C(=O)-N(R) zx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R zx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R zx )-、-P(=O)(R zx )-、-N(R zx -SO2-, -SO2-N(R) zx -, -C(=S)-, -C(=NR) zx -, -N = N-, -C(=N2)-、C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are each independently bounded by one or more R groups. zx replace;

[0381] Each R wa Each Rwb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -S(O)R r -S(O)2R r -C(O)N(R) ra (R) rb -SO2N(R) ra (R) rb -OC(O)R r -N(R) ra SO2R rb C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl, the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Each cycloalkenyl group is independently bounded by one or more R groups. r replace;

[0382] Each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl;

[0383] yn is 0, 1, 2, 3, 4 or 5;

[0384] yp is 0 or 1;

[0385] g, h, and i are each independently selected from integers from 0 to 20;

[0386] The L3 and L3' are each independently selected from a bond or a peptide residue consisting of 2 to 7 natural or non-natural amino acids;

[0387] L4 and L4' are each independently a bond or selected from any of the structures shown in formulas (L4-1) to (L4-13):

[0388] in:

[0389] X is independently selected from O, S, sulfone, sulfoxide, carbonyl, and NR. xa alkylene and cycloalkyl;

[0390] R ua and R ub Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl;

[0391] Each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl;

[0392] Each n is independently selected from 0 or 1.

[0393] In some embodiments, the polypeptide-drug conjugate has a structure as shown in formula (II).

[0394] in:

[0395] E is as defined in any of the above items;

[0396] A' and A” are cytotoxic agents independently of each other;

[0397] Each wavy line in formula (II) indicates that L1 or L1' is covalently attached to a side chain residue or terminal group of E;

[0398] m and p are independent of each other and can be 0, 1, 2, 3, 4 or 5;

[0399] L1 and L1' are each independently selected from formula (L1-I):

[0400] in:

[0401] T 1 T 2 T 3 T 4 T 5 Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, -S(=NR t )-、-C(=NR t )-、-C(R t (=N)-、-P(R) t )-、-P(=O)(R t )-、C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are each independently bounded by one or more R groups. t replace;

[0402] Each R t Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and OR. s -SR s -N(R) sa (R) sb -C(O)R s -CO2R s -C(O)C(O)R s-C(O)CH2C(O)R s -S(O)R s -S(O)2R s C(O)N(R) sa (R) sb -SO2N(R) sa (R) sb -OC(O)R s -N(R)SO2R s C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are bound by one or more R groups. s replace;

[0403] Each R s Each R sa Each R sb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl;

[0404] b, c, d, e, and f are each independently 0, 1, or 2;

[0405] L2 and L2' are each independently a bond or selected from formula (L2-I):

[0406] in:

[0407] Each W is independently selected from -C(R) wa (R) wb )-、-N(R wx -C(=O)-、-C(=O)-N(R) wx)-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R wx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R wx )-、-P(=O)(R wx )-、-N(R wx -SO2-, -SO2-N(R) wx -, -C(=S)-, -C(=NR) wx -, -N = N-, -C(=N2)-、C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are each independently bounded by one or more R groups. wx replace;

[0408] Y is -(OCH2CH2) yn -O yp -;

[0409] Each Z is independently selected from -C(R) za (R) zb )-、-N(R zx -C(=O)-、-C(=O)-N(R) zx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R zx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R zx )-、-P(=O)(R zx )-、-N(R zx -SO2-, -SO2-N(R) zx -, -C(=S)-, -C(=NR) zx -, -N = N-, -C(=N2)-、C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are each independently bounded by one or more R groups. zx replace;

[0410] Each R wa Each R wbEach R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -S(O)R r -S(O)2R r -C(O)N(R) ra (R) rb -SO2N(R) ra (R) rb -OC(O)R r -N(R) ra SO2R rb C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl, the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Each cycloalkenyl group is independently bounded by one or more R groups. r replace;

[0411] Each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl;

[0412] yn is 0, 1, 2, 3, 4 or 5;

[0413] yp is 0 or 1;

[0414] g, h, and i are each independently selected from integers from 0 to 10;

[0415] The L3 and L3' are each independently selected from a bond or a peptide residue consisting of 2 to 7 natural or non-natural amino acids;

[0416] L4 and L4' are each independently a bond or selected from any of the structures shown in formulas (L4-1) to (L4-13):

[0417] in:

[0418] X is independently selected from O, S, sulfone, sulfoxide, carbonyl, and NR. xa alkylene and cycloalkyl;

[0419] R ua and R ub Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl;

[0420] Each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl;

[0421] Each n is independently selected from 0 or 1.

[0422] In some implementations, the T 1 The T 2 The T 3 The T 4 The T 5Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, -S(=NR t )-、-C(=NR t )-、-C(R t (=N)-、-P(R) t )-、-P(=O)(R t )-、C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. t replace.

[0423] In some implementations, the T 1 The T 2 The T 3 The T 4 The T 5 Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, S(=O)-, -SO2-, -C(=NR t )-、-C(R t (=N)-、C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. t replace.

[0424] In some implementations, each R tEach is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and OR. s -SR s -N(R) sa (R) sb -C(O)R s -CO2R s -C(O)C(O)R s -C(O)CH2C(O)R s -S(O)R s -S(O)2R s C(O)N(R) sa (R) sb -SO2N(R) sa (R) sb -OC(O)R s -N(R)SO2R s C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are bound by one or more R groups. s replace.

[0425] In some implementations, each R t Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, and OR. s -N(R) sa (R) sb -C(O)R s -CO2R s -C(O)C(O)R s -C(O)CH2C(O)R s C(O)N(R) sa (R) sb -OC(O)R s C 1-3 Alkyl, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C1-3 Alkyl, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are bound by one or more R groups. s replace.

[0426] In some implementations, L1 and L1' are each independently selected from any of the following structures:

[0427] Wherein, the R t As defined in any of the above items.

[0428] In some implementations, each R s Each R sa Each R sb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl.

[0429] In some implementations, each R s Each R sa Each R sb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -OH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -OC(O)H, C 1-3 Alkyl and C 3-6 Cycloalkyl.

[0430] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-、C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 3-6 cycloalkyl, C3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. wx replace.

[0431] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, C 3-6 cycloalkyl and 6-8 aryl, wherein C 3-6 Cycloalkyl and 6-8 aryl groups are bound by one or more R wx replace.

[0432] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-、C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. wx replace.

[0433] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, C 3-6 cycloalkyl and 6-8 aryl, wherein C 3-6 Cycloalkyl and 6-8 aryl groups are bound by one or more R wx replace.

[0434] In some implementations, each Z is independently selected from -C(R) za (R) zb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-、C 3-6 cycloalkyl, C 3-6Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. wx replace.

[0435] In some implementations, each Z is independently selected from -C(R) za (R) zb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, Or C 3-6 cycloalkyl, the C 3-6 cycloalkyl groups are formed by one or more R groups zx replace.

[0436] In some implementations, each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -OC(O)R r C 1-6 Alkyl and C 3-6 cycloalkyl, the C 1-6 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace.

[0437] In some implementations, each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, and -OR. r -N(R) ra(R) rb -C(O)R r C 1-3 Alkyl and C 3-6 cycloalkyl, the C 1-3 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace.

[0438] In some implementations, each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0439] In some implementations, each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -OH, -NH2, -C(O)H, C 1-3 Alkyl and C 3-6 Cycloalkyl.

[0440] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-、C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. wx replace;

[0441] Each Z is independently selected from -C(R) za (R) zb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-、C 3-6 cycloalkyl, C 3-6Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 3-6 cycloalkyl, C 3-6 Cycloalkenyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are each independently bound by one or more R groups. wx replace;

[0442] Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -OC(O)R r C 1-6 Alkyl and C 3-6 cycloalkyl, the C 1-6 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace;

[0443] Each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0444] In some implementations, each W is independently selected from -C(R) wa (R) wb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, C 3-6 cycloalkyl and 6-8 aryl, wherein C 3-6 Cycloalkyl and 6-8 aryl groups are bound by one or more R wx replace;

[0445] Each Z is independently selected from -C(R) za (R) zb )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, Or C 3-6 cycloalkyl, the C 3-6 cycloalkyl groups are formed by one or more R groups zx replace;

[0446] Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, and -OR. r -N(R) ra (R) rb -C(O)R r C 1-3 Alkyl and C 3-6 cycloalkyl, the C 1-3 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace;

[0447] Each R r Each R ra Each R rb Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -OH, -NH2, -C(O)H, C 1-3 Alkyl and C 3-6 Cycloalkyl.

[0448] In some embodiments, L3 and L3' are each independently selected from bonds, dipeptide residues consisting of natural or non-natural amino acids, tripeptide residues consisting of natural or non-natural amino acids, and tetrapeptide residues consisting of natural or non-natural amino acids.

[0449] In some embodiments, the peptide residues of L3 and L3' are amino acid residues formed from amino acids selected from phenylalanine, glycine, valine, citrulline, alanine, lysine, serine, glutamic acid, aspartic acid, and 1-aminocyclobutanecarboxylic acid.

[0450] In some embodiments, the peptide residues of L3 and L3' are amino acid residues formed from amino acids selected from phenylalanine, glycine, valine, citrulline, alanine, and 1-aminocyclobutanecarboxylic acid.

[0451] In some embodiments, L3 and L3' are each independently selected from a key or from any of the structures shown in formulas (L3-1) to (L3-6):

[0452] In some embodiments, each of X is independently selected from O, S, sulfone, sulfoxide, carbonyl, and NR. xa C 1-6 Alkylene and C 3-10 Cycloalkyl.

[0453] In some embodiments, each of X is independently selected from O, carbonyl, NR. xa C 1-3 Alkylene and C 3-6 Cycloalkyl.

[0454] In some embodiments, X is independently selected from carbonyl and NR. xa .

[0455] In some implementations, the R ua The R ub Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl.

[0456] In some implementations, the R ua The R ub Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl and C 3-6 Cycloalkyl.

[0457] In some implementations, the R ua The R ub Each is independently selected from hydrogen, deuterium, methyl, and ethyl.

[0458] In some implementations, each R xaEach is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl.

[0459] In some implementations, each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl and C 3-6 Cycloalkyl.

[0460] In some implementations, each R xa Each is independently selected from hydrogen, deuterium, methyl, and ethyl.

[0461] In some embodiments, A' and A" are each independently selected from paclitaxel or paclitaxel derivatives, salitoxin derivatives, doxorubicin or doxorubicin derivatives, camptothecin derivatives, or maytansin or maytansin analogs.

[0462] In some embodiments, A' and A” are each independently selected from any of the structures shown in formulas (A-1) to (A-21):

[0463] in:

[0464] R aa Selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0465] R ab Selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0466] R acSelected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0467] R ad Selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0468] R ae Selected from H, D, halogen, amino, alkyl, haloalkyl, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0469] In some implementations, the R aa Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0470] In some implementations, the R aa Selected from H, D, halogens, hydroxyl, carboxyl and C 1-6 alkyl.

[0471] In some implementations, the R aa Selected from carboxyl and C 1-3 alkyl.

[0472] In some implementations, the R aa Selected from methyl and carboxyl groups.

[0473] In some implementations, the R ab Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0474] In some implementations, the R ab Selected from H, D, halogen, hydroxyl, amino, carboxyl and C 1-6 alkyl.

[0475] In some implementations, the R ab Selected from H, D and hydroxyl groups.

[0476] In some implementations, the R ab Selected from H and hydroxyl groups.

[0477] In some implementations, the R ac Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0478] In some implementations, the R acSelected from H, D, halogens, hydroxyl groups and C 1-6 alkyl.

[0479] In some implementations, the R ac Selected from H, D and C 1-3 alkyl.

[0480] In some implementations, the R ac Selected from H and methyl;

[0481] In some implementations, the R ad Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0482] In some implementations, the R ad Selected from H, D, halogens, hydroxyl groups, C 1-6 Alkoxy group and 6-10 membered aryl group.

[0483] In some implementations, the R ad Selected from C 1-4 Alkoxy and 6-8 membered aryl.

[0484] In some implementations, the R ad Selected from phenyl and tert-butoxy.

[0485] In some implementations, the R ae Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0486] In some implementations, the R ae Selected from H, D, halogens, hydroxyl groups and C 1-6 alkyl.

[0487] In some implementations, the R ae Selected from H, D and C 1-3 alkyl.

[0488] In some implementations, the R ae Selected from H and methyl.

[0489] In some implementations, the R aa Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0490] The R ab Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0491] The R acSelected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0492] The R ad Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl;

[0493] The R ae Selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0494] In some implementations, the R aa Selected from H, D, halogen, hydroxyl, amino, carboxyl and C 1-6 alkyl;

[0495] The R ab Selected from H, D, halogen, hydroxyl, amino, carboxyl and C 1-6 alkyl;

[0496] The R ac Selected from H, D, halogens, hydroxyl groups and C 1-6 alkyl;

[0497] The R ad Selected from H, D, halogens, hydroxyl groups, C 1-6 Alkoxy and 6-10 membered aryl;

[0498] The R ae Selected from H, D, halogens, hydroxyl groups and C 1-6 alkyl.

[0499] In some implementations, the R aa Selected from carboxyl and C 1-3 alkyl;

[0500] The R ab Selected from H, D and hydroxyl groups;

[0501] The R ac Selected from H, D and C 1-3 alkyl;

[0502] The R ad Selected from C 1-4 Alkoxy and 6-8 membered aryl;

[0503] The R ae Selected from H, D and C 1-3 alkyl.

[0504] In some implementations, the R aa Selected from methyl and carboxyl groups;

[0505] The Rab Selected from H and hydroxyl groups;

[0506] The R ac Selected from H and methyl;

[0507] The R ad Selected from phenyl and tert-butoxy;

[0508] The R ae Selected from H and methyl.

[0509] In some embodiments, the polypeptide-drug conjugate is selected from:

[0510] On the other hand, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide-drug conjugate or its stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts as described in any of the preceding claims, as well as one or more pharmaceutically acceptable carriers, diluents or excipients.

[0511] The formulations of the compositions according to the invention can be prepared in a variety of ways by combining them with the pharmaceutically acceptable carriers described above. For example, for oral administration, the compositions can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, rice paper wafers, etc. For injections, the compositions can be formulated into single-dose ampoules or multi-dose containers. The compositions can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0512] In addition, the pharmaceutical compositions of the present invention can be prepared from any formulation type selected from tablets, pills, powders, granules, capsules, suspensions, oral liquid drugs, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations and suppositories.

[0513] In addition, the composition can be formulated into a unit dosage form suitable for the patient’s body, and preferably into a formulation useful for the conjugate drug according to typical methods in the pharmaceutical field, for administration via oral or parenteral routes, such as through the skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastric, local, sublingual, vaginal, or rectal routes, but is not limited thereto.

[0514] The total effective dose of the compositions of the present invention can be administered to the patient in a single dose or in multiple doses over a long period of time according to a fractionated treatment protocol. In the pharmaceutical compositions of the present invention, the content of one or more active ingredients can be varied according to the severity of the disease. Specifically, the total daily dose of the conjugates of the present invention can be from about 0.0001 mg to 500 mg per 1 kg of patient body weight. However, in addition to the route of administration and treatment frequency of the pharmaceutical composition, various factors including the patient's age, weight, health status, sex, disease severity, diet, and excretion rate are considered in determining the effective dose of the conjugates. In this respect, those skilled in the art can readily determine the effective dose suitable for the specific use of the pharmaceutical compositions of the present invention. The pharmaceutical compositions according to the present invention are not particularly limited in formulation and route of administration or method, as long as they exhibit the effects of the present invention.

[0515] On the other hand, the present invention provides the use of any of the preceding polypeptide-drug conjugates or their stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or any of the preceding pharmaceutical compositions, in the treatment and / or prevention of diseases or conditions overexpressing Sortilin, or in the preparation of medicaments for the prevention and / or treatment of diseases or conditions overexpressing Sortilin.

[0516] On the other hand, the present invention provides the use of any of the preceding polypeptide-drug conjugates or their stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts, or any of the preceding pharmaceutical compositions, in the treatment and / or prevention of cancer, or in the preparation of medicaments for the treatment and / or prevention of cancer.

[0517] In some embodiments, the cancers include adrenocortical carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, rhabdoid tumor, skin cancer, testicular cancer, thyroid cancer, and melanoma.

[0518] In some embodiments, the cancers include ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, colorectal cancer, and melanoma.

[0519] On the other hand, the present invention provides a method for treating and / or preventing cancer, comprising administering to a patient a therapeutically effective amount of a polypeptide-drug conjugate or its stereoisomers, tautomers, solvates, hydrates, prodrugs, stable isotope derivatives and pharmaceutically acceptable salts as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0520] In some embodiments, the cancers include adrenocortical carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, rhabdoid tumor, skin cancer, testicular cancer, thyroid cancer, and melanoma.

[0521] In some embodiments, the cancers include ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, colorectal cancer, and melanoma.

[0522] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0523] In the following embodiments,

[0524] "Room temperature" is not a specific temperature value, but refers to a temperature range of 10-30℃.

[0525] The structure of the compound was determined by mass spectrometry (MS).

[0526] MS determination was performed using (Agilent 6125B(ESI));

[0527] The HPLC determination was performed using an Agilent 1260DAD high-performance liquid chromatograph (Gemini-NX-C18 4.6×250mm, 5μm).

[0528] In this invention, some commonly used abbreviations have the following meanings:

[0529] Fmoc refers to fluorenylmethoxycarbonyl;

[0530] HBTU refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethylurea hexafluorophosphate;

[0531] DCM refers to dichloromethane;

[0532] DMF stands for N,N-dimethylformamide;

[0533] NMM refers to N-methylmorpholine;

[0534] TFA refers to trifluoroacetic acid;

[0535] EDT stands for 1,2-ethylenedithiol;

[0536] EDCI refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0537] MMAE refers to methylauratestatin E;

[0538] HOBT refers to 1-hydroxybenzotriazole;

[0539] DIEA refers to N,N-diisopropylethylamine;

[0540] EEDQ refers to 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline;

[0541] DCC refers to N,N'-dicyclohexylcarbodiimide;

[0542] HOSu refers to N-hydroxysuccinimide;

[0543] DSC refers to N,N' disuccinimide carbonate;

[0544] DIC stands for N,N'-diisopropylcarbodiimide.

