Three-branched hydrophilic linker and protein-drug conjugate thereof

Abstract

The present application belongs to the field of biopharmaceutics, and relates to a three-branched hydrophilic linker and a protein-drug conjugate thereof, and in particular, to a three-branched hydrophilic linker, a linker-payload thereof or a protein-drug conjugate thereof, preparation methods therefor, and pharmaceutical use thereof.

Description

Three-branched hydrophilic linkers and their protein drug conjugates

[0001] Cross-references to related applications

[0002] This disclosure claims the benefit and priority of Chinese invention patent applications with application numbers 202510055336.6, 202510127540.4, 202510518517.8, 202510961554.6 and 202610019960.5 filed with the State Intellectual Property Office of the People's Republic of China on January 13, 2025, January 27, 2025, April 23, 2025, July 11, 2025 and January 8, 2026, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of biomedicine and relates to three-branched hydrophilic linkers and their protein drug conjugates. Specifically, it relates to three-branched hydrophilic linkers, linker-load compound containing the linker and drug conjugate (e.g., protein drug conjugate), as well as their preparation methods and pharmaceutical uses. Background Technology

[0004] Protein drug conjugates, such as antibody-drug conjugates (ADCs), are novel targeted therapies that deliver cytotoxic drugs to disease sites via appropriate carriers (e.g., antibodies), potentially reducing off-target side effects and / or toxicity and improving the therapeutic index. For example, in ADC design, the linker is not only the molecular part that forms a covalent link between the antibody and the drug, but also a key element determining the efficacy and safety of ADCs in targeted drug therapy. Hydrophilic linkers appear to contribute to the stability and effectiveness of ADCs.

[0005] Therefore, the continuous development of hydrophilic linkers for protein drug conjugates (such as ADCs) is an indispensable part of the drug design and development process and has important clinical value and significance. Summary of the Invention

[0006] This disclosure provides a three-branched hydrophilic linker, a linker-payload containing the linker, and a protein drug conjugate. The protein drug conjugate, under the specific targeting effect of the protein, can achieve the effective release of small molecule drugs at the target site to treat related diseases.

[0007] In one aspect, this disclosure provides a linker segment of Formula I, or a stereoisomer thereof.

[0008] Among them, L 1A L1C and L 2 Each can be an independent interval zone, either the same or different.

[0009] L 1B It is a peptide unit;

[0010] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0011] R 1’ This is a linker fragment that connects to a protein-targeting ligand;

[0012] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0013] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0014] r, p, and q are each independently selected from 0, 1, 2, 3, 4, or 5;

[0015] m1 and m2 are each independently selected from 0, 1, 2 or 3;

[0016] The condition is that when m2 is 0, L 1B When connected to the payload, and m2 is not 0, L 1C Connect to the payload.

[0017] In one aspect, this disclosure provides a linker segment of formula I-0, or a stereoisomer thereof.

[0018] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0019] L 1B It is a peptide unit;

[0020] R 1’ This is a linker fragment that connects to a protein-targeting ligand;

[0021] R A and R BEach of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0022] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0023] r, p, and q are each independently selected from 0, 1, 2, 3, 4, or 5;

[0024] m1 and m2 are each independently selected from 0, 1, 2 or 3;

[0025] The condition is that when m2 is 0, L 1B When connected to the payload, and m2 is not 0, L 1C Connect to the payload.

[0026] In one aspect, this disclosure provides a linker intermediate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the linker intermediate comprises a linker segment represented by Formula I or Formula I-0.

[0027] In one aspect, this disclosure provides a linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the linker-payload comprises a linker fragment or a stereoisomer fragment of Formula I or Formula I-0, and the payload is a bioactive molecular fragment.

[0028] In one aspect, this disclosure provides a linker-payload of formula II-0, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0029] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0030] L 1B It is a peptide unit;

[0031] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0032] R 1 For connector unit;

[0033] R A and R BEach of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0034] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0035] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0036] m1 and m2 are each independently 0, 1, 2 or 3;

[0037] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0038] D represents a bioactive molecular fragment.

[0039] In one aspect, this disclosure provides a linker-payload of Formula II, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0040] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0041] L 1B It is a peptide unit;

[0042] R 1 For connector unit;

[0043] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0044] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0045] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0046] m1 and m2 are each independently 0, 1, 2 or 3;

[0047] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0048] D represents a bioactive molecular fragment.

[0049] In one aspect, this disclosure provides a protein drug conjugate of Formula IV, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, Ab-(LD). n IV

[0050] Among them, Ab is a protein-targeting ligand;

[0051] L is the linker segment or its stereoisomer segment shown in Formula I or Formula I-0;

[0052] D represents a bioactive molecular fragment;

[0053] n is an integer or decimal greater than 0 and less than or equal to 20. For example, n is an integer or decimal selected from 2 to 8.

[0054] In one aspect, this disclosure provides the use of the linker fragments, linker intermediates, linker-payloads, stereoisomers thereof, or pharmaceutically acceptable salts thereof in the preparation of protein drug conjugates.

[0055] In one aspect, this disclosure provides linker fragments, linker intermediates, linker-loads, stereoisomers thereof, or pharmaceutically acceptable salts thereof for the preparation of protein drug conjugates.

[0056] In one aspect, this disclosure provides a method for preparing a protein drug conjugate, comprising:

[0057] The protein-targeting ligand and the bioactive molecular fragment are coupled via the linker fragment disclosed herein; or

[0058] The protein-targeting ligand and the bioactive molecular fragment are coupled via the linker intermediate disclosed herein; or

[0059] The protein targeting ligand is coupled to the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in this disclosure.

[0060] In one aspect, this disclosure provides a pharmaceutical composition comprising the protein drug conjugate of this disclosure, its stereoisomer, or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition of this disclosure further includes a pharmaceutically acceptable excipient.

[0061] In one aspect, this disclosure provides the use of the protein drug conjugate of this disclosure (e.g., antibody-drug conjugate), its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of this disclosure in the preparation of a medicament for treating a disease, wherein the disease is associated with a protein specifically bound to a protein-targeting ligand contained in the protein drug conjugate. In another aspect, this disclosure provides the use of the protein drug conjugate of this disclosure (e.g., antibody-drug conjugate), its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of this disclosure in the preparation of a medicament for treating a disease associated with a protein specifically bound to a protein-targeting ligand contained in the protein drug conjugate.

[0062] In one aspect, this disclosure provides the use of the protein drug conjugates of this disclosure (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions of this disclosure in the preparation of a medicament for treating tumors.

[0063] In one aspect, this disclosure provides a protein-drug conjugate of the present disclosure (e.g., an antibody-drug conjugate), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure for treating a disease, wherein the disease is associated with a protein in which a protein-targeting ligand contained in the protein-drug conjugate specifically binds.

[0064] In one aspect, this disclosure provides protein drug conjugates (e.g., antibody-drug conjugates), stereoisomers thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for the treatment of tumors.

[0065] In one aspect, this disclosure provides a method of treating a disease, comprising administering to a subject in need a therapeutically effective amount of the disclosed protein-drug conjugate (e.g., an antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this disclosure, wherein the disease is associated with a protein specifically bound to a protein-targeting ligand contained in the protein-drug conjugate.

[0066] In one aspect, this disclosure provides a method of treating tumors, comprising administering to a subject in need a therapeutically effective amount of the disclosed protein-drug conjugate (e.g., an antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this disclosure.

[0067] Connector fragments

[0068] Linker fragments in protein drug conjugates (such as antibody-drug conjugates) are used to link protein-targeting ligands to bioactive molecular fragments to release bioactive molecules at the target site. Therefore, linker fragments need to have stability, release specificity, and reduced immunogenicity to reduce systemic toxicity and improve efficacy.

[0069] This disclosure provides a linker segment of Formula I, or a stereoisomer segment thereof.

[0070] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0071] L 1B It is a peptide unit;

[0072] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0073] R 1’ This is a linker fragment that connects to a protein-targeting ligand;

[0074] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0075] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0076] r, p, and q are each independently selected from 0, 1, 2, 3, 4, or 5;

[0077] m1 and m2 are each independently selected from 0, 1, 2 or 3;

[0078] The condition is that when m2 is 0, L 1B When connected to the payload, and m2 is not 0, L 1C Connect to the payload.

[0079] This disclosure provides a linker segment of formula I-0 or a stereoisomer segment thereof.

[0080] Among them, L 1A L1C and L 2 Each can be an independent interval zone, either the same or different.

[0081] L 1B It is a peptide unit;

[0082] R 1’ This is a linker fragment that connects to a protein-targeting ligand;

[0083] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0084] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0085] r, p, and q are each independently selected from 0, 1, 2, 3, 4, or 5;

[0086] m1 and m2 are each independently selected from 0, 1, 2 or 3;

[0087] The condition is that when m2 is 0, L 1B When connected to the payload, and m2 is not 0, L 1C Connect to the payload.

[0088] In some embodiments, this disclosure provides a linker intermediate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the linker intermediate comprises a linker fragment or a stereoisomer thereof represented by Formula I or Formula I-0. The linker intermediates described herein may be intermediate compounds or portions thereof (e.g., portions with one or more leaving groups removed).

[0089] In some implementations, r, p, and q are each independently selected from 1, 2, 3, or 4.

[0090] In some implementations, r, p, and q are each independently selected from 1, 2, or 3.

[0091] In some implementations, m1 and m2 are each independently selected from 0, 1, or 2.

[0092] In some implementations, m1 and m2 are each independently selected from 0 or 1.

[0093] In some embodiments, the linker segment or its stereoisomer segment shown in Formula I or Formula I-0 is selected from the linker segment or its stereoisomer segment shown in Formula I-1 or Formula I-2.

[0094] Among them, L 1A L1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 m1 and m2 are as defined in this disclosure.

[0095] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ It is a linker fragment used to connect to the amino or thiol group of an amino acid residue, or the azide group of a non-natural amino acid residue.

[0096] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ It is a linker fragment used to attach to the amino group of lysine residues, the thiol group of cysteine ​​residues, or the azide group of non-natural amino acid residues.

[0097] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ Selected from Wherein, -PG is an amino linking group.

[0098] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ Selected from Where -PG is an amino linker, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L 2 .

[0099] In some embodiments, -PG is selected from C1-C6 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.

[0100] In some embodiments, -PG is selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxetyl, aziridine, piperidinyl, phenyl, or pyridinyl.

[0101] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0102] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0103] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0104] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 For C1-C 10 Alkylene, C2-C 10 alkenyl, C2-C 10 alkynyl group, C1-C 10 alkylene oxide C1-C 10 Alkylene, (C1-C 10 (alkylene oxide) x (C1-C) 10 (alkylene oxide) x C1-C 10 alkyleneamine, C1-C 10 alkylene amide group (C1-C) 10 (alkylene oxide) x C1-C 10 Alkylene, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic, C6-C 10 Aryl, 5-10 heteroaryl, -O-, -S-, -NH-, -NHC(O)-, -OC(O)-, And any combination thereof, where x is independently selected from 0, 1, 2, 3, 4, 5 or 6.

[0105] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 It can be C1-C6 alkylene, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyleneoxy, C1-C6 alkylene, or (C1-C6 alkyleneoxy) x (C1-C6 alkylene oxides) x C1-C6 alkylene amino, C1-C6 alkylene amide (C1-C6 alkylene oxy) x C1-C6 alkylene or Each x is independently selected from 0, 1, 2, 3, 4, or 5.

[0106] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene amide (C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0107] In this disclosure, (C1-C 10 (alkylene oxide) x It covers all of C1-C. 10 In cases where the alkylene groups are the same or different (e.g., ethoxyethoxy, methoxyethoxy), and (C1-C6 alkylene groups) x (C1-C4 alkylene oxides) x They are in the same situation.

[0108] In some embodiments, the “amide group” described in this disclosure is -C(O)NH- or -NHC(O)-.

[0109] In some embodiments, the “carboxylic acid ester group” described in this disclosure is -C(O)-O- or -OC(O)-.

[0110] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene-C(O)NH-(C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0111] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 for

[0112] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 2 It is a C1-C6 alkylene group. In some embodiments, L 2 The derivatives are methylene, ethylene, propylene, butylene, and pentylene. In some embodiments, L... 2 for In some implementations, L 2 for

[0113] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0114] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 8-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 8-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-8 cycloalkyl- and -C(O)-NH-C 4-8 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0115] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0116] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0117] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 heteroatoms selected from N, O or S.

[0118] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4- 6-cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0119] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 For single bond,

[0120] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 For single bond,

[0121] In some embodiments, in the connector segment shown in Formula I or Formula I-2, L 3 It is a single key.

[0122] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R A and R BEach of the C1-C4 alkyl groups is independently substituted with H, C1-C4 alkyl, C1-C4 haloalkyl or hydroxyl.

[0123] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R A and R B Each is independently H or C1-C4 alkyl. In some embodiments, R A and R B Each is independently H or C1-C3 alkyl. In some embodiments, R A and R B Each is independently H, methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, R A and R B Each is independently H, methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently represented by H.

[0124] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each of these is independently PEG (e.g., PEG2 to PEG24) or its derivatives (e.g., methoxy PEG (mPEG), carboxyl PEG (PEG-COOH), amino PEG (NH2-PEG), PEG-NHS ester, azide PEG (PEG-N3), glucose-modified azide PEG), azides and their derivatives (e.g., sugar-modified azides, such as glucose-modified azides), sugars (e.g., glucose or galactose) and their derivatives (e.g., sorbitol), cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin) and their derivatives, polyarginine and its derivatives, or polyglycine and its derivatives.

[0125] By including hydrophilic unit R 2 and R 3 It can achieve effects such as balancing the hydrophobicity of bioactive molecules, increasing DAR value, increasing solubility, reducing the aggregation of protein drug conjugates (such as antibody-drug conjugates), reducing immunogenicity, prolonging the half-life of protein drug conjugates (such as antibody-drug conjugates), and optimizing pharmacokinetics.

[0126] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each independently -(C1-C6 alkylene C(O)NR 4 ) y C(O)C1-C6 alkyl, Where y is an integer from 1 to 24, G represents either glucosyl or galactosyl, CD represents either α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and R... 4 Each occurrence is independently of H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxy-substituted C1-C6 alkyl, z is independently an integer from 1 to 5, and k is independently an integer from 1 to 10.

[0127] In some embodiments, the α-cyclodextrin group has the following structure:

[0128] In some embodiments, the β-cyclodextrin group has the following structure:

[0129] In some embodiments, the γ-cyclodextrin group has the following structure:

[0130] In some implementations, CD each independently represents a β-cyclodextrin group.

[0131] In some embodiments, when multiple Gs are present in the linker fragments shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, the multiple Gs are linked by glycosidic bonds.

[0132] In some implementations, the R 2 and R 3 Each component independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl. In some embodiments, the Rs... 2 and R 3 Each of the Gs independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl, and the glucosyl groups, galactosyl groups, or galactose and glucose are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0133] In some embodiments, G represents glucose. In some embodiments, R... 2 and R 3Each of the components independently contains multiple Gs, where each G represents a glucose unit, and the multiple glucose units are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0134] In some embodiments, G represents galactosyl. In some embodiments, R... 2 and R 3 Each of the multiple Gs independently represents a galactosyl group, and the multiple galactosyl groups are linked by β-1,4 glycosidic bonds, α-1,4 glycosidic bonds, α-1,6 glycosidic bonds, β-1,6 glycosidic bonds, α-1,3 glycosidic bonds, or β-1,3 glycosidic bonds.

[0135] In some implementations, the R 4 Each instance is independently H or C1-C6 alkyl. In some embodiments, the R... 4 Each instance is independently H or C1-C5 alkyl. In some embodiments, the R... 4 Each of these substances, when it appears, is independently methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, the R... 4 Each of these compounds is independently methyl, ethyl, or propyl, depending on its appearance.

[0136] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each independently Where y is an integer from 1 to 24, and k is an integer from 1 to 10.

[0137] In some implementations, y is an integer from 2 to 12. In some implementations, y is an integer from 3 to 12. In some implementations, y is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0138] In some implementations, z is independently 1, 2, 3, or 4. In some implementations, z is independently 1, 2, or 3. In some implementations, z is independently 1 or 2.

[0139] In some implementations, k is an independent integer from 1 to 8. In some implementations, k is an independent integer from 1 to 6. In some implementations, k is an independent integer from 1 to 5. In some implementations, k is an independent integer of 1, 2, 3, or 4. In some implementations, k is an independent integer of 1, 2, or 3.

[0140] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each independently

[0141] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each independently In each of these numbers, y is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0142] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, R 2 and R 3 Each independently

[0143] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1A Each independently Each h is an integer from 0 to 10 (preferably an integer from 1 to 8, 2 to 7, or 3 to 6, such as 2, 3, 4, 5, 6, 7, or 8).

[0144] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1A Each independently

[0145] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently Where f is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4). In some implementations, R 5 Each is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, -NO2, or halogen. In some embodiments, R 5Each is independently selected from methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoromethoxy, -NO2, fluorine, or chlorine.

[0146] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently

[0147] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each is independently -CH2-NH-.

[0148] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently Or -CH2-NH-*, where * represents a combination with L 1B The ends are connected.

