Dolastatin analogue, ligand-drug conjugate thereof, preparation method therefor, and use thereof

Abstract

The present invention pertains to the technical field of medicine. Specifically disclosed are a dolastatin analogue, a ligand-drug conjugate thereof, a preparation method therefor, and use thereof. According to the present invention, a toxin, a linker, and a linker moiety are separately optimized, and the compound is prepared by means of organic synthesis. Also disclosed is use of the compound and the ligand-drug conjugate thereof in preparing a medicament for preventing and treating diseases, the diseases including but not limited to hyperproliferative diseases and angiogenic diseases, such as cancer, chronic metabolic diseases, and cardiovascular diseases. Further disclosed is a drug / pharmaceutical composition. The ligand-drug conjugate of the present invention offers the following effects: high stability, reduced off-target-induced toxicity, an expanded therapeutic window, a good tumor tissue-targeting property, and an excellent in vivo anti-tumor effect, achieving reduced toxicity and enhanced efficacy. The present invention provides a research foundation for preparing ligand-drug conjugates with high efficacy and low toxicity, having broad application prospects.

Description

Sea hare toxin analogues and their ligand-conjugated drugs, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and provides compounds, pharmaceutical compositions thereof, and methods of use that have pharmaceutical, biological effect, and tumor cell killing or inhibiting properties. Specifically, this invention relates to novel toxic molecules for treating tumors and cancers, their derivatives and conjugates, as well as methods for preparing these compounds and compositions containing these compounds. Background Technology

[0002] Cancer is a major social, public health, and economic problem in the 21st century; it is reported that in 2022, there were nearly 20 million new cancer cases worldwide (including non-melanoma skin cancer), and 9.7 million cancer deaths (including non-melanoma) (CA Cancer J Clin. 2024, 74, 229–263.). Chemotherapy drugs commonly used as first-line cancer treatment, such as cytotoxic drugs like paclitaxel, vincristine, and irinotecan, lack targeted therapy and, while effective, can cause serious toxic side effects, such as gastrointestinal reactions, neurotoxicity, hematologic toxicity, and liver damage.

[0003] Antibody-drug conjugates (ADCs) are typically composed of monoclonal antibodies, linkers, and bioactive molecules (primarily cytotoxic agents that kill tumor cells). They represent a novel cancer therapy following traditional chemotherapy and macromolecular antibody drugs. Combining the targeting specificity of antibodies with the high activity of cytotoxic drugs, they offer better selectivity and stronger efficacy than traditional chemotherapy, and have become a hot topic in current anti-tumor drug development.

[0004] As of December 2023, a total of 15 antibody-drug conjugates (ADCs) had been launched globally; among them, antibody-drug conjugates using monomethylolpropionate E / F as the toxin molecule include... and This accounts for 40% (Chem. Soc. Rev. 2019, 48, 4361-4374). These drugs are widely used to treat relapsed or refractory breast cancer, Hodgkin's lymphoma, urothelial carcinoma, lymphoma, ovarian cancer, leukemia, and gastric cancer that have previously received other therapies (Blood Advances. 2021, 5, 5098–5106; Drugs. 2020, 80, 1607–1613; Drugs. 2021, 81, 2141–2147; Drugs. 2019, 79, 1467–1475; Drugs. 2021, 81, 1929–1935). However, with the advancement of clinical research, antibody-drug conjugates using monomethylolpropionate (MOP) E / F as the toxic molecule have gradually revealed some problems, such as a narrow therapeutic window, off-target toxicity, and a high incidence of adverse events. The maximum dosage range of these drugs is 1.25 mg / kg to 2.5 mg / kg, which is much lower than that of other types of antibody-drug conjugates. At the maximum dosage, approximately 50% of patients experience adverse reactions such as ocular toxicity, peripheral sensory neuropathy, neutropenia, anemia, acute respiratory distress syndrome, diarrhea, vomiting, nausea, fever, and demyelinating nerve damage (Cancers. 2023, 15, 713; Drug safety. 2019, 42, 295-314; Invest New Drugs. 2018, 36, 121-135).

[0005] In summary, ADCs based on monomethylolpropionate (e / f) have limitations such as a narrow therapeutic window and a high incidence of adverse events. There is an urgent need in the field to develop antibody-drug conjugates (ADCs) with lower off-target toxicity, better efficacy, and a wider therapeutic window. Therefore, this invention focuses on: developing new ADCs based on the discovery of novel, low-toxicity sea haretoxin derivatives through structural optimization; developing new ADCs by cyclizing and attenuating linear sea haretoxin derivatives using a cyclic peptide strategy; and developing new ADCs based on linker optimization strategies for novel cyclic peptide ADCs. Summary of the Invention

[0006] This invention proposes a class of conjugates of bioactive molecules (also known as ligand-conjugated drugs), including but not limited to antibody-conjugated drugs, small molecule conjugates, protein-conjugated drugs, peptide-conjugated drugs, carbohydrate-conjugated drugs, and nucleic acid-conjugated drugs. Its technical advantage lies in its high efficiency and low toxicity. In vitro enzymatic digestion experiments show that the ligand-conjugated drugs of this invention are cleaved by cathepsins at a slower rate, indicating increased stability and effectively avoiding off-target toxicity caused by premature release of toxin molecules. Regarding in vitro safety, the ligand-conjugated drugs of this invention are virtually non-toxic to normal human cells such as NCM460 colonic epithelial cells, HUVEC umbilical vein endothelial cells, and FHC colonic epithelial cells, while the control group kills normal human cells at low nanomolar concentrations. In mouse toxicology experiments, the ligand-conjugated drugs of this invention have no effect on mouse body weight at a dose of 100 mg / kg, while the MMAF control group ligand-conjugated drugs significantly reduce mouse body weight at a dose of 50 mg / kg. Furthermore, the ligand-conjugated drug of the present invention has no platelet toxicity, while the platelet count in the control group was significantly reduced. In vivo pharmacodynamic experiments showed that the ligand-conjugated drug of the present invention significantly inhibited tumor growth, and the tumor inhibition rate in the SKOV3 ovarian cancer xenograft model was significantly better than that in the control group. Therefore, the present invention can improve the safety and therapeutic window of ligand-conjugated drugs, reduce off-target toxicity, and achieve the effect of reducing toxicity and increasing efficacy. The present invention provides one or more compounds of the following formulas (A), (B), (T) and (C), pharmaceutically acceptable salts and / or solvates (e.g., hydrates), pharmaceutical formulations thereof, and their use in the treatment of cancer and tumors.

[0007] To address the shortcomings of existing technologies, this invention provides a compound, which is a sea haretoxin derivative or any form of cyclic derivative thereof, selected from the structure shown in formula (DA) or formula (DB):

[0008] Among them, R a Choose from one of the following groups: optional substitution Optional replacement Optional replacement Among them, R 4 Selected from any one of the following groups: hydrogen, optionally substituted alkyl groups of C1-C8 and optionally substituted alkoxy groups of C1-C8, wherein each of the substituted alkyl group and the substituted alkoxy group is independently and optionally substituted by one or more substituents selected from deuterium, halogen, hydroxyl, carbonyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; wherein R 5Selected from any one of the following groups: hydrogen, optional substituted alkyl group of C1-C8, optional substituted alicyclic group of C1-C8, optional substituted aliheterocyclic group of C1-C8, optional substituted aryl group of C1-C8, optional substituted heteroaryl group of C1-C8, or R. 5 The nitrogen atom bonded to it forms an alicyclic structure, wherein the heteroaryl group and the heteroatom in the alicyclic structure are selected from one or more of O, S, and N; wherein R 6 Selected from any one of the following groups: C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alicyclic, C1-C8 optionally substituted aliheterocyclic, C1-C8 optionally substituted aryl, C1-C8 optionally substituted heteroaryl, or R 6 The carbon atoms bonded to it form an alicyclic / heterocyclic structure, wherein the heteroatom of the alicyclic or heteroaryl group is selected from one or more of O, S, and N; R b Optionally substituted with one or more substituents selected from deuterium, carbonyl, halogen, hydroxyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; R c The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3- 15 PEG with alcohol and amino protected ends 3-15The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms; the aryl group is a C6-C10 aryl group, wherein the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms; the substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; the R d The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, and the heterocyclic aryl group is a C5-C6 aryl group containing oxygen, a C5-C6 aryl group containing nitrogen, or a C5-C6 aryl group containing sulfur. The substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoyl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; wherein, R... eThe alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-1 5. An alkyl group of amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, I, or a PEG-containing acyl group when halogenated. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms, and the substituent of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group is a C3-C15 alkyl group, a C3-C15 alkenyl group, a C3-C15 alkoxy group, a nitro group, a diethylamino group, a cyano group, a haloalkyl group, a morpholinyl group, a piperazine group, or a halogenated substituted aryl group; n is 0 or 1.

[0009] The present invention also provides a compound comprising the structure shown in formula (T): LC(T);

[0010] Wherein, L is an optional substituted linking group;

[0011] Wherein, C is a bioactive molecule or a precursor structure containing a bioactive molecule.

[0012] The present invention also provides a compound comprising the structure shown in formula (B): QLC (B);

[0013] Where Q represents the connector unit;

[0014] Wherein, L is an optional substituted linking group;

[0015] Wherein, C is a bioactive molecule or a precursor structure containing a bioactive molecule.

[0016] This invention also provides a compound (also called a conjugate) containing a bioactive molecular precursor structure, an optionally substituted linker group, a linker unit, and a ligand, wherein the ligand portion forms a targeted conjugate with the linker unit via an active group (such as a thiol group, which is a group inherent to the ligand). The compound comprises the structure shown in formula (A):

[0017] in,

[0018] Q stands for connector unit;

[0019] L is an optional substituted linking group;

[0020] C is a bioactive molecule or a precursor structure containing a bioactive molecule;

[0021] P is the ligand, and m is any integer or decimal between 1 and 20.

[0022] In formula (B) of the present invention, in some preferred embodiments, P is any one of small molecule ligands, protein ligands, polypeptide ligands, carbohydrate ligands, nucleic acid ligands, antibody or antigen-binding fragments, etc.

[0023] In some preferred embodiments of the present invention, P is an antibody or antigen-binding fragment.

[0024] In some preferred embodiments of the present invention, P is an antibody, wherein the antibody is selected from one of the following groups: murine antibodies, chimeric antibodies, humanized antibodies, and fully humanized antibodies; and / or, the antibody comprises bispecific antibodies, multispecific antibodies, etc. More preferably, the antibody is any one of sacituzumab, trastuzumab, pertuzumab, enfortumab, and patritumab; and / or, the antigen-binding fragment comprises any one of the antigen-binding fragments of the aforementioned monoclonal antibodies.

[0025] In this invention, when P is an antigen-binding fragment, in some preferred embodiments, the antigen-binding fragment is selected from one of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, ScFv, di-scFv, VHH and dAb, etc., or antigen-binding fragments thereof.

[0026] In this invention, when P is an antibody or antigen-binding fragment, in some preferred embodiments, the antibody or antigen-binding fragment is selected from any one of the following groups: anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7H3 antibody, anti-BCMA antibody, anti-CDH6 antibody, anti-EGFR antibody, anti-Nectin-4 antibody, anti-TIM1 antibody, anti-PSMA antibody, anti-EpCAM antibody, anti-MUC1 antibody, anti-FGF2 antibody, anti-c-MET antibody, anti-GFR antibody, anti-EphA2 antibody, anti-ROR1 antibody, anti-PD-L1 antibody, anti-5T4 antibody, anti-NaPi2b antibody, anti-STEAP antibody, anti-BCMA antibody, anti-CEACAM5 antibody, anti-SC-16 antibody, anti-Delt-like antibody, etc. Protein 3 antibody, anti-Claudin 18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD48 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD84 antibody, anti-CD138 antibody, anti-CD147 antibody, anti-CD166 antibody, anti-CD223 antibody, anti-CD229 antibody, anti-C D244 antibody, anti-CD319 antibody, anti-MUC16 antibody, anti-ASCT2 antibody, anti-CD324 antibody, anti-CD352 antibody, anti-CD48a antibody, anti-CS1 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-ETBR antibody, anti-FGFR2 antibody, anti-FLT3 antibody, anti-LAMP-1 antibody, anti-Ly6-E antibody, anti-NOTCH3 antibody, anti-PRLR antibody, anti-RNF43 antibody, and / or, the antigen-binding fragment is selected from the antigen-binding fragments corresponding to the above targets, etc.

[0027] In this invention, when P is an antibody, in some preferred embodiments, the antibody includes a heavy chain variable region VH and a light chain variable region VL, etc.

[0028] In this invention, when P is an antibody, in some preferred embodiments, the antibody comprises a heavy chain and a light chain, etc.; the antibody comprises heavy chains HCDR1, HCDR2, HCDR3 and light chains LCDR1, LCDR2, LCDR3, etc.

[0029] In this invention, when P is an antibody, in some preferred embodiments, the antibody includes any one of Sacituzumab, Trastuzumab, Pertuzumab, Enfortumab, Patritumab, etc.

[0030] In some preferred embodiments of the present invention, P is a small molecule ligand or polypeptide ligand, which includes any one of folic acid derivatives, glutamate urea derivatives, dermalin analogs, growth hormone inhibitory analogs, RGD peptides, anthocyanin dyes, or IR783.

[0031] In formula (A) or formula (B) of the present invention, in some preferred embodiments, Q is selected from any one of the following groups: optionally substituted azide, optionally substituted... Optional replacement Optional replacement

[0032] In some preferred embodiments of formula (A), (B), or (T) of the present invention, L is L1-C(=O)-L2-C(=O)-;

[0033] In some preferred embodiments, L1 is selected from any one of the following groups: optionally substituted alkylene, optionally substituted alkenyl, optionally substituted polyethylene glycol, optionally substituted alicyclic, optionally substituted alicyclic heterocyclic, optionally substituted aryl, optionally substituted heterocyclic; or L1 is selected from any one of the following groups: optionally substituted methylene, optionally substituted propylene, optionally substituted pentylene, optionally substituted cyclopropylene, optionally substituted cyclobutylene, optionally substituted cyclohexylene, optionally substituted diethylene glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted pentaethylene glycol, optionally substituted hexaethylene glycol, optionally substituted heptaethylene glycol, optionally substituted octaethylene glycol.

[0034] In some preferred embodiments, L2 is any one of the following: an optionally substituted natural amino acid, a non-natural amino acid, a di-to-tetrapeptide condensed from a natural or non-natural amino acid, a C2-C15 alkyl carboxylic acid, a C2-C15 oxacarboxylic acid, or a C2-C15 azacarboxylic acid.

[0035] In some preferred embodiments, when L2 contains lysine residues, the lysine residues are replaced by a structure R containing polyethylene glycol residues. 2 Replace; wherein, the R 2 For optional replacement Where n1 is an integer from 1 to 30, R 3 It is any one of the following groups: hydroxyl, optionally substituted amino, tert-butoxycarbonyl-protected amino, optionally substituted alkoxy, optionally substituted alkylamine, optionally substituted cycloalkylamine.

[0036] In formula (A), (B), or (T) of the present invention, in some preferred embodiments, C is selected from formula (DA), formula (DB), or optionally substituted. Or optional replacement

[0037] In some preferred embodiments, -X- is selected from any one of the following group: optional substitutions Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement

[0038] Wherein X1 is selected from one of the following group: carbonyl, C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alkoxy, straight-chain heteroalkyl containing 1-8 carbon atoms, C1-C8 optionally substituted alicyclic group, C1-C8 optionally substituted alicyclic heteroalkyl group, C1-C8 optionally substituted aryl group, C1-C8 optionally substituted heterocyclic group, wherein the heteroatom of the heteroalkyl, alicyclic, or heteroaryl group is selected from one or more of N, O, and S; and / or,

[0039] X2 is selected from any one of the following groups: hydrogen, optional alkyl group of C1-C8, optional alkenyl group of C1-C8, optional polyethylene glycol group of C1-C30, optional alicyclic group of C1-C8, optional aliheterocyclic group of C1-C8, optional aryl group of C1-C8, optional heterocyclic group of C1-C8, wherein the heteroatom of the optional alicyclic group of C1-C8, the optional heterocyclic group of C1-C8, is selected from one or more of N, O, S, wherein the substituted alkyl group, substituted alkenyl group, substituted polyethylene glycol group, substituted alicyclic group, substituted aliheterocyclic group, substituted aryl group and substituted heteroaryl group can be independently and optionally substituted by one or more substituents selected from hydrogen atom, deuterium atom, halogen, haloalkyl group, alkoxy group, hydroxyl group, amino group, alkylamino group, carboxyl group, sulfonic acid group, cyano group, nitro group, aryl group, heteroaryl group, cycloalkyl group or heterocyclic group;

[0040] X3 is selected from any one of the following groups: hydrogen, optional substituted alkyl groups of C1-C8, optional substituted polyethylene glycol groups of C1-C30, and optional substituted linear heteroalkyl groups of C1-C8, wherein the heteroalkyl heteroatom is selected from one or more of N, O, and S, and wherein the substituted alkyl group, substituted polyethylene glycol group, and substituted linear heteroalkyl group can be independently substituted by one or more substituents selected from hydrogen atom, deuterium atom, carbonyl group, halogen, haloalkyl group, alkoxy group, hydroxyl group, amino group, alkylamino group, carboxyl group, sulfonic acid group, cyano group, nitro group, aryl group, heteroaryl group, cycloalkyl group, or heterocyclic group;

[0041] Where R 1 Selected from one of the following groups: hydrogen, C1-C8 alkyl, optional substituted straight-chain heteroalkyl containing 1-8 carbon atoms, wherein the heteroatom in the heteroalkyl is selected from one or more of N, O, and S;

[0042] In some preferred embodiments of the present invention, X3 is one of the following group: optionally substituted Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Where n is an integer from 1 to 30;

[0043] In this invention, C is a bioactive molecule. In some preferred embodiments, C includes linear olistatin compounds and cyclic olistatin compounds.

[0044] In this invention, C is a bioactive molecule, and in some preferred embodiments, C comprises the structures shown in formulas (DA) and (DB):

[0045] Among them, R a Choose from one of the following groups: optional substitution Optional replacement Optional replacement

[0046] Among them, R 4 Selected from one of the following groups: hydrogen, optional substituted alkyl groups of C1-C8 and optional substituted alkoxy groups of C1-C8, more preferably, the substituted alkyl groups and the substituted alkoxy groups are each independently and optionally substituted by one or more substituents selected from deuterium, halogen, hydroxyl, carbonyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl;

[0047] Among them, R5 Selected from one of the following groups: hydrogen, optionally substituted alkyl group of C1-C8, optionally substituted alicyclic group of C1-C8, optionally substituted aliheterocyclic group of C1-C8, optionally substituted aryl group of C1-C8, optionally substituted heteroaryl group of C1-C8, or R. 5 The nitrogen atom attached thereto forms an alicyclic group, which may include, for example, a five-membered alicyclic group, a six-membered alicyclic group, etc.; the heteroatom of the alicyclic group or heteroaryl group is selected from one or more of O, S, and N;

[0048] Among them, R 6 Selected from one of the following groups: C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alicyclic, C1-C8 optionally substituted aliheterocyclic, C1-C8 optionally substituted aryl, C1-C8 optionally substituted heteroaryl, or R 6 The carbon atoms bonded to it form a cyclic alicyclic structure (or alicyclic heterocyclic structure), such as a five-membered alicyclic (five-membered alicyclic heterocyclic) or a six-membered alicyclic (six-membered alicyclic heterocyclic), etc., wherein the heteroatom of the alicyclic group or heteroaryl group is selected from one or more of O, S, and N; more preferably, the R 6 It is any one of methyl, isopropyl, or optionally substituted benzyl;

[0049] Among them, R b Optionally substituted with one or more substituents selected from deuterium, carbonyl, halogen, hydroxyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; more preferably, R b It is replaced by one or more of the following: fluorine, chlorine, bromine, iodine, and amino groups.

[0050] Among them, R c The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3- 15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms; the aryl group is a C6-C10 aryl group, wherein the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms; the substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogen-substituted aryl group; preferably, R c Selected from hydrogen, deuterium, alkyl, substituted alkyl, and acyl groups.

[0051] Where R d The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, and the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms; the substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; preferably, R d Selected from amides, any substituted amides, and aromatic heterocycles.

[0052] Wherein, the R e The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing a nitrogen atom, an oxygen atom, and a sulfur atom, the aryl group is a C6-C10 aryl group, the heterocyclic aryl group is a C5-C6 aryl group containing an oxygen atom, a C5-C6 aryl group containing a nitrogen atom, or a C5-C6 aryl group containing a sulfur atom, and the substituent of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group is a C3-C15 alkyl group, a C3-C15 alkenyl group, a C3-C15 alkoxy group, a nitro group, a diethylamino group, a cyano group, a haloalkyl group, a morpholinyl group, a piperazine group, or a halogenated substituted aryl group.

[0053] Where n is 0 or 1.

[0054] In some preferred embodiments of the present invention, C comprises any one of the following structures:

[0055] In some preferred embodiments of the present invention, the compound of formula (T) is selected from any one of the following groups of structures:

[0056] In some preferred embodiments of the present invention, the compound of formula (B) is selected from any one of the following groups of structures:

[0057] Where n2 is any integer between 1 and 20.