[0545] TBTU refers to 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate.

[0546] Example 1: Method for preparing a polypeptide having the sequence SEQ ID NO: 92

[0547] A solid-phase organic synthesis method was adopted, utilizing the Fmoc-protected amino acid strategy and solid-phase synthesis technology to synthesize linear peptides, which were then cleaved and purified to obtain the target product.

[0548] Weigh 1 mmol of each of the amino acids with protecting groups required for synthesis and dissolve them in DMF solution (63.4 mL) to prepare a 0.3 mmol / mL amino acid mixed solution for later use.

[0549] Pour the Fmoc-amino acid-wangresin starting resin into the reaction column, add DCM and soak for 30 minutes, then dry. Add an appropriate amount of deprotection solution (20% hexahydropyridine + 80% DMF) to the reaction column, purge with nitrogen and stir for 30 minutes, then dry. Measure an appropriate amount of the next amino acid mixture solution or weigh the same amount of the protected amino acid Fmoc-Ile-OH, and weigh an appropriate amount of HBTU, for later use. Add an appropriate amount of DMF to the reaction column, purge with nitrogen for 2 minutes, then dry. Repeat this process 6 times. Add the prepared amino acid mixture solution or the protected amino acid and HBTU to the reaction column, then add 6 times the molar amount of NMM to the resin, and purge with nitrogen for 30 minutes. Dry the solution in the reaction column, add an appropriate amount of DMF to wash, purge with nitrogen for 2 minutes, then dry. Repeat this process 3 times. Take an appropriate amount (10-20 pieces) of resin in a small test tube, and add two drops each of buffer A (80% phenol + 20% anhydrous ethanol), buffer B (re-distilled pyridine), and buffer C (5 g ninhydrin + 100 mL anhydrous ethanol). Place the tube in a dry heater and heat for 3 minutes (110 °C). If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete, and the next amino acid can be ligated. Repeat the above steps until the last amino acid, Pro, is ligated. Then remove Fmoc and cap the ends with acetic anhydride Ac. Finally, wash with DMF and methanol, and dry the resin to obtain the resin peptide.

[0550] The dried resin was placed into a suitable round-bottom flask, and an appropriate amount of the prepared cutting fluid (1g / 10ml, 87.5% TFA + 5% anisole + 2.5% phenol + 2.5% EDT + 2.5% H2O) was added. The flask was then placed in a constant-temperature shaker at 25°C and shaken for 2 hours.

[0551] Filter the resin particles using a 50ml sintered glass funnel, then pour the filtrate into a 100ml centrifuge tube. Add 6-8 times the volume of anhydrous diethyl ether while stirring. After a white solid precipitates, seal the centrifuge tube and centrifuge at 4000 rpm for 3 minutes. Remove the tube, discard the supernatant, add more diethyl ether, stir well with a glass rod, and centrifuge again. Repeat this process 5 times.

[0552] The peptide, after being washed five times, was placed in a vacuum desiccator and dried under vacuum for 24 hours. The resulting white powder was the crude peptide, which was weighed and purified by preparative liquid chromatography-HPLC to obtain a pure peptide with the sequence SEQ ID NO: 92. The molecular weight was calculated to be 2137.19 by LCMS [(M+2) / 2].+ :1069.8.

[0553] The polypeptide synthesis method of Example 1 is applicable to the preparation of linear polypeptide molecules containing sequences of SEQ ID NO: 1 to SEQ ID NO: 108.

[0554] Example 2: Method for preparing a polypeptide having the sequence SEQ ID NO: 223

[0555] A solid-phase organic synthesis method was adopted, utilizing the Fmoc-protected amino acid strategy and solid-phase synthesis technology to synthesize linear peptides, which were then cleaved and cyclized to obtain cyclic peptides, and purified to obtain the target product.

[0556] Weigh 1 mmol of each of the amino acids with protecting groups required for synthesis and dissolve them in DMF solution (63.4 mL) to prepare a 0.3 mmol / mL amino acid mixed solution for later use.

[0557] Pour the Fmoc-amino acid-wangresin starting resin into the reaction column, add DCM and soak for 30 minutes, then dry. Add an appropriate amount of deprotection solution (20% hexahydropyridine + 80% DMF) to the reaction column, purge with nitrogen and stir for 30 minutes, then dry. Measure an appropriate amount of the next amino acid mixture solution or weigh the same amount of the protected amino acid Fmoc-Ile-OH, and weigh an appropriate amount of HBTU, for later use. Add an appropriate amount of DMF to the reaction column, purge with nitrogen for 2 minutes, then dry. Repeat this process 6 times. Add the prepared amino acid mixture solution or the protected amino acid and HBTU to the reaction column, then add 6 times the molar amount of NMM to the resin, and purge with nitrogen for 30 minutes. Dry the solution in the reaction column, add an appropriate amount of DMF to wash, purge with nitrogen for 2 minutes, then dry. Repeat this process 3 times. Take an appropriate amount (10-20 pieces) of resin in a small test tube, and add two drops each of buffer A (80% phenol + 20% anhydrous ethanol), buffer B (re-distilled pyridine), and buffer C (5 g ninhydrin + 100 mL anhydrous ethanol). Place the tube in a dry heater and heat for 3 minutes (110°C). If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete, and the next amino acid can be ligated. Repeat the above steps until the last amino acid (Cys) is ligated. Then remove the fmoc and cap the ends with acetic anhydride (Ac). Finally, wash with DMF and methanol, and dry the resin to obtain the resin peptide.

[0558] The dried resin was placed into a suitable round-bottom flask, and an appropriate amount of the prepared cutting fluid (1g / 10ml, 87.5% TFA + 5% anisole + 2.5% phenol + 2.5% EDT + 2.5% H2O) was added. The flask was then placed in a constant-temperature shaker at 25°C and shaken for 2 hours.

[0559] Filter the resin particles using a 50ml sintered glass funnel, then pour the filtrate into a 100ml centrifuge tube. Add 6-8 times the volume of anhydrous diethyl ether while stirring. Once a white solid precipitates, seal the centrifuge tube and centrifuge at 4000 rpm for 3 minutes. Remove the tube, discard the supernatant, add diethyl ether again to wash, stir well with a glass rod, and centrifuge again. Repeat this washing process 5 times.

[0560] The polypeptide, after being washed five times, was placed in a vacuum desiccator and dried under vacuum for 24 hours. The resulting white powder was the crude product of the desired polypeptide, which was weighed.

[0561] The crude peptide was dissolved in pure water (1 g / L), the pH was adjusted to 7.5-8.0, and stirred for 24 h. The pH was then adjusted to 7.0, and the solution was purified by preparative HPLC. The purified peptide with the sequence SEQ ID NO: 223 was obtained by preparative HPLC purification. The molecular weight was calculated to be 1366.71 by LCMS, [(M+2) / 2]. + :684.5.

[0562] The polypeptide synthesis method of Example 2 is applicable to the preparation of cyclic peptide molecules containing the sequences of SEQ ID NO: 109 to SEQ ID NO: 234.

[0563] Example 3: Method for preparing a polypeptide having the sequence SEQ ID NO: 220

[0564] The preparation method is the same as in Example 2.

[0565] LCMS analysis calculated the molecular weight to be 1295.63, [M / 2+H]. + :649.0.

[0566] Example 4: Method for preparing a polypeptide having the sequence SEQ ID NO: 225

[0567] The preparation method is the same as in Example 2.

[0568] LCMS analysis calculated the molecular weight to be 1295.63, [M / 2+H]. + :649.0.

[0569] Example 5: Method for preparing a polypeptide having the sequence SEQ ID NO: 228

[0570] The preparation method is the same as in Example 2.

[0571] LCMS analysis calculated the molecular weight to be 1435.78, [M / 2+H]. + :719.0.

[0572] Example 6: Method for preparing a polypeptide having the sequence SEQ ID NO: 231

[0573] The preparation method is the same as in Example 2.

[0574] LCMS analysis calculated the molecular weight to be 1629.88, [M / 2+H]. + :816.65.

[0575] Example 7: Method for preparing a polypeptide having the sequence SEQ ID NO: 233

[0576] The preparation method is the same as in Example 2.

[0577] LCMS analysis calculated the molecular weight to be 1310.64, [M+H]. + :1311.95.

[0578] Example 8: Preparation of Coupling 1

[0579] A polypeptide (10 mg, 1 eq) with the sequence SEQ ID NO: 92, INT1 (13.2 mg, 2.2 eq, from Shanghai WuXi AppTec New Drug Development Co., Ltd., ES27189-1), and N,N-diisopropylethylamine (5.2 μL, 4 eq) were added to a 200 μL DMF solution. The reaction mixture was stirred at 25 °C for 16 hours. LC-MS detected the target compound conjugate 1. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-performance liquid chromatography (HPLC). Lyophilization yielded conjugate 1 (10.8 mg) as a white solid. LC-MS analysis calculated the molecular weight to be 4602.67, [M / 3+H]. + :1535.7.

[0580] Example 9: Preparation of Coupling 2

[0581] A polypeptide (15 mg, 1 eq) with the sequence SEQ ID NO: 92, INT2 (19.5 mg, 2.1 eq, from Shanghai WuXi AppTec New Drug Development Co., Ltd., ES27189-2), and N,N-diisopropylethylamine (5.1 μL, 4 eq) were added to a DMF solution (500 μL). The reaction mixture was stirred at 25 °C for 16 hours. The target compound conjugate 2 was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-pressure preparative HPLC and lyophilized to obtain conjugate 2 (9.9 mg) as a white solid. The molecular weight was calculated to be 4768.75 by LC-MS [M / 3+H]. + :1591.3.

[0582] Example 10: Preparation of Coupling 3

[0583] first step:

[0584] A polypeptide (12.5 mg, 1 eq) with the sequence SEQ ID NO: 92, INT3 (20 mg, 2.1 eq, from Shanghai WuXi AppTec New Drug Development Co., Ltd., ES27189-4), and N,N-diisopropylethylamine (4 μL, 4 eq) were added to a DMF solution (500 μL). The reaction mixture was stirred at 25 °C for 16 hours. The target compound conjugate 3-1 was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-pressure preparative HPLC and lyophilized to obtain conjugate 3-1 (15.5 mg) as a white solid. The molecular weight was calculated to be 5260.8 by LC-MS [M / 4+H]. + :1316.4.

[0585] Step Two:

[0586] Conjugate 3-1 (18 mg, 1 eq) and LiOH (1 M, 113 μL, 10 eq) were added to a tetrahydrofuran solution (300 μL). The reaction mixture was stirred at 25 °C for 4 hours. The target compound, conjugate 3, was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-performance liquid chromatography (HPLC). Lyophilization yielded conjugate 3 (5.6 mg) as a white solid. LC-MS analysis calculated the molecular weight to be 5016.72, [M / 3+H]. + :1674.1.

[0587] Example 11: Preparation of INT-4

[0588] first step:

[0589] INT-4-1 (docetaxel) (600 mg, 0.74 mmol, from Shaoyuan Chemical Technology (Shanghai) Co., Ltd., T0186), succinic anhydride (111 mg, 1.11 mmol), and triethylamine (300 mg, 2.97 mmol) were added to a tetrahydrofuran solution (6 mL). The reaction mixture was stirred at 25 °C for 16 hours. TLC monitoring showed the disappearance of the reactants, and the reaction mixture was concentrated. It was then diluted with dichloromethane (200 mL), washed with dilute hydrochloric acid (0.5 M, 100 mL), and then washed with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate, then filtered and concentrated to obtain crude INT-4-2 (480 mg, crude product) as a pale yellow solid.

[0590] Step Two:

[0591] INT-4-2 (400 mg, 441 μmmol), N-hydroxysuccinimide (76.1 mg, 661 μmol), and EDCI (127 mg, 661 μmol) were added to a dichloromethane solution (4 mL). The reaction mixture was stirred at 25 °C for 16 hours. LC-MS detected the target product peak INT-4 as the main peak. The reaction mixture was concentrated, purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain INT-4 (160 mg, 159.2 μmol) as a white solid. LC-MS analysis determined the molecular weight to be 1004.38, [M+2H]. + :1006.5.

[0592] Example 12: Preparation of Coupling 4

[0593] INT-4 (20 mg, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 92 (21 mg, 1.0 eq), and N,N-diisopropylethylamine (3.5 μL, 2 eq) were added to DMSO solution (4 mL). The reaction mixture was stirred at 25 °C for 16 hours. The target compound conjugate 4 was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-performance liquid chromatography (HPLC). Lyophilization yielded conjugate 4 (5.6 mg) as a white solid. LC-MS analysis calculated the molecular weight to be 3915.89, [M / 2+H]. + :1959.9.

[0594] Example 13: Preparation of Coupling 5

[0595] INT-4 (40 mg, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (27.2 mg, 1.0 eq), and N,N-diisopropylethylamine (7 μL, 2 eq) were added to DMSO solution (4 mL). The reaction mixture was stirred at 25 °C for 16 hours. The target compound conjugate 5 was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by preparative HPLC and lyophilized to obtain conjugate 5 (13.3 mg) as a white solid. The molecular weight was calculated to be 3145.41 by LC-MS [M / 2+H]. + :1574.6.

[0596] Example 14: Preparation of intermediate INT-5

[0597] first step:

[0598] INT-5-1 (700 mg, 1.03 mmol, from Shanghai WuXi AppTec New Drug Development Co., Ltd., EC21914-7), MMAE (738 mg, 1.03 mmol), HOBT (166 mg, 1.23 mmol), and DIEA (199 mg, 1.54 mmol) were added to a DMF solution (7 mL). The reaction mixture was stirred at 25 °C for 3 hours. Then, TEA (508 mg, 5.03 mmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-5-2 (777 mg, 0.75 mmol). The molecular weight was calculated to be 1036.66 by LCMS [M+H]. + :1037.8.

[0599] Step Two:

[0600] Bis(2,5-dioxopyrrolidone-1-yl)succinate (602 mg, 1.93 mmol) and DIEA (50 mg, 0.385 mmol) were added to a 2 mL DMSO solution. Then, a 2 mL DMSO solution containing INT-5-2 (200 mg, 0.192 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred at 20 °C for 0.5 hours. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-5 (140 mg, 0.109 mmol) as a white solid. The molecular weight was calculated to be 1233.69 by LCMS [M+H]. + :1235.0.

[0601] Example 15: Preparation of Coupling 6

[0602] INT-5 (30 mg, 24.3 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (15.2 mg, 12.2 μmol, 1.0 eq), and N,N-diisopropylethylamine (8.47 μL, 48.6 μmol, 4 eq) were added to DMSO solution (0.3 mL). The reaction mixture was stirred at 25 °C for 4 hours. The target compound conjugate 6 was detected by LC-MS. The reaction mixture was filtered to remove insoluble matter, and the filtrate was purified by high-performance liquid chromatography (HPLC). Lyophilization yielded conjugate 6 (10.4 mg, 2.88 μmol) as a white solid. LC-MS analysis calculated the molecular weight to be 3604.04, [M / 3+H]. + :1203.2.

[0603] Example 16: Preparation of Coupling 7

[0604] INT-5 (28.2 mg, 22.9 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 233 (14.8 mg, 11.4 μmol, 1.0 eq), and N,N-diisopropylethylamine (5.97 μL, 34.3 μmol, 3 eq) were added to DMSO solution (0.1 mL). The reaction mixture was stirred at 25 °C for 2 hours. Isopropyl ether (15 mL) was added to the reaction mixture for recrystallization. The mixture was centrifuged and filtered to obtain a crude product, which was purified by preparative HPLC and lyophilized to obtain conjugate 7 (13.5 mg, 3.8 μmol) as a white solid. The molecular weight was calculated to be 3547.97 by LCMS [M / 3+H]. + :1184.4.

[0605] Example 17: Preparation of intermediate INT-6

[0606] first step:

[0607] INT-6-1 (2 g, 4.39 mmol, from Shanghai WuXi AppTec New Drug Development Co., Ltd., EC21914-11) was added to a DCM solution (2 mL). Then, a DCM solution (24 mL) containing EEDQ (2.17 g, 8.78 mmol) and 4-(tert-butoxy)-4-oxobutyric acid (1.15 g, 6.59 mmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 18 hours. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. The reaction mixture was washed with saturated sodium bicarbonate aqueous solution (100 mL) and then extracted with dichloromethane (50 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE 10%–30%) to obtain INT-6 (1.84 g, 3.01 mmol) as a white solid. LCMS analysis calculated the molecular weight to be 611.22, [M+H]. + :612.4.

[0608] Step Two:

[0609] INT-6-2 (2 g, 3.27 mmol), DIEA (0.845 g, 6.54 mmol), and phenyl p-nitrochloroformate (1.99 g, 6.54 mmol) were added to a DCM solution (20 mL). The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with water (200 mL) and then extracted with dichloromethane (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE 10%–30%) to give INT-6-3 (2.3 g, 2.87 mmol) as a white solid. The molecular weight was calculated to be 776.23 by LCMS, [M+Na]. + 799.5.

[0610] Step 3:

[0611] INT-6-3 (700 mg, 0.9 mmol), MMAE (647 mg, 0.9 mmol), HOBT (146 mg, 1.08 mmol), and DIEA (174 mg, 1.35 mmol) were added to a 7 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was then directly purified by high-performance liquid chromatography (HPLC) to obtain INT-6-4 (900 mg, 0.657 mmol). The molecular weight was calculated to be 1354.7 by LCMS [M+H]. + :1355.9.