[0149] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently Where * represents L 1B The ends are connected.

[0150] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently Where s and t are each independently an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, s is 2, 3, 4, or 5. In some embodiments, t is 1, 2, 3, or 4. In some embodiments, s is 2, 3, or 4, and t is 1 or 2. In some embodiments, s is 3. In some embodiments, t is 1.

[0151] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently

[0152] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1C Each independently Where * represents L 1B The ends are connected.

[0153] In some embodiments, in the connector segments shown in Equations I, I-0, I-1, or I-2 above, m1 and m2 may be the same or different, each being an independent integer from 0 to 3, such as 0 or 1. In some embodiments, in the connector segments shown in Equations I, I-0, I-1, or I-2 above, m1 is 0. In some embodiments, in the connector segments shown in Equations I, I-0, I-1, or I-2 above, m2 is 1.

[0154] In some embodiments, in the connector segment shown in Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1B It is a peptide unit that can be specifically cleaved by cathepsins.

[0155] In some embodiments, in the connector segment shown by Formula I, Formula I-0, Formula I-1 or Formula I-2, L 1B It is a short peptide composed of natural amino acids or non-natural amino acids and their analogues. In some embodiments, L 1B It is a dipeptide, tripeptide, tetrapeptide, or a modified product thereof. In some embodiments, L 1B For Val-Cit, Val-Ala, Val-Lys, Val-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Phe-Lys, Ala-Ala, Phe-Cit, Gly- Gly, Gly-Arg, Arg-Arg, Gly-Gly-Val-Cit, Val-Ala-Gly-Gly, Ala-Leu-Ala-Leu, Ser-Ser-Tyr-Ser, Val-N 6 N 6 -Dipropyllysine. In some embodiments, L 1B For Val-Cit. In some implementations, L 1B It is Gly-Gly-Phe-Gly.

[0156] Connector - Payload

[0157] The payload exerts the intracellular cytotoxic activity of protein drug conjugates. Payloads with the following characteristics can be selected: sufficiently high cytotoxicity; sufficiently low immunogenicity; high stability; a modifiable functional set without significantly affecting efficacy; bystander-kill effect; appropriate water solubility; and an intracellular target (e.g., most ADCs release their payload only after entering tumor cells). Prodrugs constructed by linking the payload to suitable linkers can facilitate specific release of the payload at the target lesion site, thereby reducing systemic toxicity and improving efficacy. Challenges such as solvent effects and poor sample stability must be avoided in linker-payload development.

[0158] In some embodiments, this disclosure relates to a linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the linker-payload comprises a linker fragment or a stereoisomer fragment of Formula I or Formula I-0, and the payload is a bioactive molecular fragment.

[0159] In some embodiments, this disclosure relates to a linker-payload of formula II-0, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0160] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0161] L 1B It is a peptide unit;

[0162] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0163] R 1 For connector unit;

[0164] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0165] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0166] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0167] m1 and m2 are each independently 0, 1, 2 or 3;

[0168] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0169] D represents a bioactive molecular fragment.

[0170] In some embodiments, this disclosure relates to a linker-payload of Formula II, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0171] Among them, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0172] L 1B It is a peptide unit;

[0173] R 1 For connector unit;

[0174] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0175] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0176] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0177] m1 and m2 are each independently 0, 1, 2 or 3;

[0178] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0179] D represents a bioactive molecular fragment.

[0180] In some implementations, r, p, and q are each independently selected from 1, 2, 3, or 4.

[0181] In some implementations, r, p, and q are each independently selected from 1, 2, or 3.

[0182] In some implementations, m1 and m2 are each independently selected from 0, 1, or 2.

[0183] In some implementations, m1 and m2 are each independently selected from 0 or 1.

[0184] In some embodiments, this disclosure relates to a linker-payload of formula II-1 or II-2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0185] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 m1, m2, and D are as defined in this disclosure.

[0186] In some embodiments, the bioactive molecule is selected from cytotoxic drugs, immune agonists (such as STING agonists, Toll receptor agonists (e.g., TLR7 agonists, TLR8 agonists, or TLR9 agonists), glucocorticoid receptor modulators, Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, proteasome inhibitors (Carmaphycins), tyrosine kinase inhibitors, or radionuclide drugs.

[0187] In some embodiments, the cytotoxic drug is selected from one or more of the following: DNA alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, or RNA polymerase II (RNApol II) inhibitors.

[0188] In some embodiments, the cytotoxic drug is selected from camptothecin drugs (e.g., ethanotecan or its derivatives) or taxane drugs (e.g., paclitaxel or its derivatives, docetaxel or its derivatives).

[0189] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 1 It can form covalent bonds with the amino or thiol groups of amino acid residues, or the azide groups of non-natural amino acid residues, through chemical reactions (such as addition reactions).

[0190] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 1It can form covalent bonds with the amino group of lysine residues, the thiol group of cysteine ​​residues, or the azide group of non-natural amino acid residues through chemical reactions (such as addition reactions).

[0191] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 1 Selected from Wherein, -PG is an amino linking group.

[0192] In some embodiments, -PG is selected from C1-C6 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.

[0193] In some embodiments, -PG is selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxetyl, aziridine, piperidinyl, phenyl, or pyridinyl.

[0194] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 1 for In some implementation schemes, R 1 for

[0195] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 For C1-C 10 Alkylene, C2-C 10 alkenyl, C2-C 10 alkynyl group, C1-C 10 alkylene oxide C1-C 10 Alkylene, (C1-C 10 (alkylene oxide) x (C1-C) 10 (alkylene oxide) x C1-C 10 alkyleneamine, C1-C 10 alkylene amide group (C1-C) 10 (alkylene oxide) x C1-C 10 Alkylene, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic, C6-C 10 Aryl, 5-10 heteroaryl, -O-, -S-, -NH-, -NHC(O)-, -OC(O)-, And any combination thereof, where x is independently selected from 0, 1, 2, 3, 4, 5 or 6.

[0196] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 It can be C1-C6 alkylene, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyleneoxy, C1-C6 alkylene, or (C1-C6 alkyleneoxy) x (C1-C6 alkylene oxides) x C1-C6 alkylene amino, C1-C6 alkylene amide (C1-C6 alkylene oxy) x C1-C6 alkylene or Each x is independently selected from 0, 1, 2, 3, 4, or 5.

[0197] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene amide (C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0198] In this article, (C1-C 10 (alkylene oxide) x It covers all of C1-C. 10 In cases where the alkylene groups are the same or different (e.g., ethoxyethoxy, methoxyethoxy), and (C1-C6 alkylene groups) x (C1-C4 alkylene oxides) x They are in the same situation.

[0199] In some embodiments, the “amide group” described in this disclosure is -C(O)NH- or -NHC(O)-.

[0200] In some embodiments, the “carboxylic acid ester group” described in this disclosure is -C(O)-O- or -OC(O)-.

[0201] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy).x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene-C(O)NH-(C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0202] In some preferred embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 for

[0203] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 2 It is a C1-C6 alkylene group. In some embodiments, L 2 The derivatives are methylene, ethylene, propylene, butylene, and pentylene. In some embodiments, L... 2 for In some implementations, L 2 for

[0204] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0205] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 8-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 8-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-8 cycloalkyl- and -C(O)-NH-C 4-8 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0206] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0207] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0208] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 heteroatoms selected from N, O or S.

[0209] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0210] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 For single bond,

[0211] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 For single bond,

[0212] In some embodiments, in the connector segment shown in Formula II-0 or Formula II-2, L 3 It is a single key.

[0213] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R A and R B Each of the C1-C4 alkyl groups is independently substituted with H, C1-C4 alkyl, C1-C4 haloalkyl or hydroxyl.

[0214] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R A and R B Each is independently H or C1-C4 alkyl. In some embodiments, R A and R B Each is independently H or C1-C3 alkyl. In some embodiments, R A and R B Each is independently H, methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, R A and R B Each is independently H, methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently represented by H.

[0215] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each of these is independently PEG (e.g., PEG2 to PEG24) or its derivatives (e.g., methoxy PEG (mPEG), carboxyl PEG (PEG-COOH), amino PEG (NH2-PEG), PEG-NHS ester, azide PEG (PEG-N3), glucose-modified azide PEG), azides and their derivatives (e.g., sugar-modified azides, such as glucose-modified azides), sugars (e.g., glucose or galactose) and their derivatives (e.g., sorbitol), cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin) and their derivatives, polyarginine and its derivatives, or polyglycine and its derivatives.

[0216] In the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, the inclusion of a hydrophilic unit R... 2 and R 3 It can achieve effects such as balancing the hydrophobicity of bioactive molecules, increasing DAR value, increasing solubility, reducing ADC aggregation, reducing immunogenicity, prolonging ADC half-life, and optimizing pharmacokinetics.

[0217] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each independently -(C1-C6 alkylene C(O)NR 4 ) y C(O)C1-C6 alkyl, Where y is an integer from 1 to 24, G represents either glucosyl or galactosyl, CD represents either α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin, and R... 4 Each occurrence is independently of H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxy-substituted C1-C6 alkyl, z is independently an integer from 1 to 5, and k is independently an integer from 1 to 10.

[0218] In some implementations, CD each independently represents a β-cyclodextrin group.

[0219] In some embodiments, when a plurality of Gs are present in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, the plurality of Gs are linked by glycosidic bonds.

[0220] In some implementations, the R 2 and R 3 Each component independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl. In some embodiments, the Rs... 2 and R 3 Each of the Gs independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl, and the glucosyl groups, galactosyl groups, or galactose and glucose are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0221] In some embodiments, G represents glucose. In some embodiments, R... 2 and R 3 Each of the components independently contains multiple Gs, where each G represents a glucose unit, and the multiple glucose units are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0222] In some embodiments, G represents galactosyl. In some embodiments, R... 2 and R 3 Each of the multiple Gs independently represents a galactosyl group, and the multiple galactosyl groups are linked by β-1,4 glycosidic bonds, α-1,4 glycosidic bonds, α-1,6 glycosidic bonds, β-1,6 glycosidic bonds, α-1,3 glycosidic bonds, or β-1,3 glycosidic bonds.

[0223] In some implementations, the R 4 Each instance is independently H or C1-C6 alkyl. In some embodiments, the R... 4 Each instance is independently H or C1-C5 alkyl. In some embodiments, the R... 4 Each of these substances, when it appears, is independently methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, the R... 4 Each of these compounds is independently methyl, ethyl, or propyl, depending on its appearance.

[0224] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each independently Where y is an integer from 1 to 24, and k is an integer from 1 to 10.

[0225] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, y is each independently an integer from 2 to 12. In some embodiments, y is each independently an integer from 3 to 12. In some embodiments, y is each independently 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0226] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, z is independently 1, 2, 3, or 4. In some embodiments, z is independently 1, 2, or 3. In some embodiments, z is independently 1 or 2.

[0227] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, k is each independently an integer from 1 to 8. In some embodiments, k is each independently an integer from 1 to 6. In some embodiments, k is each independently an integer from 1 to 5. In some embodiments, k is each independently 1, 2, 3, or 4. In some embodiments, k is each independently 1, 2, or 3.

[0228] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each independently

[0229] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each independently In each of these numbers, y is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0230] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, R 2 and R 3 Each independently

[0231] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1A Each independently Each h is an integer from 0 to 10 (preferably an integer from 1 to 8, 2 to 7, or 3 to 6, such as 2, 3, 4, 5, 6, 7, or 8).

[0232] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1A Each independently

[0233] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently Where f is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4). In some implementations, R 5 Each is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, -NO2, or halogen. In some embodiments, R 5 Each is independently selected from methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoromethoxy, -NO2, fluorine, or chlorine.

[0234] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently

[0235] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each is independently -CH2-NH-.

[0236] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently Or -CH2-NH-*, where * represents a combination with L 1B The ends are connected.

[0237] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently Where * represents L 1B The ends are connected.

[0238] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently Where s and t are each independently an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, s is 2, 3, 4, or 5. In some embodiments, t is 1, 2, 3, or 4. In some embodiments, s is 2, 3, or 4, and t is 1 or 2. In some embodiments, s is 3. In some embodiments, t is 1.

[0239] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1CEach independently

[0240] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1C Each independently Where * represents L 1B The ends are connected. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, m1 and m2 may be the same or different, each independently being an integer from 0 to 3, for example, 0 or 1. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, m1 is 0. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1, or Formula II-2, m2 is 1.

[0241] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1B It is a peptide unit that can be specifically cleaved by cathepsins.

[0242] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1B A short peptide composed of natural amino acids or non-natural amino acids and their analogues. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1B It is a dipeptide, tripeptide, tetrapeptide, or a modified product thereof. In some embodiments, L 1B For Val-Cit, Val-Ala, Val-Lys, Val-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Phe-Lys, Ala-Ala, Phe-Cit, Gly- Gly, Gly-Arg, Arg-Arg, Gly-Gly-Val-Cit, Val-Ala-Gly-Gly, Ala-Leu-Ala-Leu, Ser-Ser-Tyr-Ser, Val-N 6 N 6 -Dipropyllysine. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1B Val-Cit. In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, L 1B It is Gly-Gly-Phe-Gly.

[0243] In some embodiments, in the compounds represented by Formula II, Formula II-0, Formula II-1 or Formula II-2, the linker fragment (especially L) 1B (when m2 is 0) or L 1C (When m2 is not 0) it is linked to the amino group in D.

[0244] This disclosure relates to a linker-payload of formula III-A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0245] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are defined as above.

[0246] This disclosure relates to a linker-payload of formula III-A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0247] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0248] This disclosure relates to a linker-payload of formula III-2A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0249] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3r, p, q, m1, and m2 are defined as above.

[0250] This disclosure relates to a linker-payload of formula III-2A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0251] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0252] This disclosure relates to a linker-payload of formula III-1A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0253] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0254] This disclosure relates to a linker-payload of formula III-1A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0255] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L1B The ends are connected.

[0256] This disclosure relates to a linker-payload of formula III-3A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0257] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0258] This disclosure relates to a linker-payload of formula III-3A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0259] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 m1 and m2 are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected. This disclosure relates to a connector of formula III-B—a payload, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0260] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are defined as above.

[0261] This disclosure relates to a linker-payload of formula III-2B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0262] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are defined as above.

[0263] This disclosure relates to a linker-payload of formula III-3B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0264] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are defined as above.

[0265] This disclosure relates to a linker-payload of formula III-1B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0266] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0267] This disclosure relates to a linker-payload of formula III-4B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0268] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0269] This disclosure relates to a linker-payload of formula III-5B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0270] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0271] This disclosure relates to a linker-payload of formula III-C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0272] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0273] This disclosure relates to a linker-payload of formula III-2C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0274] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 r, p, q, m1, and m2 are defined as above.

[0275] This disclosure relates to a linker-payload of formula III-3C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0276] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R BR 2 R 3 r, p, q, m1, and m2 are defined as above.

[0277] This disclosure relates to a linker-payload of formula III-1C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0278] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0279] This disclosure relates to a linker-payload of formula III-4C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0280] Among them, L 1A L 1C L 2 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0281] This disclosure relates to a linker-payload of formula III-5C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0282] Among them, L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 m1 and m2 are defined as above.

[0283] This disclosure relates to a connector-payload of formula V, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0284] Among them, R 6 For hydrogen, methyl, D, L3 p, q, R A R B R 2 and R 3 As defined in this article.

[0285] In some embodiments, D is derived from paclitaxel or docetaxel. In some embodiments, D is...

[0286] In some embodiments, this disclosure relates to a linker-payload of formula V-1 or V-2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0287] Among them, R 6 For hydrogen, methyl, L 3 p, q, R A R B R 2 and R 3 As defined in this article.

[0288] In some implementations, in formula V, formula V-1 or formula V-2, R 6 Methyl or In some implementations, in formula V, formula V-1 or formula V-2, p and q are each independently 0, 1, 2 or 3, preferably p and q are each independently 0, 1 or 2.

[0289] In some implementations, in formula V, formula V-1 or formula V-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl groups, or -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl group contains 1-3 (preferably 1-2) heteroatoms selected from N, O, or S. In some embodiments, L 3 Each is independently selected from a single bond, -C(O)-6-membered heterocyclic alkyl-, or -C(O)-NH-C6 cycloalkyl-, wherein the heterocyclic alkyl contains 1-3 (preferably 1-2) atoms selected from N atoms.

[0290] In some implementations, in formula V, formula V-1 or formula V-2, R A and R B Each is independently H or C1-C3 alkyl. In some embodiments, R A and R B Each is independently represented by H.

[0291] In some implementations, in formula V, formula V-1 or formula V-2, R2 and R 3 Each independently Where y is an independent integer from 1 to 24. In some implementations, R 2 and R 3 Each independently In this context, each y is an independent integer from 2 to 12.

[0292] In some embodiments, this disclosure relates to a linker-payload of formula V-1A, V-1B, V-1C, V-2A, V-2B, or V-2C, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0293] Among them, R 6 For hydrogen, methyl, Each y is an independent integer from 2 to 12.

[0294] In some implementation schemes, R 6 Methyl or

[0295] In some implementations, y is an integer independently of 3-10, 4-9, 5-8, 6-8, or 7-8. In some implementations, y is 8.