[0058] In some preferred embodiments of the present invention, the compound of formula (A) is selected from any one of the following groups of structures:

[0059] Where Ab is an antibody or antigen-binding fragment, -S- is the group attached to Ab, m is any decimal or integer between 1 and 20, and n2 is any integer between 1 and 20.

[0060] On the other hand, the present invention also provides a compound, wherein the compound is selected from any one of compounds with the structure shown in formula (A) or formula (B) or formula (T) or formula (DA) or formula (DB), or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or pharmaceutically acceptable salt, prodrug, or solvate thereof.

[0061] On the other hand, the present invention also provides a method for preparing the conjugate (compound) shown in formula (A), wherein a pair of cysteine ​​residues are generated by reducing the disulfide bond of the ligand, and the thiol group in the cysteine ​​residue undergoes a substitution reaction with the linker unit in the compound shown in formula (B) of the present invention, for example, an addition reaction with the maleimide group, thereby attaching the compound shown in formula (B) of the present invention to the thiol group of the ligand to obtain the conjugate shown in formula (A). The DAR (m in the present invention) of ligand-drug conjugation is controlled according to the reaction conditions, and is commonly an integer or decimal between 1 and 8. Compared with conventional ligand-conjugated drugs, the present invention has found that the ligand-conjugated drug of the present invention has significantly improved stability in plasma. Therefore, the ligand-conjugate of the present invention is more stable and less prone to premature breakage.

[0062] In this invention, the method for preparing the compound represented by formula (A) includes the following steps:

[0063] Step 1: The ligand undergoes a reduction reaction with a reducing agent to obtain the reduced ligand;

[0064] Step 2: The reduced ligand obtained in Step 1 reacts with the compound shown in Formula (B) in an organic solvent or buffer solution to obtain the conjugate shown in Formula (A).

[0065] In this invention, in the first step, in some preferred embodiments, the method includes reacting a ligand with a reducing agent, wherein the disulfide bond of the ligand is reduced by the reducing agent to form a cysteine ​​residue, thereby obtaining the reduced ligand; preferably, the reducing agent is any one of tri-(2-carboxyethyl)phosphine hydrochloride, β-mercaptoethanol, and dithiothreitol.

[0066] In step 1, in some preferred embodiments, the molar ratio of the ligand to the reducing agent is 1:1 to 1:10; preferably, the molar ratio of the ligand to the reducing agent is 1:2 to 1:8.

[0067] In the first step, in some preferred embodiments, the ligand reacts with the reducing agent at a temperature of 10–37°C; preferably, the reaction temperature is 37°C.

[0068] In step 1, in some preferred embodiments, the reaction time between the ligand and the reducing agent is 1 to 24 hours; preferably, the reaction time between the ligand and the reducing agent is 0 to 4 hours; more preferably, the reaction time is 2 to 4 hours.

[0069] In this invention, in step 2, in some preferred embodiments, the thiol group of the cysteine ​​residue in the ligand reduced in step 1 reacts with the linker unit in the compound shown in formula (B) to generate the conjugate shown in formula (B).

[0070] In this invention, in step 2, in some preferred embodiments, the method includes coupling the thiol group of the cysteine ​​residue of the reduced ligand with the compound shown in formula (B) at 0–37°C; preferably, the coupling temperature is 37°C.

[0071] In this invention, in step 2, in some preferred embodiments, the method includes reacting the ligand with the compound represented by formula (B) for 1 to 24 hours; preferably, the reaction time is 4 to 10 hours.

[0072] In this invention, in step 2, in some preferred embodiments, the method includes reacting the ligand with the compound of formula (B) in an organic solvent; preferably, the organic solvent is selected from any one and any combination thereof of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (e.g., methanol, ethanol).

[0073] In this invention, in step 2, in some preferred embodiments, the method includes reacting the ligand with the compound of formula (B) in a buffer solution; preferably, the buffer solution is selected from any one or any combination thereof: potassium dihydrogen phosphate-sodium hydroxide / sodium chloride-diethyltriaminepentaacetic acid buffer (DTPA), disodium hydrogen phosphate-citric acid / sodium chloride-diethyltriaminepentaacetic acid buffer (DTPA), histidine-sodium hydroxide / sodium chloride / diethyltriaminepentaacetic acid buffer (DTPA), and phosphate buffered saline (PBS) / diethyltriaminepentaacetic acid buffer (DTPA), etc.

[0074] In this invention, in step 2, in some preferred embodiments, the method includes a molar ratio of the ligand to the compound represented by formula (B) of 1:1 to 1:20; preferably, the molar ratio of the ligand to the compound represented by formula (B) of 1:1 to 1:10.

[0075] In this invention, the ligand includes any one of small molecule ligands, protein ligands, polypeptide ligands, carbohydrate ligands, nucleic acid ligands, antibodies, or antigen-binding fragments. In some preferred embodiments, the ligand is an anti-HER2 monoclonal antibody or an anti-Trop2 monoclonal antibody, or an antigen-binding fragment thereof. Preferably, the anti-HER2 monoclonal antibody is trastuzumab or pertuzumab, and the anti-Trop2 monoclonal antibody is sacituzumab, etc.

[0076] In some preferred embodiments of the present invention, the method further includes the step of purifying the coupling product; preferably, the coupling product is purified by one or more of ion exchange chromatography, hydrophobic chromatography, reverse chromatography or affinity chromatography.

[0077] In some preferred embodiments of the present invention, m is determined by one or more of the following methods: hydrophobic chromatography, sodium dodecyl sulfonate polyacrylamide gel electrophoresis, or liquid chromatography-mass spectrometry, etc.

[0078] On the other hand, the present invention also provides a method for preparing the compound represented by formula (B), the method comprising the following steps:

[0079] Step 1: Contact the amino acid active ester of N1 with an amino protecting group with an amino acid to obtain intermediate M1;

[0080] Step 2: In the presence of a condensing agent, the intermediate M1 obtained in the first step is contacted with p-aminobenzyl alcohol or p-aminobenzyl alcohol containing a tert-butoxycarbonyl protecting group to obtain intermediate M2.

[0081] Step 3: Remove N1 from intermediate M2 obtained in step 2 to obtain intermediate M3;

[0082] Step 4: The intermediate M3 obtained in step 3 is contacted with a compound containing a maleimide group to obtain intermediate M4;

[0083] Step 5: The intermediate M4 obtained in step 4 is contacted with bis(4-nitrophenyl) carbonate to obtain intermediate M5;

[0084] Step 6: The intermediate M5 obtained in step 5 is contacted with the bioactive molecule C to obtain intermediate M6 or the compound shown in formula (B);

[0085] Step 7: Remove the tert-butyloxycarbonyl protecting group from intermediate M6 obtained in step 6 to obtain intermediate M7;

[0086] Step 8: In the presence of a condensing agent, the intermediate M7 obtained in step 7 undergoes intramolecular condensation or condensation with the corresponding carboxylic acid to obtain the compound shown in formula (B).

[0087] In the first step, in some preferred embodiments, the molar ratio of the amino acid active ester of N1 with an amino protecting group to the amino acid is 1:1 to 1:1.2; preferably, the molar ratio of the amino acid active ester of N1 with an amino protecting group to the amino acid is 1:1.2.

[0088] In the first step, in some preferred embodiments, the amino acid active ester of N1 with an amino protecting group reacts with the amino acid at 0–37°C; preferably, the reaction temperature is 37°C.

[0089] In the first step, in some preferred embodiments, the reaction time between the amino acid active ester of N1 with the amino protecting group and the amino acid is 0 to 24 hours; preferably, the reaction time is 24 hours.

[0090] In the second step, in some preferred embodiments, the condensing agent is any one or more of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), etc.

[0091] In the second step, in some preferred embodiments, the molar ratio of intermediate M1 to p-aminobenzyl alcohol or p-aminobenzyl alcohol containing a tert-butyloxycarbonyl protecting group is 1:1 to 1:1.5; preferably, the molar ratio of intermediate M1 to p-aminobenzyl alcohol or p-aminobenzyl alcohol containing a tert-butyloxycarbonyl protecting group is 1:1.5.

[0092] In the second step, in some preferred embodiments, the reaction temperature of the intermediate M1 with the p-aminobenzyl alcohol containing the tert-butoxycarbonyl protecting group or the p-aminobenzyl alcohol is 0 to 37°C; preferably, the reaction temperature is 37°C.

[0093] In the second step, in some preferred embodiments, the reaction time of the intermediate M1 with the p-aminobenzyl alcohol containing the tert-butyloxycarbonyl protecting group is 0 to 24 hours; preferably, the reaction time is 24 hours.

[0094] In the third step, in some preferred embodiments, the N1 of the intermediate M2 is removed at 0–37°C; preferably, the removal temperature is 0°C.

[0095] In the third step, in some preferred embodiments, the N1 of the intermediate M2 is removed for 0 to 4 hours; preferably, the removal time is 4 hours.

[0096] In the fourth step, in some preferred embodiments, the molar ratio of intermediate M3 to the compound containing maleimide groups is 1:1 to 1:1.5; preferably, the molar ratio of intermediate M3 to the compound containing maleimide groups is 1:1.2.

[0097] In the fourth step, in some preferred embodiments, the reaction temperature of the intermediate M3 with the compound containing the maleimide group is 0–37°C; preferably, the reaction temperature is 37°C.

[0098] In the fourth step, in some preferred embodiments, the reaction time between the intermediate M3 and the compound containing the maleimide group is 0 to 24 hours; preferably, the reaction time is 24 hours.

[0099] In the fifth step, in some preferred embodiments, the molar ratio of intermediate M4 to bis(4-nitrophenyl) carbonate is 1:1 to 1:1.5; preferably, the molar ratio of intermediate M4 to bis(4-nitrophenyl) carbonate is 1:1.5.

[0100] In the fifth step, in some preferred embodiments, the reaction temperature of the intermediate M4 with the bis(4-nitrophenyl) carbonate is 0–37°C; preferably, the reaction temperature is 37°C.

[0101] In the fifth step, in some preferred embodiments, the reaction time between the intermediate M4 and the bis(4-nitrophenyl) carbonate is 0 to 24 hours; preferably, the reaction time is 24 hours.

[0102] In the sixth step, in some preferred embodiments, the molar ratio of intermediate M5 to bioactive molecule D is 1:1 to 1:1.5; preferably, the molar ratio of intermediate M5 to bioactive molecule D is 1:1.

[0103] In the sixth step, in some preferred embodiments, the reaction temperature between the intermediate M5 and the bioactive molecule D is 0–37°C; preferably, the reaction temperature is 37°C.

[0104] In the sixth step, in some preferred embodiments, the reaction time between the intermediate M5 and the bioactive molecule D is 0 to 24 hours; preferably, the reaction time is 24 hours.

[0105] In the seventh step, in some preferred embodiments, the tert-butyloxycarbonyl protecting group of the intermediate M6 is removed at 0–37°C; preferably, the removal temperature is 0°C.

[0106] In the seventh step, in some preferred embodiments, the tert-butyloxycarbonyl protecting group of the intermediate M6 is removed for 0 to 4 hours; preferably, the removal time is 4 hours.

[0107] In the eighth step, in some preferred embodiments, the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), etc.

[0108] In the eighth step, in some preferred embodiments, the reaction temperature for the intramolecular condensation or amide condensation reaction of the intermediate M7 is 0–37°C; preferably, the reaction temperature is 37°C.

[0109] In the eighth step, in some preferred embodiments, the reaction time for the intermediate M7 to undergo intramolecular condensation or amide condensation is 0 to 4 hours; preferably, the reaction time is 4 hours.

[0110] The present invention also provides a drug / drug composition comprising the compounds described above.

[0111] In some preferred embodiments of the present invention, the drug / drug composition is used alone and / or in combination with one or more of other therapeutic agents, including chemotherapy, radiotherapy, immunotherapy agents, anti-autoimmune disease drugs, anti-infective drugs or other antibody-drug conjugates.

[0112] In some preferred embodiments of the present invention, the drug / drug composition is used alone or in combination with one or more other drugs, including nivolumab, pembrolizumab, etc.

[0113] This invention provides the use of the compounds described above, or the preparation methods described above, or the drugs / drug compositions described above in the preparation of treatments for diseases related to abnormal cell activity, tumor diseases, autoimmune diseases, infectious diseases, inflammatory diseases, etc.

[0114] In some preferred embodiments of the present invention, the tumor is selected from any one of the following groups of tumors associated with the expression of the target: HER2, HER3, TROP2, B7H3, BCMA, CDH6, EGFR, Nectin-4, TIM1, PSMA, EpCAM, MUC1, FGF2, c-MET, GFR, EphA2, ROR1, PD-L1, 5T4, NaPi2b, STEAP, BCMA, CEACAM5, SC-16, Delt-like protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD48, CD56, CD70, CD73, CD74, CD79b, CD84, CD138, CD147, CD166, CD22 3. CD229, CD244, CD319, MUC16, ASCT2, CD324, CD352, CD48a, CS1, FGFR2, FGFR3, ETBR, FGFR2, FLT3, LAMP-1, Ly6-E, NOTCH3, PRLR, RNF43, etc.

[0115] In some preferred embodiments of the present invention, the tumor associated with target expression includes tumors that highly express the target and / or tumors that are positive for the target.

[0116] In some preferred embodiments of the present invention, the tumor comprises a solid tumor or a hematopoietic tumor.

[0117] In some preferred embodiments of the present invention, the tumor is selected from one of the following groups: breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, or any other tumor that grows and divides at an uncontrollable rate.

[0118] In some preferred embodiments of the present invention, the diseases associated with abnormal cell activity include hereditary hemoglobinopathies, muscular dystrophy, etc.

[0119] In some preferred embodiments of the present invention, the autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, psoriasis, etc.

[0120] In some preferred embodiments of the present invention, the inflammatory disease includes enteritis, etc.

[0121] The conjugates described in this invention achieve at least one of the following technical effects: Through in-depth research on ligand-conjugated drugs, this invention innovatively proposes a class of conjugates with lower off-target toxicity and significantly improves the therapeutic window and safety. The ligand-conjugates described in this invention achieve at least one of the following technical effects: The ligand-conjugates possess high stability, reducing off-target toxicity; the ligand-conjugated drugs have a high therapeutic window; the ligand-conjugates exhibit good tumor tissue targeting; and the ligand-conjugates possess excellent in vivo antitumor effects, achieving a reduction in toxicity and enhancement of efficacy. Attached Figure Description

[0122] Figure 1 shows the size exclusion chromatography (HIC-HPLC) chromatogram of the MMAF control ADC of the present invention;

[0123] Figure 2 shows the hydrophobic interaction chromatography (SEC-HPLC) chromatogram of the MMAF control ADC of the present invention;

[0124] Figure 3 shows the size exclusion chromatography (HIC-HPLC) chromatogram of A2 of the present invention;

[0125] Figure 4 shows the hydrophobic interaction chromatography (SEC-HPLC) chromatogram of A2 of the present invention;

[0126] Figure 5 shows the size exclusion chromatography (HIC-HPLC) chromatogram of A3 of the present invention;

[0127] Figure 6 shows the hydrophobic interaction chromatography (SEC-HPLC) chromatogram of A3 of the present invention;

[0128] Figure 7 shows the size exclusion chromatography (HIC-HPLC) chromatogram of A16 of the present invention;

[0129] Figure 8 shows the hydrophobic interaction chromatography (SEC-HPLC) chromatogram of A16 of the present invention;

[0130] Figure 9 shows the results of the in vitro HCC1954 cell (human breast cancer cell) proliferation inhibition activity test of some of the antibody-drug conjugates of the present invention;

[0131] Figure 10 shows the results of the in vitro assay of the antibody-drug conjugates of the present invention to inhibit the proliferation of SK-OV-3 cells (human ovarian cancer cells);

[0132] Figure 11 shows the results of the antibody-drug conjugate of the present invention in vitro on the proliferation inhibition activity of HCT116 cells (human colorectal cancer cells);

[0133] Figure 12 shows the results of in vitro toxicity tests of some of the antibody-drug conjugates of the present invention on MRC5 cells (normal human lung fibrosis cells);

[0134] Figure 13 shows the in vitro toxicity test results of some of the antibody-drug conjugates of the present invention in NCM460 (normal human colonic epithelial cells);

[0135] Figure 14 shows the results of in vitro HUVEC (human normal umbilical vein endothelial cells) toxicity test of some of the antibody-drug conjugates of the present invention;

[0136] Figure 15 shows the results of mouse weight changes in some of the antibody-drug conjugates of the present invention in an acute toxicity experiment.

[0137] Figure 16 shows the changes in platelet count in mice during acute toxicity experiments of some of the antibody-drug conjugates of the present invention.

[0138] Figure 17 shows the results of rat weight changes in some of the antibody-drug conjugates of the present invention during acute toxicity experiments;

[0139] Figure 18 shows the stability test results of some of the antibody-drug conjugates of the present invention in plasma;

[0140] Figure 19 shows the results of the anti-tumor growth assay of some of the antibody-drug conjugates of the present invention in the HCC1954 human breast cancer model.

[0141] Figure 20 shows the anti-tumor growth experiment results of some of the antibody-drug conjugates of the present invention in the pharmacological efficacy experiment of the SK-OV-3 human ovarian cancer model.

[0142] Figure 21 shows the in vitro inhibition of tubulin polymerization by MMAE, where from top to bottom they represent Taxol-2.5 μM, Control, MMAE-0.32 μM, MMAE-0.97 μM, MMAE-1.25 μM, and MMAE-1.875, respectively.

[0143] Figure 22 shows the in vitro inhibition of tubulin polymerization by compound D8, where from top to bottom, they represent Taxol-2.5 μM, Control, D8-0.3125 μM, D8-0.937 μM, D8-1.25 μM, and D8-1.875 μM, respectively. Detailed Implementation

[0144] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0145] The present invention will be further described in detail below with reference to the following embodiments. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for carrying out the present invention, except for those specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particularly limiting content.

[0146] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0147] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0148] This invention belongs to the field of pharmaceutical technology, specifically disclosing compounds with sea hare toxin-like compounds as bioactive molecules, their ligand-conjugated drugs, preparation methods, and applications. This invention optimizes the toxin, linker, and terminator components, and prepares the compounds through organic synthesis. This invention also discloses the use of the compounds and their ligand-conjugated drugs in the preparation of drugs for the prevention and treatment of diseases, including but not limited to proliferative and angiogenic diseases such as cancer, chronic metabolic diseases, and cardiovascular diseases. This invention also discloses a drug / drug composition. The ligand-conjugated drugs of this invention have the following effects: high stability, reducing off-target toxicity; a high therapeutic window; good tumor tissue targeting; and excellent in vivo antitumor effects, achieving a reduction in toxicity and enhancement of efficacy. This invention provides a research basis for the preparation of highly effective and low-toxicity ligand-conjugated drugs and has broad application prospects.

[0149] Terminology Definition

[0150] In this invention, the term "ligand" generally refers to a macromolecular, polypeptide, or small molecule ligand that can recognize and bind to antigens or receptors associated with a target cell. The role of a ligand can be to deliver a drug layer to a receptor that is bound to it; these ligands include, but are not limited to, protein hormones, polypeptides, small molecules, lectins, growth factors, antibodies, or other molecules that can bind to a receptor / antigen. In this invention, the ligand can be represented as P. The ligand forms a linking bond with the linking unit through a heteroatom on the ligand. When the ligand is an antibody or antigen-binding fragment, the antibody is selected from chimeric antibodies, humanized antibodies, and fully human antibodies. The antibody can be a monoclonal antibody, a bispecific antibody, or a nanobody. For example, the antibody can be an antibody or antigen-binding fragment selected from the following targets: HER2, HER3, TROP2, B7H3, CDH6, EGFR, Nectin-4, TIM1, PSMA, EpCAM, MUC1, FGF2, c-MET, GFR, EphA2, ROR1, PD-L1, 5T4, NaPi2b, STEAP, BCMA, CEACAM5, SC-16, Delt-like. protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD70, CD73, CD74, CD79b, CD138, CD147, CD166 , CD223, MUC16, ASCT2, CD324, CD352, CD48a, CS1, FGFR2, FGFR3, ETBR, FGFR2, FLT3, LAMP-1, Ly6-E, NOTCH3, PRLR and RNF43.

[0151] In this invention, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and can be a straight-chain or branched group containing 1-20 carbon atoms, for example, containing 1-20 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, and propyl. The alkyl group can be substituted or unsubstituted, or alternative or non-alternative. For example, when substituted, the substituent can be substituted at any usable connection point. The substituent can be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heterocycloyl, cycloalkoxy, and heterocycloalkoxy. For example, it can be hydrogen, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -COOH, -C(O)C(O)H, -S(O)2H, -CONH2, -OC(O)H, or C1-6 aliphatic groups.

[0152] In this invention, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It may contain a straight-chain or branched group of 1-20 carbon atoms. For example, the term "methylene" may refer to a residue derived from the removal of two hydrogen atoms from a one-carbon group. The methylene group may be substituted or unsubstituted, substituted or non-substituted; for example, containing 1-12 carbon atoms, such as an alkylene group containing 1-6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-)1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH3-), etc. The alkylene group can be substituted or unsubstituted, or alternatively substituted. For example, when substituted, the substituent can be replaced at any usable connection point. The substituent can be independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heterocycloyl, cycloalkoxy, and heterocycloalkoxy groups. For example, it can be hydrogen, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -COOH, -C(O)C(O)H, -S(O)2H, -CONH2, -OC(O)H, or a C1-6 aliphatic group. The methylene group can be substituted or unsubstituted.