[0612] Step 4:

[0613] INT-6-4 (400 mg, 0.295 mmol) and SnCl4 (1 M, 2.4 mL, 60%) were added to a DCM solution (4 mL). The reaction mixture was stirred at 25 °C for 0.5 h. The reaction mixture was diluted with water (50 mL) and then extracted with ethyl acetate (20 mL * 3). The organic phase was dried over anhydrous sodium sulfate, then filtered and concentrated to give INT-6-5 (480 g, crude product) as a white solid. LCMS analysis calculated the molecular weight to be 1298.64, [M+H]. + :1299.8.

[0614] Step 5:

[0615] INT-6-5 (460 mg, 0.353 mmol), DCC (146 mg, 0.707 mmol), and HOSu (81.5 mg, 0.707 mmol) were added to a DMF solution (0.46 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. The reaction mixture was filtered and purified directly by high-performance liquid chromatography (HPLC) to obtain INT-6 (253 mg, 0.181 mmol) as a white solid. LCMS analysis calculated the molecular weight to be 1395.66, [M+Na]. + :1419.4.

[0616] Example 18: Preparation of Coupling 8

[0617] first step:

[0618] INT-6 (110 mg, 78.8 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (54 mg, 39.4 μmol, 1.0 eq), and N,N-diisopropylethylamine (34.3 μL, 197 μmol, 5 eq) were added to 1.1 mL of DMSO solution. The reaction mixture was stirred at 25 °C for 3 hours. Isopropyl ether (15 mL) was added to the reaction mixture for recrystallization. The mixture was centrifuged and filtered to obtain the crude product, which was purified by preparative HPLC and lyophilized to obtain conjugate 8-1 (45.7 mg, 11.6 μmol) as a white solid. The molecular weight was calculated to be 3929.82 by LCMS [M / 3+H]. + :1311.5.

[0619] Step Two:

[0620] Coupling 8-1 (5 mg, 1.9 μmol, 1.0 eq) and lithium hydroxide monohydrate (37.9 μL, 37.9 μmol, 20 eq, 1 M) were added to a THF solution (50 μL). The reaction mixture was stirred at 0 °C for 1 hour. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 37.9 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 100 μL). The solution was purified by preparative HPLC and lyophilized to obtain coupling 8 (0.5 mg, 0.137 μmol) as a white solid. The molecular weight was calculated to be 3647.88 by LCMS [M / 3+H]. + :1218.4.

[0621] Example 19: Preparation of intermediate INT-7

[0622] first step:

[0623] INT-6-3 (700 mg, 0.9 mmol), Exatecan (479 mg, 0.9 mmol), HOBT (146 mg, 1.08 mmol), and DIEA (232 mg, 1.8 mmol) were added to a DMF solution (7 mL). The reaction mixture was stirred at 25 °C for 3 hours. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. Isopropyl ether (70 mL) was added to the reaction mixture, followed by filtration to obtain crude INT-7-1 (1.1 g, 0.85 mmol), which was used directly in the next step. LCMS analysis calculated the molecular weight to be 1072.36, [M+H]. + :1073.4.

[0624] Step Two:

[0625] INT-7-1 (800 mg, 0.618 mmol) and TFA (153 mg, 1.35 mmol, 100 μL) were added to a DCM solution (7 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the starting materials and the detection of the product peak. Isopropyl ether (70 mL) was added to the reaction mixture, and the mixture was stirred and filtered. The filtrate was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-7-2 (270 mg, 0.265 mmol) as a white solid. LCMS analysis determined the molecular weight to be 1016.30, [M+H]. + :1017.3.

[0626] Step 3:

[0627] INT-7-2 (270 mg, 0.265 mmol), DCC (109 mg, 0.531 mmol), and HOSu (61.1 mg, 0.531 mmol) were added to a 2.7 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. After filtration, the reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain a white solid of INT-7 (190 mg, 0.17 mmol). The molecular weight was calculated to be 1113.31 by LCMS [M+H]. + :1114.5.

[0628] Example 20: Preparation of Coupling 9

[0629] first step:

[0630] INT-7 (110 mg, 98.7 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (67.5 mg, 49.4 μmol, 1.0 eq), and N,N-diisopropylethylamine (43 μL, 247 μmol, 5 eq) were added to 1.1 mL of DMSO solution. The reaction mixture was stirred at 25 °C for 3 hours. The target compound conjugate 9-1 was detected by LC-MS. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, and filtered to obtain crude conjugate 9-1 (102 mg, 49.4 μmol), a brown oily product. LC-MS analysis calculated the molecular weight to be 3366.76, [M / 3+H]. + :1124.1.

[0631] Step Two:

[0632] Conjugate 9-1 (102 mg, 49.2 μmol, 1.0 eq) and lithium hydroxide monohydrate (985 μL, 985 μmol, 20 eq, 1 M) were added to a THF solution (1 mL). The reaction mixture was stirred at 0 °C for 1 hour. The target compound, conjugate 9, was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 985 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 9 (1.47 mg, 0.476 μmol) as a white solid. The molecular weight was calculated to be 3083.19 by LC-MS analysis, [M / 3+H]. + :1029.3.

[0633] Example 21: Preparation of Coupling 10

[0634] INT-4 (27.9 mg, 27.8 μmol, 1.2 eq), a peptide with the sequence SEQ ID NO: 220 (30 mg, 23.2 μmol, 1.0 eq), and N,N-diisopropylethylamine (12 mg, 16.1 μL, 4 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 10 (21.5 mg, 9.84 μmol) as a white solid. The molecular weight was calculated to be 2184.99 by LCMS [M / 2+H]. + :1093.9.

[0635] Example 22: Preparation of Coupling 11

[0636] INT-4 (30.8 mg, 30.6 μmol, 1 eq), a polypeptide with the sequence SEQ ID NO: 225 (39.7 mg, 30.6 μmol, 1.0 eq), and N,N-diisopropylethylamine (15.8 mg, 21.3 μL, 4 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 11 (21 mg, 9.59 μmol) as a white solid. The molecular weight was calculated to be 2184.99 by LCMS [M / 2+H]. + :1094.5.

[0637] Example 23: Preparation of intermediate INT-8

[0638] first step:

[0639] INT-5-1 (600 mg, 881 μmol, from Shanghai WuXi AppTec Co., Ltd.), MMAE (632 mg, 881 μmol), HOBT (142 mg, 1.06 mmol), and DIEA (227 mg, 1.76 mmol) were added to a 12 mL DMF solution. The reaction mixture was stirred at 25 °C for 3 hours. LCMS monitoring showed the disappearance of the starting materials and the detection of the product peak INT-8-1. LCMS analysis calculated the molecular weight to be 1258.7, [M+H]. + :1259.8. The reaction solution is used directly in the next reaction step.

[0640] Step Two:

[0641] TEA (2.18 g, 21.5 mmol) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 3 hours. Isopropyl ether (115 mL) was added to the reaction solution for recrystallization, followed by centrifugation and filtration. The solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-8-2 (405 mg, 385 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1036.6, [M+H]. + :1037.6.

[0642] Step 3:

[0643] Bis(2,5-dioxopyrrolidone-1-yl)3,3'-oxydipropionate (532 mg, 1.49 mmol) and DIEA (38.6 mg, 298 μmol) were added to a 7 mL DMF solution. Then, a 7 mL DMF solution containing INT-8-2 (155 mg, 149 μmol) was added to the reaction mixture. The reaction mixture was stirred at 0 °C for 10 minutes. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain a white solid of INT-8 (104 mg, 79.9 μmol). The molecular weight was calculated to be 1277.7 by LCMS [M+H]. + :1278.4.

[0644] Example 24: Preparation of Coupling 12

[0645] INT-8 (56.2 mg, 44.0 μmol, 2.4 eq), a peptide with the sequence SEQ ID NO: 223 (25.0 mg, 18.3 μmol, 1.0 eq), and N,N-diisopropylethylamine (11.8 mg, 91.4 μmol, 5 eq) were added to DMSO solution (0.1 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 12 (12 mg, 3.24 μmol) as a white solid. LCMS analysis determined the molecular weight to be 3692.09, [M / 3+H]. + :1232.3.

[0646] Example 25: Preparation of intermediate INT-9

[0647] Bis(2,5-dioxopyrrolidone-1-yl)3,3'-(ethane-1,2-diylbis(oxy))dipropionate (617 mg, 1.54 mmol) and DIEA (39.8 mg, 308 μmol) were added to a DMF solution (8 mL). Then, a DMF solution containing INT-8-3 (160 mg, 154 μmol) was added to the reaction mixture. The reaction mixture was stirred at 0 °C for 10 minutes. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-9 (89.1 mg, 65.5 μmol) as a white solid. LCMS analysis calculated the molecular weight to be 1321.7, [M+H]. + :1322.8.

[0648] Example 26: Preparation of Coupling 13

[0649] INT-9 (56.2 mg, 44.0 μmol, 2.4 eq), a polypeptide with the sequence SEQ ID NO: 223 (25.0 mg, 18.3 μmol, 1.0 eq), and N,N-diisopropylethylamine (11.8 mg, 91.4 μmol, 5 eq) were added to DMSO solution (0.1 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 13 (13 mg, 3.44 μmol) as a white solid. LCMS analysis determined the molecular weight to be 3780.14, [M / 3+H]. + :1261.7.

[0650] Example 27: Preparation of Coupling 14

[0651] INT-5 (51.4 mg, 41.7 μmol, 1.2 eq), a peptide with the sequence SEQ ID NO: 220 (45.0 mg, 34.7 μmol, 1.0 eq), and N,N-diisopropylethylamine (17.9 mg, 139 μmol, 4 eq) were added to DMSO solution (0.4 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 14 (14 mg, 5.79 μmol) as a white solid. The molecular weight was calculated to be 2414.30 by LCMS [M / 2+H]. + :1209.0.

[0652] Example 28: Preparation of intermediate INT-10

[0653] first step:

[0654] In a mixed solution of DCM and MeOH (DCM:MeOH = 3:1, 9 mL), INT-10-1 (1.00 g, 2.02 mmol, from Shanghai WuXi AppTec New Drug Development Co., Ltd., MD00130-1), 4-aminobenzyl alcohol (0.50 g, 4.04 mmol), and EEDQ (1.00 g, 2.02 mmol) were added sequentially. The reaction solution was stirred at 25 °C for 4.5 h. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. The reaction solution was washed with saturated sodium bicarbonate aqueous solution (100 mL) and then extracted with dichloromethane (50 mL * 3). The organic phase was dried over anhydrous sodium sulfate, then filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE 10%–50%) to obtain INT-10-2 (1.5 g, 2.29 mmol) as a yellow solid. LCMS analysis calculated the molecular weight to be 599.27, [M+H]. + :600.2.

[0655] Step Two:

[0656] INT-10-2 (1.5 g, 2.29 mmol), DIEA (0.59 g, 4.59 mmol), and di(p-nitrobenzene) carbonate (1.39 g, 4.59 mmol) were added to a DMF solution (10 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was diluted with water (200 mL) and then extracted with dichloromethane (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE 10%–50%) to give INT-10-3 (0.96 g, 1.22 mmol) as a white solid. The molecular weight was calculated to be 764.28 by LCMS analysis, [M+H]. + :765.0.

[0657] Step 3:

[0658] INT-10-3 (0.96 g, 1.22 mmol), MMAE (0.88 g, 1.22 mmol), HOBT (0.20 g, 1.46 mmol), and DIEA (0.31 g, 2.44 mmol) were added to a 12 mL DMF solution. The reaction mixture was stirred at 25 °C for 7 hours. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak INT-10-4. LCMS analysis calculated the molecular weight to be 1342.76, [M+H]. + 1344.9. The reaction solution is used directly in the next reaction step.

[0659] Step 4:

[0660] TEA (3.00 mL) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 13 hours. Isopropyl ether (115 mL) was added to the reaction solution for recrystallization, followed by centrifugation and filtration. The solution was purified by preparative HPLC and lyophilized to obtain INT-10-5 (1.13 g, 932 μmol) as a white solid. LCMS analysis calculated the molecular weight to be 1120.69, [M / 2+H]. + :561.3.

[0661] Step 5:

[0662] N,N'-(succinyldioxy)disuccinimide (525 mg, 1.65 mmol) and DIEA (85.1 mg, 660 μmol) were added to a DMSO solution (4 mL). Then, a DMSO solution containing INT-10-5 (400 mg, 330 μmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-10 (141 mg, 80.2 μmol) as a white solid. The molecular weight was calculated to be 1317.72 by LCMS [M+H]. + :1319.0.

[0663] Example 29: Preparation of Coupling 15

[0664] INT-10 (67.5 mg, 51.2 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (25.0 mg, 18.3 μmol, 1.0 eq), and N,N-diisopropylethylamine (9.46 mg, 73.2 μmol, 4 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 15 (11 mg, 2.91 μmol) as a white solid. The molecular weight was calculated to be 3772.10 by LCMS [M / 3+H]. + :1259.2.

[0665] Example 30: Preparation of Intermediate 11

[0666] first step:

[0667] INT-11-1 (500 mg, 1.60 mmol), MMAE (1.04 g, 1.44 mmol), DIEA (414 mg, 3.2 mmol), and HATU (730 mg, 1.92 mmol) were added to a 5 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the starting materials and the detection of the product peak INT-11-2. LCMS analysis calculated the molecular weight to be 1010.61, [M+H]. + :1011.5. The reaction solution is used directly in the next reaction step.

[0668] Step Two:

[0669] TEA (908 mg, 8.98 mmol) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 4 hours. The reaction solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-11-3 (1.13 g, 1.25 mmol) as a white solid. The molecular weight was calculated to be 788.54 by LCMS [M+H]. + :789.5.

[0670] Step 3:

[0671] INT-11-3 (1.13 g, 1.25 mmol), Fmoc-L-valine (473 mg, 1.39 mmol), DIEA (540 mg, 4.18 mmol), and HATU (636 mg, 1.67 mmol) were added to a DMF solution (10 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak INT-11-4. The reaction mixture was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain a white solid of INT-11-4 (1.01 g, 773 μmol). LCMS analysis determined the molecular weight to be 1109.68, [M+H]. + :1110.7.

[0672] Step 4:

[0673] INT-11-4 (500 mg, 450 μmol) and TEA (1.82 g, 17.9 mmol) were added to 10 mL of DMF solution, and the reaction mixture was stirred at 25 °C for 4 hours. The reaction mixture was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-11-5 (466 mg, 465 μmol) as a white solid. The molecular weight was calculated to be 887.61 by LCMS [M+H]. + :888.6.

[0674] Step 5:

[0675] N,N'-(succinyldioxy)disuccinimide (1.05 g, 3.38 mmol) and DIEA (87.3 mg, 675 μmol) were added to 1.5 mL of DMSO solution. Then, 1 mL of DMSO solution containing INT-11-5 (300 mg, 337 μmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-11 (243 mg, 211 μmol) as a white solid. The molecular weight was calculated to be 1084.64 by LCMS [M+H]. + :1085.7.

[0676] Example 31: Preparation of Coupling 16

[0677] INT-10 (57 mg, 47.4 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (30.0 mg, 23.7 μmol, 1.0 eq), and N,N-diisopropylethylamine (18.4 mg, 143 μmol, 3 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 16 (14 mg, 3.43 μmol) as a white solid. LCMS analysis determined the molecular weight to be 3305.94, [M / 3+H]. + :1103.7.

[0678] Example 32: Preparation of Coupling 17

[0679] first step:

[0680] DSC (70.2 mg, 274 μmol, 2.5 eq), a peptide with the sequence SEQ ID NO:223 (150 mg, 120 μmol, 1.0 eq), and N,N-diisopropylethylamine (42.5 mg, 329 μmol, 3 eq) were added to a 1.5 mL DMF solution. The reaction mixture was stirred at 0 °C for 1 hour. The target compound conjugate 17-1 was detected by LC-MS. The reaction mixture was purified by preparative HPLC and lyophilized to obtain conjugate 17-1 (12.6 mg, 7.64 μmol) as a white solid. The molecular weight was calculated to be 1648.72 by LC-MS [M / 2+H]. + :825.8.

[0681] Step Two:

[0682] INT-8-3 (16.6 mg, 16.0 μmol, 2.1 eq), coupling compound 17-1 (12.6 mg, 7.64 μmol, 1.0 eq), and N,N-diisopropylethylamine (1.98 mg, 15.3 μmol, 2 eq) were added to a DMF solution (0.15 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain coupling compound 17 (10.1 mg, 2.89 μmol) as a white solid. LCMS analysis determined the molecular weight to be 3491.98, [M / 3+H]. + :1165.9.

[0683] Example 33: Preparation of Coupling 18

[0684] INT-5 (22.0 mg, 18.1 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 92 (18.4 mg, 8.60 μmol, 1.0 eq), and N,N-diisopropylethylamine (4.49 μL, 25.8 μmol, 3 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The target compound conjugate 18 was detected by LC-MS. The reaction mixture was purified by preparative HPLC and lyophilized to obtain conjugate 18 (14 mg, 3.20 μmol) as a white solid. The molecular weight was calculated to be 4374.51 by LC-MS [M / 3+H]. + :1459.8.

[0685] Example 34: Preparation of Intermediate 12

[0686] first step:

[0687] INT-12-1 (1.50 g, 2.06 mmol, from Shanghai WuXi AppTec New Drug Development Co., Ltd., MD00139-10), Exatecan (1.00 g, 1.85 mmol), HOBT (306 mg, 2.26 mmol), DIEA (477 mg, 3.70 mmol), and DIC (466 mg, 3.70 mmol) were added to a DMF solution (15 mL). The reaction mixture was stirred at 25 °C for 18 hours. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. The reaction mixture was recrystallized with isopropyl ether (70 mL), centrifuged, filtered, purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain INT-12-2 (0.9 g, 0.84 mmol) as a yellow solid. LCMS analysis calculated the molecular weight to be 1062.39, [M+H]. + :1063.4.