[0296] In some embodiments, the connector-load capacity shown in Formula V-1C or Formula V-2C is the connector-load capacity shown in Formula V-1C1 or Formula V-2C1:

[0297] Among them, R 6 And y are as defined above.

[0298] In some embodiments, the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof may be selected from:

[0299] Protein drug conjugates

[0300] Protein drug conjugates (e.g., antibody-drug conjugates) are obtained by conjugating a protein-targeting ligand (e.g., an antibody) to a payload portion via a linker fragment. This allows for the selective delivery of the payload to the target lesion site, thereby reducing toxicity and improving pharmacokinetics.

[0301] In some embodiments, this disclosure relates to a protein drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the protein drug conjugate comprises a linker fragment of Formula I or Formula I-0 or a stereoisomer fragment thereof.

[0302] In some embodiments, this disclosure relates to a protein drug conjugate of Formula IV, its stereoisomer, or a pharmaceutically acceptable salt thereof, Ab-(LD). n IV

[0303] Among them, Ab is a protein-targeting ligand;

[0304] L is the linker segment or its stereoisomer segment shown in Formula I;

[0305] D represents a bioactive molecular fragment;

[0306] n is an integer or decimal selected from 0 and less than or equal to 20.

[0307] In some implementations, n is an integer or decimal selected from 2 to 8.

[0308] In some implementations, n is an integer or decimal selected from 2-8, 2-6, 2-4, 4-8, 6-8, or 7-8.

[0309] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0310] Among them, Ab is a protein-targeting ligand;

[0311] L 1A L 1C and L 2Each can be an independent interval zone, either the same or different.

[0312] L 1B It is a peptide unit;

[0313] R 1’ For the connector segment;

[0314] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0315] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0316] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0317] m1 and m2 are each independently 0, 1, 2 or 3;

[0318] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0319] D represents a bioactive molecular fragment;

[0320] n is an integer or decimal between 2 and 8.

[0321] In some implementations, r, p, and q are each independently selected from 1, 2, 3, or 4.

[0322] In some implementations, r, p, and q are each independently selected from 1, 2, or 3.

[0323] In some implementations, m1 and m2 are each independently selected from 0, 1, or 2.

[0324] In some implementations, m1 and m2 are each independently selected from 0 or 1.

[0325] In some implementations, n is an integer or decimal selected from 2-8, 2-6, 2-4, 4-8, 6-8, or 7-8.

[0326] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-3, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0327] Among them, Ab is a protein-targeting ligand;

[0328] L 1A L 1C and L2 Each can be an independent interval zone, either the same or different.

[0329] L 1B It is a peptide unit;

[0330] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0331] R 1’ For the connector segment;

[0332] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0333] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0334] r, p, and q are each independently 0, 1, 2, 3, 4, or 5;

[0335] m1 and m2 are each independently 0, 1, 2 or 3;

[0336] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0337] D represents a bioactive molecular fragment;

[0338] n is an integer or decimal between 2 and 8.

[0339] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-2, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0340] Among them, Ab is a protein-targeting ligand;

[0341] L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0342] L 1B It is a peptide unit;

[0343] R1’ For the connector segment;

[0344] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0345] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0346] m1 and m2 are each independently 0, 1, 2 or 3;

[0347] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0348] D represents a bioactive molecular fragment;

[0349] n is an integer or decimal between 2 and 8.

[0350] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-4, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0351] Among them, Ab is a protein-targeting ligand;

[0352] L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0353] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0354] L 1B It is a peptide unit;

[0355] R 1’ For the connector segment;

[0356] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0357] R 2 and R3 Each can be an independent hydrophilic unit, either the same or different.

[0358] m1 and m2 are each independently 0, 1, 2 or 3;

[0359] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0360] D represents a bioactive molecular fragment;

[0361] n is an integer or decimal between 2 and 8.

[0362] In some embodiments, the protein-targeting ligand is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof targeting one or more (e.g., 1-2, 1-3) targets selected from: HER2, HER3, EGFR, ROR1, CLDN18.2, CLDN6, B7-H3, B7-H4, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, GPC3, DLL3, FRα, TF, Nectin-4, CDH17, FGFR2b, PD-1, and PD-L1.

[0363] In some embodiments, the antibody or its antigen-binding fragment is an anti-HER2 antibody or its antigen-binding fragment, an anti-HER3 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, an anti-ROR1 antibody or its antigen-binding fragment, an anti-CLDN18.2 antibody or its antigen-binding fragment, an anti-CLDN6 antibody or its antigen-binding fragment, an anti-B7-H3 antibody or its antigen-binding fragment, an anti-B7-H4 antibody or its antigen-binding fragment, an anti-TROP-2 antibody or its antigen-binding fragment, an anti-CD20 antibody or its antigen-binding fragment, an anti-CD22 antibody or its antigen-binding fragment, an anti-CD30 antibody or its antigen-binding fragment, an anti-CD33 antibody or its antigen-binding fragment, an anti-CD47 antibody or its antigen-binding fragment, an anti-CD56 antibody or its antigen-binding fragment, or an anti-CD7 antibody. Antibody or its antigen-binding fragment, anti-CD79b antibody or its antigen-binding fragment, anti-VEGF antibody or its antigen-binding fragment, anti-VEGFR antibody or its antigen-binding fragment, anti-MUC1 antibody or its antigen-binding fragment, anti-c-MET antibody or its antigen-binding fragment, anti-RET antibody or its antigen-binding fragment, anti-LIV-1 antibody or its antigen-binding fragment, anti-GPC3 antibody or its antigen-binding fragment, anti-DLL3 antibody or its antigen-binding fragment, FRα antibody or its antigen-binding fragment, anti-TF antibody or its antigen-binding fragment, anti-Nectin-4 antibody or its antigen-binding fragment, anti-CDH17 antibody or its antigen-binding fragment, anti-FGFR2b antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, or anti-PD-L1 antibody or its antigen-binding fragment.

[0364] In some embodiments, the antibody or its antigen-binding fragment is selected from trastuzumab or its antigen-binding fragment.

[0365] In some embodiments, the bioactive molecule is selected from cytotoxic drugs, immune agonists (such as STING agonists, Toll receptor agonists (e.g., TLR7 agonists, TLR8 agonists, or TLR9 agonists)), glucocorticoid receptor modulators, Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, proteasome inhibitors (Carmaphycins), tyrosine kinase inhibitors, or radionuclide drugs.

[0366] In some embodiments, the cytotoxic drug is selected from one or more of the following: DNA alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, or RNA polymerase II (RNApol II) inhibitors.

[0367] In some embodiments, the cytotoxic drug is selected from camptothecin drugs (e.g., ethanotecan or its derivatives) or taxane drugs (e.g., paclitaxel or its derivatives, docetaxel or its derivatives).

[0368] In some embodiments, the cytotoxic drug is selected from ethatecan, paclitaxel, or docetaxel.

[0369] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ It is a linker fragment used to connect to the amino or thiol group of an amino acid residue, or the azide group of a non-natural amino acid residue.

[0370] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ It is a linker fragment used to attach to the amino group of lysine residues, the thiol group of cysteine ​​residues, or the azide group of non-natural amino acid residues.

[0371] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ Selected from Wherein, -PG is an amino linking group.

[0372] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ Selected from Where -PG is an amino linker, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L 2 .

[0373] In some embodiments, -PG is selected from C1-C6 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.

[0374] In some embodiments, -PG is selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxetyl, aziridine, piperidinyl, phenyl, or pyridinyl.

[0375] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0376] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0377] In some embodiments, in the compounds represented by formula IV-1, IV-2, IV-3 or IV-4, R 1’ Selected from In this context, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 .

[0378] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 For C1-C 10 Alkylene, C2-C 10 alkenyl, C2-C 10 alkynyl group, C1-C 10 alkylene oxide C1-C 10 Alkylene, (C1-C 10 (alkylene oxide) x (C1-C) 10 (alkylene oxide) x C1-C 10 alkyleneamine, C1-C 10 alkylene amide group (C1-C) 10 (alkylene oxide) x C1-C 10 Alkylene, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic, C6-C 10 Aryl, 5-10 heteroaryl, -O-, -S-, -NH-, -NHC(O)-, -OC(O)-, And any combination thereof, where x is independently selected from 0, 1, 2, 3, 4, 5 or 6.

[0379] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 It can be C1-C6 alkylene, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyleneoxy, C1-C6 alkylene, or (C1-C6 alkyleneoxy) x (C1-C6 alkylene oxides) x C1-C6 alkylene amino, C1-C6 alkylene amide (C1-C6 alkylene oxy) x C1-C6 alkylene or Each x is independently selected from 0, 1, 2, 3, 4, or 5.

[0380] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene amide (C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0381] In this article, (C1-C 10 (alkylene oxide) x It covers all of C1-C. 10 In cases where the alkylene groups are the same or different (e.g., ethoxyethoxy, methoxyethoxy), and (C1-C6 alkylene groups) x (C1-C4 alkylene oxides) x They are in the same situation.

[0382] In some embodiments, the “amide group” described in this disclosure is -C(O)NH- or -NHC(O)-.

[0383] In some embodiments, the “carboxylic acid ester group” described in this disclosure is -C(O)-O- or -OC(O)-.

[0384] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene-C(O)NH-(C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4.

[0385] In some preferred embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 for

[0386] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 It is a C1-C6 alkylene group. In some embodiments, L 2 The derivatives are methylene, ethylene, propylene, butylene, and pentylene. In some embodiments, L... 2 for In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 2 for

[0387] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0388] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 Each is independently selected from single bonds, -C(O)-4 to 8-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 8-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-8 cycloalkyl- and -C(O)-NH-C 4-8 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0389] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S.

[0390] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0391] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 heteroatoms selected from N, O or S.

[0392] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 (preferably 1-2) N atoms.

[0393] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 For single bond,

[0394] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 For single bond,

[0395] In some embodiments, in the connector segment shown in Formula IV-3 or Formula IV-4, L 3 It is a single key.

[0396] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R A and RB Each of the C1-C4 alkyl groups is independently substituted with H, C1-C4 alkyl, C1-C4 haloalkyl or hydroxyl.

[0397] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R A and R B Each is independently H or C1-C4 alkyl. In some embodiments, R A and R B Each is independently H or C1-C3 alkyl. In some embodiments, R A and B Each is independently H, methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, R A and R B Each is independently H, methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently methyl, ethyl, or propyl. In some embodiments, R A and R B Each is independently represented by H.

[0398] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each of these is independently PEG (e.g., PEG2 to PEG24) or its derivatives (e.g., methoxy PEG (mPEG), carboxyl PEG (PEG-COOH), amino PEG (NH2-PEG), PEG-NHS ester, azide PEG (PEG-N3), glucose-modified azide PEG), azides and their derivatives (e.g., sugar-modified azides, such as glucose-modified azides), sugars (e.g., glucose or galactose) and their derivatives (e.g., sorbitol), cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin) and their derivatives, polyarginine and its derivatives, or polyglycine and its derivatives.

[0399] In the compounds represented by Formula IV, Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, the inclusion of a hydrophilic unit (e.g., R in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4) 2 and R 3 It can achieve effects such as balancing the hydrophobicity of bioactive molecules, increasing DAR value, increasing solubility, reducing ADC aggregation, reducing immunogenicity, prolonging ADC half-life, and optimizing pharmacokinetics.

[0400] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each independently -(C1-C6 alkylene C(O)NR 4 ) y C(O)C1-C6 alkyl, Where y is an integer from 1 to 24, G represents either glucosyl or galactosyl, CD represents either α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin (e.g., β-cyclodextrin), and R... 4 Each occurrence is independently of H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxy-substituted C1-C6 alkyl, z is independently an integer from 1 to 5, and k is independently an integer from 1 to 10.

[0401] In some implementations, CD each independently represents a β-cyclodextrin group.

[0402] In some embodiments, when a plurality of Gs are present in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, the plurality of Gs are linked by glycosidic bonds.

[0403] In some implementations, the R 2 and R 3 Each component independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl. In some embodiments, the Rs... 2 and R 3 Each of the Gs independently contains one or more Gs, and the Gs may be the same as or different from each other, wherein the Gs represent glucosyl or galactosyl, and the glucosyl groups, galactosyl groups, or galactose and glucose are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0404] In some embodiments, G represents glucose. In some embodiments, R... 2 and R 3 Each of the components independently contains multiple Gs, where each G represents a glucose unit, and the multiple glucose units are linked by α-1,4-glycosidic bonds, β-1,4-glycosidic bonds, α-1,6-glycosidic bonds, β-1,6-glycosidic bonds, α-1,3-glycosidic bonds, β-1,3-glycosidic bonds, α-1,2-glycosidic bonds, or β-1,2-glycosidic bonds.

[0405] In some embodiments, G represents galactosyl. In some embodiments, R... 2 and R 3 Each of the multiple Gs independently represents a galactosyl group, and the multiple galactosyl groups are linked by β-1,4 glycosidic bonds, α-1,4 glycosidic bonds, α-1,6 glycosidic bonds, β-1,6 glycosidic bonds, α-1,3 glycosidic bonds, or β-1,3 glycosidic bonds.

[0406] In some implementations, the R 4 Each instance is independently H or C1-C6 alkyl. In some embodiments, the R... 4 Each instance is independently H or C1-C5 alkyl. In some embodiments, the R... 4 Each of these substances, when it appears, is independently methyl, ethyl, propyl, butyl, or pentyl. In some embodiments, the R... 4 Each of these compounds is independently methyl, ethyl, or propyl, depending on its appearance.

[0407] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each independently Where y is an integer from 1 to 24, and k is an integer from 1 to 10.

[0408] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, y is each independently an integer from 2 to 12. In some embodiments, y is each independently an integer from 3 to 12. In some embodiments, y is each independently 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0409] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, z is independently 1, 2, 3, or 4. In some embodiments, z is independently 1, 2, or 3. In some embodiments, z is independently 1 or 2.

[0410] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, k is each independently an integer from 1 to 8. In some embodiments, k is each independently an integer from 1 to 6. In some embodiments, k is each independently an integer from 1 to 5. In some embodiments, k is each independently 1, 2, 3, or 4. In some embodiments, k is each independently 1, 2, or 3.

[0411] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each independently

[0412] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each independently In each of these numbers, y is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0413] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, R 2 and R 3 Each independently

[0414] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1A Each independently Each h is an integer from 0 to 10 (preferably an integer from 1 to 8, 2 to 7, or 3 to 6, such as 2, 3, 4, 5, 6, 7, or 8).

[0415] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1A Each independently

[0416] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently Where f is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4). In some implementations, R 5Each is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, -NO2, or halogen. In some embodiments, R 5 Each is independently selected from methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoromethoxy, -NO2, fluorine, or chlorine.

[0417] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently

[0418] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each is independently -CH2-NH-.

[0419] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently Or -CH2-NH-*, where * represents a combination with L 1B The ends are connected.

[0420] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently Where * represents L 1B The ends are connected.

[0421] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently Where s and t are each independently an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, s is 2, 3, 4, or 5. In some embodiments, t is 1, 2, 3, or 4. In some embodiments, s is 2, 3, or 4, and t is 1 or 2. In some embodiments, s is 3. In some embodiments, t is 1.

[0422] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently

[0423] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1C Each independently Where * represents L 1B The ends are connected.

[0424] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, m1 and m2 may be the same or different, each being an independent integer from 0 to 3, for example, 0 or 1. In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, m1 is 0. In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3, or Formula IV-4, m2 is 1.

[0425] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1B It is a peptide unit that can be specifically cleaved by cathepsins.

[0426] In some embodiments, in the compounds represented by Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1B A short peptide composed of natural amino acids or non-natural amino acids and their analogues. In some embodiments, in the compounds shown in Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, L 1B It is a dipeptide, tripeptide, tetrapeptide, or a modified product thereof. In some embodiments, L 1B For Val-Cit, Val-Ala, Val-Lys, Val-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Phe-Lys, Ala-Ala, Phe-Cit, Gly- Gly, Gly-Arg, Arg-Arg, Gly-Gly-Val-Cit, Val-Ala-Gly-Gly, Ala-Leu-Ala-Leu, Ser-Ser-Tyr-Ser, Val-N 6 N 6 -Dipropyllysine. In some embodiments, in the compounds shown in Formula IV-1, IV-2, IV-3 or IV-4, L 1B For Val-Cit. In some embodiments, in the compounds shown in Formula IV-1, IV-2, IV-3 or IV-4, L 1B It is Gly-Gly-Phe-Gly.

[0427] In some embodiments, in the compounds represented by Formula IV, Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, the linker fragment (especially the L) 1B (when m2 is 0) or L1C (When m2 is not 0) it is linked to the amino group in D.

[0428] In some embodiments, in the antibody-drug conjugates shown in Formula IV, Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, the linker (especially R therein) 1’ It is linked to a protein-targeting ligand (such as an antibody or its antigen-binding fragment) via a thioether bond.