[0153] In this invention, the term "alkenyl" generally refers to a branched hydrocarbon group containing one or more double bonds. Exemplary examples of alkenyl groups include allyl, homoallyl, vinyl, crotonyl, butenyl, and pentenyl. Exemplary examples of C2-6 chain alkenyl groups having one or more double bonds include butadienyl, pentadienyl, hexadienyl, and their branched forms. The unsaturated bond (double bond) can be located at any position on the carbon chain, and the alkenyl group can be substituted or unsubstituted.

[0154] In this invention, the term "alkenyl" generally refers to a residue derived from a carbon atom of an olefin by removing two hydrogen atoms. Examples include allylene, vinylene, butenylene, pentenylene, and hexenylene. Alkenyl groups can be substituted or unsubstituted.

[0155] In this invention, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group. Examples include ethynyl, 1-propynyl, propynyl, butynyl, etc. The alkynyl group can be substituted or unsubstituted.

[0156] In this invention, the term "ynynyl" generally refers to a residue derived from a carbon atom of an alkene by removing two hydrogen atoms. It can be ethynyl, propynyl, propynyl, or butynyl. The ynynyl group can be substituted or unsubstituted.

[0157] In this invention, the term "aryl" generally refers to a residue derived from the removal of a hydrogen atom from an aromatic ring. The term "aromatic ring" can refer to a 6-14 member all-carbon monocyclic or fused polycyclic ring with a conjugated π-electron system, and can be 6 to 10 members, such as benzene and naphthalene. The aromatic ring can be a fused heterocyclic group or a heteroaryl group, wherein the ring connected to the parent structure is an aryl ring. The aryl group can be substituted or unsubstituted, and when substituted, it can be independently selected from the group consisting of: alkyl, alkynyl, alkenyl, alkoxy, alkylamine, halogen, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heterocyclic, cycloalkoxy, and heterocyclic epoxy.

[0158] In this invention, the term "arylene" generally refers to a derived residue having two hydrogen atoms removed from a carbon atom of an aromatic heterocyclic ring. Examples include arylene furanyl, arylethienyl, pyridinyl, pyrimidinyl, pyrazinyl, etc. The heterocyclic group can be substituted or unsubstituted.

[0159] In this invention, the term "alicyclic group" generally refers to a residue derived from the removal of hydrogen atoms from the same or multiple different carbon atoms of an aliphatic ring. The term "cycloalkane" generally refers to a saturated or unsaturated monocyclic or polycyclic hydrocarbon, the carbon ring comprising 3 to 20 carbon atoms. Non-limiting examples of alicyclic groups include cyclopropane, cyclobutane, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, and cyclooctane. Alicyclic groups can be substituted or unsubstituted. Polycyclic carbon rings include spirocyclic, bridged, and fused ring carbon rings.

[0160] In this invention, the term "cycloene alicyclic group" generally refers to a residue derived from the removal of two hydrogen atoms from a carbon atom of an alicyclic ring. Examples include cycloene propane, cycloene butane, cycloene pentane, cycloene hexenyl, cycloene heptanyl, and cycloene heptatrienyl. Cycloene alicyclic groups can be substituted or unsubstituted.

[0161] In this invention, the term "aliphatic heterocyclic group" generally refers to a non-aromatic 3- to 7-membered monocyclic carbon ring structure, or a fused 7- to 10-membered bicyclic heterocyclic structure. These cyclic structures can be saturated or partially saturated. In addition to the carbon atom, these cyclic structures contain at least one heteroatom, wherein the heteroatom can be selected from the group consisting of oxygen, sulfur, and nitrogen atoms. For example, an aliphatic heterocyclic group can include "heterocyclic alkyl group," which can refer to a stable, non-aromatic 3- to 7-membered monocyclic alkane structure, or a fused 7- to 10-membered bicyclic heterocyclic structure, in addition to the carbon atom. These cyclic structures contain at least one heteroatom, wherein the heteroatom can be selected from the group consisting of oxygen, sulfur, and nitrogen atoms. Heterocyclic alkyl groups can be substituted or unsubstituted, and aliphatic heterocyclic groups can be substituted or unsubstituted.

[0162] In this invention, the term "allelic heterocyclic group" generally refers to a residue derived from the removal of two hydrogen atoms from a carbon atom of an alicyclic ring. Allelic heterocyclic groups can be substituted or unsubstituted.

[0163] In this application, the terms “optional” or “optionally” generally refer to events or circumstances described below that may but do not necessarily occur, and this description includes situations in which such events or circumstances may or may not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not necessarily exist, and this description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0164] In this invention, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, being independently substituted by the corresponding substituents. Substituents are only considered in their possible chemical positions. Those skilled in the art can determine possible or impossible substitutions without much effort. For example, a hydroxyl or amino group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (double bond).

[0165] In this invention, as those skilled in the art will know, terms such as "alkyl" and "alkenyl" can be preceded by an identifier to indicate the number of atoms present in the group under specific conditions, such as C1-C4 alkyl, C3-C7 cycloalkyl, etc., and the subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with 3 carbon atoms; C1-10 refers to a group with 1-10 carbon atoms, with at least 1 carbon atom and at most 10 carbon atoms.

[0166] One or more hydrogen atoms in a group, for example, up to five, such as 1-3 hydrogen atoms, are independently replaced by the corresponding number of substituents. The substituents are only in their possible positions, and those skilled in the art can determine possible or impossible substitutions without much effort. For example, a hydroxyl or amino group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (double bond).

[0167] In this invention, the term "compound" generally refers to a substance having two or more different elements. For example, the compounds of this invention can be organic compounds, compounds with a molecular weight of less than 500, compounds with a molecular weight of less than 1000, or compounds with a molecular weight of more than 1000. In this invention, compounds can be compounds linked by chemical bonds, for example, compounds in which one or more molecules with a molecular weight of less than 1000 are linked by chemical bonds to biological macromolecules, such as polysaccharides, proteins, nucleic acids, etc.

[0168] In this invention, the terms "comprising" and "including" generally refer to including specific features but not excluding other elements, and the terms "above" and "below" generally refer to including the number itself.

[0169] In this invention, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, for example, a variation within a range of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% above or below a specified value.

[0170] In this invention, the compounds comprise tautomers, meso compounds, racemic compounds, enantiomers, or diastereomers of the compound. In this invention, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point and boiling point. In this invention, the term "tautomer" generally refers to an isomer that can be converted into different energy structural isomers through a low energy barrier. In this invention, the term "meso compound" generally refers to a molecule containing symmetry elements (such as symmetry axes) that result in an overall optical rotation of zero. The term "racemic compound" generally refers to a mixture composed of equimolar amounts of two enantiomers.

[0171] In this invention, certain atoms in the compounds of this invention can appear in more than one isotopic form; for example, hydrogen may appear as protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon can exist in the form of H, and it may exist in three different isotopes (H). 12 C 13 C14 C) Naturally occurring. Examples of isotopes that can be incorporated into the compounds of this invention include, but are not limited to, those found in nature. 15 N、 18 O、 18 F, 32 P, 33 P, 129 I, 131 I, or similar isotopes.

[0172] In this invention, the term "pharmaceutical composition" generally refers to a mixture of one or more compounds described in this invention, or their physiologically usable salts or prodrugs, with other chemical components, such as pharmaceutically usable carriers and excipients. Pharmaceutical compositions can facilitate administration to organisms, promote the absorption of the active ingredient, and thereby exert its biological activity.

[0173] In this invention, the term "pharmaceutically acceptable salt" generally refers to a salt of the compounds or antibody-drug conjugates of this invention, or a salt of the compounds described in this invention, which may be safe or effective when used in mammals. The antibody-drug conjugates of this invention can form salts with acids, and non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, sulfate, hydrogen sulfate, citrate, acetate, succinate, oxalate, hydrogen phosphate, dihydrogen phosphate, salicylate, tartrate, maleate, fumarate, methanesulfonate, and p-toluenesulfonate.

[0174] In this invention, the term "coupled compound" generally refers to a compound prepared by one or more chemical reactions, or formed by one or more structures such as bridging groups, spacer groups, or connecting portions.

[0175] In this invention, the term "TROP2" generally refers to a single-pass transmembrane type I cell membrane protein. In this invention, the term "Trop2" may also encompass Trop2 homologs and isoforms. The term "Trop2" also includes proteins having one or more sequences from Trop2 homologs, as well as fragments of those sequences, provided they are variant proteins. For example, uniprot accession number P09758 provides a description of Trop2 and its sequence.

[0176] In this invention, the term "HER2" generally refers to human epidermal growth factor receptor 2 (HER2). For example, the term "HER2" refers to HER2 of any human origin. The term also covers full-length and unprocessed HER2 protein, and it also covers natural variants of HER2. For example, uniprot accession number P04626 provides a description of HER2 and its sequence.

[0177] In this invention, the term "chimeric antibody" generally refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. This can mitigate the immune response induced by murine antibodies. In this invention, the term "humanized antibody," also known as a CDR transplanted antibody, generally refers to an antibody formed by transplanting a murine CDR sequence into a human variable region framework. This can overcome the immune response induced by chimeric antibodies carrying a large amount of murine components. Such framework sequences can be obtained from public DNA libraries containing germline antibody gene sequences or from publicly available references.

[0178] In this invention, the term "fully human antibody" is also called "fully human monoclonal antibody," meaning that both its variable and constant regions are human-derived, and it is immune to immunogenicity. The antibody described in this invention can be a fully human monoclonal antibody. Fully human antibody preparation techniques can include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, etc.

[0179] In this invention, the term "CDR" generally refers to one of the six hypervariable regions within the antibody variable domain that bind to the antigen. The most commonly used definitions of the six CDRs are provided by Kabat EA et al., Chothia et al., and MacCallum et al. As used in this invention, the Kabat definition of CDR can be applied to the light chain variable domains CDR1, CDR2, and CDR3 (L1, L2, L3), and the heavy chain domains CDR1, CDR2, and CDR3 (H1, H2, H3).

[0180] In this invention, the term "group capable of coupling with a thiol group" generally refers to compound A having a thiol group, compound B having a group capable of coupling with a thiol group, and compound B being able to react with the thiol group of compound A, thereby achieving the connection between compound A and compound B.

[0181] In this invention, the term "connector unit" generally refers to a group having a linking group attached to another group. For example, a compound having a linking group can achieve the connection between the compound and another group through a coupling reaction between the linking group and another group. For example, the connector unit can be the structure shown in Q of this invention.

[0182] In this invention, the term "bioactive molecule" generally refers to a portion that is directly or indirectly conjugated with an antibody or antibody fragment to form an antibody-drug conjugate. For example, bioactive molecules include, but are not limited to, the aforementioned antitumor active compounds. For instance, a bioactive molecule can be the structure shown in D of this invention.

[0183] In some embodiments of the present invention, the bioactive molecule in the conjugate is a compound with antitumor activity, such as: radioactive isotopes, including but not limited to At211, I131, Y90, Re186, Re188, Sm153, Bi212, etc.; metal complexes, such as platinum complexes (oxaliplatin); glycopeptide antibiotics, such as bleomycin; topoisomerase inhibitors; compounds that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytarabine, gemcitabine, etc.; microtubule-acting drugs, such as vincristine, taxanes, maytansine, auristatin; tumor signaling pathway inhibitors, such as serine / acid kinase inhibitors, tyrosine kinase inhibitors, etc.; and epigenetic target inhibitors.

[0184] In this invention, the term "microtubule inhibitor" includes, but is not limited to, taxanes, maytansines, and auristatins. Taxanes include paclitaxel and docetaxel; maytansines include DM1 and DM4; auristatins mainly include monosaurus toxin 10, methylauristatin F (MMAF), and monomethylauristatin E (MMAE).

[0185] In this invention, the term "monomethylaurestatin F analogue" generally refers to compounds that are structurally similar to or derived from monomethylaurestatin F. For example, the CAS Registry Number for monomethylaurestatin F is 745017-94-1.

[0186] In this invention, the term "disease associated with the expression of a target" generally refers to a disease whose occurrence or progression is related to the expression level of that target. For example, the expression level of cells from a specific patient's tissue or organ is increased relative to the expression level in normal tissues or organs, i.e., high expression. Or, for example, the expression level of cells from a specific patient's tissue or organ is decreased relative to the expression level in normal tissues or organs, i.e., low expression. Or, for example, a target is expressed in a specific patient's tissue or organ, i.e., positive; or, for example, a target is not expressed in a specific patient's tissue or organ, i.e., negative. For example, the characteristics of target expression can be determined by standards known in the art.

[0187] In this invention, the term "effective amount" generally refers to the amount of a therapeutic agent used to treat, alleviate, or prevent a target disease, or the amount that exhibits a detectable therapeutic or preventative effect. The precise effective amount for a given subject depends on the subject's body size or health condition, the nature of the disease, and the therapeutic agent chosen to be administered.

[0188] Unless otherwise specified, all compounds mentioned in this invention include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds. In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or S configuration, or a mixture of R and S configurations.

[0189] As used herein, the term "compound of the invention" refers to the compound of the invention. This term also includes various crystal forms, pharmaceutically acceptable salts, solvates, etc., of the compounds of the invention.

[0190] When the invention uses a trade name, the trade name includes the product formulation of that trade name, its corresponding generic drug, and the active ingredient of the product of that trade name.

[0191] As used in this invention, "antibody" is used in its broadest sense and covers monoclonal antibodies, polyclonal antibodies, dimers, multimeric multispecific antibodies, and antibody fragments, provided they exhibit the desired biological activity. Antibodies are proteins produced by the immune system capable of recognizing and binding to specific antigens. Target antigens generally have a CDR composed of multiple antibodies. S Numerous binding sites are identified, i.e., antigenic epitopes. An antigen has more than one corresponding antibody. Such targets include, but are not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins are derived from humans, mice, or rabbits. An "antibody fragment" may contain a portion of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; biantibodies; nanobodies, etc. Fragments prepared from Fab expression libraries; multispecific antibodies formed from Fab fragments. The antibodies in the antibody-drug conjugates of this application can retain the ability to bind to wild-type antigens. Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell proliferation regulators, lymphokines, cytokines, and molecules related to angiogenesis. Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known to those skilled in the art and can be obtained through antibody preparation methods well known in the industry.

[0192] In this invention, the term "enrofloxacin" generally refers to an antibody targeting Nectin-4. For example, enrofloxacin may be described in patent WO2017042210A1. In this invention, enrofloxacin may refer to any antibody or antigen-binding fragment containing the heavy chain variable regions CDR1-3 and light chain variable regions of enrofloxacin. In this invention, enrofloxacin may refer to any antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of enrofloxacin.

[0193] In this invention, the term "pertuzumab" generally refers to an antibody targeting HER2. For example, pertuzumab can be described in WO2014172371A2. In this invention, pertuzumab can refer to any antibody or antigen-binding fragment containing the heavy chain variable regions CDR1-3 and light chain variable regions of pertuzumab.

[0194] In this invention, the term "trastuzumab" generally refers to an antibody targeting HER2. For example, trastuzumab can be described in US20060275305A1. In this invention, trastuzumab can refer to any antibody or antigen-binding fragment comprising the heavy chain variable regions CDR1-3 and light chain variable regions of trastuzumab. In this invention, trastuzumab can refer to any antibody or antigen-binding fragment comprising the heavy chain variable regions and light chain variable regions of trastuzumab.

[0195] In this invention, the term "patritumab" generally refers to an antibody targeting HER3. For example, palitumab may be described in CN102174105B. In this invention, palitumab may refer to any antibody or antigen-binding fragment containing the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 of palitumab. In this invention, palitumab may refer to any antibody or antigen-binding fragment containing the heavy chain variable regions and light chain variable regions of palitumab.

[0196] In this invention, the term "peptide residue" generally refers to a residue consisting of one or more amino acid residues linked together. For example, one or more amino acids in a polypeptide residue may be optionally substituted. For example, the polypeptide residues of this invention may be selected from the group consisting of: phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), valine-citrulline (Val-Cit), glutamic acid-valine-citrulline (Glu-Val-Cit), valine-lysine (val-Lys), alanine-alanine-alanine (Val-Val-Val), and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0197] In this invention, the term "polyethylene glycol group" generally refers to a residue consisting of one or more polyethylene glycol residues linked together. For example, a polyethylene glycol group may contain -(CH2CH2O). P-, where p is a number that is at least 1. For example, the polyethylene glycol group in this invention may be optionally substituted.

[0198] In this invention, the term "glycol group" generally refers to a polyethylene glycol group. For example, the glycol group in this invention may be optionally substituted. For example, the number preceding the glycol group may indicate the number of ethylene glycol units in the glycol group; for example, a diethylene glycol group may refer to two ethylene glycol polymerized residues.

[0199] In this invention, the term "sodium dodecyl sulfate polyacrylamide gel electrophoresis" generally refers to a material analysis and characterization technique. For example, ammonium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can detect the molecular weight of a substance.

[0200] In this invention, the term "hydrophobic chromatography" generally refers to an analytical technique based on the differences in hydrophobicity between substances.

[0201] In this invention, the term "liquid chromatography-mass spectrometry" generally refers to an analytical method for identifying the components of a substance. For example, liquid chromatography-mass spectrometry can analyze the molecular weight of an analyte using liquid chromatography-mass spectrometry coupling.

[0202] In this invention, the term "tumor" generally refers to any new pathological tissue proliferation. For the purposes of this invention, angiogenesis is part of the characteristics of a tumor. Tumors may be benign or malignant. The term "tumor" generally refers to a benign or malignant tumor, while the term "cancer" is generally used to refer to a malignant tumor, which may be metastatic or non-metastatic. Tumors that can be diagnosed using the methods of this invention are selected from the group consisting of: breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, progressive large cell lymphoma, multiple myeloma, non-small cell lung cancer, small cell lung cancer, glioblastoma, renal cell carcinoma, pancreatic cancer, prostate cancer, etc. For research purposes, these tissues can be isolated from readily available resources using methods well known to those skilled in the art.

[0203] In this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0204] Example

[0205] Example 1: Synthesis and preparation of compounds represented by formulas (DA) and (DB)

[0206] The raw materials described in this invention are commercially available products, or can be prepared by methods known in the art or according to the methods described herein. Wherein Fmoc is a 9-fluorenylmethyloxycarbonyl protecting group, Boc is a tert-butoxycarbonyl protecting group, and TBDMS / TBS is a tert-butyldimethylsilyl protecting group.

[0207] Example 1-1 Synthesis of Compound D1

[0208] Synthesis route:

[0209] Step 1: Synthesis of Intermediate 1-1

[0210] AHPA (10.0 g, 51.28 mmol, 1 eq) was suspended in a mixed solution of tetrahydrofuran (100 mL) and water (50 mL), stirred, and cooled to 0 °C. First, 1N sodium hydroxide (5.12 g, 128.2 mmol, 2.5 eq) was added dropwise to obtain a clear solution, followed by the addition of Boc anhydride (1.44 g, 56.41 mmol, 1.1 eq). The reaction was allowed to proceed overnight. After the reaction was complete, the tetrahydrofuran solution was concentrated under reduced pressure to remove the precipitate. The solution was then placed in an ice bath, and the pH was adjusted to 2.0 with 1N hydrochloric acid. The solution was extracted three times with dichloromethane (3 × 120 mL). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to give compound 1-1 (13.0 g, 86% yield) as a white solid. 1 H NMR(400MHz,DMSO)δ7.28(t,J=7.3Hz,2H),7.24–7.17(m,3H),6.40(d,J=9.5Hz,1H),4.0 6–3.94(m,1H),3.86(d,J=2.6Hz,1H),2.86–2.76(m,1H),2.74–2.63(m,1H),1.30(s,9H).

[0211] Step 2: Synthesis of intermediates 1-2

[0212] 1-1 (1.48 g, 5.0 mmol, 1 eq), 1-hydroxybenzotriazole (HOBt) (0.743 g, 5.5 mmol, 1.1 eq) and o-phenylenediamine (0.54 g, 5.0 mmol, 1 eq) were dissolved in ultra-dry DMF (30 mL), purged with nitrogen, and stirred in an ice bath. Then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.25 g, 6.5 mmol, 1.3 eq) was added, and the reaction was allowed to proceed overnight. After the reaction was completed, the reaction solution was poured into ice water, extracted three times with ethyl acetate (3×60mL), washed twice with water (2×60mL), washed once with saturated sodium chloride solution (1×60mL), dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. The crude product was then subjected to silica gel column chromatography (dichloromethane:methanol = 200:1 to 100:1) to give compound 1-2 (1.5g, yield 78%) as a gray solid. 1 H NMR (500MHz, DMSO) δ9.03 (s, 1H), 7.34–7.24 (m, 4H), 7.20 (t, J = 6.8Hz, 1H), 7.1 2(d,J=7.8Hz,1H),6.90(t,J=7.6Hz,1H),6.72(d,J=7.9Hz,1H),6.52(t,J=7.5H z,1H),6.47(d,J=9.4Hz,1H),5.76(d,J=6.4Hz,1H),4.79(s,2H),4.15–4.07(m ,1H),4.00(d,J=6.3Hz,1H),2.92–2.85(m,1H),2.80–2.71(m,1H),1.28(s,9H).