[0688] Step Two:

[0689] INT-12-2 (0.9 g, 820 μmol) and TEA (1.82 g, 17.9 mmol) were added to a DMF solution (10 mL), and the reaction mixture was stirred at 25 °C for 3 hours. The reaction mixture was recrystallized with isopropyl ether (70 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain INT-12-3 (323 mg, 373 μmol) as a yellow solid. The molecular weight was calculated to be 840.32 by LCMS [M+H]. + :841.3.

[0690] Step 3:

[0691] N,N'-(succinyldioxy)disuccinimide (267 mg, 837 μmol) and DIEA (55.4 μL, 335 μmol) were added to 13 mL of DMSO solution. Then, 2 mL of DMSO solution containing INT-12-3 (150 mg, 167 μmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 0.5 hours. After filtration, the reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain a white solid of INT-12 (87 mg, 44.4 μmol). The molecular weight was calculated to be 1037.36 by LCMS [M+H]. + :1037.8.

[0692] Example 35: Preparation of Coupling 19

[0693] INT-12 (34.0 mg, 15.9 μmol, 2.0 eq), a polypeptide with the sequence SEQ ID NO: 223 (10.0 mg, 7.95 μmol, 1.0 eq), and N,N-diisopropylethylamine (3.33 mg, 25.8 μmol, 3 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was recrystallized with isopropyl ether (15 mL), centrifuged, filtered, purified by preparative HPLC, and lyophilized to obtain conjugate 19 (4.3 mg, 1.34 μmol) as a white solid. The molecular weight was calculated to be 3211.37 by LCMS [M / 2+H]. + :1607.6.

[0694] Example 36: Preparation of Coupling 20

[0695] INT-5 (28.4 mg, 22.9 μmol, 2.2 eq), a peptide with the sequence SEQ ID NO: 228 (15.0 mg, 10.4 μmol, 1.0 eq), and N,N-diisopropylethylamine (5.40 mg, 41.8 μmol, 4 eq) were added to a DMF solution (0.4 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 20 (11.0 mg, 2.99 μmol) as a white solid. The molecular weight was calculated to be 3673.10 by LCMS [M / 3+H]. + :1226.1.

[0696] Example 37: Preparation of Coupling 21

[0697] INT-5 (24.9 mg, 20.2 μmol, 2.2 eq), a peptide with the sequence SEQ ID NO: 231 (15.0 mg, 9.19 μmol, 1.0 eq), and N,N-diisopropylethylamine (4.75 mg, 36.7 μmol, 4 eq) were added to a DMF solution (0.4 mL). The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was directly purified by preparative HPLC and lyophilized to obtain conjugate 21 (13.0 mg, 3.36 μmol) as a white solid. The molecular weight was calculated to be 3867.21 by LCMS [M / 3+H]. + :1290.9.

[0698] Example 38: Preparation method of polypeptide P-1 having the sequence SEQ ID NO: 233

[0699] The preparation method is the same as in Example 2.

[0700] LCMS analysis calculated the molecular weight to be 1588.78, [M+H]. + :1589.80.

[0701] Example 39: Preparation method of polypeptide P-2 having the sequence SEQ ID NO: 233

[0702] The preparation method is the same as in Example 2.

[0703] LCMS analysis calculated the molecular weight to be 1588.78, [M+H]. + :1589.80.

[0704] Example 40: Preparation of Coupling 22

[0705] first step:

[0706] INT-5 (46.6 mg, 37.7 μmol, 1.0 eq), peptide P-1 (60.0 mg, 37.7 μmol, 1.0 eq) with the sequence SEQ ID NO: 223, and N,N-diisopropylethylamine (7.32 mg, 56.6 μmol, 1.5 eq) were added to a DMF solution (0.6 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the starting materials and the detection of the peak of conjugate 22-1. The molecular weight was calculated to be 2707.44 [M / 2+H] by LCMS. + :1355.0, the reaction solution is used directly in the next step.

[0707] Step Two:

[0708] TEA (0.15 mL) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 3 hours. The reaction solution was purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain conjugate 22-2 (9.6 mg, 3.86 μmol) as a white solid. The molecular weight was calculated to be 2485.37 by LCMS [M / 2+H]. + :1243.9.

[0709] Step 3:

[0710] INT-12 (10.3 mg, 9.92 μmol, 2.56 eq), coupling compound 22-2 (9.6 mg, 3.86 μmol, 1.0 eq), and N,N-diisopropylethylamine (1.50 mg, 11.6 μmol, 3 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain coupling compound 22 (7.00 mg, 2.05 μmol) as a white solid. The molecular weight was calculated to be 3407.70 by LCMS [M / 3+H]. + :1137.4.

[0711] Example 41: Preparation of Coupling 23

[0712] INT-4 (30.7 mg, 30.6 μmol, 2.00 eq), a peptide with the sequence SEQ ID NO: 231 (25.0 mg, 15.3 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.96 mg, 30.6 μmol, 2 eq) were added to a DMF solution (0.1 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 23 (10.00 mg, 2.93 μmol) as a white solid. The molecular weight was calculated to be 3408.59 by LCMS [M / 2+H]. + :1707.2.

[0713] Example 42: Preparation of Coupling 24

[0714] INT-10 (34.0 mg, 19.3 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (15.0 mg, 9.2 μmol, 1.00 eq), and N,N-diisopropylethylamine (12.0 mg, 92.0 μmol, 10 eq) were added to a DMF solution (0.15 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 24 (10.3 mg, 2.55 μmol) as a white solid. The molecular weight was calculated to be 4035.27 by LCMS [M / 3+H].+ :1347.2.

[0715] Example 43: Preparation of Intermediate 13

[0716] N,N'-(succinyldioxy)disuccinimide (556 mg, 1.78 mmol) and DIEA (118 μL, 712 μmol) were added to an 8 mL DMF solution. Then, a 2 mL DMF solution containing INT-13-1 (400 mg, 356 μmol, from Shanghai WuXi AppTec Co., Ltd., Wuxi: VC-PABc-MMAE) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 0.5 hours. After filtration, the reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-13 (241.7 mg, 183 μmol) as a white solid. The molecular weight was calculated to be 1319.74 by LCMS [M+H]. + :1320.8.

[0717] Example 44: Preparation of Coupling 25

[0718] INT-13 (17.0 mg, 12.9 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (10.0 mg, 6.13 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.96 mg, 30.6 μmol, 5 eq) were added to a DMF solution (0.1 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 25 (13.0 mg, 3.21 μmol) as a white solid. The molecular weight was calculated to be 4039.31 by LCMS [M / 3+H]. + :1348.4.

[0719] Example 45: Preparation of Intermediate 14

[0720] first step:

[0721] Terephthalic acid (100 mg, 601 μmmol), N-hydroxysuccinimide (173 mg, 1.51 mmol), and EDCI (288 mg, 1.51 mmol) were added to a DMF solution (1 mL). The reaction mixture was stirred at 25 °C for 16 hours. LCMS detected the main peak as the target product peak INT-14-1. The reaction mixture was diluted with water (200 mL) and then extracted with dichloromethane (50 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA / PE 0%–80%) to obtain INT-14-1 (35 mg, 97.1 μmol) as a white solid. LCMS analysis calculated the molecular weight to be 360.06, [M + Na]. + :383.0.

[0722] Step Two:

[0723] INT-14-1 (35.0 mg, 97.1 μmol) and DIEA (8.84 mg, 68.4 μmol) were added to a DMF solution (0.3 mL). Then, a DMF solution (2 mL) containing INT-5-2 (35.0 mg, 34.2 μmol) was added to the reaction mixture. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-14 (16.0 mg, 11.9 μmol) as a white solid. The molecular weight was calculated to be 1281.69 by LCMS [M+H]. + :1284.1.

[0724] Example 46: Preparation of Coupling 26

[0725] INT-14 (16.0 mg, 11.9 μmol, 2.2 eq), a peptide with the sequence SEQ ID NO: 231 (9.0 mg, 5.51 μmol, 1.00 eq), and N,N-diisopropylethylamine (1.42 mg, 11.0 μmol, 2 eq) were added to a DMF solution (0.1 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 26 (11.0 mg, 2.74 μmol) as a white solid. The molecular weight was calculated to be 3963.21 by LCMS [M / 3+H]. + :1322.8.

[0726] Example 47: Preparation of Intermediate 15

[0727] first step:

[0728] Monomethyl malonate (102 mg, 867 μmol), INT-5-2 (600 mg, 578 μmol), N,N-diisopropylethylamine (224 mg, 1.74 mmol), and EDCI (166 mg, 867 μmol) were added to a DMF solution (6 mL). The reaction mixture was stirred at 25 °C for 1 hour. LC-MS analysis revealed the target product peak, INT-15-1, as the main peak. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-15-1 (382 mg, 336 μmol) as a white solid. LC-MS analysis calculated the molecular weight to be 1136.67, [M+H]. + :1137.7.

[0729] Step Two:

[0730] INT-15-1 (413 mg, 363 μmol, 1.0 eq) and lithium hydroxide monohydrate (726 μL, 726 μmol, 2 eq, 1 M) were added to a THF solution (4 mL). The reaction mixture was stirred at 25 °C for 0.5 h. The target compound INT-15-2 was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 985 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-15-2 (325.8 mg, 290 μmol) as a white solid. The molecular weight was calculated to be 1122.66 by LC-MS analysis, [M+H]. + :1123.7.

[0731] Step 3:

[0732] INT-15-2 (325.8 mg, 290 μmol), N-hydroxysuccinimide (100 mg, 0.87 mmol), and EDCI (249.9 mg, 1.31 mmol) were added to a DCM solution (3 mL). The reaction mixture was stirred at 25 °C for 16 hours, and LC-MS detected the target product peak INT-15 as the main peak. The reaction mixture was diluted with water (20 mL) and then extracted with dichloromethane (5 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by high-performance liquid chromatography (HPLC) to obtain INT-15 (159 mg, 130 μmol) as a white solid. LC-MS analysis showed a molecular weight of 1219.67, [M+H]. + :1220.8.

[0733] Example 48: Preparation of Coupling 27

[0734] INT-15 (58.0 mg, 38.4 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (25.0 mg, 18.3 μmol, 1.00 eq), and N,N-diisopropylethylamine (11.8 mg, 91.5 μmol, 5 eq) were added to a DMF solution (0.25 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 27 (10.5 mg, 2.93 μmol) as a white solid. The molecular weight was calculated to be 3576.00 by LCMS [M / 3+H]. + :1194.0.

[0735] Example 49: Preparation of Intermediate 16

[0736] Disuccinimidyl glutarate (315.0 mg, 964 μmol) and DIEA (74.8 mg, 578 μmol) were added to a 2.0 mL DMF solution. Then, a 1 mL DMF solution containing INT-5-2 (200.0 mg, 193 μmol) was added to the reaction mixture. The reaction mixture was stirred at 0 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-16 (163.0 mg, 131 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1247.71, [M+H]. + :1249.9.

[0737] Example 50: Preparation of Coupling 28

[0738] INT-16 (38.3 mg, 30.7 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (20.0 mg, 14.6 μmol, 1.00 eq), and N,N-diisopropylethylamine (9.45 mg, 73.1 μmol, 5 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 28 (19.0 mg, 5.23 μmol) as a white solid. The molecular weight was calculated to be 3632.07 by LCMS [M / 3+H]. + :1212.7.

[0739] Example 51: Preparation of Coupling 29

[0740] INT-15 (48.6 mg, 32.1 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (25.0 mg, 15.3 μmol, 1.00 eq), and N,N-diisopropylethylamine (9.87 mg, 76.5 μmol, 5 eq) were added to a DMF solution (0.25 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 29 (11.0 mg, 2.86 μmol) as a white solid. The molecular weight was calculated to be 3839.18 by LCMS [M / 3+H]. + :1281.5.

[0741] Example 52: Preparation of Coupling 30

[0742] INT-16 (32.1 mg, 25.7 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (20.0 mg, 12.3 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.17 mg, 24.6 μmol, 2 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 30 (11.0 mg, 2.82 μmol) as a white solid. The molecular weight was calculated to be 3895.24 by LCMS [M / 3+H]. + :1300.4.

[0743] Example 53: Preparation of Intermediate 17

[0744] first step:

[0745] Monomethyl trans-1,3-cyclobutanedicarboxylate (16.8 mg, 106 μmol), INT-5-2 (100 mg, 96.4 μmol), N,N-diisopropylethylamine (18.7 mg, 145 μmol), and HATU (44.0 mg, 116 μmol) were added to a DMF solution (1 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS detected the main peak as the target product peak, INT-17-1. The reaction mixture was diluted with water (10 mL) and then extracted with dichloromethane (5 mL * 3). The organic phase was dried over anhydrous sodium sulfate, then filtered and concentrated to obtain crude INT-17-1 (155 mg), which was directly used in the next reaction. LCMS analysis showed a molecular weight of 1176.70, [M+H]. + :1178.1.

[0746] Step Two:

[0747] INT-15-1 (114 mg, 96.4 μmol, 1.0 eq) and lithium hydroxide monohydrate (193 μL, 193 μmol, 2 eq, 1 M) were added to a THF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The target compound INT-17-2 was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 200 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain a white solid of INT-17-2 (78.2 mg, 67.2 μmol). The molecular weight was calculated to be 1162.69 by LC-MS analysis. [M+H] + :1164.2.

[0748] Step 3:

[0749] INT-17-2 (78.2 mg, 67.2 μmol), N-hydroxysuccinimide (23.2 mg, 202 μmol), and EDCI (51.5 mg, 269 μmol) were added to a DMF solution (0.8 mL). The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS detected the target product peak INT-17 as the main peak. The reaction mixture was diluted with water (20 mL) and then extracted with dichloromethane (5 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by high-performance liquid chromatography (HPLC) to obtain INT-17 (54.3 mg, 43.1 μmol) as a white solid. LC-MS analysis showed a molecular weight of 1259.71, [M+H]. + :1261.9.

[0750] Example 54: Preparation of Coupling 31

[0751] INT-17 (24.3 mg, 19.3 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (15.0 mg, 9.20 μmol, 1.00 eq), and N,N-diisopropylethylamine (5.34 mg, 41.4 μmol, 4.5 eq) were added to a DMF solution (0.15 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 31 (16.0 mg, 4.079 μmol) as a white solid. The molecular weight was calculated to be 3919.24 by LCMS [M / 3+H]. + :1308.4.

[0752] Example 55: Preparation of Intermediate 18

[0753] first step:

[0754] Monomethyl trans-1,4-cyclohexanedicarboxylate (43.1 mg, 231 μmol), INT-5-2 (200 mg, 192 μmol), N,N-diisopropylethylamine (37.3 mg, 289 μmol), and HATU (87.9 mg, 231 μmol) were added to a 2 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. LC-MS analysis showed that the main peak was the target product peak, INT-18-1. The reaction mixture was diluted with water (10 mL) and then extracted with dichloromethane (5 mL x 3). The organic phase was dried over anhydrous sodium sulfate, then filtered and concentrated to obtain crude INT-18-1 (290 mg), which was directly used in the next reaction. LC-MS analysis showed a molecular weight of 1204.70, [M+H]. + :1207.1.

[0755] Step Two:

[0756] INT-18-1 (290 mg, 240 μmol, 1.0 eq) and lithium hydroxide monohydrate (962 μL, 962 μmol, 4 eq, 1 M) were added to a 2 mL THF solution. The reaction mixture was stirred at 25 °C for 4 hours. The target compound INT-18-2 was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 200 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by preparative HPLC and lyophilized to obtain a white solid of INT-18-2 (138 mg, 112 μmol). The molecular weight was calculated to be 1190.72 by LC-MS analysis. [M+H] + :1193.0.

[0757] Step 3:

[0758] INT-18-2 (138 mg, 112 μmol), N-hydroxysuccinimide (19.9 mg, 173 μmol), and EDCI (33.3 mg, 173 μmol) were added to a DMF solution (2 mL). The reaction mixture was stirred at 25 °C for 1 hour, and LC-MS detected the target product peak INT-18 as the main peak. The reaction mixture was diluted with water (20 mL) and then extracted with dichloromethane (5 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by high-performance liquid chromatography (HPLC) to obtain INT-18 (88.0 mg, 67.6 μmol) as a white solid. LC-MS analysis showed a molecular weight of 1287.74, [M+H]. + :1288.5.

[0759] Example 56: Preparation of Coupling 32

[0760] INT-18 (33.2 mg, 25.7 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (20.0 mg, 12.30 μmol, 1.00 eq), and N,N-diisopropylethylamine (4.75 mg, 36.8 μmol, 3 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 32 (12.0 mg, 3.016 μmol) as a white solid. The molecular weight was calculated to be 3975.30 by LCMS [M / 3+H]. + :1327.0.

[0761] Example 57: Preparation of Coupling 33

[0762] INT-17 (29 mg, 23.0 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (15.0 mg, 11.0 μmol, 1.00 eq), and N,N-diisopropylethylamine (5.67 mg, 43.9 μmol, 4 eq) were added to a DMF solution (0.15 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 33 (17.0 mg, 4.65 μmol) as a white solid. The molecular weight was calculated to be 3656.07 by LCMS [M / 3+H]. + :1220.4.

[0763] Example 58: Preparation of Coupling 34

[0764] INT-14 (30.9 mg, 24.1 μmol, 2.2 eq), a peptide with the sequence SEQ ID NO: 223 (18.0 mg, 10.9 μmol, 1.00 eq), and N,N-diisopropylethylamine (7.08 mg, 54.8 μmol, 5 eq) were added to a DMF solution (0.2 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 34 (10.5 mg, 2.59 μmol) as a white solid. The molecular weight was calculated to be 3700.04 by LCMS [M / 3+H]. + :1235.0.