[0429] In some embodiments, in the antibody-drug conjugates shown in Formula IV, Formula IV-1, Formula IV-2, Formula IV-3 or Formula IV-4, the linker fragment (especially the R) 1’ The -(succinimide-3-yl-N)- in ) (i.e., The 3-position of -(succinimide-3-yl-N)- is linked to the antibody or its antigen-binding fragment via a thioether bond. In some specific embodiments, the 3-position of -(succinimide-3-yl-N)- is linked to the antibody or its antigen-binding fragment via a thioether bond.

[0430] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-5, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0431] Among them, Ab is a protein-targeting ligand;

[0432] L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0433] L 1B It is a peptide unit;

[0434] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0435] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0436] m1 and m2 are each independently 0, 1, 2 or 3;

[0437] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0438] D represents a bioactive molecular fragment;

[0439] n is an integer or decimal between 2 and 8.

[0440] In some embodiments, in the compounds represented by Formula IV-5, Ab, L 1A L 1C L 2 L 1B R 2 R 3 The definitions of m1 and m2, D and n are as defined above for the compounds shown in formula IV-1, IV-2, IV-3 or IV-4.

[0441] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-6, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0442] Among them, Ab is a protein-targeting ligand;

[0443] L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different.

[0444] L 1B It is a peptide unit;

[0445] L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-;

[0446] R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl;

[0447] R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different.

[0448] m1 and m2 are each independently 0, 1, 2 or 3;

[0449] The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D;

[0450] D represents a bioactive molecular fragment;

[0451] n is an integer or decimal between 2 and 8.

[0452] In some embodiments, in the compounds represented by Formula IV-6, Ab, L 1A L 1C L 2 L 3 L 1B R 2 R 3 The definitions of m1 and m2, D and n are as defined above for the compounds shown in formula IV-3 or IV-4.

[0453] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-1A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0454] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0455] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-1A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0456] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0457] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-4A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0458] Among them, Ab and L 1A L 1CL 2 L 3 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0459] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-4A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0460] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are as defined above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0461] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-2A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0462] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0463] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-2A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0464] Among them, Ab and L 1A L 1C L 2 L 1B R 1’R A R B R 2 R 3 m1, m2, and n are defined as above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0465] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-5A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0466] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0467] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-5A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0468] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0469] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-3A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0470] Among them, Ab and L 1A L 1C L2 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0471] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-3A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0472] Among them, Ab and L 1A L 1C L 2 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0473] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-6A, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0474] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0475] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-6A', its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0476] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R A R B R 2 R 3m1, m2, and n are defined as above. In some implementations, L 1C Each is independently -CH2-NH-. In some implementations, L 1C Each independently -CH2-NH-*, where * represents the relationship with L 1B The ends are connected.

[0477] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-1B or IV-1B1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0478] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0479] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-4B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0480] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0481] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-2B or IV-2B1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0482] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0483] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-5B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0484] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0485] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-3B or IV-3B1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0486] Among them, Ab and L 1A L 1C L 2 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0487] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-6B, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0488] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0489] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-1C or IV-1C1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0490] Among them, Ab and L 1A L 1C L 2 L 1B R1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0491] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-4C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0492] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1, m2, n are defined as above.

[0493] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-2C or IV-2C1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0494] Among them, Ab and L 1A L 1C L 2 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0495] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-5C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0496] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 m1, m2, and n are defined as above.

[0497] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-3C or IV-3C1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0498] Among them, Ab and L 1A L 1C L 2 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0499] In some embodiments, this disclosure relates to a protein drug conjugate of formula IV-6C, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0500] Among them, Ab and L 1A L 1C L 2 L 3 L 1B R A R B R 2 R 3 m1, m2, and n are defined as above.

[0501] In some embodiments, this disclosure provides a protein drug conjugate of Formula VI, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0502] Among them, R 6 For hydrogen, methyl, Ab、L 3 R A R B R 2 R 3 p, q, and n are defined as above.

[0503] In some embodiments, D is derived from paclitaxel or docetaxel. In some embodiments, D is...

[0504] In some embodiments, this disclosure provides a protein drug conjugate of formula VI-1 or formula VI-2, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0505] Among them, R 6 For hydrogen, methyl, Ab、L 3 p, q, n, R A R B R 2 and R 3 As defined in this article.

[0506] In some implementations, in formula VI, formula VI-1 or formula VI-2, R 6 Methyl or

[0507] In some implementations, in formula VI, formula VI-1 or formula VI-2, p and q are each independently 0, 1, 2 or 3, preferably p and q are each independently 0, 1 or 2.

[0508] In some implementations, in formula VI, formula VI-1 or formula VI-2, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl groups, or -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl group contains 1-3 (preferably 1-2) heteroatoms selected from N, O, or S. In some embodiments, L 3 Each is independently selected from a single bond, -C(O)-6-membered heterocyclic alkyl-, or -C(O)-NH-C6 cycloalkyl-, wherein the heterocyclic alkyl contains 1-3 (preferably 1-2) atoms selected from N atoms.

[0509] In some implementations, in formula VI, formula VI-1 or formula VI-2, R A and R B Each is independently H or C1-C3 alkyl. In some embodiments, R A and R B Each is independently represented by H.

[0510] In some implementations, in formula VI, formula VI-1 or formula VI-2, R 2 and R 3 Each independently Where y is an independent integer from 1 to 24. In some implementations, R 2 and R 3 Each independently In this context, each y is an independent integer from 2 to 12.

[0511] In some embodiments, this disclosure provides a protein drug conjugate of formula VI-1A, VI-1B, VI-1C, VI-2A, VI-2B, or VI-2C, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0512] Among them, R 6 For hydrogen, methyl, Each y is an independent integer from 2 to 12, and Ab and n are defined as above.

[0513] In some implementation schemes, R 6 Methyl or

[0514] In some implementations, y is an integer independently of 3-10, 4-9, 5-8, 6-8, or 7-8. In some implementations, y is 8.

[0515] In some embodiments, the protein drug conjugate of formula VI-1C or formula VI-2C, its stereoisomer, or a pharmaceutically acceptable salt thereof is the protein drug conjugate of formula VI-1C1 or formula VI-2C1, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0516] Among them, Ab, n, R 6 And y are as defined above.

[0517] In some embodiments, this disclosure provides protein (e.g., antibody) drug conjugates, their stereoisomers, or pharmaceutically acceptable salts thereof.

[0518] The number of bioactive molecules (e.g., cytotoxic drugs) linked to the protein-targeting ligand (e.g., antibody or its antigen-binding fragment) in the protein drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt provided in this disclosure can vary, such that the protein drug conjugate (ADC) can be homogeneous or heterogeneous. The heterogeneous type is that the protein drug conjugate includes protein-targeting ligands (e.g., antibodies or their antigen-binding fragments) linked to different numbers of bioactive molecules (e.g., cytotoxic drugs). For example, one molecule of antibody or its antigen-binding fragment may be linked to 0 (i.e., without cytotoxic drugs), 1, 2, 3, 4, 5, 6, 7, 8, or more molecules of cytotoxic drugs.

[0519] In this document, in the protein drug conjugates described herein, n represents the average molar ratio of the bioactive molecule (e.g., a cytotoxic drug) to the protein-targeting ligand (e.g., an antibody or its antigen-binding fragment) in the ADC, for example, the Drug-to-Antibody Ratio (DAR), which represents the average number of cytotoxic drugs conjugated per molecule of antibody or its antigen-binding fragment. In some embodiments, the DAR value (i.e., n) is an integer or decimal of 2-8, such as 2-6, 2-4, 4-8, 6-8, or 7-8. For example, "DAR of 4" means that such an antibody-drug conjugate, its stereoisomers, or its pharmaceutically acceptable salts comprise a heterogeneous mixture in which each antibody or its antigen-binding fragment molecule is linked with the same or different numbers of cytotoxic drugs (e.g., each antibody or its antigen-binding fragment is linked with 0, 1, 2, 3, 4, 5, 6, 7, and / or 8 cytotoxic drugs), but the average number of cytotoxic drugs linked per molecule of antibody or its antigen-binding fragment is 4. Similarly, "DAR 8" means that the average number of cytotoxic drugs attached to each molecule of antibody or its antigen-binding fragment in the antibody-drug conjugate, its stereoisomer, or its pharmaceutically acceptable salt is 8.

[0520] The DAR values ​​(i.e., average drug / antibody ratio) in this article can be obtained by calculation using results from conventional analytical methods known in the art (e.g., reversed-phase LC-MS mass spectrometry and / or HIC-HPLC).

[0521] Pharmaceutical Composition

[0522] In some embodiments, this disclosure relates to a pharmaceutical composition comprising the protein drug conjugate of this disclosure, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0523] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0524] The pharmaceutical compositions described herein can be formulated in any manner known in the art and can be formulated to suit any route of administration (e.g., non-gastrointestinal administration). The pharmaceutical compositions described herein contain therapeutically effective amounts of the aforementioned components.

[0525] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0526] In some embodiments, the pharmaceutical composition is administered via a non-gastrointestinal route.

[0527] In some embodiments, the pharmaceutical composition may be in unit dose form to facilitate administration to the patient at the desired dosage. In some embodiments, a single dose of the pharmaceutical composition may be 0.01-2000 mg, or a single dose may be 0.02-1000 mg.

[0528] In some embodiments, a single-dose pharmaceutical composition comprises 0.01-2000 mg (or 0.02-1000 mg) of a protein drug conjugate (e.g., an antibody-drug conjugate), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0529] The dosage range of the pharmaceutical compositions described in this disclosure can be determined by clinicians based on experience, taking into account factors such as the method of administration (including administration time, administration interval, and route of administration), the patient's age, weight, sex or pathological condition, diet, excretion rate, and sensitivity to the drug.

[0530] The pharmaceutical compositions described herein may be included in containers, packages or dispensers together with the instructions for use.

[0531] Connector segments and the purpose of connector-payload

[0532] In some embodiments, this disclosure relates to the use of the linker fragments of this disclosure or their stereoisomers, the linker intermediates of this disclosure, their stereoisomers, or their pharmaceutically acceptable salts, or the linker-payloads of this disclosure, their stereoisomers, or their pharmaceutically acceptable salts in the preparation of protein drug conjugates (e.g., antibody-drug conjugates).

[0533] In some embodiments, this disclosure relates to linker fragments of this disclosure, or stereoisomer fragments thereof, or linker intermediates of this disclosure, stereoisomers thereof, or pharmaceutically acceptable salts thereof, linker-payloads of this disclosure, stereoisomers thereof, or pharmaceutically acceptable salts thereof, for the preparation of protein drug conjugates (e.g., antibody-drug conjugates).

[0534] In some embodiments, the linker fragments or stereoisomer fragments described herein, or the linker-payload fragments, stereoisomers, or pharmaceutically acceptable salts thereof described herein, constitute part of a protein-drug conjugate (e.g., an antibody-drug conjugate).

[0535] Medical Use

[0536] In some embodiments, this disclosure relates to the use of the protein-drug conjugates of this disclosure (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of this disclosure in the preparation of a medicament for treating a disease, wherein the disease is associated with a protein in which a protein-targeting ligand contained in the protein-drug conjugate specifically binds.

[0537] In some embodiments, this disclosure relates to the use of the protein-drug conjugates of this disclosure (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of this disclosure in the preparation of a medicament for treating tumors.

[0538] In some embodiments, this disclosure relates to protein drug conjugates of this disclosure (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of this disclosure for the treatment of diseases, wherein the disease is associated with a protein in which a protein-targeting ligand contained in the protein drug conjugate specifically binds.

[0539] In some embodiments, this disclosure relates to protein drug conjugates (e.g., antibody-drug conjugates), stereoisomers thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, for the treatment of tumors.

[0540] In some embodiments, this disclosure relates to a method of treating a disease, comprising administering to a subject in need a therapeutically effective amount of the protein-drug conjugate of this disclosure (e.g., an antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this disclosure, wherein the disease is associated with a protein specifically bound to a protein-targeting ligand contained in the protein-drug conjugate.

[0541] In some embodiments, the method includes contacting tumor cells with the protein drug conjugates (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition described herein, thereby killing tumor cells or inhibiting tumor cell growth.

[0542] In some embodiments, the method includes contacting tumor cells with the protein drug conjugate (e.g., antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described herein, and simultaneously or sequentially contacting the tumor cells with one or more additional therapeutic agents, thereby killing tumor cells or inhibiting tumor cell growth.

[0543] In some embodiments, the method includes administering to a subject a therapeutically effective amount of a protein drug conjugate (e.g., an antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present disclosure, which can kill tumor cells or inhibit tumor cell growth.

[0544] In some embodiments, the method includes administering to a subject a therapeutically effective amount of the protein drug conjugate of this disclosure (e.g., an antibody-drug conjugate), its stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more additional therapeutic agents that can kill tumor cells or inhibit tumor cell growth. In some embodiments, administering to a subject a therapeutically effective amount of the pharmaceutical composition of this disclosure and one or more additional therapeutic agents can kill tumor cells or inhibit tumor cell growth.

[0545] In some embodiments, this disclosure relates to a method of treating tumors, including administering to a subject in need a therapeutically effective amount of the protein-drug conjugate of this disclosure (e.g., an antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of this disclosure.

[0546] In some embodiments, the patient is given a therapeutically effective amount of the protein drug conjugate (e.g., antibody-drug conjugate), its stereoisomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, as described in this disclosure.

[0547] In some embodiments, this disclosure relates to the use of the protein drug conjugate of this disclosure (e.g., antibody-drug conjugate), its stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of this disclosure for the treatment of diseases associated with proteins that specifically bind to a protein-targeting ligand contained in the protein drug conjugate.

[0548] In some embodiments, this disclosure relates to the use of the protein-drug conjugates (e.g., antibody-drug conjugates), their stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions of this disclosure for the treatment of tumors.

[0549] In some implementations, the tumor is cancer.

[0550] Therapeutic doses can be administered via single or multiple doses. For example, therapeutic doses of the protein drug conjugates described herein (e.g., antibody-drug conjugates), their stereoisomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions, can be formulated into unit formulations to facilitate administration to patients in need.

[0551] The protein drug conjugates (e.g., antibody-drug conjugates), their stereoisomers, or pharmaceutically acceptable salts thereof described herein, or pharmaceutical compositions, may be formulated into any dosage form known in the art, such as non-oral dosage forms.

[0552] The protein-drug conjugates (e.g., antibody-drug conjugates), their stereoisomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions described herein, are suitable for non-gastrointestinal administration. The antibody-drug conjugates, their stereoisomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions described herein, may be in solution form.

[0553] The patients described herein may include those at risk of developing cancer. Administration of the protein drug conjugates (e.g., antibody-drug conjugates), their stereoisomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions described herein may reduce the rate of increase in tumor volume over time in a patient, reduce the risk of metastasis, or reduce the risk of recurrence in a subject; or may prevent, slow, delay, or inhibit cancer progression; or may cause a reduction in the severity and / or duration of one or more symptoms of cancer in a patient; or may kill or reduce the number of tumor cells in a patient.

[0554] Methods for preparing antibody-drug conjugates

[0555] In one aspect, this disclosure relates to a method for preparing protein-drug conjugates (e.g., antibody-drug conjugates), comprising:

[0556] The protein-targeting ligand (e.g., an antibody or its antigen-binding fragment) and a fragment of a bioactive molecule (e.g., a cytotoxic drug) are conjugated via a linker fragment disclosed herein; or

[0557] The protein-targeting ligand (e.g., an antibody or its antigen-binding fragment) and a fragment of a bioactive molecule (e.g., a cytotoxic drug) are conjugated via a linker intermediate disclosed herein; or

[0558] The protein-targeting ligand (e.g., an antibody or its antigen-binding fragment) is coupled to the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in this disclosure.

[0559] In some implementations, prior to coupling, the protein-targeting ligand (e.g., an antibody or its antigen-binding fragment) is placed under reducing conditions to produce one or more free cysteine ​​residues.

[0560] In some implementations, a reducing agent (e.g., DDT, 2-mercaptoethanol, or TCEP) is used to reduce the protein-targeting ligand (e.g., an antibody or its antigen-binding fragment).

[0561] In this paper, the prepared ADC can be purified using any purification method known in the art, such as, but not limited to, affinity chromatography, ion exchange chromatography, size exclusion chromatography, etc. Beneficial effects

[0562] The three-branched hydrophilic linker provided in this disclosure allows for flexible covalent coupling of protein-targeting ligands (e.g., antibodies or their antigen-binding fragments) to payload molecules. Protein-drug conjugates (e.g., antibody-drug conjugates) constructed using this linker exhibit high purity and non-aggregation, possessing not only excellent hydrophilicity and stability but also significant cytotoxic activity (e.g., NCI-N87 and NCI-H2170 cells). Furthermore, it demonstrates favorable properties in one or more aspects, including pharmacokinetics, bioavailability, safety, and in vitro / in vivo efficacy.

[0563] In some embodiments of this disclosure, the antibody-drug conjugate has a maximum killing rate of >60% against NCI-N87 cells, preferably >70%, more preferably >80%, and even more preferably >90%. In some embodiments of this disclosure, the antibody-drug conjugate has a maximum killing rate of >80% against NCI-H2170 cells, preferably >90%. Detailed Implementation

[0564] The present application has been described in detail below with reference to embodiments, but this does not imply any adverse limitations on the present application. The present application has been described in detail herein, and specific embodiments thereof have been disclosed. For those skilled in the art, various changes and modifications to the specific implementation methods of the present application without departing from the spirit and scope of the present application are also included within the scope of protection of the present application.