[0213] Step 3: Synthesis of intermediates 1-3

[0214] Compounds 1-2 (1.0 g, 2.60 mmol, 1 eq) and dextrorotatory camphor sulfonic acid (1.0 g, 0.52 mmol, 0.2 eq) were suspended in ultra-dry toluene (40 mL), purged three times with nitrogen, and then stirred overnight in an oil bath at 80 °C. After the reaction was complete, the toluene was removed by concentration under reduced pressure; the residue was subjected to silica gel column chromatography (dichloromethane:methanol = 200:1 to 100:4) to give compounds 1-3 (0.65 g, yield 68%) as a pale yellow bubbly solid. 1H NMR (500MHz, DMSO) δ12.28(s,1H),7.55(d,J=7.3Hz,1H),7.44(d,J=7.2Hz,1H),7.30–7.20(m,4H),7.19–7.15(m,1H),7.14–7.09(m,2H) ,6.43(d,J=9.2Hz,1H),6.11(d,J=5.3Hz,1H),4.83–4.77(m,1H),4.13–4.03(m,1H),2.94–2.85(m,1H),2.62–2.54(m,1H),1.23(s,9H).

[0215] Step 4: Synthesis of intermediates 1-4

[0216] Compounds 1-3 (0.55 g, 1.5 mmol, 1 eq) were dissolved in dichloromethane (8 mL) and stirred in an ice bath. Trifluoroacetic acid (2 mL) was slowly added dropwise, and the reaction was allowed to proceed for 2 h. After the reaction was complete, the mixture was concentrated under reduced pressure to remove the organic solvent. Toluene (4 mL) was then added and the mixture was concentrated again to remove the trifluoroacetic acid. This process was repeated twice, and the mixture was evaporated to dryness for later use.

[0217] Step 5: Synthesis of intermediates 1-6

[0218] Compounds 1-5 (0.43 g, 1.5 mmol, 1 eq) were dissolved in ultradry DMF (30 mL), purged three times with nitrogen, and then placed in an ice bath. DEPC (0.294 g, 1.8 mmol, 1.2 eq) and DIPEA (1.94 g, 15.0 mmol, 10 eq) were then added dropwise, and the mixture was stirred for 10 min. The residue (0.40 g, 1.5 mmol, 1 eq) was then dissolved in ultradry DMF (10 mL) and slowly added dropwise, and the reaction was allowed to proceed overnight. After the reaction was complete, the reaction mixture was poured into ice water, extracted three times with ethyl acetate (3 × 60 mL), washed twice with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. The crude product was then subjected to silica gel column chromatography (dichloromethane:methanol = 200:1–100:1) to give compounds 1-6 (0.55 g, yield 68%) as a pale yellow solid. 1HNMR (500MHz, DMSO) δ12.21(s,1H),7.59(s,1H),7.47(d,J=7.6Hz,1H),7.42(d,J=7.5Hz,1H),7.27(d,J=4 .3Hz,4H),7.18(d,J=3.7Hz,1H),7.14–7.04(m,2H),6.23(s,1H),4.83(d,J=19.3Hz,1H),4.40(s,1H),3.70 (d,J=8.5Hz,0.4H),3.50(d,J=8.9Hz,0.6H),3.33(s,2H),3.21(s,3H),3.12–2.98(m,1H),2.91(s,1H),2.6 7–2.57(m,1H),2.13(s,1H),1.71–1.42(m,3H),1.37(d,J=11.6Hz,9H),1.24(s,1H),0.86(d,J=6.3Hz,3H).

[0219] Step 6: Synthesis of intermediates 1-7

[0220] Dissolve 1-6 (0.214 g, 0.4 mmol, 1 eq) in dichloromethane (8 mL) and stir in an ice bath. Slowly add trifluoroacetic acid (2 mL) and react for 2 h. After the reaction is complete, concentrate under reduced pressure to remove the organic solvent, then add toluene (4 mL) and concentrate again to remove trifluoroacetic acid. Repeat this operation twice, then evaporate to dryness and proceed to the next reaction step.

[0221] Step 7: Synthesis of intermediates 1-8

[0222] Compounds 1-7 (0.43 g, 0.4 mmol, 1 eq) and the residue from the previous step (0.43 g, 0.4 mmol, 1 eq) were dissolved in ultradry DMF (8 mL), purged three times with nitrogen, and placed in an ice bath. DIPEA (1.94 g, 15.0 mmol, 10 eq) was then added dropwise, and the mixture was stirred for 10 min. HATU (0.19 g, 1.5 mmol, 1 eq) was then slowly added, and the reaction was allowed to proceed overnight. After the reaction was complete, the reaction mixture was poured into ice water, extracted three times with ethyl acetate (3 × 50 mL), washed twice with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. This crude product was then subjected to silica gel column chromatography (dichloromethane:methanol = 100:1–100:4) to give compounds 1-8 (0.55 g, 45% yield in both steps), as a pale yellow solid. 1H NMR (500MHz, DMSO) δ12.17(d,J=44.4Hz,1H),7.90(d,J=7.3Hz,3H),7.74(d,J=8.2Hz,1H ),7.63(d,J=6.6Hz,1.5H),7.58–7.46(m,1.5H),7.41(t,J=6.7Hz,3H),7.36–7.24(m,6H ),7.20–7.04(m,2.5H),6.99(t,J=7.3Hz,0.5H),6.30–6.19(m,1H),4.92–4.70(m,1.5H) ,4.54(d,J=6.8Hz,1.5H),4.42(s,2H),4.37–4.20(m,2.5H),4.13–4.04(m,0.5H),3.97( s,1H),3.77(d,J=9.2Hz,0.5H),3.59(s,1H),3.25(d,J=11.9Hz,3H),3.21(d,J=7.5Hz,2 H),3.16(d,J=14.3Hz,3H),3.06–2.93(m,3H),2.89–2.77(m,3H),2.75–2.68(m,0.5H),2 .66–2.59(m,0.5H),2.46–2.29(m,2H),2.25–2.09(m,2H),2.09–1.93(m,1.5H),1.88(s, 0.5H), 1.72 (d, J = 48.0Hz, 1H), 1.61–1.50 (m, 2H), 1.40–1.21 (m, 2H), 1.05–0.67 (m, 24H).

[0223] Step 8: Synthesis of intermediates 1-9

[0224] Compounds 1-8 (0.43 g, 0.4 mmol, 1 eq) and 1-7 (0.43 g, 0.4 mmol, 1 eq) were dissolved in ultradry DMF (8 mL), purged three times with nitrogen, and then placed in an ice bath. DIPEA (1.94 g, 15.0 mmol, 10 eq) was then added dropwise, and the mixture was stirred for 10 min. HATU (0.19 g, 1.5 mmol, 1 eq) was then slowly added, and the reaction was allowed to proceed overnight. After the reaction was complete, the reaction mixture was poured into ice water, extracted three times with ethyl acetate (×50 mL), washed twice with water, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain the crude product. The crude product was then subjected to silica gel column chromatography (dichloromethane:methanol = 100:1–100:4) to give compounds 1-9 (0.55 g, two-step yield 45%) as a pale yellow solid. 1H NMR (500MHz, DMSO) δ12.17(d,J=44.4Hz,1H),7.90(d,J=7.3Hz,3H),7.74(d,J=8.2Hz,1H ),7.63(d,J=6.6Hz,1.5H),7.58–7.46(m,1.5H),7.41(t,J=6.7Hz,3H),7.36–7.24(m,6H ),7.20–7.04(m,2.5H),6.99(t,J=7.3Hz,0.5H),6.30–6.19(m,1H),4.92–4.70(m,1.5H) ,4.54(d,J=6.8Hz,1.5H),4.42(s,2H),4.37–4.20(m,2.5H),4.13–4.04(m,0.5H),3.97( s,1H),3.77(d,J=9.2Hz,0.5H),3.59(s,1H),3.25(d,J=11.9Hz,3H),3.21(d,J=7.5Hz,2 H),3.16(d,J=14.3Hz,3H),3.06–2.93(m,3H),2.89–2.77(m,3H),2.75–2.68(m,0.5H),2 .66–2.59(m,0.5H),2.46–2.29(m,2H),2.25–2.09(m,2H),2.09–1.93(m,1.5H),1.88(s, 0.5H), 1.72 (d, J = 48.0Hz, 1H), 1.61–1.50 (m, 2H), 1.40–1.21 (m, 2H), 1.05–0.67 (m, 24H).

[0225] Step 9: Synthesis of compound D1

[0226] Compounds 1-9 (0.211 g, 0.2 mmol, 1 eq) were dissolved in dichloromethane (8 mL) and stirred in an ice bath. Diethylamine (2 mL) was slowly added dropwise, and the reaction was allowed to proceed for 4 h. After the reaction was complete, the organic solvent was removed by concentration under reduced pressure. The crude product was first subjected to silica gel column chromatography (dichloromethane:methanol = 100:2.5–100:7.5) to remove small polar impurities, and then prepared by reverse C18 column chromatography (acetonitrile:water = 10:90–80:20). After lyophilization, compound D1 (86.6 mg, yield 55%) was obtained as a white foamy solid. 1H NMR (500MHz, DMSO) δ12.16 (d, J=45.0Hz, 1H), 8.02 (dd, J=24.7, 8.3Hz, 1H), 7.75 (d, J= 8.1Hz,0.5H),7.54(dd,J=17.8,8.2Hz,1H),7.46(d,J=6.7Hz,0.5H),7.40(d,J=7.4Hz ,1H),7.34(s,2H),7.27(d,J=3.6Hz,2H),7.18(s,1H),7.13–7.04(m,1.5H),6.98(t,J =7.1Hz,0.5H),6.25(dd,J=19.4,4.7Hz,1H),4.88–4.67(m,2H),4.64–4.53(m,1.5H), 4.42(s,0.5H),3.97(s,1H),3.76(d,J=9.0Hz,0.5H),3.58(s,0.5H),3.26–3.14(m,6H ),3.06–2.89(m,3H),2.80–2.67(m,2H),2.66–2.57(m,1H),2.46–2.39(m,1H),2.34(d ,J=16.0Hz,1H),2.19(d,J=27.4Hz,5H),2.07–1.93(m,1H),1.91–1.61(m,3H),1.60–1 .45(m,2H),1.39–1.22(m,2H),0.96–0.83(m,18H),0.81–0.72(m,6H); HR-MS(ESI)m / z C 46 H 71 N7O7[M+Na] + Calculated value: 856.5307, measured value: 856.5310.

[0227] Synthesis of Compound D2 in Examples 1-2

[0228] The experimental procedure was the same as that for compound D1, yielding compound D2 (0.13 g, yield 76%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ11.88(d,J=85.3Hz,1H),8.01(d,J=8.8Hz,0.4H),7.94(d,J=9.0Hz, 0.6H),7.67(d,J=8.5Hz,0.6H),7.54(d,J=8.6Hz,0.4H),7.34(t,J=5.2Hz,3H),7.25(t, J=6.8Hz,2H),7.20–7.11(m,2H),6.16(d,J=5.2Hz,1H),4.83–4.69(m,2H),4.67–4.49(m ,1.5H),4.42–4.34(m,0.5H),3.93(s,1H),3.78(d,J=9.3Hz,0.5H),3.63(d,J=6.0Hz,0. 5H),3.27(d,J=16.6Hz,3H),3.17(t,J=9.1Hz,4H),3.02–2.90(m,3H),2.78–2.68(m,1H) ,2.68–2.55(m,1H),2.33(s,1H),2.27(d,J=3.3Hz,3H),2.22(d,J=11.5Hz,4H),2.15(d, J=6.8Hz,4H),2.09–2.01(m,1H),1.99–1.92(m,1H),1.88(s,1H),1.80–1.66(m,2H),1.6 3–1.43(m,2H),1.40–1.21(m,2H),0.93–0.81(m,17H),0.81–0.71(m,7H); HR-MS(ESI)m / z C 48 H 75 N7O7[M+Na] + Calculated value: 884.5620, measured value: 884.5617.

[0229] Synthesis of compound D3 in Examples 1-3

[0230] The experimental procedure was the same as that for compound D1, yielding compound D3 (0.14 g, yield 80%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ12.36(d,J=43.9Hz,1H),8.01(d,J=8.9Hz,0.4H),7.92(d,J=9.0Hz,0.6H), 7.77(d,J=8.4Hz,0.5H),7.63–7.53(m,1H),7.48–7.33(m,4H),7.28(d,J=4.1Hz,1.5H),7.22–7. 14(m,1H),6.34(dd,J=43.3,4.7Hz,1H),4.81(s,1H),4.73–4.65(m,2H),4.58(t,J=8.6Hz,0.6H ),4.46(d,J=8.1Hz,0.4H),4.07–3.99(m,1H),3.95(s,0.5H),3.70(d,J=9.7Hz,0.5H),3.30(d,J =2.2Hz,3H),3.21(d,J=7.6Hz,2H),3.16(d,J=8.8Hz,2H),3.02–2.90(m,3H),2.79–2.73(m,1H) ,2.71(d,J=6.1Hz,1H),2.69–2.63(m,1H),2.38–2.27(m,1H),2.26–2.11(m,5H),2.09–2.01(m,1 H),1.99–1.92(m,1H),1.85(s,1H),1.80–1.66(m,2H),1.64–1.51(m,1H),1.51–1.44(m,1H),1.4 0–1.20(m,2H),0.97–0.74(m,20H),0.67(d,J=6.5Hz,2H),0.62(d,J=6.5Hz,2H); HR-MS(ESI)m / z C 46 H 69 F2N7O7[M+Na] + Calculated value: 892.5119, measured value: 892.5118.

[0231] Synthesis of compound D4 in Examples 1-4

[0232] The experimental procedure was the same as that for compound D1, yielding compound D4 (93 mg, yield 60%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ8.01(t,J=8.3Hz,1.5H),7.70(d,J=9.2Hz,0.5H),7.34– 7.21(m,4H),7.21–7.13(m,1H),5.72(d,J=5.3Hz,0.5H),5.61(d,J=5.4Hz,0. 5H),4.74–4.52(m,2H),4.40–4.25(m,1H),4.10–3.94(m,2H),3.79–3.72(m, 1H),3.69–3.60(m,1H),3.58–3.46(m,3H),3.28(s,1.5H),3.23(s,1.5H),3.1 7(s,1.5H),3.15(s,1.5H),3.11(s,1.5H),2.97(s,1.5H),2.88–2.82(m,1H) ,2.73–2.63(m,2H),2.48–2.38(m,1H),2.35–2.26(m,1H),2.26–2.13(m,4H), 2.02–1.92(m,1H),1.92–1.68(m,5H),1.68–1.55(m,1H),1.36–1.21(m,2H),0 .94(d,J=6.6Hz,2H),0.91–0.81(m,19H),0.79–0.73(m,3H); HR-MS(ESI)m / zC 41 H 69 N5O9[M+Na] + Calculated value: 798.4988, measured value: 798.4986.

[0233] Synthesis of compound D5 in Examples 1-5

[0234] The experimental procedure was the same as that for compound D1, yielding compound D5 (0.14 g, yield 85%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ9.48(d,J=59.2Hz,1H),8.00(t,J=10.1Hz,1H),7.87(d,J=8.8Hz,0 .5H),7.66(d,J=7.9Hz,1H),7.60(d,J=7.5Hz,1.5H),7.38–7.10(m,7H),7.08–6.96(m, 1H),6.36(d,J=90.2Hz,1H),4.81–4.67(m,1H),4.66–4.54(m,1H),4.49(d,J=7.4Hz,0. 5H),4.36(d,J=7.3Hz,0.5H),4.04(d,J=31.1Hz,1H),3.96(s,1H),3.78(d,J=9.3Hz,0.5 H),3.64(s,0.5H),3.24(d,J=13.2Hz,3H),3.21–3.12(m,6H),2.97(s,1H),2.93–2.85( m,1H),2.82–2.74(m,1H),2.72–2.63(m,1H),2.42–2.28(m,1H),2.27–2.13(m,5H),2.08 –1.99(m,1H),1.98–1.90(m,1H),1.84(s,1H),1.80–1.68(m,1H),1.66–1.49(m,3H),1. 30(s,1H),1.24–1.11(m,1H),0.95–0.81(m,21H),0.77(d,J=3.8Hz,3H); HR-MS(ESI)m / z C 46 H 72 N6O8[M+Na] + Calculated value: 859.5304, measured value: 859.5303.

[0235] Synthesis of compound D6 in Examples 1-6

[0236] The experimental procedure was the same as that for compound D1, yielding compound D6 (0.118 g, yield 69%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ10.08(s,1H),9.30(d,J=19.6Hz,1H),8.19(d,J=7.5Hz,0.5 H),8.06(d,J=7.6Hz,0.5H),8.04–7.96(m,1H),7.92(d,J=9.0Hz,0.5H),7.63(d, J=8.6Hz,0.5H),7.29(s,4H),7.18(s,1H),6.92–6.73(m,3H),6.60(d,J=54.9Hz ,1H),4.79–4.68(m,1H),4.67–4.54(m,1.5H),4.49(d,J=7.1Hz,0.5H),4.05(d,J =36.3Hz,1H),3.96(s,1H),3.72(d,J=8.8Hz,0.4H),3.57(s,0.6H),3.26(s,2H) ,3.14(t,J=22.3Hz,6H),2.96(s,1H),2.87(s,1H),2.80–2.64(m,2H),2.44–2.31 (m,1H),2.29–2.12(m,4H),2.07(d,J=6.3Hz,1H),1.99–1.89(m,1H),1.85–1.69( m,2H),1.67–1.41(m,2H),1.41–1.10(m,2H),1.07–0.61(m,24H); HR-MS(ESI)m / z C 46 H 73 N6O9[M+H] + Calculated value: 853.5434, measured value: 853.5434.

[0237] Synthesis of Compound D7 in Examples 1-7

[0238] The experimental procedure was the same as that for compound D1, yielding compound D7 (0.17 g, yield 93%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ9.42(d,J=18.0Hz,1H),7.97(dd,J=13.4,9.2Hz,1H),7.86(d,J=8 .9Hz,0.5H),7.52(d,J=8.9Hz,0.5H),7.35–7.26(m,4H),7.23–7.15(m,1H),7.10(d,J= 7.5Hz,2H),6.42(d,J=5.8Hz,0.5H),6.28(d,J=5.7Hz,0.5H),4.77–4.65(m,1H),4.64– 4.52(m,1H),4.48–4.36(m,1H),3.98(s,1H),3.96–3.86(m,1H),3.78(dd,J=9.7,1.6Hz ,0.5H),3.70(s,3H),3.65(s,3H),3.60(s,1.5H),3.56(s,1.5H),3.49(d,J=6.8Hz,0. 5H),3.21–3.13(m,7H),3.04–2.93(m,2H),2.91–2.84(m,1H),2.80–2.72(m,1H),2.68( dd,J=23.8,6.1Hz,1H),2.32–2.25(m,1H),2.24–2.13(m,5H),2.04–1.89(m,2H),1.83– 1.50(m,5H),1.35–1.14(m,2H),1.03–0.81(m,21H),0.80–0.72(m,3H); HR-MS(ESI)m / z C 49 H 78 N6O 11 [M+Na] + Calculated value: 949.5632, measured value: 949.5621.

[0239] Synthesis of Compound D8 in Examples 1-8

[0240] The experimental procedure was the same as that for compound D1, yielding compound D8 (0.13 g, yield 77%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ11.29(s,1H),8.02(t,J=9.1Hz,1H),7.96(d,J=9.2Hz,0.5H),7.6 8(d,J=9.0Hz,0.5H),7.43(dd,J=12.9,3.5Hz,1H),7.27(d,J=4.7Hz,4H),7.18(d,J=4. 1Hz,1H),7.15(dd,J=15.7,3.5Hz,1H),6.25(d,J=82.4Hz,1H),4.78–4.54(m,2H),4.52 –4.38(m,1H),4.22(d,J=25.7Hz,1H),3.89(d,J=35.0Hz,1H),3.74(d,J=9.6Hz,0.4H), 3.52(d,J=9.7Hz,0.6H),3.24–3.12(m,7H),3.08(s,1H),3.02–2.93(m,2H),2.93–2.8 4(m,1H),2.77(dd,J=14.0,6.9Hz,1H),2.74–2.66(m,1H),2.43(d,J=16.4Hz,0.5H),2. 32(d,J=15.7Hz,0.5H),2.29–2.07(m,5H),2.05–1.91(m,1H),1.80–1.56(m,4H),1.46– 1.34(m,1H),1.31–1.16(m,3H),0.97–0.79(m,20H),0.78–0.70(m,4H); HR-MS(ESI)m / z C 43 H 69 N7O8S[M+Na] + Calculated value: 866.4821, measured value: 866.4831.