[0765] Example 59: Preparation of Coupling 35

[0766] INT-13 (60.9 mg, 46.0 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (30.0 mg, 22.0 μmol, 1.00 eq), and N,N-diisopropylethylamine (17.0 mg, 132 μmol, 6 eq) were added to a DMF solution (0.15 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 35 (10.0 mg, 2.64 μmol) as a white solid. The molecular weight was calculated to be 3776.13 by LCMS [M / 3+H]. + :1260.8.

[0767] Example 60: Preparation of Coupling 36

[0768] INT-18 (59.3 mg, 46.1 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 223 (30.0 mg, 22.0 μmol, 1.00 eq), and N,N-diisopropylethylamine (11.3 mg, 87.7 μmol, 6 eq) were added to a DMF solution (0.30 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 36 (15.1 mg, 3.76 μmol) as a white solid. The molecular weight was calculated to be 3712.13 by LCMS [M / 3+H]. + :1239.4.

[0769] Example 61: Preparation of Coupling 37

[0770] first step:

[0771] INT-5 (30.0 mg, 24.3 μmol, 1.00 eq), peptide P-2 (45.0 mg, 24.3 μmol, 1.00 eq) with the sequence SEQ ID NO: 223, and N,N-diisopropylethylamine (20 μL, 121 μmol, 5 eq) were added to a DMF solution (0.45 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the starting materials and detection of the conjugate peak 37-1. The calculated molecular weight by LCMS was 2970.62, [M / 2+H]. + :1486.8, the reaction solution is used directly in the next step.

[0772] Step Two:

[0773] TEA (0.12 mL) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 5 hours. The reaction solution was purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain conjugate 37-2 (33.6 mg, 12.2 μmol) as a white solid. The molecular weight was calculated to be 2748.55 by LCMS [M / 2+H]. + :1375.6.

[0774] Step 3:

[0775] INT-12 (32.5 mg, 31.4 μmol, 2.00 eq), coupling compound 37-2 (33.6 mg, 12.2 μmol, 1.00 eq), and N,N-diisopropylethylamine (8.07 μL, 48.9 μmol, 4.00 eq) were added to a DMF solution (0.33 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain coupling compound 37 (17.00 mg, 4.63 μmol) as a white solid. The molecular weight was calculated to be 3670.88 by LCMS [M / 3+H]. + :1226.0.

[0776] Example 62: Preparation of Coupling 38

[0777] first step:

[0778] INT-5 (30.0 mg, 24.3 μmol, 1.00 eq), peptide P-1 (45.0 mg, 24.3 μmol, 1.00 eq) with the sequence SEQ ID NO: 223, and N,N-diisopropylethylamine (20 μL, 121 μmol, 5 eq) were added to a DMF solution (0.45 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS monitoring showed the disappearance of the starting materials and the detection of the conjugate peak 38-1. The calculated molecular weight by LCMS was 2970.62, [M / 2+H]. + :1486.8, the reaction solution is used directly in the next step.

[0779] Step Two:

[0780] TEA (0.12 mL) was added to the reaction solution from the previous step, and the reaction solution was stirred at 25 °C for 5 hours. The reaction solution was purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain conjugate 38-2 (35.4 mg, 12.9 μmol) as a white solid. LCMS analysis determined the molecular weight to be 2748.55, [M / 2+H]. + :1375.6.

[0781] Step 3:

[0782] INT-12 (34.2 mg, 33.0 μmol, 2.00 eq), coupling compound 38-2 (35.4 mg, 12.9 μmol, 1.00 eq), and N,N-diisopropylethylamine (8.07 μL, 48.9 μmol, 4.00 eq) were added to a DMF solution (0.33 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain coupling compound 38 (14.00 mg, 3.81 μmol) as a white solid. The molecular weight was calculated to be 3670.88 by LCMS [M / 3+H]. + :1226.0.

[0783] Example 63: Preparation of Intermediate 19

[0784] first step:

[0785] Monomethyl malonate (21.1 mg, 178 μmol), INT-10-5 (100 mg, 81.1 μmol), HOBt (28.9 mg, 214 μmol), N,N-diisopropylethylamine (88.4 μL, 535 μmol), and EDCI (51.0 mg, 268 μmol) were added to a 2 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was then purified directly by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-19-1 (68.00 mg, 55.6 μmol) as a white solid. The molecular weight was calculated to be 1220.71 by LCMS [M+H]. + :1221.9.

[0786] Step Two:

[0787] INT-19-1 (68 mg, 55.6 μmol, 1.0 eq) and lithium hydroxide monohydrate (112 μL, 112 μmol, 4 eq, 1 M) were added to a THF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 15 min. The target compound INT-19-2 was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 200 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by preparative HPLC and lyophilized to obtain INT-19-2 (56 mg, 46.3 μmol) as a white solid. The molecular weight was calculated to be 1206.69 by LC-MS analysis, [M+H]. + :1207.8.

[0788] Step 3:

[0789] INT-19-2 (56 mg, 46.3 μmol), N-hydroxysuccinimide (32.0 mg, 27.8 μmol), and EDCI (53.0 mg, 27.8 μmol) were added to a DCM solution (0.5 mL). The reaction solution was stirred at 25 °C for 3 hours. LCMS detected the target product peak INT-19 as the main peak. The reaction solution was directly purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain a white solid of INT-19 (38.4 mg, 29.4 μmol). LCMS analysis calculated the molecular weight to be 1303.71, [M+H]. + :1304.9.

[0790] Example 64: Preparation of Coupling 39

[0791] INT-19 (38.4 mg, 12.9 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (10.0 mg, 6.13 μmol, 1.00 eq), and N,N-diisopropylethylamine (8.11 μL, 49.0 μmol, 8 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 39 (10.2 mg, 2.46 μmol) as a white solid. The molecular weight was calculated to be 4007.24 by LCMS [M / 3+H]. + :1337.7.

[0792] Example 65: Preparation of Intermediate 20

[0793] first step:

[0794] Monomethyl malonate (21.1 mg, 178 μmol), INT-13-1 (100 mg, 89 μmol, from Shanghai WuXi AppTec Co., Ltd., Wuxi: VC-PABc-MMAE), HOBt (28.9 mg, 214 μmol), N,N-diisopropylethylamine (88.4 μL, 535 μmol), and EDCI (51.0 mg, 268 μmol) were added to a 2 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was then purified directly by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-20-1 (70.50 mg, 57.6 μmol) as a white solid. The molecular weight was calculated to be 1222.72 by LCMS [M+H]. + :1224.0.

[0795] Step Two:

[0796] INT-20-1 (70.5 mg, 57.6 μmol, 1.0 eq) and lithium hydroxide monohydrate (115 μL, 115 μmol, 2 eq, 1 M) were added to a THF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 15 min. The target compound INT-20-2 was detected by LC-MS. The reaction mixture was neutralized with dilute hydrochloric acid (1 M, 200 μL), concentrated, and then diluted with acetonitrile aqueous solution (50%, 2 mL). The solution was purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-20-2 (61 mg, 50.4 μmol) as a white solid. The molecular weight was calculated to be 1208.71 by LC-MS analysis. [M+H] + :1210.9.

[0797] Step 3:

[0798] INT-20-2 (61 mg, 50.4 μmol), N-hydroxysuccinimide (23.2 mg, 20.1 μmol), and EDCI (38.5 mg, 20.1 μmol) were added to a DCM solution (0.5 mL). The reaction solution was stirred at 25 °C for 3 hours. LCMS detected the target product peak INT-20 as the main peak. The reaction solution was directly purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain INT-20 (38 mg, 29.1 μmol) as a white solid. LCMS analysis calculated the molecular weight to be 1304.73, [M+H]. + :1306.9.

[0799] Example 66: Preparation of Coupling 40

[0800] INT-19 (38 mg, 19.4 μmol, 2.1 eq), a peptide with the sequence SEQ ID NO: 231 (15.0 mg, 9.19 μmol, 1.00 eq), and N,N-diisopropylethylamine (9.12 μL, 55.1 μmol, 6 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 40 (24.3 mg, 5.88 μmol) as a white solid. The molecular weight was calculated to be 4011.27 by LCMS [M / 3+H]. + :1339.1.

[0801] Example 67: Preparation of Intermediate 21

[0802] first step:

[0803] Fmoc-O-tert-butyl-L-glutamic acid (379 mg, 890 μmol), INT-13-1 (1.00 g, 890 μmol, from Shanghai WuXi AppTec Co., Ltd., Wuxi: VC-PABc-MMAE), HOBt (265 mg, 1.96 mmol), N,N-diisopropylethylamine (690 mg, 5.34 mmol), and DIC (449 mg, 3.56 mmol) were added to a 10 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was then purified directly by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-21-1 (972 mg, 635 μmol) as a white solid. The molecular weight was calculated to be 1529.88 by LCMS [M+H]. + :1531.3.

[0804] Step Two:

[0805] INT-21-1 (972 mg, 635 μmol, 1.0 eq) and triethylamine (1.45 g, 14.4 mmol, 22.6 eq) were added to a DMF solution (8 mL). The reaction mixture was stirred at 25 °C for 4 hours. The target compound INT-21-2 was detected by LC-MS. Isopropyl ether was added to the reaction mixture, the solid was filtered off, and dried to obtain a white solid of INT-21-2 (734 mg, 561 μmol). The molecular weight was calculated to be 1307.81 by LC-MS [M+H]. + :1309.0.

[0806] Step 3:

[0807] INT-21-2 (634 mg, 484 μmol), succinic anhydride (58.1 mg, 581 μmol), and N,N-diisopropylethylamine (125 mg, 968 μmol) were added to a DMF solution (6 mL). The reaction mixture was stirred at 25 °C for 1 hour. LC-MS analysis showed that the main peak was the target product INT-21-3. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain INT-21-3 (540 mg, 383 μmol) as a white solid. LC-MS analysis calculated the molecular weight to be 1407.83, [M+H]. + :1410.1.

[0808] Step 4:

[0809] INT-21-3 (540 mg, 383 μmol), N-hydroxysuccinimide (88.2 mg, 766 μmol), EDCI (147 mg, 766 μmol), and DMAP (4.68 mg, 38.3 μmol) were added to a mixed solution of DCM (3 mL) and DMF (3 mL). The reaction solution was stirred at 25 °C for 5 hours. LCMS detected the main peak as the target product peak INT-21. After adding 1 M hydrochloric acid aqueous solution, the organic phase was separated, concentrated to obtain a crude product, purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain INT-21 (428 mg, 284 μmol) as a white solid. LCMS analysis calculated the molecular weight to be 1504.84, [M / 2+H]. + :753.8.

[0810] Example 68: Preparation of Coupling 41

[0811] first step:

[0812] INT-21 (60.9 mg, 40.5 μmol, 2.2 eq), a peptide with the sequence SEQ ID NO: 231 (30.0 mg, 18.4 μmol, 1.00 eq), and N,N-diisopropylethylamine (14.3 mg, 40.5 μmol, 2.2 eq) were added to a DMF solution (1 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 41-1 (44.6 mg, 10.1 μmol) as a white solid. The molecular weight was calculated to be 4409.52 by LCMS [M / 3+H]. + :1472.0.

[0813] Step Two:

[0814] Coupling 41-1 (44.6 mg, 10.1 μmol, 1.0 eq), trifluoroacetic acid (1.23 g, 10.8 mmol, 107 eq), and triisopropylsilane (30.8 mg, 195 μmol, 19.3 eq) were added to a DCM solution (0.76 mL). The reaction mixture was stirred at 0 °C for 5 hours. Isopropyl ether was added to the reaction mixture, and the solid was filtered off. The solid was dissolved in 10% potassium carbonate and acetonitrile, purified by preparative HPLC, and lyophilized to obtain a white solid of coupling 41 (20.0 mg, 4.65 μmol). The molecular weight was calculated to be 4297.39 by LCMS [M / 3+H]. + :1434.7.

[0815] Example 69: Preparation of Intermediate 22

[0816] first step:

[0817] The preparation method of INT-22-1 is the same as in Example 2.

[0818] LCMS analysis calculated the molecular weight to be 487.26, [M+H]. + :488.2.

[0819] Step Two:

[0820] INT-22-1 (1.00 g, 2.05 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (0.61 g, 2.46 mmol), and p-aminobenzyl alcohol (0.30 g, 2.46 mmol) were added to a 10 mL DCM solution. The reaction mixture was stirred at 25 °C for 3 hours. Isopropyl ether was added to the reaction mixture, and the solid was filtered off to obtain a brown solid, INT-22-2 (0.81 g, 1.37 mmol). The molecular weight was calculated to be 592.32 by LCMS [M+H]. + :593.4.

[0821] Step 3:

[0822] INT-22-2 (810 mg, 1.37 mmol), p-nitrophenylchloroformic acid (831 mg, 2.73 mmol), and DIEA (353 mg, 2.73 mmol) were added to a 10 mL DMF solution. The reaction mixture was stirred at 25 °C for 2 hours. Isopropyl ether was added to the reaction mixture, and the solid was filtered off to obtain a yellow solid, INT-22-3 (0.86 g, 1.14 mmol). The molecular weight was calculated to be 757.33 by LCMS [M+H]. + :758.4.

[0823] Step 4:

[0824] INT-22-3 (861 mg, 1.14 mmol), MMAE (816 mg, 1.14 mmol), HOBT (169 mg, 1.25 mmol), and DIEA (294 mg, 2.27 mmol) were added to a 10 mL DMF solution. The reaction mixture was stirred at 25 °C for 20 hours. The reaction mixture was then directly purified by high-performance liquid chromatography (HPLC) to obtain INT-22-4 (921 mg, 0.689 mmol) as a white solid. The molecular weight was calculated to be 1335.81 by LCMS [M+H]. + :1337.0.

[0825] Step 5:

[0826] INT-22-4 (500 mg, 0.374 mmol) and trifluoroacetic acid (3.84 g, 33.7 mmol) were added to a DCM solution (2.5 mL). The reaction mixture was stirred at 0 °C for 5 hours. Icy-isopropyl ether was added to the reaction mixture, and the solid was filtered off. The solid was dissolved in 10% potassium carbonate and acetonitrile, purified by preparative HPLC, and lyophilized to obtain INT-22-5 (219 mg, 171 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1279.74, [M / 2+H]. + :641.4.

[0827] Step 6:

[0828] INT-22-5 (100 mg, 78.1 μmol), HOSu (22.5 mg, 195 μmol), EDCI (37.4 mg, 195 μmol), and DMAP (954 μg, 7.81 μmol) were added to a mixed solution of DMF (1 mL) and DCM (0.5 mL). The reaction solution was stirred at 25 °C for 7 hours. LCMS monitoring showed the disappearance of the reactants and the detection of the product peak. 1 M aqueous hydrogen chloride solution and DCM were added to the reaction solution, the organic phase was separated, dried, and concentrated to obtain a yellow oily crude product of INT-22 (274 mg). LCMS analysis calculated the molecular weight to be 1376.76, [M+Na]. + :1400.1.

[0829] Example 70: Preparation of Coupling 42

[0830] INT-22 (193 mg, 140 μmol, 11.4 eq), a peptide with the sequence SEQ ID NO: 231 (20.0 mg, 12.3 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.17 mg, 24.5 μmol, 2.00 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 4 hours. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 42 (12.4 mg, 2.98 μmol) as a white solid. The molecular weight was calculated to be 4153.35 by LCMS [M / 3+H]. + :1386.6.

[0831] Example 71: Preparation of Coupling 43

[0832] first step:

[0833] Add peptide P-2 (30 mg, 16.2 μmol), N-succinimide acetate (3.05 mg, 19.4 μmol), and DIEA (5.35 μL, 32.4 μmol) with the sequence SEQ ID NO: 231 to a DMF (0.5 mL) solution. Stir the reaction mixture at 25 °C for 1 hour. The reaction mixture was then purified directly by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 43-1 (14.3 mg, 7.54 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1893.96 [M+H]. + :1895.2.

[0834] Step Two:

[0835] Conjugate 43-1 (14.3 mg, 7.54 μmol, 1.0 eq) and triethylamine (0.2 mL) were added to a DMF solution (0.8 mL). The reaction mixture was stirred at 25 °C for 5 hours. The target conjugate 43-2 was detected by LC-MS. Isopropyl ether was added to the reaction mixture, the solid was filtered off, and dried to obtain conjugate 43-2 (12.6 mg, 7.53 μmol) as a white solid. The molecular weight was calculated to be 1671.90 by LC-MS, [M+H]. + :1674.6.

[0836] Step 3:

[0837] Conjugate 43-2 (12.6 mg, 7.53 μmol, 1.0 eq), INT-20 (14.6 mg, 7.53 μmol, 1.00 eq), and N,N-diisopropylethylamine (2.49 μL, 15.1 μmol, 2.00 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 43 (12.5 mg, 4.36 μmol) as a white solid. The molecular weight was calculated to be 2862.59 by LCMS [M / 3+H]. + :955.8.

[0838] Example 72: Preparation of Coupling 44

[0839] first step:

[0840] Add peptide P-1 (30 mg, 16.2 μmol), N-succinimide acetate (3.05 mg, 19.4 μmol), and DIEA (5.35 μL, 32.4 μmol) with the sequence SEQ ID NO: 231 to a DMF (0.5 mL) solution. Stir the reaction mixture at 25 °C for 1 hour. The reaction mixture was then purified directly by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 44-1 (16.8 mg, 8.86 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1893.96 [M+H]. + :1895.2.