[0565] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Perbal, A Practical Guide to Molecular Cloning (1984). A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its common meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0566] In this document, unless the context clearly indicates otherwise, the term "or" is intended to include "and" and vice versa. In this document, unless otherwise stated, singular terms cover plural referents and vice versa.

[0567] In this document, unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (e.g., contain, containing, include, including) are open-ended expressions and should be understood as “including but not limited to”, meaning that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be covered.

[0568] The term “about” means that the object it modifies has an experimental error acceptable in the art, and when used in conjunction with a numerical value, it means any value within ±1%, such as ±0.5% or ±0.1%, of the specified value.

[0569] Unless otherwise stated, the term "patient" as used herein may be used interchangeably with "subject" and covers any vertebrate, such as mammals, including humans, non-human primates, sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc. Preferably, the subject in this application is a human.

[0570] Unless otherwise stated, the terms "optional" or "optionally" as used herein mean that the object or event it modifies exists or does not exist, or occurs or does not occur. For example, "optionally contains A" means that A is included or not included.

[0571] The term “independently” means that when a plurality of substituents are selected from many possible groups, these substituents may be the same or different each time they appear.

[0572] C in this article m-n This means that the part has an integer or fractional number of carbon atoms within a given range. For example, "C 1-6 "" means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

[0573] The term "alkyl" refers to a compound with the general formula C1. n H 2n+1 The alkyl group. This alkyl group can be straight-chain or branched. For example, the term "C 1-6 "Alkyl" refers to an alkyl group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.). The term "alkylene" refers to the group obtained by further removing one hydrogen atom from an alkyl group.

[0574] The term "alkyleneamino" refers to -alkylene-NH-.

[0575] The term "heterocyclic group" refers to a non-aromatic ring that is fully saturated or partially unsaturated (but not fully unsaturated) and can exist as a monocyclic, bridged (including fused) or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 20-membered ring or a 3- to 15-membered ring (e.g., 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered), 4- to 8-membered, 5- to 8-membered, or 5- to 6-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, nitrogen, phosphorus, silicon, and / or boron (preferably 1 or 2 heteroatoms). Non-limiting examples of heterocyclic groups include, but are not limited to, ethylene oxide, tetrahydrofuranyl, dihydrofuranyl, pyrrolyl, N-methylpyrrolyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, and tetrahydrothiopheneyl. Preferred heterocyclic groups have a single 4- to 6-membered ring, especially a 5- to 6-membered ring.

[0576] The term "heterocyclic alkyl" refers to a fully saturated cyclic group that may exist as a monocyclic, bridged (including fused) or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 20-membered ring, a 3- to 15-membered ring, a 3- to 10-membered ring, a 5- to 10-membered ring, a 3- to 7-membered ring, a 4- to 8-membered ring, a 5- to 8-membered ring, or a 5- to 6-membered ring containing 1 to 3 heteroatoms independently selected from sulfur, oxygen, nitrogen, phosphorus, silicon, and / or boron (preferably 1 or 2 heteroatoms). Examples of 3-membered heterocyclic alkyl groups include, but are not limited to, ethylene oxide, cyclothioethylene, and cycloazoethylene; non-limiting examples of 4-membered heterocyclic alkyl groups include, but are not limited to, acridine, oxadiazolyl, and thiobutyl; examples of 5-membered heterocyclic alkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, and tetrahydropyrazolyl; examples of 6-membered heterocyclic alkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, piperazine, 1,4-thiaoxane, 1,4-dioxane, thiomorpholinyl, 1,3-dithiaalkyl, and 1,4-dithiaalkyl; and examples of 7-membered heterocyclic alkyl groups include, but are not limited to, azirheptanyl, oxeheptanyl, and thioheptanyl. Monocyclic heterocyclic alkyl groups having 5 or 6 ring atoms are preferred.

[0577] The term "heteroaryl" or "heterocyclic aryl" refers to a monocyclic or fused polycyclic system containing at least one (e.g., 1-3, 1-2) ring atoms selected from N, O, and S, with the remaining ring atoms being C, and having at least one aromatic ring. Preferred heteroaryls have a single 5- to 8-membered ring (e.g., 5-, 6-, 7-, or 8-membered), or multiple fused rings containing 6 to 20, 6 to 14, and especially 6 to 10 ring atoms (e.g., 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrole, furanyl, thiophene, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothiophene, indolyl, isoindolyl, pyridopyrroleyl, etc.; for example, 6-membered heteroaryl groups include pyridinyl, pyrazinyl, pyrimidinyl, or pyridazinyl.

[0578] The term "cycloalkyl" refers to a fully saturated carbon ring that may exist as a monocyclic, bridged (including fused) or spirocyclic ring. Unless otherwise indicated, cycloalkyl is preferably a 4- to 6-membered ring (e.g., a 5- to 6-membered ring). Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0579] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration representing the center of a solid.

[0580] In some embodiments, sugars include, but are not limited to, monosaccharides, oligosaccharides (polymers composed of 2-10 monosaccharides), polysaccharides (long-chain polymers composed of 11 or more monosaccharides), and their derivatives. In some embodiments, the monosaccharides and their derivatives include, but are not limited to, glucose, galactose, mannose, fucose, sialic acid, N-acetylglucosamine, N-acetylglucosamine, sugar alcohols, sugar acids, deoxyglucoses, or amino sugars. In some embodiments, the oligosaccharides and their derivatives include, but are not limited to, trehalose, sucrose, lactose, and oligosaccharide fragments derived from hyaluronic acid, chondroitin sulfate, heparin, or heparan sulfate. In some embodiments, the polysaccharides and their derivatives include, but are not limited to, starch, glycogen, or cellulose.

[0581] The term "linker" refers to a chemical molecule or portion that is linked to an antibody at one end and to a payload at the other. The linker can be cleavable or non-cleavable; a cleavable linker is one that can break within target cells to release the cytotoxic drug.

[0582] The term "linker-drug" refers to a molecule or part consisting of a linker compound linked to a biologically active drug (payload).

[0583] The term "antibody-drug conjugate" refers to an antibody linked to a biologically active drug (payload) via a stable linker. The linker-drug and the antibody can be linked using conventional methods in the art, including but not limited to lysine conjugation, light-heavy chain reducing disulfide bond conjugation, and directional conjugation.

[0584] The term "antibody" is used in its broadest sense to refer to a polypeptide encoded by an immunoglobulin gene that is capable of binding antigens, including but not limited to monoclonal antibodies, polyclonal antibodies, and various antibody structures such as bispecific antibodies and trispecific antibodies, as long as they exhibit the desired antigen-binding activity.

[0585] The term “antigen-binding fragment” refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen. Examples covered by the term “antigen-binding fragment” include: (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments connected by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody single arm; (v) dAb fragments consisting of VH domains (see Ward et al., Nature. 341:544-546 (1989)); (vi) separated complementarity-determining regions (CDRs); (vii) nanobodies; and (viii) single-chain Fv (scFv).

[0586] The antibodies or antigen-binding fragments thereof disclosed herein may be IgG1, IgG2, IgG3, or IgG4 isotypes. The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene. In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are IgG1 isotypes. The antibodies or antigen-binding fragments thereof disclosed herein may be derived from any species, including but not limited to mice, rats, rabbits, non-human primates (such as chimpanzees, cynomolgus monkeys, spider monkeys, and rhesus monkeys), llamas, and humans. The antibodies or antigen-binding fragments thereof disclosed herein may be murine antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0587] The term "spacer" is a component of the linker, whose main function is to connect the antibody and the payload, and to regulate the length of the linker, as well as the drug release mechanism and stability, thereby improving drug targeting, increasing ADC stability, promoting drug release, and reducing the hydrophobicity of the ADC.

[0588] The term "hydrophilic unit" refers to a chemical group or structural unit in a linker that has hydrophilic properties. By introducing hydrophilic groups or molecules, the hydrophobicity of ADCs can be reduced, thereby reducing the tendency to aggregate, improving blood circulation stability, and enhancing the therapeutic effect.

[0589] The term "connector unit" refers to a group or linker unit in a linker used to connect biological ligands (such as antibodies), which can modulate ADC stability, optimize ADC pharmacokinetics, and improve drug release.

[0590] The term “treatment” means administering the compound described in this disclosure or its protein drug conjugate or formulation to improve or eliminate a disease or one or more symptoms associated with said disease, and includes: (i) suppressing the disease or disease state, i.e., curbing its development; and (ii) alleviating the disease or disease state, even if the disease or disease state subsides.

[0591] The term “prevention” means administering the compound described in this disclosure or its protein drug conjugate or formulation to prevent a disease or one or more symptoms associated with said disease, including: preventing the occurrence of a disease or disease state in mammals, particularly when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state.

[0592] For the prevention and treatment of diseases, the effective dose for prevention or treatment can be determined by clinicians using routine methods or experience based on the individual condition of the subject, the severity of the disease, gender, age, weight, and method of administration.

[0593] The term "pharmaceutical acceptable" refers to compounds or their protein-drug conjugates, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0594] The term "pharmaceutically acceptable salt" refers to a salt of a compound (such as the compound disclosed herein or its protein drug conjugate) that is safe and effective in mammals and has the intended biological activity. For example, it may be a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, or a salt formed with a basic or acidic amino acid.

[0595] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their protein drug conjugates or salts thereof with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of the present disclosure or their protein drug conjugates to an organism.

[0596] The term "single-dose pharmaceutical composition" refers to the smallest packaged unit containing a certain amount of pharmaceutical product. For example, if a box of medicine contains seven capsules, then each capsule is a single-dose pharmaceutical composition; or each vial of injection solution is a single-dose pharmaceutical composition; or each vial of inhalation spray solution is a single-dose pharmaceutical composition. In this disclosure, the terms "single-dose pharmaceutical composition" and "unit-dose pharmaceutical composition" have the same meaning and are used interchangeably.

[0597] The term "excipient" refers to any ingredient other than the active ingredient (e.g., the compound of this disclosure or its protein drug conjugate). The selection of excipients will largely depend on factors such as the specific route of administration, the efficacy of the excipient in terms of solubility and stability, and the nature of the dosage form.

[0598] In this document, the compounds disclosed herein include intermediate compounds, linker-loador or protein drug conjugates (e.g., antibody-drug conjugates).

[0599] The compounds disclosed herein may also exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, in which a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0600] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0601] Certain isotope-labeled compounds of this disclosure (e.g., using...) 3 H and 14 Those labeled with C can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0602] In addition, heavier isotopes (such as deuterium) are used. 2 H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, wherein deuterium substitution can be partial or complete, and partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium, and all such compounds are included within the scope of this disclosure.

[0603] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0604] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include enantiomers and diastereomers. The compounds containing asymmetric carbon atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0605] The pharmaceutical compositions disclosed herein can be prepared by combining the compounds of the disclosed invention with suitable pharmaceutically acceptable excipients.

[0606] The pharmaceutical compositions disclosed herein can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0607] The single dose of the pharmaceutical composition disclosed herein may be 0.01-2000 mg or 0.02-1000 mg.

[0608] In all methods of administration of the compounds described herein, the daily dose is from 0.001 to 2000 mg / kg body weight. The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this disclosure.

[0609] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0610] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. To obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0611] The following abbreviations are used in this disclosure: PEG represents polyethylene glycol, NHS represents N-hydroxysuccinimide, and Fmoc represents N-fluorenemethyloxycarbonyl.

[0612] The technical solutions of this disclosure will be described below with the help of different embodiments, but those skilled in the art will understand that the scope of protection of this disclosure is not limited thereto.

[0613] Example

[0614] Unless otherwise specified, the experimental methods in the following examples were performed under standard conditions. Unless otherwise stated, reagents, materials, or instruments used in the following examples that do not specify the manufacturer are all commercially available products.

[0615] The heavy chain amino acid sequence of the trastuzumab used below is shown in SEQ ID NO:1, and the light chain amino acid sequence is shown in SEQ ID NO:2. The amino acid K at the C-terminus of SEQ ID NO:1 may or may not be present.

[0616] SEQ ID NO:1

[0617] SEQ ID NO:2

[0618] Example 1

[0619] Step 1:

[0620] Compound 1-1 (CAS: 150114-97-9, 10.0 g), 4-aminobenzyl alcohol (3.60 g), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (9.10 g) were added to a reaction flask, along with dichloromethane (150.0 mL) and methanol (150.0 mL). The reaction was carried out at 25 °C for 12 hours. The solvent was evaporated, and methyl tert-butyl ether (100.0 mL) and ethyl acetate (100.0 mL) were added and the mixture was stirred. The mixture was filtered and dried to obtain compound 1-2, totaling 8.30 g. LC-MS: m / z 516.3 (M+H) + .

[0621] Step 2:

[0622] Compounds 1-2 (600.0 mg) and pyridine (230.0 mg) were added to a reaction flask, followed by 50.0 mL of tetrahydrofuran solution. Phenyl 4-nitrochloroformate (352.0 mg) was added at 0-5 °C, and the mixture was stirred at 25 °C for 2 hours. 100 mL of ethyl acetate was added, and the mixture was washed with saturated brine (50 mL × 2). The mixture was dried over anhydrous sodium sulfate, and the solvent was evaporated. A slurry was then prepared by adding methyl tert-butyl ether (50.0 mL) and ethyl acetate (50.0 mL), followed by filtration and drying to obtain compounds 1-3, totaling 707.0 mg. LC-MS: m / z 681.3 (M+H) + .

[0623] Step 3:

[0624] Compounds 1-3 (700.0 mg) were added to a reaction flask, followed by 25.0 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (469.0 mg) and ethatecan mesylate (537.8 mg) were then added at 0-5 °C. The mixture was stirred at 0-5 °C for 1 hour, then reacted at 25 °C for 12 hours. Purification was performed by HPLC (YMC TAC18, 50*250 mm, 10 μm; mobile phase: A: 0.1% trifluoroacetic acid-water, B: methanol; gradient: 60%-100% B 0-60 min, wavelength 254 nm, v = 60 mL / min, rt 32 min) to obtain compounds 1-4, totaling 635.0 mg. LC-MS: m / z 977.4 (M+H) + .

[0625] 1 H NMR(500MHz,DMSO-d6)δ9.99(s,1H),8.17(d,1H),8.05(d,1H),7.88(d,2H),7.80– 7.70(m,3H),7.60(d,2H),7.48–7.28(m,8H),5.44(s,2H),5.35–5.20(d,3H),5.08( s,2H),4.45–4.18(m,4H),3.96–3.88(m,1H),3.29–3.19(m,1H),3.16–3.06(m,1H), 2.37(s,3H),2.25–2.10(m,2H),2.08–1.79(m,3H),1.30(d,3H),0.92–0.82(m,9H).

[0626] Step 4:

[0627] Compound tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (CAS: 175724-30-8, 5.0 g) and compounds 1-5 (CAS: 55750-53-3, 2.50 g) were added to a reaction flask, followed by 80.0 mL of N,N-dimethylformamide. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.70 g) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (3.80 g) was added dropwise. The reaction was stirred at 0-5 °C for 1 hour, then at 25 °C for 12 hours. 200 mL of saturated brine was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The extract was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain compounds 1-6, totaling 5.90 g. LC-MS: m / z 699.4 (M+H) + .

[0628] Step 5:

[0629] Compounds 1-6 (5.90 g) were added to a reaction flask, followed by 20.0 mL of dichloromethane. Trifluoroacetic acid (20.0 mL) was added at 25 °C, and the mixture was stirred at 25 °C for 12 hours. The solvent was evaporated, and the mixture was purified by HPLC (YMC AQ C18, 30*250 mm, 10 μm; mobile phase: A: 0.1% formic acid-water, B: acetonitrile; gradient: 10%-40% B 0-60 min, wavelength 210 nm, v = 50 mL / min, rt 35 min) to obtain compounds 1-7, totaling 3.0 g. LC-MS: m / z 531.3 (M+H) + .

[0630] 1 H NMR (500MHz, DMSO-d6): δ12.15(s,3H),7.00(s,2H),6.91(s,1H),3.65–3.50(m,1 2H),3.37(t,2H),2.42(t,6H),2.03(t,2H),1.52–1.40(m,4H),1.23–1.13(m,2H).

[0631] Step 6:

[0632] Compounds 1-7 (706.2 mg) and m-PEG9-Amine (CAS: 211859-73-3, 1.0 g) were added to a reaction flask, followed by 20.0 mL of N,N-dimethylformamide. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.40 g) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (739.3 mg) was added dropwise. The reaction was stirred at 0-5 °C for 1 hour, then at 25 °C for 12 hours. HPLC (YMC AQ C18, 30*250 mm, 10 μm, mobile phase: A: 0.1% formic acid-water, B: acetonitrile; gradient: 20%-50% B, 0-60 min, wavelength 210 nm, v = 50 mL / min, rt) (31.2 min) Purification yielded compounds 1-8, totaling 117.0 mg. LC-MS: m / z 1349.6 (M+H) + .