[0241] Synthesis of compound D9 in Examples 1-9

[0242] The experimental procedure was the same as that for compound D1, yielding compound D9 (0.1 g, yield 61%), which was a white, foamy solid. 1H NMR(500MHz, DMSO)δ8.05–7.91(m,1.5H),7.68(d,J=8.7Hz,0.5H),7.33–7.25(m,4H),7.22–7.14 (m,1H),6.70(s,1H),6.62(s,1H),6.52(s,0.5H),5.89(s,0.5H),4.88–4.77(m,0.5H),4.76–4.6 2(m,1.5H),4.58(t,J=8.7Hz,0.5H),4.45–4.34(m,0.5H),4.18–4.12(m,0.5H),4.08(d,J=9.1Hz ,1H),3.99(s,0.5H),3.81(s,0.5H),3.74(d,J=9.7Hz,0.5H),3.27(s,2H),3.23–3.16(m,6H),3.0 2–2.96(m,2H),2.89–2.82(m,1H),2.81–2.73(m,1H),2.71(d,J=6.2Hz,0.5H),2.66(d,J=6.3Hz, 0.5H),2.39(d,J=15.6Hz,0.5H),2.33–2.19(m,4.5H),2.16(d,J=11.2Hz,1H),2.12–2.05(m,0.5 H),2.00–1.91(m,0.5H),1.82–1.75(m,2H),1.74–1.64(m,2H),1.64–1.54(m,1H),1.47–1.37(m, 1H),1.35–1.27(m,1H),1.25–1.17(m,1H),0.93–0.82(m,18H),0.80–0.72(m,6H); HR-MS(ESI)m / z C 43 H 70 N8O8[M+Na] + Calculated value: 849.5209, measured value: 849.5202.

[0243] Synthesis of compound D10 in Examples 1-10

[0244] The experimental procedure was the same as that for compound D1, yielding compound D10 (0.114 g, yield 68%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ9.07(d,J=2.5Hz,1H),8.10–7.99(m,1H),7.93(d,J=9.1Hz,0.5H),7.66(d,J=9.2Hz,0.5H),7.27(t,J=4.9Hz,4H),7.18(td,J=8. 5,4.7Hz,1H),4.71–4.55(m,2H),4.48(dd,J=15.0,6.8Hz,1H),4.27(dd,J= 17.1,1.9Hz,1H),3.90(d,J=4.7Hz,1H),3.71(d,J=10.6Hz,0.5H),3.50–3. 46(m,0.5H),3.21–3.11(m,7H),3.06(s,1H),2.98(s,2H),2.90–2.84(m,1 H),2.82–2.67(m,2H),2.40(d,J=16.1Hz,0.5H),2.33(d,J=15.6Hz,0.5H), 2.27–2.13(m,5H),2.05–1.93(m,1H),1.87–1.63(m,5H),1.52–1.39(m,1H) ,1.34–1.16(m,2H),0.93–0.80(m,20H),0.79–0.71(m,4H); HR-MS(ESI)m / z C 42 H 67 N8O8S[MH] - Calculated value: 843.4808, measured value: 843.4810.

[0245] Synthesis of compound D11 in Examples 1-11

[0246] The experimental procedure was the same as that for compound D1, yielding compound D11 (0.14 g, yield 83%), which was a white, foamy solid. 1H NMR(500MHz,DMSO)δ9.97(d,J=52.4Hz,1H),8.05–7.95(m,1H),7.77(d,J=9.2Hz,0.5H),7.65(d ,J=2.9Hz,0.5H),7.52(t,J=6.1Hz,1H),7.39–7.36(m,0.5H),7.33–7.26(m,4.5H),7.26–7.23( m,1H),7.21–7.15(m,1H),6.32(dd,J=49.3,4.9Hz,1H),4.76(d,J=5.9Hz,0.5H),4.69(t,J=8.5 Hz,0.5H),4.63(s,0.5H),4.60–4.51(m,1H),4.41–4.33(m,0.5H),4.08(s,0.5H),4.03–3.94(m ,1.5H),3.76(d,J=9.6Hz,0.5H),3.62(d,J=5.8Hz,0.5H),3.44(d,J=5.5Hz,1H),3.25–3.14(m, 8H),3.00–2.93(m,2H),2.91–2.83(m,1H),2.79–2.66(m,2H),2.45–2.35(m,1H),2.35–2.28(m, 1H),2.27–2.13(m,5H),2.11–2.01(m,0.5H),2.00–1.90(m,0.5H),1.83–1.70(m,2H),1.65(s,1 H),1.61–1.51(m,1H),1.37–1.17(m,3H),1.03–0.80(m,21H),0.79–0.73(m,3H); HR-MS(ESI)m / z C 44 H 70 N6O8S[M+Na] + Calculated value: 865.4868, measured value: 865.4861.

[0247] Synthesis of compound D12 in Examples 1-12

[0248] The experimental procedure was the same as that for compound D1, yielding compound D12 (0.11 g, yield 66%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ8.01(t,J=9.1Hz,1H),7.93(d,J=9.3Hz,0.5H),7.66(d,J=9.1Hz,0.5H),7.3 3–7.23(m,4H),7.21–7.15(m,1H),6.73–6.60(m,1H),6.19(d,J=43.7Hz,1H),4.72–4.61(m,1.5H ),4.58(t,J=8.7Hz,0.5H),4.53–4.46(m,0.5H),4.45–4.38(m,0.5H),4.23(d,J=2.0Hz,0.5H),4 .16(d,J=2.2Hz,0.5H),3.96–3.90(m,0.5H),3.88–3.81(m,0.5H),3.78–3.72(m,0.5H),3.54(t,J =5.8Hz,0.5H),3.22–3.13(m,6.5H),3.08(s,1.5H),3.03–2.94(m,2H),2.90–2.83(m,1H),2.80– 2.73(m,1H),2.68(dd,J=20.1,6.3Hz,1H),2.47(d,J=16.4Hz,0.5H),2.31(d,J=15.6Hz,0.5H),2. 27–2.13(m,8H),2.08–1.87(m,2H),1.78–1.59(m,4H),1.55–1.45(m,0.5H),1.41–1.35(m,0.5H) ,1.32–1.25(m,2H),0.94–0.82(m,19H),0.80(d,J=6.5Hz,2H),0.78–0.72(m,3H); HR-MS(ESI)m / z C 44 H 71 N7O8S[M+Na] + Calculated value: 880.4977, measured value: 880.4974.

[0249] Synthesis of compound D13 in Examples 1-13

[0250] The experimental procedure was the same as that for compound D1, yielding compound D13 (0.126 g, yield 74%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ8.01(t,J=9.3Hz,1H),7.92(d,J=9.4Hz,0.5H),7.67(d,J=8.9Hz,0.5H),7.3 1–7.24(m,4H),7.22–7.14(m,1H),7.13–7.02(m,1H),6.26(d,J=81.1Hz,1H),4.77–4.61(m,1.5H) ,4.61–4.51(m,1H),4.44–4.35(m,0.5H),4.25(d,J=1.4Hz,0.6H),4.16(s,0.4H),3.94(s,0.4H), 3.85(s,0.6H),3.76(dd,J=9.6,1.7Hz,0.5H),3.58(t,J=5.9Hz,0.5H),3.22(s,1.5H),3.18(s,3H ),3.14(s,1.5H),3.08(s,2H),3.05–2.98(m,1H),2.97(s,1H),2.90–2.83(m,1H),2.79–2.73(m,1 H),2.68(dd,J=17.8,6.3Hz,1H),2.41(d,J=16.5Hz,0.5H),2.34–2.28(m,1.5H),2.27–2.09(m,7H ),2.09–2.03(m,0.5H),1.98–1.92(m,0.5H),1.89–1.78(m,1H),1.77–1.56(m,4H),1.50–1.37(m, 1H),1.31–1.21(m,3H),0.95–0.83(m,17H),0.83–0.78(m,4H),0.77–0.72(m,3H); HR-MS(ESI)m / z C 44 H 71 N7O8S[M+Na] + Calculated value: 880.4977, measured value: 880.4971.

[0251] Synthesis of compound D14 in Examples 1-14

[0252] The experimental procedure was the same as that for compound D1, yielding compound D14 (0.15 g, yield 82%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ10.63(s,1H),7.98(d,J=7.3Hz,1H),7.86(t,J=5.7Hz,0.5H),7.81(d,J=8.5Hz,0.5 H),7.74(t,J=5.7Hz,0.5H),7.47(d,J=8.9Hz,0.5H),7.29–7.23(m,4H),7.21(d,J=8.7Hz,1H),7.20–7. 15(m,1H),7.10–7.05(m,1H),7.04–6.98(m,1H),6.73–6.69(m,1H),6.08(d,J=6.0Hz,0.4H),5.90(d,J= 5.7Hz,0.6H),4.77–4.64(m,1H),4.61–4.51(m,1H),4.33–4.24(m,1H),3.92–3.83(m,1.5H),3.79(d,J= 9.3Hz,0.5H),3.75(d,J=4.9Hz,3H),3.69(d,J=6.0Hz,0.5H),3.54–3.48(m,0.5H),3.22(t,J=10.2Hz,5 H),3.16(s,1.5H),3.13(s,2H),3.11(s,1.5H),2.89–2.79(m,3H),2.78–2.72(m,2H),2.71–2.64(m,2H) ,2.43(s,1H),2.33–2.22(m,1H),2.16(d,J=16.2Hz,4H),2.03–1.90(m,1H),1.87–1.78(m,3H),1.77–1. 64(m,2H),1.65–1.38(m,2H),1.35–1.20(m,1H),0.93–0.81(m,19H),0.79–0.70(m,5H); HR-MS(ESI)m / z C 51 H 79 N7O9[M+Na] + Calculated value: 956.5831, measured value: 956.5822.

[0253] Synthesis of compound D15 in Examples 1-15

[0254] The experimental procedure was the same as that for compound D1, yielding compound D15 (0.128 g, 70% yield), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ7.99(t,J=9.6Hz,1H),7.77(d,J=8.5Hz,0.5H),7.72(d,J=4.5Hz,0.5H),7.61(d, J=5.0Hz,0.5H),7.45(d,J=8.9Hz,0.5H),7.30–7.20(m,4H),7.21–7.14(m,1H),5.89(d,J=5.9Hz,0.5H ),5.72(d,J=5.8Hz,0.5H),4.78–4.64(m,1H),4.57(t,J=8.7Hz,1H),4.27–4.16(m,1H),3.99(s,1H), 3.87–3.78(m,2H),3.61–3.55(m,0.5H),3.51(t,J=10.5Hz,0.5H),3.25(d,J=8.7Hz,4H),3.17(d,J=7. 4Hz,3H),3.10(s,1.5H),2.97(s,1.5H),2.88–2.72(m,1.5H),2.71–2.63(m,2H),2.58(ddd,J=11.4,7 .8,4.0Hz,0.5H),2.46–2.39(m,1H),2.34–2.26(m,1H),2.26–2.20(m,1H),2.16(d,J=14.9Hz,3H),2.0 4–1.93(m,1H),1.91–1.79(m,4H),1.77–1.68(m,2H),1.66–1.56(m,1H),1.36–1.22(m,1H),0.96–0.8 0(m,21H),0.78–0.71(m,3H),0.56–0.50(m,2H),0.48–0.44(m,1H),0.43–0.38(m,1H); HR-MS(ESI)m / z C 43 H 72 N6O8[M+Na] + Calculated value: 823.5304, measured value: 823.5316.

[0255] Synthesis of compound D16 in Examples 1-16

[0256] The experimental procedure was the same as that for compound D1, yielding compound D16 (0.137 g, yield 83%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ8.00(t,J=9.3Hz,1H),7.82(d,J=8.4Hz,0.5H),7.58(d,J=7.5Hz,0.5H),7.54 –7.48(m,1H),7.31–7.21(m,4H),7.18(d,J=6.4Hz,1H),5.95(d,J=5.0Hz,0.5H),5.73(d,J=5.2Hz, 0.5H),4.73(d,J=5.4Hz,0.5H),4.66(t,J=8.6Hz,1H),4.57(t,J=8.7Hz,0.5H),4.27–4.16(m,1H) ,4.00(s,1H),3.97–3.91(m,1H),3.84(d,J=9.0Hz,0.5H),3.82–3.75(m,1H),3.72(d,J=6.3Hz,0.5 H),3.62–3.55(m,0.5H),3.51(t,J=10.6Hz,0.5H),3.31–3.21(m,3H),3.18(d,J=1.3Hz,3H),3.10 (s,1.5H),2.97(s,1.5H),2.85–2.74(m,2H),2.71–2.63(m,2H),2.46–2.39(m,1H),2.33–2.20(m,2 H),2.16(d,J=15.3Hz,3H),2.04–1.92(m,1H),1.90–1.68(m,8H),1.65–1.54(m,3H),1.50–1.42(m ,2H),1.41–1.32(m,2H),1.32–1.24(m,1H),0.96–0.81(m,21H),0.79–0.73(m,3H); HR-MS(ESI)m / z C 45 H 75 N6O8[MH] - Calculated value: 827.5652, measured value: 827.5652.

[0257] Synthesis of compound D17 in Examples 1-17

[0258] The experimental procedure was the same as that for compound D1, yielding compound D17 (0.1 g, yield 62%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ7.99(t,J=9.1Hz,1H),7.80(d,J=8.6Hz,0.5H),7.70(t,J=5.6Hz,0.5H),7. 64(t,J=5.7Hz,0.5H),7.47(d,J=8.9Hz,0.5H),7.30–7.21(m,4H),7.20–7.15(m,1H),6.10(d,J =6.1Hz,0.5H),5.94(d,J=5.8Hz,0.5H),4.75–4.63(m,1.5H),4.57(t,J=8.7Hz,0.5H),4.27–4. 20(m,1H),3.99(s,1H),3.90–3.83(m,1H),3.78(t,J=8.5Hz,1.5H),3.69(d,J=6.5Hz,0.5H),3.6 2–3.55(m,0.5H),3.54–3.48(m,0.5H),3.24(d,J=9.5Hz,3H),3.18(d,J=2.1Hz,3H),3.15–3.08 (m,2H),3.06–2.94(m,3H),2.87–2.71(m,2H),2.70–2.63(m,2H),2.46–2.38(m,1H),2.35–2.22( m,2H),2.16(d,J=15.0Hz,3H),2.06–1.92(m,1H),1.91–1.83(m,2H),1.82–1.66(m,4H),1.65–1 .55(m,1H),1.39–1.23(m,1H),0.93–0.82(m,21H),0.76(dd,J=15.8,7.6Hz,3H); HR-MS(ESI)m / z C 42 H 72 N6O9[M+Na] + Calculated value: 827.5253, measured value: 827.5271.

[0259] Synthesis of compound D18 in Examples 1-18

[0260] The experimental procedure was the same as that for compound D1, yielding compound D18 (0.14 g, yield 82%), which was a white, foamy solid. 1H NMR (500MHz, DMSO) δ8.01(t,J=9.7Hz,1H),7.81(d,J=8.9Hz,0.5H),7.62(d,J=9.1H z,0.5H),7.31–7.21(m,4H),7.18(d,J=5.7Hz,1H),4.65(t,J=8.3Hz,1H),4.60–4.5 4(m,1H),4.44–4.34(m,2H),4.30(d,J=6.1Hz,0.6H),4.19(d,J=7.3Hz,0.4H),4.10 –3.99(m,1H),3.86–3.75(m,1H),3.71(d,J=7.0Hz,1H),3.63(s,0.4H),3.53(s,0.6H ),3.25–3.15(m,9H),3.06(s,2H),2.97(s,2H),2.92–2.86(m,1H),2.81–2.72(m,1H ),2.71–2.62(m,1H),2.44(d,J=15.2Hz,1H),2.36–2.25(m,3H),2.22–2.08(m,6H),2 .01–1.92(m,1H),1.90–1.82(m,3H),1.77–1.68(m,3H),1.66–1.54(m,1H),1.38–1. 21(m,1H),1.00–0.94(m,3H),0.92–0.79(m,21H),0.78–0.72(m,3H); HR-MS(ESI)m / z C 46 H 79 N7O8[M+Na] + Calculated value: 880.5882, measured value: 880.5892.

[0261] Synthesis of Compound D25 in Examples 1-25

[0262] Synthesis route:

[0263] Step 1: Synthesis of Intermediate 25-1

[0264] 1.0 g (3.37 mmol) of tert-butyl (2-(hydroxymethyl)-5-nitrobenzyl)(methyl)carbamate was dissolved in 10 mL of LMF. Bis(4-nitrophenyl) carbonate (1.3 g, 4.27 mmol) and DIPEA (1.55 mL, 8.90 mmol) were added separately at room temperature. The reaction was carried out under nitrogen protection for 8 hours. Methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, washed twice with diethyl ether, and dried to obtain intermediate 25-1 (1.10 g, 74% yield), which was used directly in the next reaction. Theoretical LCMS (ESI, m / z) value: 461.14, measured value: 462.14 [M+H] + .

[0265] Step 2: Synthesis of intermediate 25-2

[0266] Intermediate 25-1 (100 mg, 0.133 mmol) was dissolved in 2 mL of anhydrous DMF, followed by 200 μL of anhydrous pyridine, then MMAF (purchased from Shanghai Bied Pharmaceutical, 138.3 mg, 0.187 mmol) and HOBt (18.1 mg, 0.133 mmol). The reaction was carried out at room temperature for 24 hours. After concentration, the reaction solution was purified by preparative liquid chromatography to obtain intermediate 25-2 (87 mg, 49% yield). Theoretical LCMS (ESI, m / z) value: 1053.60, measured value: 1054.60 [M+H] + .

[0267] Step 3: Synthesis of Example D25

[0268] Intermediate 25-2 (50 mg, 0.037 mmol) was dissolved in 2 mL of dichloromethane. 0.5 mL of trifluoroacetic acid was added under ice bath conditions, and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2 mL of DMF. DIPEA (48.3 mg, 0.373 mmol) and HATU (17 mg, 0.045 mmol) were added under ice bath conditions, and the reaction was carried out at room temperature for 2 hours. After concentration, the product D25 (18 mg, 40% yield) was obtained by preparative liquid chromatography. Theoretical LCMS (ESI, m / z) value: 935.54, measured value: 936.54 [M+H] + .

[0269] Synthesis of compound D26 in Examples 1-26

[0270] Synthesis route:

[0271] Step 1: Synthesis of intermediate 26-1

[0272] MMAF (0.5 g, 0.70 mmol) was dissolved in 5 ml of methanol, and then 0.167 g of di-tert-butyl dicarbonate was added. The mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solution was concentrated to dryness to obtain intermediate 26-1. The theoretical LCMS (ESI, m / z) value was 832.09, and the measured value was 833.09 [M+H]. + .

[0273] Step 2: Synthesis of intermediate 26-3

[0274] Intermediate 43-1 was processed according to step 2 in Example T1 to obtain intermediate 26-3. The theoretical LCMS (ESI, m / z) value was 1009.61, and the measured value was 1010.61 [M+H]. + .

[0275] Step 3: Synthesis of intermediate 26-4

[0276] Intermediate 26-3 (42 mg, 0.04 mmol) was dissolved in 2 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added under ice bath conditions. The reaction was carried out at room temperature for 1 hour, and the reaction solution was concentrated under reduced pressure to obtain intermediate 26-4. The theoretical LCMS (ESI, m / z) value was 909.56, and the measured value was 910.56 [M+H]. + .

[0277] Step 4: Synthesis of compound D26

[0278] Intermediate 26-4 (42 mg, 0.04 mmol) was dissolved in 2 mL of methanol, and 4 mg of palladium on carbon was added. The reaction was carried out under a hydrogen balloon at room temperature for 1 hour. The reaction solution was filtered through a diatomaceous earth filter, and the filtrate was concentrated under reduced pressure to obtain product D26. Theoretical LCMS (ESI, m / z) value: 879.58, measured value: 880.58 [M+H] + .

[0279] Example 2 Synthesis and preparation of the compound shown in formula (T)

[0280] Example 2-1 Synthesis of compound T1

[0281] Synthesis route:

[0282] Step 1: Synthesis of intermediate 27-1

[0283] Fmoc-val-Osu (100 g, 229 mmol) was dissolved in 500 mL of tetrahydrofuran, and aqueous solutions of L-valine (26.54 g, 297.8 mmol) and sodium bicarbonate (25 g, 297.8 mmol) were added separately. The reaction was carried out at room temperature for 48 hours, and LC-MS showed that the starting material was completely consumed. The reaction solution was adjusted to pH 6 with 1 M dilute hydrochloric acid, extracted twice with 500 mL of ethyl acetate, and the organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give product 27-1 (84.6 g, 90% yield) as a white solid. Theoretical LC-MS (ESI, m / z): 410.47, Measured: 411.46 [M+H] + .

[0284] Step 2: Synthesis of intermediate 27-3

[0285] Intermediate 27-1 (55 g, 134 mmol) was dissolved in a mixture of dichloromethane and methanol (500 mL, 2:1). Intermediate 27-2 (76.5 g, 201 mmol) was added at room temperature, followed by EEDQ (49.7 g, 201 mmol). The reaction mixture was stirred at room temperature for 48 hours, and LCMS showed complete consumption of the starting material. The reaction solution was concentrated under reduced pressure, and ether was added with stirring until a solid precipitated. The solid was filtered, washed twice with ether, and dried to obtain intermediate 27-3 (80.8 g, 78% yield). LCMS (ESI, m / z) theoretical value: 773.06, measured value: 774.12 [M+H] + .