[0841] Step Two:

[0842] Conjugate 43-1 (16.8 mg, 8.86 μmol, 1.0 eq) and triethylamine (0.2 mL) were added to a DMF solution (0.8 mL). The reaction mixture was stirred at 25 °C for 5 hours. The target conjugate 44-2 was detected by LC-MS. Isopropyl ether was added to the reaction mixture, the solid was filtered off, and dried to obtain conjugate 44-2 (14.8 mg, 8.84 μmol) as a white solid. The molecular weight was calculated to be 1671.90 by LC-MS [M+H]. + :1674.3.

[0843] Step 3:

[0844] Conjugate 44-2 (14.8 mg, 8.84 μmol, 1.0 eq), INT-20 (17.2 mg, 8.84 μmol, 1.00 eq), and N,N-diisopropylethylamine (2.92 μL, 17.7 μmol, 2.00 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 44 (12.5 mg, 4.36 μmol) as a white solid. The molecular weight was calculated to be 2862.59 by LCMS [M / 3+H]. + :955.9.

[0845] Example 73: Preparation of Intermediate 23

[0846] Under ice bath conditions, INT-13-1 (50 mg, 44.5 μmol, from Shanghai WuXi AppTec Co., Ltd., Wuxi: VC-PABc-MMAE), bis(succinimide) glutarate (72.6 mg, 223 μmol), and DIEA (14.7 μL, 89.0 μmol) were added to a 0.25 mL DMF solution. The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain the intermediate INT-23 (35 mg, 26.2 μmol) as a white solid. The molecular weight was calculated to be 1333.75 by LCMS [M+H]. + :1334.9.

[0847] Example 74: Preparation of Coupling 45

[0848] first step:

[0849] The preparation method of conjugate 45-1 is the same as that in Example 1.

[0850] Step Two:

[0851] Conjugate 45-1 (1.2 g, 0.5 mmol) was added to an acetonitrile and water solution (1:2, v / v, 500 mL), and iodine in methanol (0.1 M) was added dropwise until the system turned yellow. The reaction solution was stirred at 25 °C for 2 minutes. Sodium thiosulfate aqueous solution (0.1 M) was added until the system became colorless. The reaction solution was directly purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain conjugate 45-2 (187 mg, 76 μmol) as a white solid. LCMS analysis determined the molecular weight to be 2453.3, [M / 2+H]. + :1228.6.

[0852] Step 3:

[0853] Conjugate 45-2 (20 mg, 8.15 μmol, 1.0 eq), INT-23 (10.9 mg, 8.15 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.03 μL, 18.3 μmol, 2.25 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 45 (21.4 mg, 5.38 μmol) as a white solid. The molecular weight was calculated to be 3672.03 by LCMS [M / 3+H]. + :1225.8.

[0854] Example 75: Preparation of Coupling 46

[0855] first step:

[0856] The preparation method of conjugate 46-1 is the same as that in Example 1.

[0857] Step Two:

[0858] Conjugate 46-1 (1.2 g, 0.5 mmol) was added to an acetonitrile and water solution (1:2, v / v, 500 mL), and iodine in methanol (0.1 M) was added dropwise until the system turned yellow. The reaction solution was stirred at 25 °C for 2 minutes. Sodium thiosulfate aqueous solution (0.1 M) was added until the system became colorless. The reaction solution was directly purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain conjugate 46-2 (187 mg, 76 μmol) as a white solid. LCMS analysis determined the molecular weight to be 2453.3, [M / 2+H]. + :1228.6.

[0859] Step 3:

[0860] Conjugate 46-2 (20 mg, 8.15 μmol, 1.0 eq), INT-23 (10.9 mg, 8.15 μmol, 1.00 eq), and N,N-diisopropylethylamine (3.03 μL, 18.3 μmol, 2.25 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 46 (18.0 mg, 4.66 μmol) as a white solid. The molecular weight was calculated to be 3672.03 by LCMS [M / 3+H]. + :1225.8.

[0861] Example 76: Preparation of Intermediate 24

[0862] first step:

[0863] INT-24-1 (1.00 g, 1.50 mmol, from Shanghai WuXi AppTec Co., Ltd., Wuxi: Fmoc-Cit-PAB-PNP), MMAE (968 mg, 1.35 mmol), HOBT (223 mg, 1.65 mmol), and DIEA (387 mg, 3.00 mmol) were added to a 20 mL DMF solution. The reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) to obtain INT-24-2 (1.08 g, 0.866 mmol) as a white solid. The molecular weight was calculated to be 1023.64 by LCMS [M+H]. + :1247.0.

[0864] Step Two:

[0865] INT-24-2 (1.08 g, 0.866 mmol) and trifluoroacetic acid (1.45 g, 14.4 mmol) were added to a DMF solution (8 mL). The reaction mixture was stirred at 25 °C for 5 hours. Icy-rich isopropyl ether was added to the reaction mixture, the solid was filtered off, and dried to obtain INT-24-3 (793 mg, 774 μmol) as a white solid. LCMS analysis determined the molecular weight to be 1279.74, [M+H]. + :1024.8.

[0866] Step 3:

[0867] INT-24-3 (793 mg, 774 μmol), cyclobutyl-1,1-dicarboxylic acid monoethyl ester (160 mg, 929 μmol), TBTU (298 mg, 929 μmol), and N,N-diisopropylethylamine (256 μL, 1.55 mmol) were added to a DMF solution (10 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain INT-24-4 (779 mg, 661 μmol) as a white solid. The molecular weight was calculated to be 1177.70 by LCMS [M+H]. + :1179.0.

[0868] Step 4:

[0869] INT-24-4 (748 mg, 635 μmol, 1.0 eq) and lithium hydroxide monohydrate (1.27 mL, 1.27 mmol, 2 eq, 1 M) were added to a 3 mL THF solution. The reaction mixture was stirred at 0 °C for 2 hours. The target compound INT-24-5 was detected by LC-MS. The reaction mixture was neutralized to weakly acidic with trifluoroacetic acid, purified by preparative HPLC, and lyophilized to obtain a white solid of INT-24-5 (609 mg, 529 μmol). The molecular weight was calculated to be 1149.67 by LC-MS [M+H]. + :1250.9.

[0870] Step 5:

[0871] INT-24-5 (609 mg, 529 μmol), N-hydroxysuccinimide (244 mg, 2.12 mmol), EDCI (406 mg, 2.12 mmol), and DMAP (6.46 mg, 52.9 μmol) were added to a DCM solution (6 mL). The reaction mixture was stirred at 25 °C for 1 hour. LCMS detected the main peak as the target product peak, INT-24. DCM and 1 M hydrochloric acid aqueous solution were added to the reaction mixture, the organic phase was separated, concentrated to obtain a solid, purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain a white solid of INT-24 (448.7 mg, 360 μmol). LCMS analysis calculated the molecular weight to be 1246.68, [M+H]. + :1248.0.

[0872] Example 77: Preparation of Coupling 47

[0873] INT-24 (16.8 mg, 13.5 μmol, 2.2 eq), a polypeptide with the sequence SEQ ID NO: 231 (10.0 mg, 6.13 μmol, 1.00 eq), and N,N-diisopropylethylamine (4.05 μL, 24.5 μmol, 4 eq) were added to a DMF solution (0.5 mL). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was directly purified by high-performance liquid chromatography (HPLC) and lyophilized to obtain conjugate 47 (16.5 mg, 4.24 μmol) as a white solid. The molecular weight was calculated to be 3893.20 by LCMS, and the [M / 3+H]+ was 1299.7. Example 78: Inhibitory effect of the conjugate on tumor cell proliferation

[0874] Resuspend the cells and centrifuge (1000 rpm, room temperature, 5 minutes), remove the supernatant, and dilute the cell suspension to 2500 cells / well in a 96-well plate. Dilute the conjugate to 200 μM with 40 μL of water, then dilute 3-fold to obtain 8 concentrations. Take 3 μL of each concentration of conjugate and mix with 197 μL of cell culture medium. Take 50 μL of the above diluted solution and add it to a 96-well plate containing cells, and incubate at 37°C for 3-7 days in a 5% CO2 incubator. Protect from light, and add 50 μL of [unclear text - possibly a conjugate solution] to each well. The assay reagent (Promega, Cat#G9683) was incubated at room temperature for 30 minutes. The chemiluminescence signal intensity of each well in the cell culture plate was measured using a plate reader, and the percentage inhibition rate was calculated. The positive control group contained only an equal volume of cell culture medium, while the negative control group only added cells at plate formation, without any conjugates. Inhibition rate % = (1 - (OD) / (C)) / (C) 测试组 -OD 阳性对照组 ) / (OD 阴性对照组 -OD 阳性对照组 ))×100%

[0875] The concentrations and corresponding inhibition rates of the conjugates in the test group were imported into XLfit software. The inhibition rate-concentration curve was fitted using the No.205 dose response model in the software, and the IC50 was calculated. 50 value.

[0876] Table 1. Inhibitory activity of the conjugate in the 4T1 cell line.

[0877] Table 2. Inhibitory activity of the conjugate in the MDA-MB-231 cell line.

[0878] Table 3. Inhibitory activity of the conjugate in the SK-OV-3 cell line.

[0879] Table 4. Inhibitory activity of the conjugate in the A2780-cisR cell line.

[0880] Table 5. Inhibitory activity of the conjugate in the Caov3 cell line.

[0881] The inhibitory activities of the conjugates on breast cancer cell lines 4T1 and MDA-MB-231, and ovarian cancer cell lines SK-OV-3, A2780-cisR, and Caov3 are shown in Tables 1-5. Conjugate 4 showed excellent inhibitory effects on breast cancer cell line MDA-MB-231, ovarian cancer cell lines SK-OV-3, and A2780-cisR. For the platinum-resistant A2780-cisR cell line, conjugates 4, 5, 6, 10, 11, 15, and 21 all showed significant inhibitory effects.

[0882] Example 79: Pharmacokinetic Study of the Conjugate in Mice

[0883] The conjugate was dissolved in a solvent containing 5% DMSO, 45% PEG400, and 50% pure water to prepare a 0.4 mg / mL solution. Male ICR mice (Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) aged 7-9 weeks were randomly divided into groups of 3 mice each after acclimatization. Each group received an intravenous injection (IV) of 5 mL / kg of the prepared conjugate solution (dose 2 mg / kg). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after IV administration (a total of 8 time points). Plasma was collected by centrifugation. 10 μL of blood sample was added to 2 μL of methanol and 200 μL of methanol-acetonitrile solution (1:1, v / v) containing an internal standard. After stirring, the mixture was centrifuged for 15 minutes (4000 rpm). The supernatant was analyzed by LC-MS / MS (AB Sciex Triple Quad 6500) to determine the drug concentration. The chromatographic column used was... A C18 2.6μm 100A (50mm*2.10mm) column was used; mobile phase A consisted of 0.1% formic acid and 5mM ammonium acetate aqueous solution; mobile phase B consisted of 0.1% formic acid and acetonitrile solution; the column temperature was room temperature, the injection volume was 7μL, and the gradient elution conditions are shown in Table 6; mass spectrometry was performed using an electrospray ionization source, with positive ion selected reaction monitoring (PET) mode. Compound TH1902 was prepared according to the method disclosed in WO 2021 / 108929 A1.

[0884] Table 6 Gradient elution conditions for pharmacokinetic testing

[0885] The pharmacokinetic results of some conjugates are shown in Table 7. The half-life of TH1902 in mice (t 1 / 2 The half-life (t) of the exemplary conjugate in this application in mice is 0.283 hours. 1 / 2 The stability of the above conjugates in mice was significantly better than that of TH1902, with all results exceeding 2 hours. This indicates that the stability of the above conjugates in mice is significantly better than that of TH1902. Furthermore, by measuring the concentration of free toxins in the blood, the molar ratio of free toxins to conjugates can be calculated to assess the stability and safety of the conjugates in vivo. The results are shown in Table 7. The molar ratio of free toxins to conjugates for TH1902 was 7.67%, indicating that approximately 7.67% of the conjugates broke down and released toxins in mice. The toxin release rates of the exemplary conjugates in this application were all lower than those for TH1902. These results demonstrate that the amount of toxins released by the conjugates after breaking down in mice is far less than that of TH1902, thus exhibiting better stability and higher safety.

[0886] Table 7 Results of mouse PK test

[0887] Example 80: Pharmacodynamic study of the conjugate against subcutaneous transplantation of human ovarian cancer A2780cis cells in a BALB / c nude mouse model.

[0888] Cells were placed in 5A medium containing 10% fetal bovine serum and 100 U / ml penicillin / streptomycin and cultured continuously at 37°C in a 5% CO2 incubator, passaged 2-3 times per week. During passage, the old medium was discarded, and the cells were washed with PBS, digested with trypsin, and digested with serum-containing medium to stop digestion. After centrifugation, the cells were resuspended and seeded. Cells in the logarithmic growth phase were washed with PBS, resuspended in serum-free medium, centrifuged, stained with trypan blue, and counted to ensure cell viability ≥90%. The concentration was then adjusted to 5 × 10⁶ cells / mL. 7 cells / mL, mixed with an equal volume of Matrigel to make 5 × 10⁻⁶ cells / mL 6 Prepare 0.1 mL suspension of cells and keep on ice for later use.

[0889] Female BALB / c nude mice aged 6-8 weeks were selected and acclimatized in an animal facility for one week at an ambient temperature of 20-26℃, humidity of 40-70%, and a 12-hour light cycle, with free access to food and water. Each mouse received a subcutaneous injection of 0.1 mL of cell mixture in its right axilla; the day of inoculation was designated as day 0. Tumors were allowed to develop after reaching an average size of 100 mm². 3 Around 10:00 AM, animals were divided into groups for drug administration. The experimental groups and drug administration regimens are shown in the table below. Each group of animals was administered IV, and the solvent was 5% DMSO + 20% PEG400 + 75% DI water.

[0890] During the experiment, the animals' health and mortality were observed daily, and abnormalities such as tumor ulceration were recorded. Their weight was measured twice a week. Simultaneously, the length and width of the tumor were measured twice a week using calipers, according to the formula V = 0.5 × a × b. 2 The tumor volume was calculated, followed by the relative tumor proliferation rate and tumor inhibition rate. Data were analyzed using two-way ANOVA and processed with GraphPad Prism 5. P < 0.05 was considered statistically significant. The experimental protocol was approved by IACUC and followed AAALAC guidelines.

[0891] Table 8. Experimental grouping and dosing regimen for the A2780cis animal model

[0892] Please refer to Table 9-10 and Figure 97-100 for changes in tumor volume in the human ovarian cancer model (A2780cis cells) in tumor-bearing mice. It is evident that conjugates 41, 42, 43, and 45 exhibited significant (P<0.05) antitumor effects on tumor growth in the mouse xenograft human ovarian cancer model.

[0893] Table 9. Results of anti-tumor model in human ovarian cancer model (A2780cis cells)

[0894] Table 10 Results of anti-tumor model in human ovarian cancer model (A2780cis cells)