[0633] Step 7:

[0634] Compounds 1-4 (24.4 mg) were added to a reaction flask, followed by 3.0 mL of N,N-dimethylformamide. 1,8-diazabicyclo[5.4.0]undec-7-ene (3.80 mg) was added at 0-5 °C, and the mixture was stirred at 0-5 °C for 0.5 hours to obtain the prepared solution. Compounds 1-8 (40.0 mg) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (11.4 mg) were added to 5.0 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (7.80 mg) was added dropwise at 0-5 °C, and the mixture was stirred at 0-5 °C for 15 min. The prepared solution was then added dropwise in an ice-water bath, and the mixture was stirred at 0-5 °C for 1 h, followed by a reaction at 25 °C for 12 h. The mixture was purified by HPLC (Waters CSH C18, 30*250 mm, 5 μm; mobile phase: A: 0.1% formic acid-water, B: acetonitrile; gradient: 30%-60% B 0-60 min, wavelength 254 nm, v = 15 mL / min, rt 36 min) to obtain compound 1, totaling 6.9 mg. LC-MS: m / z 2086.3 (M+H) + .

[0635] Example 2

[0636] Example 2 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), yielding compound 2. LC-MS: m / z 2172.4 (M+H) + .

[0637] Example 3

[0638] Example 3 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), and m-PEG9-Amine (CAS: 211859-73-3) in step 6 of Example 1 was replaced with 1-amino-3,6,9-triox-11-undecyl alcohol (CAS: 86770-74-3), yielding compound 3. LC-MS: m / z 1704.1 (M+H) + .

[0639] Example 4

[0640] Example 4 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), and m-PEG9-Amine (CAS: 211859-73-3) in step 6 of Example 1 was replaced with m-PEG4-Amine (CAS: 85030-56-4), yielding compound 4. LC-MS: m / z 866.8 [(M+2H) / 2] + .

[0641] Example 5

[0642] Example 5 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), and m-PEG9-Amine (CAS: 211859-73-3) in step 6 of Example 1 was replaced with m-PEG8-Amine (CAS: 869718-81-0), yielding compound 5. LC-MS: m / z 2085.4 (M+H) + .

[0643] Example 6

[0644] Example 6 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), and m-PEG9-Amine (CAS: 211859-73-3) in step 6 of Example 1 was replaced with m-PEG12-Amine (CAS: 1977493-48-3), yielding compound 6. LC-MS: m / z 2437.6 (M+H) + .

[0645] Example 7

[0646] Step 1:

[0647] Compounds 1-7 (1.59 g) and compound tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (CAS: 175724-30-8, 1.52 g) were added to a reaction flask, followed by 80.0 mL of N,N-dimethylformamide. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.71 g) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (1.55 g) was added dropwise. The reaction was continued at 0-5 °C with stirring for 1 hour, followed by a reaction at 25 °C for 1 hour, yielding a mixture of compounds 7-1. LC-MS: m / z 1018.4 (M+H) + ,spare.

[0648] Step 2:

[0649] Add m-PEG4-Amine (CAS: 85030-56-4, 1.24 g) to the mixture of compound 7-1, then add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.28 g) at 0-5℃, followed by dropwise addition of N,N-diisopropylethylamine (1.16 g). The mixture was stirred at 0-5℃ for 1 hour, then reacted at 25℃ for 12 hours. Purification was performed by HPLC (Daiso C18, 50*250 mm, 10 μm; mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 30%-90% B 0-60 min, wavelength 210 nm, rt 45 min) to obtain compound 7-2, totaling 980.0 mg. LC-MS: m / z 1397.1 (M+H) + .

[0650] Step 3:

[0651] Compound 7-2 (160.0 mg) was added to a reaction flask, followed by 3.0 mL of dichloromethane. Trifluoroacetic acid (3.0 mL) was added at 25 °C, and the mixture was stirred at 25 °C for 12 hours. The solvent was then evaporated to obtain compound 7-3. LC-MS: m / z 1228.9 (M+H). + ,spare.

[0652] Step 4:

[0653] Compound 7-4 was prepared according to Example 1, except that compound 1-1 (CAS: 150114-97-9) of Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6). Compound 7-4 (127.4 mg) was added to a reaction flask, followed by 6.0 mL of N,N-dimethylformamide. 1,8-diazabicyclo[5.4.0]undec-7-ene (18.4 mg) was added at 0-5 °C, and the mixture was stirred at 0-5 °C for 0.5 hours. LC-MS: m / z 841.5 (M+H) + The prepared solution was obtained. Compound 7-3 (147.3 mg) was added to 6.0 mL of N,N-dimethylformamide, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (68.4 mg) at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (186.0 mg) was added dropwise. The prepared solution was then added dropwise under an ice-water bath, and the mixture was stirred at 0-5 °C for 1 hour to obtain a mixture of compounds 7-5. LC-MS: m / z 2051.2 (M+H) + ,spare.

[0654] Step 5:

[0655] In a mixture of compounds 7 and 5, m-PEG4-Amine (CAS: 85030-56-4, 49.7 mg) was added, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (91.2 mg) at 0-5 °C, and then N,N-diisopropylethylamine (93.2 mg) was added dropwise. The mixture was stirred at 0-5 °C for 1 hour, then reacted at 25 °C for 12 hours. The mixture was purified by HPLC (TAC18, 50*250 mm, 10 μm, mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 30%-90% B 0-60 min, wavelength 254 nm, rt 20 min) to obtain compound 7, totaling 26.0 mg. LC-MS: m / z 2430.6 (M+H) + .

[0656] Example 8

[0657] Example 8 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), m-PEG9-Amine (CAS: 211859-73-3) in Example 1 was replaced with m-PEG8-Amine (CAS: 869718-81-0), and compound 1-5 (CAS: 55750-53-3) in Example 1 was replaced with 3-maleimide propionic acid (CAS: 7423-55-4), yielding compound 8. LC-MS: m / z 2043.1 (M+H) + .

[0658] Example 9

[0659] Example 9 was prepared according to Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), m-PEG9-Amine (CAS: 211859-73-3) in Example 1 was replaced with m-PEG8-Amine (CAS: 869718-81-0), and compound 1-5 (CAS: 55750-53-3) in Example 1 was replaced with 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionylamino)ethoxy)ethoxy)propionic acid (CAS: 756525-98-1), to obtain compound 9. LC-MS: m / z 2202.3(M+H) + .

[0660] Example 10

[0661] Example 10 was prepared following Example 1, except that compound 1-1 (CAS: 150114-97-9) in Example 1 was replaced with (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino (CAS: 159858-21-6), m-PEG9-Amine (CAS: 211859-73-3) in Example 1 was replaced with m-PEG8-Amine (CAS: 869718-81-0), and compound 1-5 (CAS: 55750-53-3) in Example 1 was replaced with 3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethoxy)propionic acid (CAS: 760952-64-5), yielding compound 10. LC-MS: m / z 2087.1 (M+H) + .

[0662] Example 11

[0663] Step 1:

[0664] Compound 11-1 (docetaxel, 4.00 g) and triethylamine (1.52 g) were added to a reaction flask, followed by 40.0 mL of dichloromethane. Phenyl 4-nitrochloroformate (1.51 g) was added at 0 °C, and the mixture was stirred at 0 °C for 5 hours. 100 mL of ethyl acetate was added, and the mixture was washed twice with 1 M potassium hydrogen sulfate (50 mL x 2) and twice with saturated brine (50 mL x 2). The mixture was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 3:7) to obtain compound 11-2, totaling 3.40 g. LC-MS: m / z 973.6 [M+H] + .

[0665] 1 H NMR (500MHz, DMSO-d6): δ8.36–8.31(m,2H),8.05–7.96(m,3H),7.71(t,1H),7.62(t,2H),7.54(d,2H) ),7.49–7.39(m,4H),7.27–7.18(m,1H),5.86(t,1H),5.41(d,1H),5.28(d,1H),5.15(t,1H),5.08(s ,1H),4.99(d,1H),4.93(d,1H),4.90(d,1H),4.48(s,1H),4.08–3.96(m,3H),3.65(d,1H),2.26(s,4 H),1.96–1.85(m,1H),1.70(s,3H),1.70–1.60(m,2H),1.52(s,3H),1.38(s,9H),1.04–0.94(m,6H).

[0666] Step 2:

[0667] ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (9H-fluorene-9-yl)methyl ester (CAS: 159858-22-7, 1.0 g), compound 11-2 (2.34 g), and 4-dimethylaminopyridine (623.2 mg) were added to a reaction flask, followed by 150 mL of dichloromethane. The reaction was carried out at 25 °C for 12 hours. Then, 200 mL of saturated brine was added, and the mixture was extracted three times with dichloromethane (50 mL × 3). The extract was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (dichloromethane / methanol = 100:5) to give compound 11-3, totaling 50.0 mg. LC-MS: m / z 1435.4 (M+H) + .

[0668] Step 3:

[0669] Compound 11-4 (CAS: 1052207-59-6, 2.0 g), D-anhydrous glucose (CAS: 50-99-7, 4.32 g), and acetic acid (24.0 mg) were added to a reaction flask, followed by 100.0 mL of methanol. The mixture was stirred at 50 °C for 1 hour. Sodium cyanoborohydride (CAS: 25895-60-7, 1.56 g) was then added, and the mixture was stirred at 50 °C for 3 hours. The mixture was purified by HPLC (Daiso C18, 50*250 mm, 10 μm; mobile phase: A: 20 μm ammonium acetate, B: acetonitrile; gradient: 5%-65% B 0-40 min, wavelength 210 nm, rt 36 min) to obtain compound 11-5, totaling 3.0 g. LC-MS: m / z 841.5 (M+H) + .

[0670] 1 H NMR (500MHz, MeOD) δ4.24–4.18(m,2H),3.96–3.78(m,6H),3.76–3.60(m,36H),3.58–3.45(m,6H),3.24(t,2H),1.46(s,9H).

[0671] Step 4:

[0672] Compound 11-5 (73.6 mg) was added to a reaction flask, followed by 3.0 mL of dichloromethane. Trifluoroacetic acid (3.0 mL) was added at 25 °C, and the mixture was stirred at 25 °C for 12 hours. The solvent was then evaporated to obtain compound 11-6. LC-MS: m / z 741.5 (M+H) + ,spare.

[0673] Step 5:

[0674] Compound 11-3 (50.0 mg) was added to a reaction flask, followed by 5.0 mL of N,N-dimethylformamide. Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (5.4 mg) was added at 0-5 °C. The mixture was stirred at 0-5 °C for 0.5 hours. LC-MS: m / z 1213.59 (M+H) + The prepared solution was obtained. Compounds 1-7 (18.6 mg) were added to 5.0 mL of N,N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (20.0 mg) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (27.2 mg) was added dropwise. The prepared solution was added dropwise in an ice-water bath, and the reaction was stirred at 0-5 °C for 1 hour to obtain a mixture of compounds 11-7. LC-MS: m / z 1725.7 (M+H) + .

[0675] Step 6:

[0676] Compounds 11-6 were added to 5.0 mL of N,N-dimethylformamide, then to a mixture of compounds 11-7, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (33.2 mg), and dropwise the addition of N,N-diisopropylethylamine (40.8 mg). The mixture was stirred at 0-5°C for 1 hour, then reacted at 25°C for 12 hours. The mixture was purified by HPLC (YMC-AQ-C18, 30*250 mm, 10 μm; mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 5%-85% B 0-60 min, wavelength 254 nm, rt 25 min) to obtain compound 11, totaling 13.5 mg. LC-MS: m / z 1586.3 [(M+2H) / 2] + .

[0677] Example 12

[0678] Example 12 was prepared following Example 11, except that ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (9H-fluorene-9-yl)methyl ester (CAS: 159858-22-7) in Example 11 was replaced with (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (CAS: 1394238-91-5), yielding compound 12. LC-MS: m / z 1543.5 [(M+2H) / 2] + .

[0679] Example 13

[0680] Example 13 was prepared following Example 11, except that compound 11-1 (CAS: 114977-28-5) in Example 11 was replaced with paclitaxel (CAS: 33069-62-4), yielding compound 13. LC-MS: m / z 1609.3 [(M+2H) / 2] + .

[0681] Example 14

[0682] The preparation of Example 14 was the same as in Example 11, except that ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (9H-fluorene-9-yl)methyl ester (CAS: 159858-22-7) in Example 11 was replaced with (9H-fluorene-9-yl)methyl ( (S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (CAS: 1394238-91-5), compound 11-1 (CAS: 114977-28-5) was replaced with paclitaxel (CAS: 33069-62-4) to give compound 14. LC-MS: m / z 1566.7 [(M+2H) / 2] + .

[0683] Example 15

[0684] Step 1:

[0685] Compound N-benzyloxycarbonyl-glycyl-glycine (CAS: 2566-19-0, 10.0 g) was added to a reaction flask, followed by 30.0 mL of acetic acid and 100.0 mL of tetrahydrofuran solution. Lead tetraacetate (CAS: 546-67-8, 20.0 g) was added at 40 °C, and the mixture was stirred at 70 °C for 5 hours. After cooling to room temperature, 200 mL of ethyl acetate was added, and the mixture was washed twice with saturated brine (50 mL * 2). The solution was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 1:2) to obtain compound 15-2, totaling 8.30 g. LC-MS: m / z 281.2 [M+H] + .

[0686] Step 2:

[0687] Compound 15-2 (8.10 g) was added to a reaction flask, along with ethylene glycol dimethyl ether (CAS: 110-71-4, 260.0 mL) and 1,3-propanediol (CAS: 504-63-2, 43.40 g). Sodium hydroxide (CAS: 1310-73-2, 1.74 g) was added at 0°C, followed by 25 mL of water. The reaction was carried out at 0°C for 2 hours. Then, 20.0 mL of acetic acid was added, and the reaction was concentrated. The mixture was purified by HPLC (YMCAQ C18, 50*250 mm, 10 μm; mobile phase: A: pure water, B: acetonitrile; gradient: 10%-40% B 0-60 min, wavelength 254 nm, rt 35.5 min) to obtain compound 15-3, totaling 5.0 g. LC-MS: m / z 297.0 [M+H]+ .

[0688] Step 3:

[0689] Compound 15-3 (5.0 g) was added to a reaction flask, followed by 50.0 mL of dichloromethane and triethylamine (5.16 g). p-Nitrobenzenesulfonyl chloride (CAS: 98-74-8, 3.94 g) and 4-dimethylaminopyridine (CAS: 1122-58-3, 1.03 g) were added at 0 °C. The mixture was stirred at 25 °C for 3 hours. Then, 100 mL of dichloromethane was added, and the mixture was washed once with saturated brine (50 mL x 1). The solution was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain compound 15-4, totaling 8.0 g. LC-MS: m / z 482.3 [M+H] + .

[0690] Step 4:

[0691] Exatecan mesylate (CAS: 169869-90-3, 2.78 g) was added to a reaction flask, followed by 50.0 mL of N-methylpyrrolidone. N,N-diisopropylethylamine (CAS: 7087-68-5, 2.03 g) was added at 25°C, and compound 15-4 (7.60 g) was added at 60°C. The mixture was stirred at 60°C for 12 hours, cooled to room temperature, and then 200 mL of ethyl acetate was added. The mixture was washed twice with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane:methanol = 25:1) to obtain compound 15-5, totaling 1.60 g. LC-MS: m / z 714.4 [M+H] + .

[0692] Step 5:

[0693] Compound 15-5 (1.60 g) was added to a reaction flask, followed by 20.0 mL of dichloromethane and 20.0 mL of methanol. Palladium on carbon (CAS: 7440-05-3, 1.06 g) was added. The mixture was stirred at 25 °C for 24 hours under a hydrogen atmosphere to obtain compound 15-6, totaling 1.50 g. LC-MS: m / z 580.3 (M+H) + .

[0694] Step 6:

[0695] Compound 15-6 (0.90 g) was added to 50.0 mL of N,N-dimethylformamide, followed by Fmoc-Gly-Gly-Phe-OH (CAS: 160036-44-2, 0.78 g), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.89 g), and N,N-diisopropylethylamine (1.0 g) was added dropwise. The mixture was stirred at 0-5 °C for 1 hour, then reacted at 25 °C for 12 hours. 100 mL of dichloromethane was added, and the mixture was washed once with saturated brine (50 mL x 2). The solution was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (dichloromethane:methanol = 100:7) to obtain compound 15-7, totaling 0.60 g. LC-MS: m / z 1063.5 [M+H] + .

[0696] Step 7:

[0697] Compound 15-7 (106.2 mg) was added to a reaction flask, followed by 5.0 mL of N,N-dimethylformamide. Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (15.3 mg) was added at 0-5 °C. The mixture was stirred at 0-5 °C for 0.5 hours to obtain a stock solution of compound 15-8. LC-MS: m / z 841.3 (M+H) + .

[0698] Step 8:

[0699] Compounds 1-7 (53.1 mg) were added to 5.0 mL of N,N-dimethylformamide, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (42.0 mg) at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (52.0 mg) was added dropwise. The prepared solution of compound 15-8 was then added dropwise in an ice-water bath, and the mixture was stirred at 0-5 °C for 1 hour to obtain a mixture of compounds 15-9. LC-MS: m / z 1353.7 (M+H) + .