[0286] Step 3: Synthesis of intermediate 27-4

[0287] Intermediate 27-3 (10 g, 12.94 mmol) was dissolved in tetrahydrofuran, and pyridinium hydrofluoride (7.86 mL, 87.24 mmol) was added under ice bath conditions. The reaction was allowed to proceed for 5 hours at room temperature until completion. The reaction solution was concentrated under reduced pressure, and diethyl ether was added with stirring until a solid precipitated. The solid was filtered, washed twice with diethyl ether, and dried to obtain intermediate 27-4 (7.5 g, 88% yield), which was used directly in the next reaction. Theoretical LCMS (ESI, m / z) value: 658.80, measured value: 659.80 [M+H] + .

[0288] Step 4: Synthesis of intermediate 27-5

[0289] Intermediate 27-4 (5 g, 7.59 mmol) was dissolved in 20 mL of DMF. Bis(4-nitrophenyl) carbonate (5.3 g, 17.43 mmol) and DIPEA (4.55 mL, 26.15 mmol) were added separately at room temperature. The reaction was carried out under nitrogen protection for 8 hours. Methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, washed twice with diethyl ether, and dried to obtain intermediate 27-5 (5.20 g, 83% yield), which was used directly in the next reaction. Theoretical LCMS (ESI, m / z): 823.90, Measured: 824.90 [M+H] + .

[0290] Step 5: Synthesis of intermediate 27-6

[0291] Intermediate 27-5 (100 mg, 0.121 mmol) was dissolved in 2 mL of anhydrous DMF, followed by 200 μL of anhydrous pyridine, then MMAF (purchased from Shanghai Bied Pharmaceutical, 138.3 mg, 0.187 mmol) and HOBt (18.1 mg, 0.133 mmol). The reaction was carried out at room temperature for 24 hours. The reaction solution was concentrated and purified by preparative liquid chromatography to obtain intermediate 1-6 (87 mg, 47% yield). Theoretical LCMS (ESI, m / z) value: 1416.77, measured value: 1417.77 [M+H] + .

[0292] Step 6: Synthesis of compound T1

[0293] Intermediate 27-6 (50 mg, 0.037 mmol) was dissolved in 2 mL of dichloromethane. 0.5 mL of trifluoroacetic acid was added under ice bath conditions, and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2 mL of DMF. DIPEA (48.3 mg, 0.373 mmol) and HATU (17 mg, 0.045 mmol) were added under ice bath conditions, and the reaction was carried out at room temperature for 2 hours. After concentration, the product T1 (18 mg, 40% yield) was obtained by preparative liquid chromatography. Theoretical LCMS (ESI, m / z) value: 1297.74, measured value: 1298.74 [M+H] + .

[0294] Example 2-2 Synthesis of compound T2

[0295] The synthesis of compound T2 was similar to that of compound T1, except that L-valine in step 1 was replaced with L-citrulline. Compound T2 was obtained after several reaction steps. Theoretical LCMS (ESI, m / z) value: 1299.57, measured value: 1300.66 [M+H] + .

[0296] Example 2-3 Synthesis of compound T3

[0297] Synthesis route:

[0298] Step 1: Synthesis of intermediate 28-1

[0299] Intermediate 27-4 (3.0 g, 4.55 mmol) was dissolved in 12 mL of dichloromethane. 3 mL of trifluoroacetic acid was added under ice bath conditions. After stirring at room temperature for 2 hours, the solution was concentrated under reduced pressure and used directly in the next reaction step. The residue was dissolved in DMF. 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid (1.46 g, 4.76 mmol), DIPEA (8.3 mL, 47.6 mmol), and HATU (1.99 g, 5.24 mmol) were added under ice bath conditions. After stirring at room temperature for 5 hours, the solution was concentrated under reduced pressure and column chromatography was used to obtain intermediate 28-1 (2.34 g, 60% yield). Theoretical LCMS (ESI, m / z) value: 803.41, measured value: 804.41 [M+H] + .

[0300] Step 2: Synthesis of intermediate 28-2

[0301] Intermediate 28-2 was obtained by following step 4 in Example T1. The theoretical LCMS (ESI, m / z) value was 968.42, and the measured value was 969.42 [M+H]. + .

[0302] Step 3: Synthesis of intermediate 28-3

[0303] Intermediate 28-3 was obtained by following the method in step 5 of Example T1. The theoretical LCMS (ESI, m / z) value is 1560.87, and the measured value is 1561.87 [M+H]. + .

[0304] Step 4: Synthesis of Example T3

[0305] Compound T3 was obtained by following step 6 in Example T1 using 28-3. The theoretical LCMS (ESI, m / z) value was 1442.81, and the measured value was 1443.81 [M+H]. + .

[0306] Example 3 Synthesis and preparation of the compound shown in formula (B)

[0307] Example 3-1 Synthesis of Compound B1

[0308] Synthesis route:

[0309] Step 1: Synthesis of intermediate 29-1

[0310] Intermediate 27-3 (30 g, 38.8 mmol) was dissolved in 300 mL of dichloromethane solution. Diethylamine (43 mL, 415.6 mmol) was added at room temperature, and the reaction was allowed to proceed for 4 hours at room temperature until the reaction was complete. The reaction solution was concentrated under reduced pressure, and a mixed solvent of diethyl ether and n-hexane was added. The mixture was stirred until a solid precipitated, filtered, and the solid was washed twice with diethyl ether and dried to obtain intermediate 29-1 (19.7 g, 92% yield). Theoretical LCMS (ESI, m / z) value: 550.82, measured value: 551.96 [M+H] + .

[0311] Step 2: Synthesis of intermediate 29-2

[0312] Intermediate 29-1 (10 g, 18.15 mmol) was dissolved in DMF, and maleimide succinimide (6.4 g, 25.42 mmol) was added at room temperature. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, washed twice with diethyl ether, and dried to obtain intermediate 29-2. LCMS (ESI, m / z) theoretical value: 687.91, measured value: 688.96 [M+H] + .

[0313] Step 3: Synthesis of intermediate 29-3

[0314] Intermediate 29-2 was followed by step 3 of Example T1 to obtain intermediate 29-3. Theoretical LCMS (ESI, m / z) value: 573.65, measured value: 574.55 [M+H] + .

[0315] Step 4: Synthesis of intermediate 29-4

[0316] Intermediate 29-3 was obtained by referring to step 4 of Example T1 to obtain intermediate 29-4. Theoretical LCMS (ESI, m / z) value: 738.75, measured value: 739.74 [M+H] + .

[0317] Step 5: Synthesis of intermediate 29-5

[0318] Intermediate 29-4 was obtained by referring to step 5 of Example T1. Theoretical LCMS (ESI, m / z) value: 1331.62, measured value: 1332.65 [M+H] + .

[0319] Step 8: Synthesis of Compound B1

[0320] Intermediate 29-5 was followed in step 6 of Example T1 to obtain product B-1 (18 mg, 40% yield). Theoretical LCMS (ESI, m / z) value: 1213.49, measured value: 1214.52 [M+H] + .

[0321] Example 3-2 Synthesis of Compound B2

[0322] The synthesis of compound B2 is similar to that of B1, except that in step 2 of B1, maleimide succinimide acetate is replaced with 6-(maleimide)hexanoate succinimide ester. Compound B2 is obtained after several reaction steps. Theoretical LCMS (ESI, m / z) value: 1213.49, Observed value: 1214.52 [M+H] + .

[0323] Example 3-3 Synthesis of Compound B3

[0324] Synthesis route:

[0325] Step 1: Synthesis of intermediate 30-1

[0326] Replacing L-valine in step 1 of Example T1 with L-citrulline yields intermediate 30-1, a white solid. Theoretical LCMS (ESI, m / z) value: 496.56, measured value: 497.57 [M+H] + .

[0327] Step 2: Synthesis of intermediate 30-2

[0328] Intermediate 30-1 was followed by step 2 of Example T1 to obtain intermediate 30-2. Theoretical LCMS (ESI, m / z) value: 859.15, measured value: 860.15 [M+H] + .

[0329] Step 3: Synthesis of intermediate 30-3

[0330] Intermediate 30-2 was followed in step 1 of Example B1 to obtain intermediate 30-3. Theoretical LCMS (ESI, m / z) value: 636.91, measured value: 637.91 [M+H] + .

[0331] Step 4: Synthesis of intermediate 30-5

[0332] Intermediate 30-3 (8 g, 12.6 mmol) was dissolved in DMF, and maleimide succinimide 30-4 (4.6 g, 15.1 mmol) was added at room temperature. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and methyl tert-butyl ether was added and stirred until a solid precipitated. The solid was filtered, washed twice with diethyl ether, and dried to obtain intermediate 30-5, which was used directly in the next reaction. Theoretical LCMS (ESI, m / z) value: 830.11, measured value: 831.11 [M+H] + .

[0333] Step 5: Synthesis of intermediate 30-6

[0334] Intermediate 30-5 was used as a reference in step 3 of Example T1 to obtain intermediate 30-6. Theoretical LCMS (ESI, m / z) value: 715.85, measured value: 716.85 [M+H] + .

[0335] Step 6: Synthesis of intermediate 30-7

[0336] Intermediate 30-6 was used as a reference in step 4 of Example T1 to obtain intermediate 30-7. Theoretical LCMS (ESI, m / z) value: 880.95, measured value: 881.95 [M+H] + .

[0337] Step 7: Synthesis of intermediate 30-8

[0338] Intermediate 30-7 was used as a reference in step 5 of Example T1 to obtain intermediate 30-8. Theoretical LCMS (ESI, m / z) value: 1403.68, measured value: 1404.68 [M+H] + .

[0339] Step 8: Synthesis of compound B3

[0340] Intermediate 30-8 was used in step 6 of Example T1 to obtain product B-3 (36 mg, 40% yield). Theoretical LCMS (ESI, m / z) value: 1269.59, measured value: 1270.60 [M+H] + .

[0341] Synthesis of Compound B4 in Examples 3-4

[0342] The synthesis of compound B4 was similar to that of compound B3, except that 30-4 in step 4 was replaced with maleimide succinimide ester. Compound B4 was obtained after several reaction steps. Theoretical LCMS (ESI, m / z) value: 1299.57, Observed value: 1300.66 [M+H] + .

[0343] Synthesis of Compound B5 in Examples 3-5

[0344] Synthesis route:

[0345] Step 1: Synthesis of intermediate 30-2

[0346] Intermediate 30-1 (Mc-GGFG-OH, purchased from Shanghai Bid Pharmaceutical, 5 g, 9.42 mmol) and intermediate 27-2 (5.38 g, 14.14 mmol) were dissolved in a mixed solvent of dichloromethane and methanol (v:v = 1:1, 100 mL). EEDQ (3.5 g, 14.14 mmol) was added at room temperature, and the mixture was stirred for 24 hours. The reaction solution was concentrated under reduced pressure, and column chromatography was used to obtain intermediate 30-2 (5.8 g, 69% yield). Theoretical LCMS (ESI, m / z) value: 892.14, measured value: 893.23 [M+H] + .

[0347] Step 2: Synthesis of intermediate 30-3

[0348] Intermediate 30-2 was used to obtain intermediate 30-3, following step 3 in Example T1. Theoretical LCMS (ESI, m / z) value: 777.88, measured value: 778.92 [M+H] + .

[0349] Step 3: Synthesis of intermediate 30-4

[0350] Intermediate 30-3 was used to obtain intermediate 30-4 in step 4 of Example T1. Theoretical LCMS (ESI, m / z) value: 942.98, measured value: 943.99 [M+H] + .

[0351] Step 4: Synthesis of intermediate 5-5

[0352] Intermediate 30-4 was obtained by referring to step 5 of Example T1 to obtain intermediate 30-5. Theoretical LCMS (ESI, m / z) value: 1550.86, measured value: 1551.86 [M+H] + .

[0353] Step 5: Synthesis of compound B5

[0354] Intermediate 30-5 was used in step 6 of Example T1 to obtain product B5. Theoretical LCMS (ESI, m / z) value: 1419.73, measured value: 1420.85 [M+H] + .

[0355] Synthesis of Compound B6 in Examples 3-6

[0356] Synthesis route:

[0357] Step 1: Synthesis of intermediate 31-3

[0358] Intermediate 31-1 (6 g, 12.5 mmol) was dissolved in a mixture of dichloromethane and methanol (V:V = 2:1, 120 mL). Intermediate 31-2 (9.43 g, 18.8 mmol) was added at room temperature, followed by EEDQ (4.64 g, 18.76 mmol). The reaction mixture was stirred at room temperature for 48 hours, and LC-MS showed complete consumption of the starting material. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain intermediate 31-3 (8.4 g, 70% yield). Theoretical LC-MS (ESI, m / z) value: 964.29, Measured value: 965.32 [M+H] + .

[0359] Step 2: Synthesis of intermediate 31-4

[0360] Intermediate 31-3 (3.6 g, 3.73 mmol) was dissolved in methanol, and 10% palladium on carbon (0.36 g) was added to replace the hydrogen gas. After reacting at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain intermediate 31-4 (2.73 g, 88% yield). Theoretical LCMS (ESI, m / z) value: 830.16, measured value: 831.20 [M+H] + .

[0361] Step 3: Synthesis of intermediate 31-5

[0362] Intermediate 31-4 was used as described in step 4 of Example B3 to obtain intermediate 31-5. The theoretical LCMS (ESI, m / z) value is 1023.36, and the measured value is 1024.44 [M+H]. + .

[0363] Step 4: Synthesis of intermediate 31-6

[0364] Intermediate 31-5 was used in step 3 of Example T1 to obtain intermediate 31-6. The theoretical LCMS (ESI, m / z) value was 909.09, and the measured value was 910.11 [M+H]. + .

[0365] Step 5: Synthesis of intermediate 31-7

[0366] Intermediate 31-6 was used as a reference in step 4 of Example T1 to obtain intermediate 31-7. The theoretical LCMS (ESI, m / z) value is 1074.20, and the measured value is 1075.33 [M+H]. + .

[0367] Step 6: Synthesis of intermediate 31-8

[0368] Intermediate 31-7 was followed in step 5 of Example T1 to obtain intermediate 31-8. Theoretical LCMS (ESI, m / z) value: 1681.96, measured value: 1682.96 [M+H] + .

[0369] Step 7: Synthesis of intermediate 31-9

[0370] Intermediate 31-8 was used as a reference in step 6 of Example T1 to obtain intermediate 31-9. Theoretical LCMS (ESI, m / z) value: 1426.81, measured value: 1427.85 [M+H] + .

[0371] Step 8: Synthesis of Compound B6

[0372] Intermediate 31-9 (50 mg, 0.035 mmol) was dissolved in 2 mL of dichloromethane. 0.4 mL of trifluoroacetic acid was added under ice bath conditions, and the reaction was carried out at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2 mL of LDMF. 2,5,8,11,14,17,20,23,26,29,32-undecanetetradecane-34-amine (16.6 mg, 0.035 mmol), DIPEA (61 μl, 0.35 mmol), and HATU (16 mg, 0.042 mmol) were added under ice bath conditions, and the reaction was carried out at room temperature for 2 hours. After concentration, the product B6 (32 mg, 50% yield) was obtained by preparative HPLC purification. Theoretical LCMS (ESI, m / z) value: 1797.29, measured value: 1798.36 [M+H] + .

[0373] Synthesis of Compound B7 in Examples 3-7

[0374] The synthesis of compound B7 was the same as that of compound B6, except that maleimide acetate succinimide was replaced with 6-(maleimino)hexanoate succinimide in step 3. Compound B7 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1725.13, Observed value: 1726.22 [M+H] + .

[0375] Synthesis of Compound B8 in Examples 3-8

[0376] The synthesis of compound B8 was the same as that of compound B3, except that in step 4, maleimide succinimide was replaced with 3-[2-[2-[2-2-[[2-(2,5-dioxopyrrolo-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]methoxy]acrylic acid. Compound B8 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1562.91, Measured value: 1563.92 [M+H] + .

[0377] Examples 3-9: Synthesis of Compound B9

[0378] The synthesis of compound B9 was the same as that of compound B3, except that in step 4, maleimide succinimide was replaced with 1-(18-oxo-3,6,9,12,15-pentoxaoxyl)-1H-pyrrole-2,5-dione. Compound B9 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1549.91, Observed value: 1550.93 [M+H] + .

[0379] Example 3-10 Synthesis of compound B10

[0380] The synthesis of compound B10 was the same as that of compound B1, except that in step 2, maleimide succinimide was replaced with 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecane-18-acid. Compound B10 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1460.78, Observed value: 1461.78 [M+H] + .

[0381] Example 3-11 Synthesis of compound B11

[0382] The synthesis of compound B11 was the same as that of compound B1, except that in step 2, maleimide succinimide was replaced with 1-(18-oxo-3,6,9,12,15-pentoxaoxyl)-1H-pyrrole-2,5-dione. Compound B11 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1447.78, Observed value: 1448.79 [M+H] + .

[0383] Example 3-12 Synthesis of compound B12

[0384] Synthetic route

[0385] Step 1: Synthesis of intermediate 32-2

[0386] Intermediates 32-1 (2.2 g, 6.52 mmol) and 31-4 (6.43 g, 8.48 mmol) were dissolved in a mixed solvent of dichloromethane and methanol (v:v = 2:1, 100 mL). EEDQ (2.09 g, 8.48 mmol) was added at room temperature, and the mixture was stirred for 24 hours at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was added to diethyl ether and stirred until a solid precipitated. The solid was filtered and dried to obtain intermediate 32-2 (4.08 g, 58% yield). Theoretical LCMS (ESI, m / z) value: 1078.39, measured value: 1079.38 [M+H] + .

[0387] Step 2: Synthesis of intermediate 32-3

[0388] Intermediate 32-2 (1.8 g, 1.67 mmol) was dissolved in methanol, and 10% palladium on carbon (0.18 g) was added. The mixture was stirred at room temperature for 8 hours under a hydrogen atmosphere. The reaction solution was filtered and concentrated under reduced pressure for use in the next step. The residue was dissolved in 20 mL of anhydrous DMF, and N-(2-aminoethyl)maleimide hydrochloride (234 mg, 1.67 mmol), DIPEA (0.58 mL, 3.34 mmol), and HATU (0.76 g, 2 mmol) were added, respectively. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 32-3 (1.27 g, 69% yield). Theoretical LCMS (ESI, m / z) value: 1110.40, measured value: 1111.52 [M+H] + .

[0389] Step 3: Synthesis of intermediate 32-4

[0390] Intermediate 32-3 was used as a reference in step 3 of Example T1 to obtain intermediate 32-4. Theoretical LCMS (ESI, m / z) value: 996.13, measured value: 997.11 [M+H] + .

[0391] Step 4: Synthesis of intermediate 32-5

[0392] Intermediate 32-4 was followed in step 4 of Example T1 to obtain intermediate 32-5. Theoretical LCMS (ESI, m / z) value: 1161.24, measured value: 1162.32 [M+H] + .

[0393] Step 5: Synthesis of intermediate 32-6

[0394] Intermediate 32-5 was used as a reference in step 5 of Example T1 to obtain intermediate 32-6. Theoretical LCMS (ESI, m / z) value: 1852.24, measured value: 1853.26 [M+H] + .

[0395] Step 6: Synthesis of product 32-7

[0396] Intermediate 32-6 was followed in step 6 of Example T1 to obtain intermediate 32-7. Theoretical LCMS (ESI, m / z) value: 1513.84, measured value: 1514.85 [M+H] + .

[0397] Step 7: Synthesis of product B12

[0398] Intermediate 32-7 (30 mg, 19.8 μmol) was dissolved in 2 mL of dichloromethane. 0.4 mL of trifluoroacetic acid was added under ice bath conditions, and the reaction was carried out at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 2 mL of LDMF. 2,5,8,11,14,17,20,23,26-nonoxynonane-29-acid (9 mg, 19.8 μmol), DIPEA (35 μL, 198 μmol), and HATU (9 mg, 24 μmol) were added under ice bath conditions, and the reaction was carried out at room temperature for 2 hours. After concentration, the product B12 (17 mg, 40% yield) was obtained by preparative liquid chromatography. Theoretical LCMS (ESI, m / z) value: 1852.24, measured value: 1853.24 [M+H] + .

[0399] Example 3-13 Synthesis of compound B13

[0400] The synthesis of compound B13 is the same as that of compound B1, using the intermediate from step 2 of T1. Replace with intermediate Compound B13 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1256.55, measured value: 1257.58 [M+H] + .

[0401] Example 3-14 Synthesis of compound B14

[0402] The synthesis of compound B14 was the same as that of compound B13, except that in step 4, maleimide succinimide was replaced with 6-(maleimide)hexanoate succinimide ester. The remaining steps were the same, and compound B14 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1256.55, measured value: 1257.58 [M+H] + .

[0403] Example 3-15 Synthesis of Compound B15

[0404] The synthesis of compound B15 is the same as that of compound B5, except that step 2 in T1 is modified. Replace with intermediate Compound B15 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1404.67, measured value: 1405.68 [M+H] + .