[0895] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

A polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof, wherein: A is a cytotoxic agent; L is a linker connecting E and A, the wavy line in formula (I) indicates that L is covalently attached to a side chain residue or a terminal group of E; a is an integer from 0 to 10; E represents a SORTILIN specific peptide ligand, wherein the peptide ligand comprises an amino acid sequence of any one of (i)-(iv) below: (i) an amino acid sequence represented by formula (E-I): R1-X1-P1-X2-P2-X3-P3-X4-P4-X5-P5-P6-B1 Formula (E-I), wherein: R1is a modification group of the N-terminal amino group or is absent; X1, X2, X3, X4, X5are independently selected from natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids, or combinations thereof; P2is selected from any one of Ala, Gly, Ile, Val, Leu, Pro, Nle, Sar, tBuA, Dpr, A2Bu, Dbu, Abu, Aib, OctG, PipAla, PirrAla, BnG, Cha, Cpa, C4al, C5al, Thea, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNle, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeAla, MeGly, MeIle, MeVal, MeLeu, MeNle, hCha, and 4-PryAla; P3is selected from any one of Asn, Gln, Trp, Arg, Lys, His, Cit, Orn, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, Cha, C4al, C5al, Thi, Tza, Mso, Aha, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, HyPro, Pip, Pzp, Ampc, Cpa, MeArg, hCha, hArg, and 4-PryAla; P4is selected from any one of Lys, Arg, His, Gln, Pro, Dab, Cit, Orn, Thi, Tza, Aha, Aoc, HyPro, hArg, 3AmiPhe, 4AmiPhe, Dimk, AcLys, MeLys, MeArg, Pip, Pzp, IPegDab, and SPegDab; P5 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, Thi, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrrl, 4-AmPyrr2, 4-PhePyrrl, 4-PhePyrr2, 5-PhePyrrl, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe and MePhe; P6 is selected from any one of Ala, Gly, Val, lie, Leu, Pro, Met, Nle, 4-AmPyrrl, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C4al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha and Mso; B1 is selected from any one of Val, lie, Leu, Nle, Aha, MeIle, MeLeu, MeVal and MeNle; (ii) an amino acid sequence represented by formula (E-II): R2-A1-P7-X6-P8-P9-X7-A2-X8-P 10 -B2 Formula (E-II), wherein: R2 is a modification group of the N-terminal amino group or is absent; X6, X7, X8 are independently selected from absent or are selected from natural amino acids, unnatural amino acids, chemical modifications of natural or unnatural amino acids or combinations thereof; A1 and A2 are each independently selected from any one of Cys, Sec and Pen; P7 is selected from absent, Ser, Thr, Tyr, Trp, Cys, Thea, His, Sec, Pen, hSer, hCys, AlloT, sBzl, tBzl and yBzl; P8 is selected from any one of Asn, Glu, Cys, Sec, Pro, Arg, Lys, His, Cit, Orn, Dab, Aha, Thea, Dimk, Isorn, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, hCys, hArg, AcLys, MeArg, MeLys, IPegDab and SPegDab; P9 is selected from any one of Phe, Tyr, Trp, His, 1-Nal, 2-Nal, 2AmPhe, 3AmPhe, 4AmPhe, 3AmiPhe, 4AmiPhe, 3GuPhe, 4GuPhe, 4-AmPyrr1, 4-AmPyrr2, 4-PhePyrr1, 4-PhePyrr2, 5-PhePyrr1, 5-PhePyrr2, 2Cl-Phe, 3Cl-Phe, 4Cl-Phe, 3,4Cl2-Phe, 3F-Phe, 4F-Phe, 4GuPhe, Bip, BnG, Bpa, C4al, C5al, Cha, Cpa, Phg, Tza, yBzl, hCha, hPhe, MePhe, Thi, Pro, Arg and Leu; P 10 selected from any one of Ala, Gly, Val, lie, Leu, Pro, Met, Nle, 4-AmPyrrl, 4-AmPyrr2, 4-PryAla, A2Bu, Abu, Aib, Cha, C4al, C5al, Dbu, Dpr, Pip, Pzp, Sar, tBuG, MeAla, MeGly, MeIle, MeLeu, MeVal, MeNle, hCha, and Mso; B2 is selected from any one of Val, lie, Leu, Trp, Nle, Aha, MeIle, MeLeu, MeVal and MeNle; (iii) an amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to an amino acid sequence as shown in formula (E-I), and which retains the activity of the amino acid sequence as shown in formula (E-I); (iv) an amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to an amino acid sequence as shown in formula (E-II), and which retains the activity of the amino acid sequence as shown in formula (E-II). The polypeptide-drug conjugate of formula (I) according to claim 1, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, characterized in that, said P1 is Asn, Glu, Arg, Lys, Cit, Orn or Dab, more preferably Cit, Orn or Arg, most preferably Arg; Preferably, said P2 is Ala, Gly, lie, Val, Leu, Pro, Nle, Dbu, Aha or Dpr, more preferably Aha, Aib, Nle, lie, Val or Leu, most preferably Val; Preferably, said P3 is Asn, Gin, Arg, Lys or His, more preferably Asn or Gin, most preferably Gin; Preferably, said P4 is Lys, Arg, His, Dab, Dbu or Aha, more preferably Lys, Dab, Dbu or Aha, most preferably Lys; Preferably, said P5 is Phe, Tyr, Trp, His, Thi or Phg, more preferably Phe, Tyr, Trp and His, preferably Tyr; Preferably, the P6 is Ala, Gly, Val, lie, Leu, Pro, Nle, Abu, Aha or Aib, more preferably Abu, Aib, Val or lie, preferably lie; Preferably, the B1 is Val, lie, Leu, Nle, Aha, MeIle, MeLeu, MeVal or MeNle, more preferably Leu, Aha or Nle, most preferably Leu; Preferably, the X1 is Ala, Gly, lie, Leu, Pro, Phe, Val, Tyr, Trp, Aib, Nle, Pip, Pzp, Sar, tBuG or a combination thereof; Preferably, the X2 is Aha, Dab, tBuG, Ala, Gly, lie, Lys, Cys, Sar, Leu, Val or a combination thereof; Preferably, the X3 is Cit, Orn, Cys, Arg or a combination thereof; Preferably, the X4 is Mso, Nle, Cys, Leu, Met, Phe, Tyr, Trp or a combination thereof; Preferably, the X5 is Pip, Pzp, Pro or a combination thereof; Preferably, the modification group of the N-terminal amino group in the R1 is selected from Ac, Cbz, Boc, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB, Bn, methoxycarbonyl and ethoxycarbonyl. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, Preferably, the X1 is 1-3 amino acid residues, preferably 2 amino acid residues, more preferably Pip-Phe, Pip-Tyr, Pip-Leu, Pip-Val, Pzp-Phe, Pzp-Tyr, Pzp-Leu, Pzp-Val, Sar-Ile, Sar-Leu, Sar-Val, tBuG-Ile, tBuG-Leu, tBuG-Val, Ala-Phe, Gly-Ile, Gly-Leu, Gly-Val, Ile-Val, Leu-Phe, Pro-Aib, Pro-Ala, Pro-Phe, Pro-Leu, Pro-Val, Pro-Trp, Pro-Tyr or Val-Phe, more preferably Ala-Phe, Gly-Val, Leu-Phe, Ile-Val, Pro-Tyr, Val-Phe, Pro-Phe or Pro-Ala; Preferably, X2 is 2 to 5 amino acid residues, preferably 4 amino acid residues, more preferably Aha-Ile-Gly-Ala, Dab-Ile-Gly-Ala, tBuG-Lys-Ala-Sar, Ala-Aha-Leu-Ile, Ala-Aha-Ala-Gly, Ala-Dab-Ala-Gly, Ala-Dab-Leu-Ile, Ala-Lys-Leu-Ile, Ala-Lys-Ala-Sar, Ala-Lys-Ala-Gly, Ala-Lys-Val-Leu, Gly-Aha-Ala-Gly, Gly-Dab-Ala-Gly, Gly-Lys-Ala-Gly, Gly-Lys-Val-Leu, Ile-Aha-Ala-Gly, Ile-Aha-Leu-Ile, Ile-Dab-Ala-Gly, Ile-Dab-Ala-Sar, Ile-Dab-Gly-Ala, Ile-Dab-Leu-Ile, Ile-Cys-Gly-Ala, Ile-Cys-Ala-Gly, Ile-Cys-Sar-Ala, Ile-Cys-Ala-tBuG, Ile-Cys-Leu-Ile, Ile-Lys-Leu-Ile, Ile-Lys-Sar-Ala, Ile-Lys-Ala-Gly, Ile-Lys-Ala-Sar, Ile-Lys-Gly-Ala, Lys-Ile-Gly-Ala or Lys-Ile-tBuG-Ala, most preferably Ala-Lys-Leu-Ile, Ala-Lys-Ala-Gly, Ala-Lys-Val-Leu, Gly-Lys-Ala-Gly, Gly-Lys-Val-Leu, Ile-Lys-Gly-Ala, Ile-Cys-Gly-Ala or Ile-Lys-Ala-Gly; Preferably, X3 is 1 to 2 amino acid residues, preferably 1 amino acid residue, more preferably Cit, Orn, Cys, Arg, most preferably Arg; Preferably, X4 is 1 to 3 amino acid residues, preferably 2 amino acid residues, more preferably Mso-Phe, Mso-Tyr, Mso-Trp, Nle-Phe, Nle-Tyr, Nle-Trp, Cys-Phe, Cys-Tyr, Cys-Trp, Leu-Phe, Leu-Tyr, Leu-Trp, Met-Phe, Me-Tyr or Met-Trp, most preferably Met-Phe, Met-Tyr or Met-Trp; Preferably, X5 is 1 to 2 amino acid residues, preferably 1 amino acid residue, more preferably Pip, Pzp, Pro, most preferably Pro. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The peptide ligand comprises an amino acid sequence represented by formula (E-I-1): R1-X 1-1 -X 1-2 -P1-X 2-1 -X 2-2 -X 2-3 -X 2-4 -P2-X3-P3-X 4-1 -X 4-2 -P4-X5-P5-P6-B1 Formula (E-I-1), wherein R1, P1, P2, P3, P4, P5, P6 and B1 are as defined in any one of claims 1-3; X 1-1 , X 1-2 , X 2-1 , X 2-2 , X 2-3 , X 2-4 , X3, X 4-1 , X 4-2 , X5 is independently selected from a natural amino acid, a non-natural amino acid, a chemical modification of a natural or non-natural amino acid. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to claim 4, wherein said X 1-1 is Pip, Pzp, Sar, tBuG, Ala, Gly, lie, Leu, Pro or Val, preferably Ala, Gly, lie, Leu, Pro or Val; Preferably, said X 1-2 is Aib, Nle, Ala, lie, Leu, Val, Tyr, Phe or Trp, preferably Ala, Phe, lie, Val or Tyr; Preferably, said X 2-1 is Aha, Dab, tBuG, Ala, Gly, lie or Lys, preferably Gly or lie; Preferably, said X 2-2 is Cys, lie, Aha, Dab or Lys, preferably Cys or Lys; Preferably, X is S, tBuG, Ala, Gly, Leu or Val, preferably Gly, Leu or Val. 2-3 Preferably, X is S, tBuG, Ala, Gly, Leu or Val, preferably Gly, Leu or Val. Preferably, X is S, tBuG, Ala, Gly, lie or Leu, preferably Ala, Gly, lie or Leu. 2-4 Preferably, X is S, tBuG, Ala, Gly, lie or Leu, preferably Ala, Gly, lie or Leu. Preferably, X3 is Cit, Orn, Cys or Arg, preferably Cys or Arg. Preferably, said X 4-1 is Mso, Nle, Cys, Leu or Met, preferably Cys or Met; Preferably, said X 4-2 is Phe, Tyr or Trp, preferably Phe or Tyr; Preferably, X5 is Pip, Pzp or Pro, preferably Pro. The polypeptide-drug conjugate of formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to claim 4 or 5, characterized in that, The peptide ligand comprises an amino acid sequence represented by formula (E-I-1-1): R1-X 1-1 -X 1-2 -Arg-X 2-1 -X 2-2 -X 2-3 -X 2-4 -Val-X3-P3-X 4-1 -X 4-2 -Lys-X5-Tyr-P6-Leu Formula (E-I-1-1), wherein R1, X 1-1 , X 1-2 , X 2-1 , X 2-2 , X 2-3 , X 2-4 , X3, P3, X 4-1 , X 4-2 , X5, P6 are as defined in claim 4 or 5. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claim 1, wherein A1 and A2 are each independently Cys or Pen, preferably Cys. Preferably, P7 is Ser, Thr, Tyr, Trp, Cys, Thea, His, Sec, Pen or absent, more preferably Ser, Thr, Tyr, Trp, Thea or His, most preferably Thr. Preferably, P8 is Asn, Glu, Cys, Pro, Arg, Lys, Cit, Orn, Dab or Aha, more preferably Arg, Cit or Orn, most preferably Arg. Preferably, P9 is Phe, Tyr, Trp, His, Phg, Tza, Thi, Pro, Arg or Leu; more preferably Phe, Tyr, Trp or His, most preferably Tyr. Preferably, said P 10 is Ala, Gly, Val, lie, Leu, Pro, Nle, Abu or Aib; more preferably lie, Abu or Aib, most preferably lie; Preferably, B2 is Leu, Trp, Nle or Aha; more preferably Leu, Trp or Nle, most preferably Leu. Preferably, X6 is Lys, Aha, Cit, Orn, Dab, Glu, Asn, Pro, Arg, Trp, Ser, Gly, Ile, Ala or a combination thereof. Preferably, X7 is absent, Abu, Aib, Dbu, Dpr, Sar, tBuG, Ala, Gly, Ile, Leu, Pro, Arg, Val or a combination thereof. Preferably, X8 is absent or Tyr, more preferably absent. Preferably, the modification group of the N-terminal amino group in R2 is selected from Ac, Cbz, Boc, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB, Bn, methoxycarbonyl and ethoxycarbonyl. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claim 7, wherein X6 is 2-5 amino acid residues, preferably 3-5 amino acid residues, more preferably Aha-Ser-Aha, Aha-Ser-Cit, Aha-Ser-Orn, Aha-Ser-Lys, Aha-Ser-Asn, Cit-Ser-Aha, Cit-Ser-Lys, Dab-Ser-Cit, Dab-Ser-Dab, Dab-Ser-Orn, Dab-Ser-Lys, Dab-Ser-Asn, Dab-Ser-Arg, Orn-Ser-Aha, Orn-Ser-Lys, Glu-Ser-Glu, Glu-Ser-Ile, Gly-Ser-Lys, Gly-Ser-Ile, Lys-Ser-Gly, Lys-Ser-Ile, Lys-Lys-Ile, Lys-Ser-Lys, Lys-Ser-Asn, Lys-Ser-Arg, Lys-Ser-Aha, Lys-Ser-Cit, Lys-Ser-Dab, Lys-Ser-Orn, Asn-Ser-Aha, Asn-Ser-Dab, Asn-Ser-Lys, Pro-Ser-Lys, Arg-Ser-Lys, Arg-Lys-Lys, Ser-Lys-Ile, Trp-Ser-Ile, Trp-Lys-Lys, Arg-Lys-Ala-Lys or Arg-Lys-Pro-Lys-Pro, most preferably Gly-Ser-Lys, Lys-Ser-Lys, Arg-Lys-Lys, Trp-Lys-Lys, Pro-Ser-Lys, Arg-Lys-Ala-Lys or Arg-Lys-Pro-Lys-Pro; Preferably, X7 is absent or 1-2 amino acid residues, preferably 1 amino acid residue, more preferably Abu, Aib, Dbu, Dpr, Sar, tBuG, Ala, Gly, Ile, Leu, Pro, Arg or Val, most preferably Ile. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to claim 7 or 8, characterized in that, The peptide ligand comprises an amino acid sequence represented by formula (E-II-1): R2-A1-P7-X 6-1 -X 6-2 -X 6-3 -X 6-4 -X 6-5 -P8-P9-X7-A2-X8-P 10 -B2 Formula (E-II-1), wherein R2, A1, P7, P8, P9, X7, A2, X8, P 10 B2 is as defined in claim 7 or 8; X 6-1 , X 6-2 , X 6-3 , X 6-4 , X 6-5 independent is selected from the absence or from a chemical modification of a natural amino acid, a non-natural amino acid, a natural or non-natural amino acid. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to claim 9, characterized in that, said X 6-1 is Aha, Cit, Dab, Orn, Glu, Gly, Lys, Asn, Pro, Arg, Ser or Trp, more preferably Lys, Arg or Trp, most preferably Lys or Arg; Preferably, said X 6-2 is Ser or Lys, more preferably Ser; Preferably, X is absent, Pro or Ala, more preferably Pro or Ala. 6-3 is absent, Pro or Ala, more preferably Pro or Ala; Preferably, said X 6-4 is Aha, Cit, Dab, Orn, Glu, Gly, lie, Lys, Asn or Arg; more preferably lie or Lys, most preferably Lys; Preferably, said X 6-5 is absent or Pro. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to claim 9 or 10, characterized in that, The peptide ligand comprises an amino acid sequence represented by formula (E-II-1-1): R2-Cys-P7-X 6-1 -Ser-X 6-3 -X 6-4 -X 6-5 -P8-P9-X7-Cys-Ile-B2 Formula (E-II-1-1), wherein R2, P7, X 6-1 , X 6-3 , X 6-4 , X 6-5 , P8, P9, X7, B2 are as defined in claim 9 or 10. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein, The peptide ligand of formula (E-I) is a linear peptide; Preferably, the peptide ligand of formula (E-II) is a cyclic peptide; Preferably, a covalent bond is formed between the amino acid at position A1 and the amino acid at position A2 to form a cyclic peptide, more preferably a disulfide bond or a diselenide bond is formed between the amino acid at position A1 and the amino acid at position A2 to form a cyclic peptide. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 1-12, characterized in that, The amino acid sequence of the peptide ligand is selected from any one of SEQ ID NO. 1 to SEQ ID NO.

234. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein, The polypeptide-drug conjugate has a structure as shown in formula (II), wherein: E is as defined in any one of claims 1-13; A' and A" are each independently a cytotoxic agent; each of the wavy lines in formula (II) indicates that L1or L1' is covalently attached to a side chain residue or a terminal group of E; m and p are each independently 0, 1, 2, 3, 4, or 5; each of the L1and the L1’ is independently selected from the group consisting of formula (L1-I): wherein: T 1 T 2 T 3 T 4 T 5 Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, -S(=NR t )-、-C(=NR t )-、-C(R t (=N)-、-P(R) t )-、-P(=O)(R t )-、C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups and natural or non-natural amino acids, wherein C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, 3-10 heterocyclic groups, and native or non-native amino acids are each independently bounded by one or more R groups. t replace; each R t is independently selected from hydrogen, protium, deuterium, tritium, halogen, -N02, -CN, OR s , -SR s , -N(R sa )(R sb ), -C(0)R s , -C02R s , -C(0)C(0)R s , -C(0)CH2C(0)R s , -S(0)R s , -S(0)2R s , C(0)N(R sa )(R sb ), -S02N(R sa )(R sb ), -OC(0)R s , -N(R)S02R s , C 1-6 1-6 alkyl, C 2-6 2-6 alkenyl, C 2-6 2-6 alkynyl, C 3-10 3-6 cycloalkyl, C 3-10 3-6 cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclyl, said C 1-6 1-6 alkyl, C 2-6 2-6 alkenyl, C 2-6 2-6 alkynyl, C 3-10 3-6 cycloalkyl, C 3-10 3-6 cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclyl being substituted with one or more R s ; each R s each R sa each R sb are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, and C 3-6 cycloalkenyl; b, c, d, e, f are each independently 0, 1, or 2; each of the L2and the L2’ is independently a bond or selected from formula (L2-I): wherein: Each W is independently selected from -C(R) wa (R) wb )-、-N(R wx -C(=O)-、-C(=O)-N(R) wx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R wx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R wx )-、-P(=O)(R wx )-、-N(R wx -SO2-, -SO2-N(R) wx -, -C(=S)-, -C(=NR) wx -, -N = N-, -C(=N2)-, C 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclyl, and natural or unnatural amino acids, each independently optionally substituted with one or more R 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclyl, and natural or unnatural amino acids, each independently optionally substituted with one or more R wx substituents; Y is -(OCH2CH2) yn -O yp -; Each Z is independently selected from -C(R) za (R) zb )-、-N(R zx -C(=O)-、-C(=O)-N(R) zx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R zx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R zx )-、-P(=O)(R zx )-、-N(R zx -SO2-, -SO2-N(R) zx -, -C(=S)-, -C(=NR) zx -, -N = N-, -C(=N2)-, C 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R zx substituents; Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -S(O)R r -S(O)2R r -C(O)N(R) ra (R) rb -SO2N(R) ra (R) rb -OC(O)R r -N(R) ra SO2R rb C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl, the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Each cycloalkenyl group is independently bounded by one or more R groups. r replace; each R r , each R ra , each R rb is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, and C 3-6 cycloalkenyl; yn is 0, 1, 2, 3, 4, or 5; yp is 0 or 1; g, h, i are each independently selected from an integer from 0 to 20; each of said L3and said L3' is independently selected from a bond or a peptide residue consisting of 2 to 7 natural or non-natural amino acids; each of the L4and the L4’is independently a bond or selected from any one of the structures shown in formulas (L4-1) to (L4-13): wherein: X is each independently selected from the group consisting of O, S, sulfone, sulfoxide, carbonyl, NR xa , alkylene, and cycloalkyl; R ua and R ub each independently is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, and C 3-10 cycloalkenyl; Each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl; each n is independently selected from 0 or 1. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein, The polypeptide-drug conjugate has a structure as shown in formula (II), wherein: E is as defined in any one of claims 1-13; A' and A" are each independently a cytotoxic agent; each of the wavy lines in formula (II) indicates that L1or L1' is covalently attached to a side chain residue or a terminal group of E; m and p are each independently 0, 1, 2, 3, 4, or 5; each of the L1and the L1’ is independently selected from the group consisting of formula (L1-I): wherein: T 1 T 2 T 3 T 4 T 5 Each is independently selected from -O-, -S-, -N(R) t )-, -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, -S(=NR t )-、-C(=NR t )-、-C(R t (=N)-、-P(R) t )-、-P(=O)(R t )-、C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups, wherein the C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 3-10 cycloalkyl, C 3-10 Cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclic groups are each independently bounded by one or more R groups. t replace; each R t is independently selected from hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, OR s , -SR s , -N(R sa )(R sb ), -C(O)R s , -CO2R s , -C(O)C(O)R s , -C(O)CH2C(O)R s , -S(O)R s , -S(O)2R s , C(O)N(R sa )(R sb ), -SO2N(R sa )(R sb ), -OC(O)R s , -N(R)SO2R s , C 1-6 1-6 alkyl, C 2-6 2-6 alkenyl, C 2-6 2-6 alkynyl, C 3-10 3-6 cycloalkyl, C 3-10 3-6 cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclyl, said C 1-6 1-6 alkyl, C 2-6 2-6 alkenyl, C 2-6 2-6 alkynyl, C 3-10 3-6 cycloalkyl, C 3-10 3-6 cycloalkenyl, 6-10 aryl, 5-10 heteroaryl, and 3-10 heterocyclyl being substituted with one or more R s ; each R s each R sa each R sb are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl and C 3-6 cycloalkenyl; b, c, d, e, f are each independently 0, 1, or 2; each of the L2and the L2’ is independently a bond or selected from formula (L2-I): wherein: Each W is independently selected from -C(R) wa (R) wb )-、-N(R wx -C(=O)-、-C(=O)-N(R) wx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R wx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R wx )-、-P(=O)(R wx )-、-N(R wx -SO2-, -SO2-N(R) wx -, -C(=S)-, -C(=NR) wx -, -N = N-, -C(=N2)-, C 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R wx substituents; Y is -(OCH2CH2) yn -O yp -; Each Z is independently selected from -C(R) za (R) zb )-、-N(R zx -C(=O)-、-C(=O)-N(R) zx )-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -N(R zx )-, -O-, -S-, -S(=O)-, -SO2-, -P(R zx )-、-P(=O)(R zx )-、-N(R zx -SO2-, -SO2-N(R) zx -, -C(=S)-, -C(=NR) zx -, -N = N-, -C(=N2)-, C 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R 3-10 cycloalkyl, C 3-10 cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, each independently substituted with one or more R zx substituents; Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -S(O)R r -S(O)2R r -C(O)N(R) ra (R) rb -SO2N(R) ra (R) rb -OC(O)R r -N(R) ra SO2R rb C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl, the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl and C 3-6 Each cycloalkenyl group is independently bounded by one or more R groups. r replace; each R r , each R ra , each R rb is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, and C 3-6 cycloalkenyl; yn is 0, 1, 2, 3, 4, or 5; yp is 0 or 1; g, h, i are each independently selected from an integer from 0 to 10; each of said L3and said L3' is independently selected from a bond or a peptide residue consisting of 2 to 7 natural or non-natural amino acids; each of the L4and the L4’is independently a bond or selected from any one of the structures shown in formulas (L4-1) to (L4-13): wherein: X is each independently selected from the group consisting of O, S, sulfone, sulfoxide, carbonyl, NR xa , alkylene, and cycloalkyl; R ua and R ub each independently is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-10 cycloalkyl, and C 3-10 cycloalkenyl; Each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-10 cycloalkyl and C 3-10 Cycloalkenyl; each n is independently selected from 0 or 1. The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, said T 1 , said T 2 , said T 3 , said T 4 , said T 5 are each independently selected from the group consisting of -0-, -S-, -N(R t )-, -C(=0)-, -C(=S)-, -S(=0)-, -S02-, -S(=NR t )-, -C(=NR t )-, -C(R t )(=N)-, -P(R t )-, -P(=0)(R t )-, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, the 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, the C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl are each independently substituted with one or more R t ; Preferably, said T 1 , said T 2 , said T 3 , said T 4 , said T 5 are each independently selected from the group consisting of -0-, -S-, -N(R t )-, -C(=0)-, S(=0)-, -S02-, -C(=NR t )-, -C(R t )(=N)-, C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 3-6 cycloalkyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, said 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, said C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 3-6 cycloalkyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl are each independently substituted with one or more R t . The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, each R t is independently selected from hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, OR s , -SR s , -N(R sa )(R sb ), -C(O)R s , -CO2R s , -C(O)C(O)R s , -C(O)CH2C(O)R s , -S(O)R s , -S(O)2R s , C(O)N(R sa )(R sb ), -SO2N(R sa )(R sb ), -OC(O)R s , -N(R)SO2R s , C 1-6 1-20 alkyl, C 2-6 2-20 alkenyl, C 2-6 2-20 alkynyl, C 3-6 3-8 cycloalkyl, C 3-6 3-8 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl, and 5-8 membered heterocyclyl, said 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S, said C 1-6 1-20 alkyl, C 2-6 2-20 alkenyl, C 2-6 2-20 alkynyl, C 3-6 3-8 cycloalkyl, C 3-6 3-8 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl, and 5-8 membered heterocyclyl being substituted with one or more R s ; Preferably, each R t Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, and OR. s -N(R) sa (R) sb -C(O)R s -CO2R s -C(O)C(O)R s -C(O)CH2C(O)R s C(O)N(R) sa (R) sb -OC(O)R s C 1-3 Alkyl, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups, wherein the 5-8 heteroaryl or 5-8 heterocyclic group contains 1-3 heteroatoms selected from N, O, and S, and the C 1-3 Alkyl, C 3-6 Cycloalkyl, 6-8 aryl, 5-8 heteroaryl, and 5-8 heterocyclic groups are bound by one or more R groups. s replace. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-17, characterized in that, each of the L1and the L1’ is independently selected from any one of the following structures: wherein R t as defined in any one of claims 14-16. The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, each R s , each R sa , each R sb is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 3-6 cycloalkyl, and C 3-6 cycloalkenyl; Preferably, each R s , each R sa , each R sb is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -OH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -OC(O)H, C 1-3 alkyl, and C 3-6 cycloalkyl. The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R wx substituents; Preferably, each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, C 3-6 cycloalkyl and 6-8 membered aryl, said C 3-6 cycloalkyl and 6-8 membered aryl are substituted by one or more R wx substituents. The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R wx substituents; Preferably, each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, C 3-6 cycloalkyl and 6-8 membered aryl, said C 3-6 cycloalkyl and 6-8 membered aryl are substituted by one or more R wx substituents. The polypeptide-drug conjugate of formula (I) according to any one of claims 14-16, or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof, wherein, each Z is independently selected from -C(R za )(R zb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, the 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, the C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, the 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, the C wx substituted; Preferably, each Z is independently selected from -C(R za )(R zb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, or C 3-6 cycloalkyl, said C 3-6 cycloalkyl is substituted by one or more R zx substituents. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-22, wherein Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -OC(O)R r C 1-6 Alkyl and C 3-6 cycloalkyl, the C 1-6 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r Substitution; preferably from hydrogen, protium, deuterium, tritium, halogens, -OR r -N(R) ra (R) rb -C(O)R r C 1-3 Alkyl and C 3-6 cycloalkyl, the C 1-3 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-23, wherein each R r each R ra each R rb is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, C 1-6 alkyl, and C 3-6 cycloalkyl; preferably from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -OH, -NH2, -C(O)H, C 1-3 alkyl, and C 3-6 cycloalkyl. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-24, wherein each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, each independently optionally substituted with one or more R wx substituents; each Z is independently selected from -C(R za )(R zb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, -S-, -S(=O)-, -C(=N2)-, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, the 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, the C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 6-8 membered aryl, 5-8 membered heteroaryl and 5-8 membered heterocyclyl, the 5-8 membered heteroaryl or 5-8 membered heterocyclyl containing 1-3 heteroatoms selected from N, O and S, the C wx substituted; Each R wa Each R wb Each R za Each R zb Each R wx Each R zx Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, and -OR. r -SR r -N(R) ra (R) rb -C(O)R r -CO2R r -C(O)C(O)R r -C(O)CH2C(O)R r -OC(O)R r C 1-6 Alkyl and C 3-6 cycloalkyl, the C 1-6 Alkyl and C 3-6 Each cycloalkyl group is independently bounded by one or more R r replace; each R r each R ra each R rb are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, C 1-6 alkyl, and C 3-6 cycloalkyl. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-25, wherein each W is independently selected from -C(R wa )(R wb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, C 3-6 cycloalkyl and 6-8 membered aryl, said C 3-6 cycloalkyl and 6-8 membered aryl are substituted by one or more R wx substituents; each Z is independently selected from -C(R za )(R zb )-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, or C 3-6 cycloalkyl, said C 3-6 cycloalkyl is substituted by one or more R zx substituents; each R wa , each R wb , each R za , each R zb , each R wx , each R zx is each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -OR r , -N(R ra )(R rb ), -C(O)R r , C 1-3 1-6alkyl, and C 3-6 3-10cycloalkyl, said C 1-3 1-6alkyl and C 3-6 3-10cycloalkyl are each independently substituted with one or more R r ; each R r each R ra each R rb is each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -OH, -NH2, -C(O)H, C 1-3 alkyl, and C 3-6 cycloalkyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-26, characterized in that, The L3 and the L3' are each independently selected from a bond, a dipeptide residue composed of natural or unnatural amino acids, a tripeptide residue composed of natural or unnatural amino acids, a tetrapeptide residue composed of natural or unnatural amino acids. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-27, characterized in that, The peptide residue of the L3 and the L3' is an amino acid residue formed from an amino acid selected from phenylalanine, glycine, norleucine, citrulline, alanine, lysine, serine, glutamic acid, aspartic acid, 1-aminocyclobutanecarboxylic acid; preferably an amino acid residue formed from an amino acid selected from phenylalanine, glycine, norleucine, citrulline, alanine, 1-aminocyclobutanecarboxylic acid. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-28, characterized in that, each of the L3and the L3’is independently selected from a bond or from any one of the structures shown in formulas (L3-1) to (L3-6): The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-29, wherein each of said X is independently selected from the group consisting of O, S, sulfone, sulfoxide, carbonyl, NR xa , C 1-6 alkylene and C 3-10 cycloalkyl; preferably from the group consisting of O, carbonyl, NR xa , C 1-3 alkylene and C 3-6 cycloalkyl; more preferably from the group consisting of carbonyl and NR xa . The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-30, wherein said R ua , said R ub are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 alkyl, C 2-4 alkenyl, C 2-4 alkynyl, C 3-6 cycloalkyl, and C 3-6 cycloalkenyl; preferably from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 alkyl, and C 3-6 cycloalkyl; more preferably from the group consisting of hydrogen, deuterium, methyl and ethyl. The polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 14-31, wherein Each R xa Each is independently selected from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl, C 2-4 alkenyl, C 2-4 alkynyl group, C 3-6 cycloalkyl and C 3-6 Cycloalkenyl groups; preferably derived from hydrogen, protium, deuterium, tritium, halogens, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H, C 1-3 Alkyl and C 3-6 Cycloalkyl; more preferably hydrogen, deuterium, methyl and ethyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-32, characterized in that, The A' and A" are each independently selected from paclitaxel or paclitaxel derivatives, a derivative of a dolastatin, a derivative of doxorubicin, a derivative of camptothecin, or a maytansine or maytansine analog. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 14-33, characterized in that, each of A' and A" is independently selected from any one of the structures shown in formulas (A-1) to (A-21): wherein: R aa selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ab selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ac selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ad selected from H, D, halogen, hydroxyl, amino, carboxyl, nitro, sulfone, sulfoxide, alkyl, alkoxy, haloalkyl, haloalkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R ae selected from H, D, halogen, amino, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. The polypeptide-drug conjugate of Formula (I) or stereoisomer, tautomer, solvate, hydrate, prodrugs, stable isotope derivative and pharmaceutically acceptable salt thereof according to claim 34, characterized in that, said R aa selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from H, D, halogen, hydroxyl, carboxyl and C 1-6 alkyl, more preferably from carboxyl and C 1-3 alkyl, most preferably from methyl and carboxyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to claim 34 or 35, characterized in that, R is selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from H, D, halogen, hydroxyl, amino, carboxyl and C ab R is selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from H, D, halogen, hydroxyl, amino, carboxyl and C 1-6 R is selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from H, D, halogen, hydroxyl, amino, carboxyl and C The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-36, characterized in that, said R ac selected from the group consisting of H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from the group consisting of H, D, halogen, hydroxyl and C 1-6 alkyl, more preferably from the group consisting of H, D and C 1-3 alkyl, most preferably from the group consisting of H and methyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-37, characterized in that, said R ad selected from the group consisting of H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from the group consisting of H, D, halogen, hydroxyl, C 1-6 alkyl, alkoxy and 6-10 membered aryl, more preferably from the group consisting of C 1-4 alkyl, alkoxy and 6-8 membered aryl, most preferably from the group consisting of phenyl and tert-butoxy. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-38, characterized in that, said R ae selected from the group consisting of H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably from the group consisting of H, D, halogen, hydroxyl and C 1-6 alkyl, more preferably from the group consisting of H, D and C 1-3 alkyl, most preferably from the group consisting of H and methyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-39, characterized in that, said R aa selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; said R ab selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; said R ac selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; said R ad selected from H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; said R ae is selected from the group consisting of H, D, halogen, hydroxyl, amino, carboxyl, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-40, characterized in that, said R aa selected from H, D, halogen, hydroxyl, amino, carboxyl, and C 1-6 alkyl; said R ab selected from H, D, halogen, hydroxyl, amino, carboxyl, and C 1-6 alkyl; said R ac selected from H, D, halogen, hydroxyl, and C 1-6 alkyl; said R ad selected from H, D, halogen, hydroxyl, C 1-6 alkoxy and 6-10 membered aryl; said R ae selected from H, D, halogen, hydroxyl, and C 1-6 alkyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-41, characterized in that, said R aa selected from carboxyl and C 1-3 alkyl; said R ab selected from H, D and hydroxyl; said R ac selected from H, D and C 1-3 alkyl; said R ad selected from C 1-4 alkoxy and 6-8 membered aryl; said R ae selected from H, D and C 1-3 alkyl. The polypeptide-drug conjugate represented by Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 34-42, characterized in that, said R aa is selected from methyl and carboxyl; said R ab selected from H and hydroxyl; said R ac is selected from H and methyl; said R ad is selected from phenyl and tert-butoxy; said R ae is selected from H and methyl. The polypeptide-drug conjugate of Formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative, and a pharmaceutically acceptable salt thereof according to any one of claims 1-43, wherein, The polypeptide-drug conjugate is selected from the group consisting of: A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide-drug conjugate represented by formula (I) or a stereoisomer, a tautomer, a solvate, a hydrate, a prodrug, a stable isotope derivative and a pharmaceutically acceptable salt thereof according to any one of claims 1-44, and one or more pharmaceutically acceptable carriers, diluents or excipients. The polypeptide-drug conjugate or stereoisomer, tautomer, solvate, hydrate, prodrug, stable isotope derivative, and pharmaceutically acceptable salt thereof of any one of claims 1-44, or the pharmaceutical composition of claim 45, for use in treating and / or preventing a disease or disorder overexpressing Sortilin, or in the manufacture of a medicament for preventing and / or treating a disease or disorder overexpressing Sortilin. The polypeptide-drug conjugate or stereoisomer, tautomer, solvate, hydrate, prodrug, stable isotope derivative, and pharmaceutically acceptable salt thereof of any one of claims 1-44, or the pharmaceutical composition of claim 45, for use in treating and / or preventing a cancer, or in the manufacture of a medicament for treating and / or preventing a cancer; preferably, the cancer comprises adrenocortical carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, rhabdoid tumor, skin cancer, testicular cancer, thyroid cancer, melanoma; more preferably, the cancer comprises ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, colorectal cancer, melanoma. A method of treating and / or preventing a cancer, comprising administering to a patient a therapeutically effective amount of the polypeptide-drug conjugate or stereoisomer, tautomer, solvate, hydrate, prodrug, stable isotope derivative, and pharmaceutically acceptable salt thereof of any one of claims 1-44, or the pharmaceutical composition of claim 45; preferably, the cancer comprises adrenocortical carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, endometrial cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neuroblastoma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, rhabdoid tumor, skin cancer, testicular cancer, thyroid cancer, melanoma; more preferably, the cancer comprises ovarian cancer, breast cancer, cervical cancer, endometrial cancer, pancreatic cancer, colorectal cancer, melanoma.

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