[0700] Step 9:

[0701] Referring to Example 11, compound 11-7 in Example 11 was replaced with compound 15-9. Specifically, compound 11-6 (0.25 mmol) was added to 5.0 mL of N,N-dimethylformamide, and then added to the mixture of compound 15-9. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (76.0 mg) was added, followed by dropwise addition of N,N-diisopropylethylamine (785.0 mg). The mixture was stirred at 0-5°C for 1 hour, then reacted at 25°C for 12 hours. The mixture was purified by HPLC (YMCAQ C18, specification: 30*150 mm, 10 μm, mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 10%-40% B 0-60 min, wavelength 254 nm, rt 32 min) to obtain compound 15, totaling 3.2 mg. LC-MS: m / z 2798.9 (M+H) + .

[0702] Example 16

[0703] Example 16 was prepared following Example 15, except that compounds 11-6 in Example 15 were replaced with aminooctaglycol monomethyl ether (CAS: 869718-81-0) to obtain compound 16. LC-MS: m / z 2084.4 [(M+H)] + .

[0704] Example 17

[0705] Step 1:

[0706] Compound N-BOC-4-piperidinic acid (CAS: 84358-13-4, 0.29 g) was added to 10.0 mL of N,N-dimethylformamide, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.69 g), and dropwise N,N-diisopropylethylamine (0.92 g) and compound 11-6 (0.74 g). The mixture was stirred at 0-5 °C for 1 hour and then reacted at 25 °C for 12 hours. The mixture was purified by HPLC (YMC*GEL ODS-AQ-HG, specification: 50*250 mm, 10 μm, mobile phase: A: 0.1% TFA water, B: acetonitrile; gradient: 0%-70% B 0-60 min, wavelength 190 nm, rt 30 min) to obtain compound 17-1, totaling 767.0 mg. LC-MS: m / z 952.8 [M+H] + .

[0707] Step 2:

[0708] Compound 17-1 (166.5 mg) was added to a reaction flask, followed by 3.0 mL of dichloromethane. Trifluoroacetic acid (3.0 mL) was added at 25 °C, and the mixture was stirred at 25 °C for 12 hours. The solvent was then evaporated to obtain compound 17-2. LC-MS: m / z 852.6 (M+H). + .

[0709] Step 3:

[0710] Compound 11-3 (100.0 mg) was added to a reaction flask, followed by 10.0 mL of N,N-dimethylformamide. Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (10.8 mg) was added at 0-5 °C. The mixture was stirred at 0-5 °C for 0.5 hours. LC-MS: m / z 1213.6 (M+H) + The prepared solution was obtained. Compounds 1-7 (37.2 mg) were added to 5.0 mL of N,N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (40.0 mg) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (54.4 mg) was added dropwise. The prepared solution was added dropwise in an ice-water bath, and the reaction was stirred at 0-5 °C for 1 hour to obtain a mixture of compounds 11-7. LC-MS: m / z 1725.7 (M+H) + .

[0711] Step 4:

[0712] Compound 17-2 was added to 5.0 mL of N,N-dimethylformamide, then to a mixture of compounds 11-7. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (66.4 mg) was added, followed by dropwise addition of N,N-diisopropylethylamine (81.6 mg). The mixture was stirred at 0-5°C for 1 hour, then reacted at 25°C for 12 hours. Purification was performed by HPLC (Waters CSH C18, 30*250 mm, 5 μm; mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 20%-70% B 0-60 min, wavelength 210 nm, rt 27 min) to obtain compound 17, totaling 4.0 mg. LC-MS: m / z 3393.09 (M+H) + .

[0713] Example 18

[0714] Step 1:

[0715] The compound trans-4-(Boc-amino)cyclohexanecarboxylic acid (CAS: 53292-89-0, 0.31 g) was added to 10.0 mL of N,N-dimethylformamide, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.69 g), and dropwise N,N-diisopropylethylamine (0.92 g) and compound 11-6 (0.74 g). The mixture was stirred at 0-5 °C for 1 h, then reacted at 25 °C for 12 h. The mixture was purified by HPLC (YMC*GEL ODS-AQ-HG, specification: 50*250 mm, 10 μm, mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 5%-55% B 0-60 min, wavelength 190 nm, rt 30 min) to obtain compound 18-1, totaling 886.0 mg. LC-MS: m / z 966.8 [M+H] + .

[0716] Step 2:

[0717] Compound 18-1 (168.9 mg) was added to a reaction flask, followed by 3.0 mL of dichloromethane. Trifluoroacetic acid (3.0 mL) was added at 25 °C, and the mixture was stirred at 25 °C for 12 hours. The solvent was then evaporated to obtain compound 18-2. LC-MS: m / z 866.7 (M+H) + ,spare.

[0718] Step 3:

[0719] Compound 11-3 (100.0 mg) was added to a reaction flask, followed by 10.0 mL of N,N-dimethylformamide. Then, 1,8-diazabicyclo[5.4.0]undec-7-ene (10.8 mg) was added at 0-5 °C. The mixture was stirred at 0-5 °C for 0.5 hours. LC-MS: m / z 1213.6 (M+H) + The prepared solution was obtained. Compounds 1-7 (37.2 mg) were added to 5.0 mL of N,N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (40.0 mg) was added at 0-5 °C. After stirring for 5 minutes at 0-5 °C, N,N-diisopropylethylamine (54.4 mg) was added dropwise. The prepared solution was added dropwise in an ice-water bath, and the reaction was stirred at 0-5 °C for 1 hour to obtain a mixture of compounds 11-7. LC-MS: m / z 1725.7 (M+H) + .

[0720] Step 4:

[0721] Compound 18-2 was added to 5.0 mL of N,N-dimethylformamide, then to a mixture of compounds 11-7, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (66.4 mg), and dropwise the addition of N,N-diisopropylethylamine (81.6 mg). The mixture was stirred at 0-5°C for 1 hour, then reacted at 25°C for 12 hours. Purification was performed by HPLC (YMCAQ C18, 30*150 mm, 10 μm, mobile phase: A: 0.1% TFA-water, B: acetonitrile; gradient: 20%-50% B 0-60 min, wavelength 254 nm, rt 25 min) to obtain compound 18, totaling 7.2 mg. LC-MS: m / z 3421.2 (M+H) + .

[0722] Example 19

[0723] Example 19 was prepared following Example 17, except that compound 11-1 (CAS: 114977-28-5) from Example 11 used in Example 17 was replaced with paclitaxel (CAS: 33069-62-4), yielding compound 19. LC-MS: m / z 3439.1 (M+H) + .

[0724] Example 20

[0725] Example 20 was prepared following Example 18, except that compound 11-1 (CAS: 114977-28-5) from Example 11 used in Example 18 was replaced with paclitaxel (CAS: 33069-62-4), yielding compound 20. LC-MS: m / z 3466.5 (M+H) + .

[0726] Experimental Example 1: Preparation of Antibody-Drug Conjugates

[0727] Experimental Example 1.1

[0728] Reagents:

[0729] The antibody was trastuzumab, and the linker-payload was the compound from Example 1.

[0730] Experimental procedure:

[0731] The antibody was transferred to histidine buffer (pH 6.0, L-histidine 1.43 mg / mL, L-histidine hydrochloride monohydrate 2.27 mg / mL) and the antibody concentration was adjusted to approximately 10 mg / mL with histidine buffer (pH 6.0). The pH was then adjusted to approximately 7.0 with 300 mM Na2HPO4 aqueous solution. 10 mM TCEP·HCl (tris(2-carboxyethyl)phosphine hydrochloride) aqueous solution was added to make the antibody to TCEP·HCl molar ratio 1:8. The solution was placed on a rotary incubator and incubated at 20 rpm and 37°C in the dark for 1 hour to obtain reaction solution 1. Reaction solution 1 was placed in an ultrafiltration tube and 10 times the volume of histidine buffer (pH 6.0) was added. After ultrafiltration 3 times, the antibody concentration was adjusted to approximately 10 mg / mL with histidine buffer (pH 6.0) again, and the pH was adjusted to approximately 7.0 with 300 mM Na2HPO4 aqueous solution to obtain reaction solution 2.

[0732] Add DMSO to reaction solution 2 to make the concentration of DMSO 5% (v / v), then add Linker-payload solution (compound of Example 1) dissolved in DMSO with a concentration of 10 mg / mL, so that the molar ratio of antibody to Linker-payload is 1:14. Place it on a rotary incubator and react at 20 rpm and 37°C in the dark for 2 hours to obtain reaction solution 3.

[0733] Place reaction solution 3 in an ultrafiltration tube, add 10 volumes of histidine buffer (pH 6.0) containing 10% (v / v) DMSO, and then replace it with histidine buffer (pH 6.0) to obtain antibody-drug conjugate ADC1.

[0734] The structure of ADC1 is as follows:

[0735] Experimental Example 1.2

[0736] The preparation of antibody-drug conjugate ADC2 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 2.

[0737] The structure of ADC2 is as follows:

[0738] Experimental Example 1.3

[0739] The preparation of antibody-drug conjugate ADC3 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 3.

[0740] The structure of ADC3 is as follows:

[0741] Experimental Example 1.4

[0742] The preparation of antibody-drug conjugate ADC4 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 4.

[0743] The structure of ADC4 is as follows:

[0744] Experimental Example 1.5

[0745] The preparation of antibody-drug conjugate ADC5 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 5.

[0746] The structure of ADC5 is as follows:

[0747] Experimental Example 1.6

[0748] The preparation of antibody-drug conjugate ADC6 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 6.

[0749] The structure of ADC6 is as follows:

[0750] Experimental Example 1.7

[0751] The preparation of antibody-drug conjugate ADC7 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 7.

[0752] The structure of ADC7 is as follows:

[0753] Experimental Example 1.8

[0754] The preparation of antibody-drug conjugate ADC8 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 8.

[0755] The structure of ADC8 is as follows:

[0756] Experimental Example 1.9

[0757] The preparation of antibody-drug conjugate ADC9 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 9.

[0758] The structure of ADC9 is as follows:

[0759] Experimental Example 1.10

[0760] The preparation of antibody-drug conjugate ADC10 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 10.

[0761] The structure of ADC10 is as follows:

[0762] Experimental Example 1.11

[0763] The preparation of antibody-drug conjugate ADC11 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 11.

[0764] The structure of ADC11 is as follows:

[0765] Experimental Example 1.12

[0766] The preparation of antibody-drug conjugate ADC12 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 12.

[0767] The structure of ADC12 is as follows:

[0768] Experimental Example 1.13

[0769] The preparation of antibody-drug conjugate ADC13 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 13.

[0770] The structure of ADC13 is as follows:

[0771] Experimental Example 1.14

[0772] The preparation of antibody-drug conjugate ADC14 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 14.

[0773] The structure of ADC14 is as follows:

[0774] Experimental Example 1.15

[0775] The preparation of antibody-drug conjugate ADC15 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 15.

[0776] The structure of ADC15 is as follows:

[0777] Experimental Example 1.16

[0778] The preparation of antibody-drug conjugate ADC16 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 16.

[0779] The structure of ADC16 is as follows:

[0780] Experimental Example 1.17

[0781] The preparation of antibody-drug conjugate ADC17 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 17.

[0782] The structure of ADC17 is as follows:

[0783] Experimental Example 1.18

[0784] The preparation of antibody-drug conjugate ADC18 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 18.

[0785] The structure of ADC18 is as follows:

[0786] Experimental Example 1.19

[0787] The preparation of antibody-drug conjugate ADC19 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 19.

[0788] The structure of ADC19 is as follows:

[0789] Experimental Example 1.20

[0790] The preparation of antibody-drug conjugate ADC20 was carried out in accordance with Test Example 1.1, except that the compound of Example 1 in Test Example 1.1 was replaced with the compound of Example 20.

[0791] The structure of ADC20 is as follows:

[0792] Experimental Example 2: Determination of DAR value (HIC) of antibody-drug conjugates

[0793] The DAR value of the ADC prepared in Example 1 was determined using hydrophobic interaction chromatography (HIC) according to one of the following two methods:

[0794] (1) The components of the sample were separated using non-porous polystyrene / divinylbenzene (PS / DVB) packing material with bonded butyl groups. The neutral high-salt mobile phase was used to enhance the hydrophobicity of protein molecules, thereby combining them with the hydrophobic bonds in the chromatographic column. Then, the elution of substances was carried out by gradually decreasing the salt concentration and gradually increasing the proportion of isopropanol. The less hydrophobic substances were eluted first, and the more hydrophobic substances were eluted later. The chromatographic column specifications were Sepax HIC-Butyl, 4.6*100mm, 5μm, and the column temperature was 25℃. Mobile phase A was 25mmol / L phosphate buffer-2mol / L ammonium sulfate, pH 7.0 (3.55g of anhydrous disodium hydrogen phosphate and 264.28g of ammonium sulfate were weighed, added to about 800mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0±0.1 with phosphoric acid, and brought to a final volume of 1L. After mixing, the solution was filtered through a 0.22μm filter membrane). Mobile phase B was 25 mmol / L phosphate buffer, pH 7.0 (3.55 g of anhydrous disodium hydrogen phosphate was weighed, added to approximately 800 mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0 ± 0.1 with phosphoric acid, and brought to a final volume of 1 L. After mixing, the solution was filtered through a 0.22 μm filter membrane). Mobile phase C was 100% isopropanol. The sample was diluted 1-fold with the initial proportioned mobile phase to prepare the test solution. The injection volume was adjusted according to the sample concentration, and 50 μg of protein was injected for detection at a wavelength of 280 nm. The flow rate was 0.5 mL / min, and gradient elution was performed for 30 min (0-15.0 min: 75% A + 25% B to 10% A + 65% B + 25% C; 15.0-15.1 min: 10% A + 65% B + 25% C to 100% B; 15.1-20.0 min: 100% B; 20.0-20.1 min: 100% B to 75% A + 25% B; 20.1-30.0 min: 75% A + 25% B).

[0795] (2) The components of the sample were separated using a non-porous hydrophilic polymer packing material with bonded phenyl groups. The neutral high-salt mobile phase was used to enhance the hydrophobicity of the protein molecules, thereby binding them with the hydrophobic bonds in the chromatographic column. Then, the elution was carried out by gradually decreasing the salt concentration and gradually increasing the proportion of isopropanol, eluting substances with lower hydrophobicity first and those with higher hydrophobicity last. The chromatographic column specifications were TSKgel HIC-ADC Phenyl, 4.6*100mm, 5μm, and the column temperature was 25℃. Mobile phase A was 25mmol / L phosphate buffer-2mol / L ammonium sulfate, pH 7.0 (3.55g of anhydrous disodium hydrogen phosphate and 198.21g of ammonium sulfate were weighed, added to about 800mL of ultrapure water, stirred until fully dissolved, adjusted to pH 7.0±0.1 with phosphoric acid, and brought to a final volume of 1L. After mixing, the solution was filtered through a 0.22μm filter membrane). Mobile phase B was 25 mmol / L phosphate buffer-25% isopropanol, pH 7.0 (weigh 3.55 g of anhydrous disodium hydrogen phosphate, add approximately 800 mL of ultrapure water, stir until fully dissolved, adjust the pH to 7.0 ± 0.1 with phosphoric acid, add ultrapure water to a final volume of 1000 mL, mix well, take 750 mL of the resulting solution, add 250 mL of isopropanol, mix well, and filter through a 0.22 μm filter membrane). The sample was diluted 1-fold with the initial proportioned mobile phase to prepare the test solution. The injection volume was adjusted according to the sample concentration, and 50 μg of protein was injected. Detection was performed at 280 nm. The flow rate was 0.5 mL / min, with gradient elution for 30 min (0-20.0 min from 100% A to 100% B, 20.0-20.1 min from 100% B to 100% A, 20.1-30.0 min at 100% A).

[0796] The results were quantitatively analyzed using the area normalization method. The peak area percentages of ADCs containing 0, 1, 2, 3, 4, 5, 6, 7, and 8 cytotoxic drugs were calculated, and the DAR values ​​were also calculated. The formula is: DAR value = (peak area percentage of ADCs containing 0 cytotoxic drugs × 0 + peak area percentage of ADCs containing 1 cytotoxic drug × 1 + peak area percentage of ADCs containing 2 cytotoxic drugs × 2 + peak area percentage of ADCs containing 3 cytotoxic drugs × 3 + peak area percentage of ADCs containing 4 cytotoxic drugs × 4 + peak area percentage of ADCs containing 5 cytotoxic drugs × 5 + peak area percentage of ADCs containing 6 cytotoxic drugs × 6 + peak area percentage of ADCs containing 7 cytotoxic drugs × 7 + peak area percentage of ADCs containing 8 cytotoxic drugs × 8) / 100%.