[0405] Example 3-16 Synthesis of compound B16

[0406] Synthesis route:

[0407] Step 1: Synthesis of intermediate 34-1

[0408] 34-A1 (8 g, 20.97 mmol) and 34-B1 (7.82 g, 29.36 mmol) were dissolved in a mixed solvent of DCM and methanol (v:v = 2:1, 160 mL). EEDQ (6.74 g, 27.3 mmol) was added at room temperature, and the mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and column chromatography was performed to give intermediate 34-1 (8.3 g, 63% yield). Theoretical LCMS (ESI, m / z): 629.76, Measured: 630.81 [M+H] + .

[0409] Step 2: Synthesis of intermediate 34-2

[0410] Intermediate 34-1 (3 g, 4.76 mmol) was dissolved in 12 mL of dichloromethane. 3 mL of trifluoroacetic acid was added under ice bath conditions. After stirring at room temperature for 2 hours, the solution was concentrated under reduced pressure and used directly in the next reaction step. The residue was dissolved in DMF. 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-acid (1.46 g, 4.76 mmol), DIPEA (8.3 mL, 47.6 mmol), and HATU (1.99 g, 5.24 mmol) were added under ice bath conditions. After stirring at room temperature for 5 hours, the solution was concentrated under reduced pressure, and column chromatography was used to obtain intermediate 34-2 (2.34 g, 60% yield). Theoretical LCMS (ESI, m / z) value: 818.97, Measured value: 630.81 [M+H] + .

[0411] Step 3: Synthesis of intermediate 34-3

[0412] Intermediate 34-2 was used to obtain intermediate 34-3, following step 4 of Example T1. Theoretical LCMS (ESI, m / z) value: 984.07, measured value: 985.11 [M+H] + .

[0413] Step 4: Synthesis of intermediate 34-4

[0414] Intermediate 34-3 was used to obtain intermediate 34-4, following step 5 in Example T1. Theoretical LCMS (ESI, m / z) value: 1576.94, measured value: 1577.96 [M+H] + .

[0415] Step 5: Synthesis of product B16

[0416] Intermediate 34-4 was used in step 6 of Example T1 to obtain product B16. Theoretical LCMS (ESI, m / z) value: 1456.83, measured value: 1457.83 [M+H] + .

[0417] Example 3-17 Synthesis of compound B17

[0418] The synthesis of compound B17 was the same as that of compound B16, except that the Mc-Val-Ala-OH amine in step 1 was replaced with Mc-Val-Cit-OH. The remaining steps were identical, and compound B17 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1530.87, measured value: 1531.83 [M+H] + .

[0419] Example 3-18 Synthesis of compound B18

[0420] The synthesis of compound B18 was the same as that of compound B16, except that the Mc-Val-Ala-OH amine in step 1 was replaced with Mc-GGFG-OH. The remaining steps were identical, and compound B18 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1605.91, measured value: 1606.92 [M+H] + .

[0421] Example 3-19 Synthesis of compound B19

[0422] The synthesis of compound B19 is the same as that of compound B1, except that step 2 in T1 is modified. Replace with intermediate The remaining steps are the same as for compound B1, and compound B19 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1256.51, measured value: 1257.60 [M+H] + .

[0423] Example 3-20 Synthesis of compound B20

[0424] The synthesis of compound B20 is the same as that of compound B19, except that L-alanine amine in step 1 of T1 is replaced with L-citrulline. The remaining steps are the same, and compound B20 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1342.60, measured value: 1343.61 [M+H] + .

[0425] Example 3-21 Synthesis of compound B21

[0426] The synthesis of compound B21 was the same as that of compound B19, except that Fmoc-Val-Ala-OH in step 2 of T1 was replaced with Fmoc-GGFG-OH. The remaining steps were identical, and compound B21 was obtained after several reaction steps. Theoretical LCMS (ESI, m / z) value: 1420.67, measured value: 1421.68 [M+H] + .

[0427] Example 3-22 Synthesis of compound B22

[0428] The synthesis of compound B22 is the same as that of compound B19, except that maleimide succinimide in step 2 of B1 is replaced with 6-(maleimide)hexanoate succinimide ester. The remaining steps are the same, and compound B22 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1312.61, measured value: 1313.73 [M+H] + .

[0429] Example 3-23 Synthesis of compound B23

[0430] The synthesis of compound B23 was the same as that of compound B20, except that maleimide succinimide in step 2 of B1 was replaced with 6-(maleimide)hexanoate succinimide ester. The remaining steps were the same, and compound B23 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1398.71, measured value: 1399.75 [M+H] + .

[0431] Example 3-24 Synthesis of compound B24

[0432] The synthesis of compound B24 was the same as that of compound B21, except that maleimide succinimide in step 2 of B1 was replaced with 6-(maleimide)hexanoate succinimide ester. The remaining steps were the same, and compound B24 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1476.78, measured value: 1477.80 [M+H] + .

[0433] Example 3-25 Synthesis of compound B25

[0434] The synthesis of compound B25 is the same as that of compound B2, except that step 2 in T1 is modified. Replace with The remaining steps are the same, and compound B25 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1269.59, measured value: 1270.62 [M+H] + .

[0435] Example 3-26 Synthesis of Compound B26

[0436] The synthesis of compound B26 is the same as that of compound B3, except that step 2 in T1 is modified. Replace with The remaining steps are the same, and compound B26 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1355.68, measured value: 1356.73 [M+H] + .

[0437] Example 3-27 Synthesis of compound B27

[0438] The synthesis of compound B27 is the same as that of compound B12, except that the intermediate in step 1 is used instead. Replace with intermediate The intermediate from step 2 Replace with intermediate The remaining steps are the same, and compound B27 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1881.29, measured value: 1882.31 [M+H] + .

[0439] Example 3-28 Synthesis of compound B28

[0440] The synthesis of compound B28 is the same as that of compound B12, except that the intermediate from step 2 is used instead. Replace with intermediate The remaining steps are the same, and compound B28 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1870.33, measured value: 1871.33 [M+H] + .

[0441] Example 3-29 Synthesis of compound B29

[0442] The synthesis of compound B29 is the same as that of compound B2, except that the intermediate from step 4 is used instead. Replace with intermediate The remaining steps are the same, and compound B29 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1342.70, measured value: 1343.71 [M+H] + .

[0443] Example 3-30 Synthesis of compound B30

[0444] The synthesis of compound B30 was the same as that of compound B29, except that the intermediate L-valine in step 1 of T1 was replaced with L-citrulline. The remaining steps were identical, and compound B30 was obtained after several steps. Theoretical LCMS (ESI, m / z) value: 1428.80, measured value: 1429.81 [M+H] + .

[0445] Example 3-31 Synthesis of compound B31

[0446] The synthesis of compound B31 is the same as that of compound B2, except that the intermediate from step 5 of B1 is used instead. Replace with The remaining steps are the same, and compound B31 is obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1299.59, measured value: 1300.63 [M+H] + .

[0447] Synthesis of compound B32 in Examples 3-32

[0448] The synthesis of compound B32 was the same as that of compound B31, except that the intermediate L-valine in step 1 was replaced with L-citrulline. The remaining steps were identical, and compound B32 was obtained after several steps of reaction. Theoretical LCMS (ESI, m / z) value: 1385.68, measured value: 1386.62 [M+H] + .

[0449] Synthesis of Reference Standard MC-Val-Ala-PAB-MMAF in Examples 3-33

[0450] Following the method described in document WO2020233174A1, Mc-Val-Ala-PAB-OH (purchased from Shanghai Bid Pharmaceutical, CAS No. 1870916-87-2) was reacted with bis(4-nitrophenyl) carbonate, and then reacted with MMAF (purchased from Shanghai Bid Pharmaceutical, CAS No. 745017-94-1) to obtain the reference standard MC-Val-Ala-PAB-MMAF.

[0451] Synthesis of compound B33 in Examples 3-34

[0452] The synthesis of compound B33 is the same as that of compound B3, except that step 2 in T1 is modified accordingly. Replace with 4-aminobenzyl alcohol

[0453] MMAF was replaced with D8, and the remaining steps were the same. After several steps of reaction and use, compound B33 (23 mg, yield 28%) was prepared as a white, fluffy solid. LC-MS (ESI) m / z C 72 H 107 N 12 O 17 S[M+H] + Calculated value: 1443.76, measured value: 1443.57.

[0454] Synthesis of Compound B34 in Examples 3-35

[0455] The synthesis of compound B34 was the same as that of compound B33, except that D8 was replaced with D11 to give compound B34 (42 mg, 44% yield), a white solid. LC-MS (ESI) m / z C 75 H 109 N 12 O 16 [M+H] + Calculated value: 1442.76, measured value: 1442.85.

[0456] Synthesis of Compound B35 in Examples 3-36

[0457] The synthesis of compound B35 was the same as that of compound B33, except that D8 was replaced with D4 to obtain compound B35 (30 mg, yield 34%), which was a white, thin, flaky solid. LC-MS (ESI) m / z C 70 H 107 N 10 O 18 [M+H]+ Calculated value: 1375.77, measured value: 1375.84.

[0458] Synthesis of Compound B36 in Examples 3-37

[0459] The synthesis of compound B36 was the same as that of compound B33, except that D8 was replaced with D1 to obtain compound B36 (26 mg, yield 36%), which was a white, foamy solid. LC-MS (ESI) m / z C 75 H 109 N 12 O 16 [M+H] + Calculated value: 1433.81, measured value: 1433.99.

[0460] Synthesis of Compound B37 in Examples 3-38

[0461] The synthesis of compound B37 was the same as that of compound B3, except that MMAF was replaced with D8, yielding compound B37 (80 mg, 42% yield), a pale yellow solid. LC-MS (ESI) m / z C 75 H 109 N 12 O 16 [M+H] + Calculated value: 1586.85, measured value: 1586.64.

[0462] Synthesis of Compound B38 in Examples 3-39

[0463] The synthesis of compound B38 was the same as that of compound B3, except that the intramolecular condensation in step 8 was replaced by a condensation with polyethylene glycol-acetic acid. The remaining steps were identical. After several steps of reaction and use, compound B38 (28 mg, two-step yield 44%) was prepared as a white, fluffy solid. LC-MS (ESI) m / z C 82 H 126 N 13 O 22 S[M+H] + Calculated value: 1676.88, measured value: 1676.98.

[0464] Synthesis of Compound B39 in Examples 3-40

[0465] Synthesis route:

[0466] The synthesis of compound B39 is the same as that of compound B3, except that step 2 in T1 is modified accordingly. Replace with 4-aminobenzyl alcohol

[0467] By replacing MMAF with D26, compound B39 was prepared through several reaction steps and usage. LC-MS (ESI) m / z C 72 H 107 N 12 O17S[M+H] + Calculated value: 1391.81, measured value: 1392.81.

[0468] Preparation of the antibody-drug conjugate shown in Formula (A) in Example 4

[0469] The antibody or antigen-binding fragment, such as the HER2-targeting Ttastuzumab stock solution, was diluted to 2 mg / mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethyltriaminepentaacetic acid (DTPA) in pH 7.0 buffer. An excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added at a 3.0 molar ratio. The reaction solution was stirred at 37°C for 2 hours.

[0470] The above reaction solution was cooled to 4°C, and an appropriate amount of unpurified dimethylacetamide (DMA) was added. Then, 6-12 times the excess molar ratio of the reference drug molecule or drug linker conjugate B1-B39 (5 ​​mg / ml of DMA dissolved in it) was added to ensure that the volume of DMA in the reaction system did not exceed 20%. The reaction was stirred at room temperature for 2 hours to carry out coupling.

[0471] The coupling reaction mixture was purified by filtration through a desalting column using an acetic acid-histidine solution at pH 6.0. The sample was collected based on the UV 280 absorption peak. It was then sterilized by filtration through a 0.22 μm filter and stored at -20°C.

[0472] Preparation of MMAF-controlled ADC

[0473] Following the method described above, a 10-fold excess of MC-Val-Ala-PAB-MMAF was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding MMAF control ADC. The drug loading (DAR) was analyzed by hydrophobic interaction liquid chromatography (HIC-HPLC), and the result was approximately 4 (see Figure 1). The polymer content was analyzed by size exclusion chromatography (SEC-HPLC), and the result was shown in Figure 2 (see Figure 2). The polymer ratio was 0.6%.

[0474] Preparation of A2

[0475] Following the method described above, a 10-fold excess of compound (B2) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A2. The drug loading (DAR) was analyzed by hydrophobic interaction liquid chromatography (HIC-HPLC), and the result was approximately 4, as shown in Figure 3. The polymer content was analyzed by size exclusion chromatography (SEC-HPLC), and the result was shown in Figure 4, indicating that the polymer ratio was 0.8%.

[0476] Preparation of A3

[0477] Following the method described above, a 10-fold excess of compound (B3) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A3. The drug loading (DAR) was analyzed by hydrophobic interaction liquid chromatography (HIC-HPLC), and the result was approximately 4, as shown in Figure 5. The polymer content was analyzed by size exclusion chromatography (SEC-HPLC), and the result was shown in Figure 6, indicating that the polymer ratio was 0.8%.

[0478] Preparation of A16

[0479] Following the method described above, a 10-fold excess of the compound (B16) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A16. The drug loading (DAR) was approximately 4, as shown in Figure 7, by hydrophobic interaction chromatography (HIC-HPLC). The polymer content was analyzed by size exclusion chromatography (SEC-HPLC), as shown in Figure 8, with a polymer ratio of 0.7%.

[0480] Preparation of A33

[0481] Following the method described above, a 10-fold excess of the compound (B33) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A33. The drug loading (DAR) was approximately 4% by hydrophobic interaction chromatography (HIC-HPLC).

[0482] Preparation of A34

[0483] Following the method described above, a 10-fold excess of the compound (B34) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A34. The drug loading (DAR) was approximately 4% by hydrophobic interaction chromatography (HIC-HPLC).

[0484] Preparation of A35

[0485] Following the method described above, a 10-fold excess of the compound (B35) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A35. The drug loading (DAR) was approximately 4% by hydrophobic interaction chromatography (HIC-HPLC).

[0486] Preparation of A36

[0487] Following the method described above, a 10-fold excess of the compound (B36) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A36. The drug loading (DAR) was approximately 4% by hydrophobic interaction chromatography (HIC-HPLC).

[0488] Preparation of A38

[0489] Following the method described above, a 10-fold excess of the compound (B38) prepared in Example 3 of this invention was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding A38. The drug loading (DAR) was approximately 4% by hydrophobic interaction chromatography (HIC-HPLC).

[0490] Preparation of MMAE-controlled ADC

[0491] Following the method described above, a 10-fold excess of MC-Val-Ala-PAB-MMAE was conjugated with the reduced Trastuzumab monoclonal antibody to obtain the corresponding MMAE control ADC. The drug loading (DAR) was analyzed by hydrophobic interaction liquid chromatography (HIC-HPLC) and found to be approximately 4%.

[0492] Example 5: In vitro antitumor activity test of some olistatin derivatives shown in formulas (DA), (DB), (T), and (B)

[0493] The cell lines used in the cytotoxicity assays included MDA-MB-231—human breast cancer cells, A549—human lung adenocarcinoma cells, MCF-7—human breast cancer cells, HCT-116—human gastric cancer cells, and SK-OV-3—human ovarian cancer cells. MDA-MB-231, MCF-7, and HCT-116 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin. SK-OV-3 cells were cultured in McCoy5A containing 10% FBS and 1% penicillin-streptomycin. A549 cells were cultured in Ham's F-12K containing 10% FBS and 1% penicillin-streptomycin. Cultured cells were digested with trypsin to achieve a cell count of 6000 cells / well and transplanted into 96-well plates. The plates were incubated at 37°C and 5% CO2 for 24 hours. Then, a 10 mM stock solution of the test compound was diluted with culture medium to the appropriate concentration and administered in triplicate wells. Control wells contained cells and culture medium but no test compound. After incubation at 37°C and 5% CO2 for 72 hours, the culture medium was aspirated, and 10% CCK8 medium was added. After incubation at 37°C for 1 hour, the absorbance at 450 nm was measured using a microplate reader. The inhibition rate and IC50 were calculated using the following formula. 50value:

[0494] Inhibition rate % = [1 - (analytical value - blank control value) / (control value - blank control value)] × 100%

[0495] Table 1. Results of in vitro antitumor activity assays for monomethyloripatine derivatives

[0496] Example 6: Inhibitory activity test of the sea hare toxin derivatives shown in formulas (DA) and (DB) against microtubule polymerization.

[0497] Prepare a 2 mM stock solution of the test compound in DMSO, and dilute it with ultrapure water to the appropriate concentration. Then, preheat the half-region 96-well plate at 37°C for 10 min in a microplate reader. Simultaneously, thaw Buffer 1, GTP stock, Tubulin Glycerol Buffer, and Tubulin stock (10 mg / mL) from the microtubule polymerization kit and place them on ice. Mix 205 μL of Buffer 1, 150 μL of Tubulin Glycerol Buffer, 4.4 μL of GTP stock, and 85 μL of Tubulin stock to prepare the microtubule polymerization reaction solution, and place it on ice. Add 5 μL of the test drug to the preheated half-region 96-well plate and continue preheating for 1 min. Then add 50 μL of the microtubule polymerization reaction solution to each well and immediately detect the fluorescence value at the excitation wavelength of 360 nm and the emission wavelength of 460 nm, detecting once per minute for 1 h; calculate the corresponding inhibition rate based on the reaction curve. The experimental results are shown in Figures 21-22. MMAE IC50 50 The IC50 of the sea haretoxin derivative D8 was 0.45 μM. 50 The value is 1.2 μM.

[0498] Example 7: In vitro cytotoxic activity test of the antibody-drug conjugate shown in formula (A)

[0499] HCC1954 human breast cancer cells and SKOV3 human ovarian cancer cells were selected as cell lines for in vitro activity assay in this experiment. The cell-killing effects of the MMAF control ADC, A2, A33, A34, A35, A36, and A38 prepared in Example 4 of this invention were observed. After adding the A2 and MMAF control ADC prepared in Example 4 of this invention, a starting concentration of 300 nM was set, and nine series of concentrations from 300 to 0.1 nM were designed. Cell-killing changes were observed after 120 hours, and the IC50 was calculated after reading the fluorescence data. 50 As shown in Table 2.

[0500] Table 2. Results of in vitro antitumor activity tests of the above antibody conjugates

[0501] As shown in Figure 9, HCC1954 cells with high HER2 expression were treated with A2 and MMAF control ADC prepared in Example 4 of the present invention. A2 prepared in Example 4 of the present invention can significantly inhibit tumor cell proliferation.

[0502] As shown in Figure 10, the A2 and MMAF control ADC prepared in Example 4 of this invention were used to treat SKOV3 cells with high HER2 expression. The antibody-drug conjugate 2 prepared in Example 2 of this invention can significantly inhibit tumor cell proliferation.

[0503] As shown in Figure 11, when HCT116 cells with low HER2 expression were treated with the A2 and MMAF control ADC prepared in Example 4 of the present invention, the MMAF control ADC had an inhibitory effect on the low-expression cells, indicating that its targeting was poor; while the A2 prepared in Example 4 of the present invention did not inhibit the low-expression cells, indicating that its targeting was good.

[0504] Example 8: In vitro safety testing of the antibody-drug conjugate shown in formula (A)

[0505] Human normal lung fibrosis cells MRC5, human normal colonic epithelial cells NCM460, and human umbilical vein endothelial cells HUVEC were selected as cell lines for this in vitro activity experiment. The killing effect of the MMAF control ADCs A2, A33, A34, A35, A36, and A38 prepared in Example 4 of this invention on normal human cells was observed. After adding the A2 and MMAF control ADCs prepared in Example 4 of this invention, a starting concentration of 300 nM was set. Nine series concentrations were designed within the range of 300–0.1 nM. The killing changes were observed after 120 hours, and the IC50 was calculated after reading the fluorescence data. 50 .

[0506] The experimental results are shown in Table 3 and Figures 12-14. The results show that when human normal lung fibrotic cells MRC5 were treated with the A2 of this invention and the MMAF control ADC, the A2 of this invention had no inhibitory effect on normal cells, indicating that its off-target toxicity was significantly less than that of the MMAF control ADC.

[0507] The results showed that when human normal colonic epithelial cells NCM460 were treated with the A2 of the present invention and the MMAF control ADC, the A2 of the present invention had no inhibitory effect on normal cells, indicating that its off-target toxicity was significantly less than that of the MMAF control ADC.

[0508] The results showed that when human umbilical vein endothelial cells (HUVECs) were treated with the A2 of the present invention and the MMAF control ADC, the A2 of the present invention had no inhibitory effect on normal cells, indicating that its off-target toxicity was significantly less than that of the MMAF control ADC.

[0509] Table 3 shows the toxicity of the above antibody-drug conjugates to normal human cells.

[0510] In vivo acute toxicity test of the antibody-drug conjugate shown in Formula (A) in Example 9

[0511] Mice were administered 50 mg / kg, 100 mg / kg of the A2 drug of this invention, and 50 mg / kg of the MMAF control ADC via tail vein injection, with PBS serving as a blank control. Mouse body weight changes (Figure 15) showed that after administration of 50 mg / kg and 100 mg / kg of the A2 drug of this invention, mouse body weight did not decrease significantly; however, after administration of the 50 mg / kg MMAF control ADC, mouse body weight decreased, indicating that the A2 drug of this invention has good tolerability and superior safety compared to the control drug.