[0797] Table 1. Drug loading distribution and DAR value of antibody-drug conjugates

[0798] Experimental Example 3: Determination of Monomer Purity of Antibody-Drug Conjugates (SEC)

[0799] Gel chromatography was used to separate the sample components. A neutral pH buffer with 10% isopropanol was used as the mobile phase for elution, and the components were eluted sequentially in descending order of molecular weight. The column used was an ACQUITY UPLC Protein BEH SEC Column. A 1.7 μm, 4.6*300 mm gel chromatographic column was used. The mobile phase was 50 mmol / L phosphate buffer-200 mmol / L sodium chloride-10% isopropanol, pH 7.0 (weigh 12.53 g of disodium hydrogen phosphate dodecahydrate, 2.33 g of sodium dihydrogen phosphate dihydrate, and 11.69 g of sodium chloride, add approximately 800 mL of ultrapure water, stir until fully dissolved, add ultrapure water to 1000 mL, and set aside. Add 100 mL of isopropanol to the above solution to 1000 mL, mix well, and filter through a 0.22 μm filter membrane). A precise 20 μg sample was injected into the liquid chromatograph and detected at 280 nm. The flow rate was 0.3 mL / min, and isocratic elution was performed for 15 min or 25 min.

[0800] Data processing employed area normalization for quantitative analysis of the results. The peak area percentages of aggregates, immunoglobulin monomers, and low molecular weight impurities were calculated separately, with the area before the main peak representing aggregates, the main peak representing immunoglobulin monomers, and the area after the main peak representing low molecular weight impurities. The proportions of monomers, aggregates, and low molecular weight impurities in the antibody-drug conjugate are shown in Table 2 below.

[0801] Table 2. Content of monomers, aggregates and low molecular weight impurities in antibody-drug conjugates

[0802] Experimental Example 4: Killing of Tumor Cells by Antibody-Drug Conjugates

[0803] To detect the killing effect of the Her2-targeting ADC of this disclosure on Her2-positive tumor cells, the killing activity was tested using NCI-N87 cells (Her2 highly expressed, source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: THu130) and NCI-H2170 cells (Her2 highly expressed, source: Yaji Biotechnology, catalog number: YS501C).

[0804] Cells in the logarithmic growth phase were harvested, and the cell density of NCI-N87 cells was adjusted to 8 × 10⁻⁶. 4 NCI-H2170 cell density up to 5 × 10⁶ cells / mL. 4Cells / mL were added to 96-well plates at 100 μL / well and cultured at 37℃ and 5% CO2 for 4-6 h. Her2-targeting ADCs were prepared using the corresponding complete culture media (RPMI 1640 + 20% FBS for NCI-H2170 cells and RPMI 1640 + 10% FBS + 1% sodium pyruvate for NCI-N87 cells) with a starting concentration of 100 nM and serially diluted 4-fold to obtain a total of 11 concentrations. Remove adherent cells from the culture plate. Add 50 μL / well of diluted Her2-targeting ADC to the experimental group, and 50 μL / well of the corresponding complete culture medium to the blank control group. After culturing for 96 h, detect the cells using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit (Vazyme, catalog number: DD1101-03). Specifically: Remove the 96-well plate, add 75 μL of CellCounting-Lite assay solution (Vazyme, catalog number: DD1101-03) to each well, vortex to mix, incubate at room temperature in the dark for 10 min, then transfer 170 μL from each well to an opaque white plate, remove air bubbles, read the chemiluminescence value using a microplate reader (PE, Envision 2105), and calculate the cell killing rate.

[0805] Experimental results showed that the antibody-drug conjugates ADC1-ADC20 disclosed herein exhibited a maximum killing rate (%) greater than 60% against NCI-N87 cells and a maximum killing rate (%) greater than 80% against NCI-H2170 cells. Maximum killing rate and EC50... 50 The calculation results are shown in Table 3.

[0806] Table 3. Killing activity of antibody-drug conjugates against tumor cells

[0807] For purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0808] Those skilled in the art will recognize that the scope of this application is not limited to the various specific implementations and embodiments described above, but rather that various modifications, substitutions, or recombinations can be made without departing from the spirit of this application, all of which fall within the protection scope of this application.

Claims

1. A linker segment of Formula I or a stereoisomer thereof, in, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different. L 1B It is a peptide unit; L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-; R 1’ This is a linker fragment that connects to a protein-targeting ligand; R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl; R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different. r, p, and q are each independently selected from 0, 1, 2, 3, 4, or 5; m1 and m2 are each independently selected from 0, 1, 2 or 3; The condition is that when m2 is 0, L 1B When connected to the payload, and m2 is not 0, L 1C Connect to the payload.

2. A linker of formula II-0 – a payload, its stereoisomer, or a pharmaceutically acceptable salt thereof. in, L 1A L 1C and L 2 Each can be an independent interval zone, either the same or different. L 1B It is a peptide unit; L 3 Each is independently selected from single bonds, -C(O)-3 to 10-membered heterocyclic groups, -C(O)-5 to 6-membered heteroaryl groups, -C(O)-NH-3 to 10-membered heterocyclic groups, -C(O)-NH-5 to 6-membered heteroaryl groups, and -C(O)-C 3-10 cycloalkyl- and -C(O)-NH-C 3-10 cycloalkyl-; R 1 For connector unit; R A and R B Each of the following is independently H, C1-C6 alkyl, C1-C6 haloalkyl or hydroxyl-substituted C1-C6 alkyl; R 2 and R 3 Each can be an independent hydrophilic unit, either the same or different. r, p, and q are each independently 0, 1, 2, 3, 4, or 5; m1 and m2 are each independently 0, 1, 2 or 3; The condition is that when m2 is 0, L 1B When connected to D, m2 is not 0, L 1C Connect to D; D represents a bioactive molecular fragment.

3. The linker fragment of claim 1 or its stereoisomer fragment, or the linker-payload of claim 2, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The R 1’ Selected from Wherein, -PG is an amino linking group; Or, the R 1’ Selected from Wherein, -PG is an amino linker, "*" indicates linkage to a protein-targeting ligand, and "#" indicates linkage to L... 2 ; The R 1 Selected from Wherein, -PG is an amino linking group; Or, R 1 for Preferably, R 1 for 4. The linker fragment or its stereoisomer fragment as described in claim 1 or 3, or the linker-payload fragment, its stereoisomer, or its pharmaceutically acceptable salt as described in claim 2 or 3, wherein, r, p, and q are each independently selected from 1, 2, 3, or 4; or r, p, and q are each independently selected from 1, 2, or 3; and / or m1 and m2 are each independently selected from 0, 1, or 2; or m1 and m2 are each independently selected from 0 or 1; and / or The -PG is selected from C1-C6 alkyl, 3-6 membered cycloalkyl, 3-6 membered heterocycloalkyl, phenyl or 5-6 membered heteroaryl; or, the -PG is selected from methyl, ethyl, n-propyl, isopropyl, cyclopropyl, oxetane, azirne, piperidinyl, phenyl or pyridinyl.

5. The linker fragment or its stereoisomer fragment as described in any one of claims 1, 3, and 4, or the linker-payload fragment, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 2-4, wherein, L 2 For C1-C 10 Alkylene, C2-C 10 alkenyl, C2-C 10 alkynyl group, C1-C 10 alkylene oxide C1-C 10 Alkylene, (C1-C 10 (alkylene oxide) x (C1-C) 10 (alkylene oxide) x C1-C 10 alkyleneamine, C1-C 10 alkylene amide group (C1-C) 10 (alkylene oxide) x C1-C 10 Alkylene, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic, C6-C 10 Aryl, 5-10 heteroaryl, -O-, -S-, -NH-, -NHC(O)-, -OC(O)-, and any combination thereof, where x is independently selected from 0, 1, 2, 3, 4, 5 or 6; Or, L 2 It can be C1-C6 alkylene, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyleneoxy, C1-C6 alkylene, or (C1-C6 alkyleneoxy) x (C1-C6 alkylene oxides) x C1-C6 alkylene amino, C1-C6 alkylene amide (C1-C6 alkylene oxy) x C1-C6 alkylene or Each x is independently selected from 0, 1, 2, 3, 4, or 5; Or, L 2 It can be C1-C6 alkylene, C3-C5 alkenyl, C3-C5 alkynyl, C1-C4 alkylamino, C1-C4 alkyleneoxy, C1-C4 alkylene, or (C1-C4 alkyleneoxy). x (C1-C4 alkylene oxides) x C1-C4 alkylene amino, C1-C6 alkylene amide (C1-C4 alkylene oxy) x C1-C4 alkylene or Each x is independently selected from 1, 2, 3, or 4; Or, L 2 for and / or L 3 Each is independently selected from single bonds, -C(O)-4 to 8-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 8-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-8 cycloalkyl- and -C(O)-NH-C 4-8 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S; Or, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S; Or, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic groups-, -C(O)-5 to 6-membered heteroaryl groups-, -C(O)-NH-4 to 6-membered heterocyclic groups-, -C(O)-NH-5 to 6-membered heteroaryl groups-, and -C(O)-C 4-6 cycloalkyl- and -C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocyclic and heteroaryl groups contain 1-3 N atoms; Or, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 heteroatoms selected from N, O, or S; Or, L 3 Each is independently selected from single bonds, -C(O)-4 to 6-membered heterocyclic alkyl-, -C(O)-5 to 6-membered heteroaryl-, -C(O)-NH-4 to 6-membered heterocyclic alkyl-, -C(O)-NH-5 to 6-membered heteroaryl-, -C(O)-C 4-6 Cycloalkyl-,-C(O)-NH-C 4-6 Cycloalkyl-, wherein the heterocycloalkyl and heteroaryl groups contain 1-3 N atoms; Or, L 3 For single bond, 6. The linker fragment or its stereoisomer fragment as claimed in any one of claims 1 and 3-5, or the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as claimed in any one of claims 2-5, wherein, R A and R B Each of the following is independently H, C1-C4 alkyl, C1-C4 haloalkyl or hydroxyl-substituted C1-C4 alkyl; Or, R A and R B Each is independently H or C1-C4 alkyl; Or, R A and R B Each is independently an H or C1-C3 alkyl group; Or, R A and R B Each can be independently H, methyl, ethyl, propyl, butyl, or pentyl; R A and R B Each independently is H; and / or R 2 and R 3 Each independently -(C1-C6 alkylene C(O)NR 4 ) y C(O)C1-C6 alkyl, Where y is an integer from 1 to 24, G represents glucosyl or galactosyl, CD represents α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, z is an integer from 1 to 5, and k is an integer from 1 to 10. R 4 Each time it appears, it is independently a C1-C6 alkyl group substituted with H, C1-C6 alkyl, C1-C6 haloalkyl, or hydroxyl, or, said R 4 Each time it appears, it is independently H or C1-C6 alkyl, or, the R 4 Each time it appears, it is independently H or C1-C5 alkyl, or, the R 4 Each time it appears, it is independently methyl, ethyl, propyl, butyl, or pentyl, or, the R 4 Each time it appears, it is independently methyl, ethyl, or propyl; Or, R 2 and R 3 Each independently Where each y is an integer from 1 to 24, each y is an integer from 1 to 24, or each y is an integer from 2 to 12, or each y is an integer from 3 to 12, or each y is 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. k can be an integer from 1 to 10, or k can be an integer from 1 to 8, or k can be 1, 2, 3 or 4, or k can be 1, 2 or 3. Optionally, the R 2 and R 3 Each independently 7. The linker fragment or its stereoisomer fragment as claimed in any one of claims 1 and 3-6, or the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as claimed in any one of claims 2-6, wherein, L 1A Each independently Where each h is an independent integer from 0 to 10; Or, L 1A Each independently and / or L 1C Each independently Or -CH2-NH-, where f is an integer from 0 to 4; R 5 Each is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C1-C6 alkoxy, -NO2 or halogen, or, R 5 Each is independently selected from methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoromethoxy, -NO2, fluorine, or chlorine; Or, L 1C Each independently Where s and t are each independent integers from 1 to 6, or s is 2, 3 or 4 and t is 1 or 2; Or, L 1C Each independently -CH2-NH-, or and / or L 1B It is a dipeptide, tripeptide, tetrapeptide, or a modified product thereof; or L 1B For Val-Cit, Val-Ala, Val-Lys, Val-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Phe-Lys, Ala-Ala, Phe-Cit, Gly- Gly, Gly-Arg, Arg-Arg, Gly-Gly-Val-Cit, Val-Ala-Gly-Gly, Ala-Leu-Ala-Leu, Ser-Ser-Tyr-Ser, Val-N 6 N 6 -Dipropyllysine; or L 1B For Val-Cit, or Gly-Gly-Phe-Gly.

8. The connector-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 2-7, wherein, The linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the linker-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof shown in Formula III-2A, Formula III-3A, Formula III-3B, Formula III-5B, Formula III-3C, or Formula III-5C. Wherein, the L 1A L 1C L 2 L 3 L 1B R 1 R A R B R 2 R 3 r, p, q, m1 and m2 as described in any one of claims 2-8.

9. The connector-payload, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 2-8, wherein, The connector-payload is selected from:

10. A protein drug conjugate of Formula IV, its stereoisomer, or a pharmaceutically acceptable salt thereof. Ab-(L-D) n IV in, Ab is a protein-targeting ligand; L is the connective fragment or its stereoisomer fragment as described in any one of claims 1 and 3-7; D represents a bioactive molecular fragment; n is an integer or decimal selected from 0 and less than or equal to 20.

11. The protein drug conjugate of claim 10, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein, The protein drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof is selected from the protein drug conjugates, stereoisomers, or pharmaceutically acceptable salts shown in Formula IV-3, Formula IV-4, Formula IV-6, Formula IV-4A, Formula IV-4B, Formula IV-4C, Formula IV-5A, Formula IV-5B, Formula IV-5C, Formula IV-6A, Formula IV-6B, or Formula IV-6C. in, The L 1A L 1C L 2 L 3 L 1B R 1’ R A R B R 2 R 3 r, p, q, m1 and m2 as described in any one of claims 1 and 3-7; Ab is a protein-targeting ligand; D represents a bioactive molecular fragment; n is an integer or decimal between 2 and 8.

12. The protein drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in claim 10 or 11, wherein, The protein targeting ligand is an antibody or its antigen-binding fragment; Optionally, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment targeting one or more targets selected from the following: HER2, HER3, EGFR, ROR1, CLDN18.2, CLDN6, B7-H3, B7-H4, TROP-2, CD20, CD22, CD30, CD33, CD47, CD56, CD70, CD79b, VEGF, VEGFR, MUC1, c-MET, RET, LIV-1, GPC3, DLL3, FRα, TF, Nectin-4, CDH17, FGFR2b, PD-1, and PD-L1; Alternatively, the antibody or its antigen-binding fragment may be an anti-HER2 antibody or its antigen-binding fragment, an anti-HER3 antibody or its antigen-binding fragment, an anti-EGFR antibody or its antigen-binding fragment, an anti-ROR1 antibody or its antigen-binding fragment, an anti-CLDN18.2 antibody or its antigen-binding fragment, an anti-CLDN6 antibody or its antigen-binding fragment, an anti-B7-H3 antibody or its antigen-binding fragment, an anti-B7-H4 antibody or its antigen-binding fragment, an anti-TROP-2 antibody or its antigen-binding fragment, an anti-CD20 antibody or its antigen-binding fragment, an anti-CD22 antibody or its antigen-binding fragment, an anti-CD30 antibody or its antigen-binding fragment, an anti-CD33 antibody or its antigen-binding fragment, an anti-CD47 antibody or its antigen-binding fragment, an anti-CD56 antibody or its antigen-binding fragment, or an anti-CD70 antibody. Or its antigen-binding fragment, anti-CD79b antibody or its antigen-binding fragment, anti-VEGF antibody or its antigen-binding fragment, anti-VEGFR antibody or its antigen-binding fragment, anti-MUC1 antibody or its antigen-binding fragment, anti-c-MET antibody or its antigen-binding fragment, anti-RET antibody or its antigen-binding fragment, anti-LIV-1 antibody or its antigen-binding fragment, anti-GPC3 antibody or its antigen-binding fragment, anti-DLL3 antibody or its antigen-binding fragment, FRα antibody or its antigen-binding fragment, anti-TF antibody or its antigen-binding fragment, anti-Nectin-4 antibody or its antigen-binding fragment, anti-CDH17 antibody or its antigen-binding fragment, anti-FGFR2b antibody or its antigen-binding fragment, anti-PD-1 antibody or its antigen-binding fragment, or anti-PD-L1 antibody or its antigen-binding fragment; Alternatively, the antibody or its antigen-binding fragment is selected from trastuzumab or its antigen-binding fragment; and / or The bioactive molecules are selected from cytotoxic drugs, immune agonists, glucocorticoid receptor modulators, Bcl-xL inhibitors, nicotinamide phosphoribosyltransferase inhibitors, proteasome inhibitors, tyrosine kinase inhibitors, or radionuclide drugs. Alternatively, the cytotoxic drug may be selected from one or more of the following: DNA alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, or RNA polymerase II inhibitors. Alternatively, the cytotoxic drug may be selected from camptothecin-based drugs or taxane-based drugs.

13. The protein drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any one of claims 10-12, wherein, The protein drug conjugate is selected from: Optionally, n is an integer or decimal of 2-8, 2-6, 2-4, 4-8, 6-8, or 7-8.

14. A pharmaceutical composition comprising the protein drug conjugate, its stereoisomer, or a pharmaceutically acceptable salt thereof, as described in any one of claims 10-13. Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

15. A protein drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as described in any one of claims 10-13, used as a protein drug conjugate, or for treating a protein-related disease or tumor that specifically binds to a protein-targeting ligand contained in the protein drug conjugate, optionally wherein the tumor is cancer.

Images