[0512] The complete blood count of mice is shown in Figure 16. Compared with the control group, there was no significant change in platelet count after administration of 50 mg / kg and 100 mg / kg of the A2 of this invention; however, after administration of 50 mg / kg of the MMAF control ADC, the platelet count of mice decreased significantly, indicating that the control group had strong platelet toxicity, while the A2 of this invention had no platelet toxicity. These results show that the A2 of this invention has excellent in vivo safety and a significantly improved therapeutic window.

[0513] Rat toxicology experiment of the antibody-drug conjugate shown in Formula (A) of Example 10

[0514] Rats were administered 10 mg / kg of the present invention's A2, 60 mg / kg of the present invention's A2, and 10 mg / kg of the MMAF control ADC via tail vein injection, with PBS serving as a blank control. The changes in rat body weight (Figure 17) showed that after administration of 10 mg / kg and 60 mg / kg of the present invention's A2, rats experienced normal weight gain, indicating good tolerability; while after administration of the 10 mg / kg MMAF control ADC, rats experienced slow weight gain, indicating poor tolerability. These results demonstrate that the present invention's A2 has good tolerability, and its safety and therapeutic window are superior to the control drug.

[0515] Plasma stability test of the antibody-drug conjugate shown in Formula (A) in Example 11

[0516] The stability of A2 prepared in plasma according to Example 4 of this invention was determined by plasma stability experiments. A2 and the MMAF control ADC prepared in Example 4 of this invention were incubated with plasma from humans, monkeys, rats, and mice, respectively. Load release was measured on days 1, 2, 3, 4, 7, 14, and 21 of incubation. The experimental results (Figure 18) showed that the MMAF control ADC exhibited significant fragmentation in rat and mouse plasma, indicating poor plasma stability; while A2 of this invention showed no significant fragmentation in plasma from any of the different species, indicating significantly improved plasma stability and lower potential off-target toxicity.

[0517] In vivo antitumor efficacy test of the antibody-drug conjugate shown in Formula (A) in Example 12

[0518] The efficacy of the drug combination of the present invention can be determined by in vivo pharmacodynamic experiments, namely by implanting allogeneic or xenogeneic cancer cells into rodents and treating the animals with the combination. Test mice are treated with the drugs or controls, and monitored for several weeks or longer to measure the time to tumor doubling and the tumor inhibition rate.

[0519] HER2-overexpressing HCC1954 human breast cancer cells (ATCC) were suspended in PBS solution and subcutaneously seeded into the right side of female nude mice at a ratio of 4 x 10⁴ cells / day. 7 Tumor cells were randomly assigned to groups on day 6. The grouping day was designated as day 0. On day 0, MMAF control ADC, the present invention A2, the present invention A3, and trastuzumab were injected intravenously via the tail vein at a dose of 6 mg / kg. PBS served as the blank control group.

[0520] The results are shown in Figure 19. For HCC1954 tumors with high HER2 expression, administration of 6 mg / kg of MMAF control ADC, the A2 and A3 of this invention all significantly inhibited tumor growth, with the efficacy of A2 being comparable to that of A3.

[0521] In vivo antitumor efficacy test of the antibody-drug conjugate shown in Formula (A) in Example 13

[0522] HER2-overexpressing SKOV3 human breast cancer cells (ATCC) were suspended in PBS solution and subcutaneously seeded into the right side of female nude mice at a dose of 4 x 10⁴ cells / day. 7 Tumor cells were randomly assigned to groups on day six. The grouping day was designated as day 0. On day 0, the A2 of this invention, the MMAF control ADC, and trastuzumab were injected intravenously via the tail vein at a dose of 6 mg / kg. PBS served as the blank control group.

[0523] The results are shown in Figure 20. For SKOV3 tumors with high HER2 expression, both 6 mg / kg of the MMAF control ADC and the A2 of the present invention significantly inhibited tumor growth. Among them, the anti-tumor effect of the A2 of the present invention was significantly better than that of the MMAF control ADC, with tumor inhibition rates of 86.6% and 49.7%, respectively.

[0524] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A compound, characterized in that, The compound is a sea haretoxin derivative or any form of cyclic derivative thereof, selected from the structure shown in formula (DA) or formula (DB): Among them, R a Choose from one of the following groups: optional substitution Optional replacement Optional replacement Among them, R 4 Selected from any one of the following groups: hydrogen, optionally substituted alkyl groups of C1-C8 and optionally substituted alkoxy groups of C1-C8, wherein each of the substituted alkyl group and the substituted alkoxy group is independently and optionally substituted by one or more substituents selected from deuterium, halogen, hydroxyl, carbonyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; wherein R 5 Selected from any one of the following groups: hydrogen, optional substituted alkyl group of C1-C8, optional substituted alicyclic group of C1-C8, optional substituted aliheterocyclic group of C1-C8, optional substituted aryl group of C1-C8, optional substituted heteroaryl group of C1-C8, or R. 5 The nitrogen atom bonded to it forms an alicyclic structure, wherein the heteroaryl group and the heteroatom in the alicyclic structure are selected from one or more of O, S, and N; wherein R 6 Selected from any one of the following groups: C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alicyclic, C1-C8 optionally substituted aliheterocyclic, C1-C8 optionally substituted aryl, C1-C8 optionally substituted heteroaryl, or R 6 The carbon atoms bonded to it form an alicyclic / heterocyclic structure, wherein the heteroatom of the alicyclic or heteroaryl group is selected from one or more of O, S, and N; R b Optionally substituted with one or more substituents selected from deuterium, carbonyl, halogen, hydroxyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; R c The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3- 15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms; the aryl group is a C6-C10 aryl group, wherein the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms; the substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; the R d The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, and the heterocyclic aryl group is a C5-C6 aryl group containing oxygen, a C5-C6 aryl group containing nitrogen, or a C5-C6 aryl group containing sulfur. The substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoyl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; wherein, R... e The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-1 5. An alkyl group of amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, I, or a PEG-containing acyl group when halogenated. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms, and the substituent of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group is a C3-C15 alkyl group, a C3-C15 alkenyl group, a C3-C15 alkoxy group, a nitro group, a diethylamino group, a cyano group, a haloalkyl group, a morpholinyl group, a piperazine group, or a halogenated substituted aryl group; n is 0 or 1.

2. A compound, characterized in that, The compound comprises the structure shown in formula (T): LC(T); Wherein, L is an optional substituted linking group; Wherein, C is a bioactive molecule or a precursor structure containing a bioactive molecule.

3. A compound, characterized in that, The compound comprises the structure shown in formula (B): QLC (B); Where Q represents the connector unit; Wherein, L is an optional substituted linking group; Wherein, C is a bioactive molecule or a precursor structure containing a bioactive molecule.

4. A compound, characterized in that, The compound comprises the structure shown in formula (A): Where Q represents the connector unit; Wherein, L is an optional substituted linking group; Wherein, C is a bioactive molecule or a precursor structure containing a bioactive molecule; Where P is the ligand and m is any integer or decimal between 1 and 20.

5. The compound according to claim 4, characterized in that, The P includes any one of small molecule ligands, protein ligands, polypeptide ligands, carbohydrate ligands, nucleic acid ligands, antibodies, or antigen-binding fragments.

6. The compound according to claim 5, characterized in that, The antibody is selected from one of the following groups: murine antibodies, chimeric antibodies, humanized antibodies, and fully humanized antibodies; and / or, The antibodies include bispecific antibodies, multispecific antibodies; and / or, The antibody comprises the heavy chains HCDR1, HCDR2, and HCDR3 and the light chains LCDR1, LCDR2, and LCDR3; and / or, The antibody comprises a heavy chain variable region VH and a light chain variable region VL.

7. The compound according to claim 5, characterized in that, The antibody or antigen-binding fragment comprises any one of sacituzumab, trastuzumab, pertuzumab, enfortumab, and paltritumab; and / or, the antigen-binding fragment comprises any one of the antigen-binding fragments of the aforementioned monoclonal antibodies.

8. The compound according to claim 5, characterized in that, The antigen-binding fragment is selected from one of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, ScFv, di-scFv, VHH, and dAb.

9. The compound according to claim 5, characterized in that, The antibody or antigen-binding fragment is selected from one of the following groups: anti-HER2 antibody, anti-HER3 antibody, anti-TROP2 antibody, anti-B7H3 antibody, anti-CDH6 antibody, anti-EGFR antibody, anti-Nectin-4 antibody, anti-TIM1 antibody, anti-PSMA antibody, anti-EpCAM antibody, anti-MUC1 antibody, anti-FGF2 antibody, anti-c-MET antibody, anti-GFR antibody, anti-EphA2 antibody, anti-ROR1 antibody, anti-PD-L1 antibody, anti-5T4 antibody, anti-NaPi2b antibody, anti-STEAP antibody, anti-BCMA antibody, anti-CEACAM5 antibody, anti-SC-16 antibody, anti-Delt-like antibody. Protein3 antibody, anti-Claudin18.2 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD45 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD74 antibody, anti-CD79b antibody, anti-CD138 antibody, anti-CD147 antibody, anti-CD166 antibody, anti-CD223 antibody, anti-MUC16 antibody, anti-ASCT2 antibody, anti-CD324 antibody, anti-CD352 antibody, anti-CD48a antibody, anti-CS1 antibody, anti-FGFR2 antibody, anti-FGFR3 antibody, anti-ETBR antibody, anti-FGFR2 antibody, anti-FLT3 antibody, anti-LAMP-1 antibody, anti-Ly6-E antibody, anti-NOTCH3 antibody, anti-PRLR antibody, anti-RNF43 antibody, and / or, the antigen-binding fragment is selected from the antigen-binding fragments corresponding to the above targets.

10. The compound according to claim 5, characterized in that, The small molecule ligand or polypeptide ligand comprises one of the following: folic acid derivative, glutamate urea derivative, dermalin analog, growth hormone inhibitor analog, RGD peptide, anthocyanin dye, or IR783.

11. The compound according to claim 4, characterized in that, The m is determined by one or more methods selected from the group consisting of hydrophobic chromatography, sodium dodecyl sulfonate polyacrylamide gel electrophoresis, or liquid chromatography-mass spectrometry.

12. The compound according to claim 3 or 4, characterized in that, The Q is selected from any one of the following groups: optionally substituted azide, substituted alkynyl, substituted chloroacetyl, disulfide-substituted derivative, substituted hydrazine, substituted aldehyde, substituted ketone, substituted amino, substituted carboxylic acid, substituted alkoxyamino or N-hydroxysuccinimide ester, optionally substituted Optional replacement Optional replacement And / or, the L is L1-C(=O)-L2-C(=O)-; And / or, the C is selected from formula (DA), formula (DB), or optionally substituted. Or optional replacement Wherein, -X- is selected from any of the following groups: optional substitution Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement 13. The compound according to claim 12, characterized in that, The L1 is selected from any one of the following groups: optionally substituted alkylene, optionally substituted alkenyl, optionally substituted polyethylene glycol, optionally substituted alicyclic, optionally substituted alicyclic heterocyclic, optionally substituted aryl, optionally substituted heterocyclic; or the L1 is selected from any one of the following groups: optionally substituted methylene, optionally substituted propylene, optionally substituted pentylene, optionally substituted cyclopropylene, optionally substituted cyclobutylene, optionally substituted cyclohexylene, optionally substituted diethylene glycol, optionally substituted triethylene glycol, optionally substituted tetraethylene glycol, optionally substituted pentaethylene glycol, optionally substituted hexaethylene glycol, optionally substituted heptaethylene glycol, optionally substituted octaethylene glycol; and / or, the L2 is any one of the following: optionally substituted natural amino acids, non-natural amino acids, di- to trioctapeptides condensed from natural or non-natural amino acids, C2-C15 alkylcarboxylic acids, C2-C15 oxacarboxylic acids, C2-C15 azacarboxylic acids.

14. The compound according to claim 13, characterized in that, When L2 contains a lysine residue, the lysine residue is optionally substituted by a structure R2 containing a polyethylene glycol residue; R2 is an optional substitution. Where n1 is an integer from 1 to 30, R 3 It is one of the following groups: hydroxyl, optionally substituted amino, tert-butoxycarbonyl-protected amino, optionally substituted alkoxy, optionally substituted alkylamine, optionally substituted cycloalkylamine.

15. The compound according to claim 12, characterized in that, The X1 is selected from one of the following groups: carbonyl, C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alkoxy, straight-chain heteroalkyl containing 1-8 carbon atoms, C1-C8 optionally substituted alicyclic group, C1-C8 optionally substituted alicyclic heteroalkyl group, C1-C8 optionally substituted aryl group, C1-C8 optionally substituted heterocyclic group, wherein the heteroatom of the heteroalkyl, alicyclic, or heteroaryl group is selected from one or more of N, O, and S; and / or, X2 is selected from any one of the following groups: hydrogen, optionally substituted alkyl groups of C1-C8, optionally substituted alkenyl groups of C1-C8, optionally substituted polyethylene glycol groups of C1-C30, optionally substituted alicyclic groups of C1-C8, optionally substituted aliheterocyclic groups of C1-C8, optionally substituted aryl groups of C1-C8, optionally substituted heterocyclic groups of C1-C8, wherein the heteroatoms of the optionally substituted alicyclic groups of C1-C8 are selected from one or more of N, O, and S, wherein the substituted alkyl, substituted alkenyl, substituted polyethylene glycol, substituted alicyclic, substituted aliheterocyclic, substituted aryl, and substituted heteroaryl groups may be independently and optionally substituted by one or more substituents selected from hydrogen, deuterium, halogen, haloalkyl, alkoxy, hydroxyl, amino, alkylamino, carboxyl, sulfonic acid, cyano, nitro, aryl, heteroaryl, cycloalkyl, or heterocyclic groups; and / or, X3 is selected from any one of the following groups: hydrogen, optional substituted alkyl groups of C1-C8, optional substituted polyethylene glycol groups of C1-C30, and optional substituted linear heteroalkyl groups of C1-C8, wherein the heteroalkyl heteroatom is selected from one or more of N, O, and S, and wherein the substituted alkyl group, substituted polyethylene glycol group, and substituted linear heteroalkyl group can be independently substituted by one or more substituents selected from hydrogen atom, deuterium atom, carbonyl group, halogen, haloalkyl group, alkoxy group, hydroxyl group, amino group, alkylamino group, carboxyl group, sulfonic acid group, cyano group, nitro group, aryl group, heteroaryl group, cycloalkyl group, or heterocyclic group; Or X3 is one of the following groups: optional substitution Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Optional replacement Where n is an integer from 1 to 30; and / or, R 1 Selected from one of the following groups: hydrogen, C1-C8 alkyl, optional substituted straight-chain heteroalkyl containing 1-8 carbon atoms, wherein the heteroatom in the heteroalkyl is selected from one or more of N, O, and S; And / or, C is a bioactive molecule comprising linear olistatin derivatives and cyclic olistatin derivatives.

16. The compound according to claim 12, characterized in that, The structures shown in equations (DA) and (DB) are as follows: Among them, R a Choose from one of the following groups: optional substitution Optional replacement Optional replacement Among them, R 4 Selected from any one of the following groups: hydrogen, optionally substituted alkyl groups of C1-C8 and optionally substituted alkoxy groups of C1-C8, wherein each of the substituted alkyl group and the substituted alkoxy group is independently and optionally substituted by one or more substituents selected from deuterium, halogen, hydroxyl, carbonyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; wherein R 5 Selected from any one of the following groups: hydrogen, optional substituted alkyl group of C1-C8, optional substituted alicyclic group of C1-C8, optional substituted aliheterocyclic group of C1-C8, optional substituted aryl group of C1-C8, optional substituted heteroaryl group of C1-C8, or R. 5 The nitrogen atom bonded to it forms an alicyclic structure, wherein the heteroaryl group and the heteroatom in the alicyclic structure are selected from one or more of O, S, and N; wherein R 6 Selected from any one of the following groups: C1-C8 optionally substituted alkyl, C1-C8 optionally substituted alicyclic, C1-C8 optionally substituted aliheterocyclic, C1-C8 optionally substituted aryl, C1-C8 optionally substituted heteroaryl, or R 6 The carbon atoms bonded to it form an alicyclic / heterocyclic structure, wherein the heteroatom of the alicyclic or heteroaryl group is selected from one or more of O, S, and N; R b Optionally substituted with one or more substituents selected from deuterium, carbonyl, halogen, hydroxyl, cyano, nitro, amino, alkyl, carboxyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; R c The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3- 15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms; the aryl group is a C6-C10 aryl group, wherein the heterocyclic aryl group is a C5-C6 aryl group containing oxygen atoms, a C5-C6 aryl group containing nitrogen atoms, or a C5-C6 aryl group containing sulfur atoms; the substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; the R d The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing nitrogen, oxygen, and sulfur atoms, the aryl group is a C6-C10 aryl group, and the heterocyclic aryl group is a C5-C6 aryl group containing oxygen, a C5-C6 aryl group containing nitrogen, or a C5-C6 aryl group containing sulfur. The substituents of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoyl group are C3-C15 alkyl, C3-C15 alkenyl, C3-C15 alkoxy, nitro, diethylamino, cyano, haloalkyl, morpholinyl, piperazine, or halogenated aryl group; wherein, R... e The alkyl group is selected from hydrogen, deuterium, alkyl, substituted alkyl, acyl, substituted acyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, aryl, substituted aryl, arylbenzyl, substituted arylbenzyl, heterocyclic benzyl, substituted heterocyclic benzyl, heterocyclic benzoaryl, and substituted heterocyclic benzoaryl; wherein the alkyl group is a C3-C15 alkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing alkyl group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-1 5. An alkyl group of amine, wherein the acyl group is a C3-C15 acyl group, and the substituted acyl group is selected from any one of F, Cl, Br, I, or a PEG-containing acyl group when halogenated. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The acyl group of the amine, wherein the cycloalkyl group is a C3-C15 cycloalkyl group, and the substituted alkyl group is selected from any one of F, Cl, Br, and I when halogenated, or is a PEG-containing group. 3-15 alcohols, PEG 3-15 PEG with amine and hydroxyl protected ends 3-15 PEG with alcohol and amino protected ends 3-15 The alkyl group of the amine, wherein the heterocyclic alkyl group is a C3-C15 cycloalkyl group containing a nitrogen atom, an oxygen atom, and a sulfur atom; the aryl group is a C6-C10 aryl group; the heterocyclic aryl group is a C5-C6 aryl group containing an oxygen atom, a C5-C6 aryl group containing a nitrogen atom, or a C5-C6 aryl group containing a sulfur atom; the substituent of the substituted aryl group, substituted aryl benzyl group, substituted heterocyclic benzyl group, or substituted heterocyclic benzoaryl group is a C3-C15 alkyl group, a C3-C15 alkenyl group, a C3-C15 alkoxy group, a nitro group, a diethylamino group, a cyano group, a haloalkyl group, a morpholinyl group, a piperazine group, or a halogenated substituted aryl group; and / or, n is 0 or 1.

17. The compound according to claim 1 or 16, characterized in that, The compound represented by formula (DA) or formula (DB) is selected from any one of the following structures:

18. The compound according to claim 2, characterized in that, The compound represented by formula (T) is selected from any one of the following structures:

19. The compound according to claim 3, characterized in that, The compound represented by formula (B) is selected from any one of the following structures: Where n2 is any integer between 1 and 20.

20. The compound according to claim 4, characterized in that, The compound shown in formula (A) contains any one of the following structures: Wherein, Ab is an antibody or antigen-binding fragment, and -S- is a group inherent to Ab; where m is any number between 1 and 20; where n2 is any integer between 1 and 20.

21. A compound, characterized in that, The compound comprises the compound according to any one of claims 1-4, or a tautomer, meso compound, racemic compound, enantiomer, diastereomer, or pharmaceutically acceptable salt, prodrug, or solvate thereof.

22. A method for preparing the compound according to claim 4, characterized in that, The preparation method is as follows: a pair of cysteine ​​residues are generated by reducing the disulfide bond of the ligand, and the thiol group in the cysteine ​​residues undergoes a substitution reaction with the linker unit in the compound of formula (B) of the present invention, thereby connecting the compound of formula (B) of the present invention to the thiol group of the ligand to obtain the conjugate of formula (A); The preparation method specifically includes the following steps: Step 1: The ligand undergoes a reduction reaction with a reducing agent to obtain the reduced ligand; Step 2: The reduced ligand obtained in Step 1 reacts with the compound shown in Formula (B) in an organic solvent or buffer solution to obtain the conjugate shown in Formula (A).

23. The preparation method according to claim 22, characterized in that, In step 1, the disulfide bond of the ligand is reduced by the reducing agent to form a cysteine ​​residue; and / or, the reducing agent is any one of tri-(2-carboxyethyl)phosphonic acid hydrochloride, β-mercaptoethanol, and dithiothreitol; and / or, the molar ratio of the ligand to the reducing agent is 1:1 to 1:10; and / or, the ligand and the reducing agent react at 10 to 37°C; and / or, the reaction time of the ligand and the reducing agent is 1 to 24 hours; and / or, In step 2, the thiol group of the cysteine ​​residue in the reduced ligand from step 1 reacts with the linker unit in the compound shown in formula (B) to generate the conjugate shown in formula (B); and / or, the reduced ligand reacts with the compound shown in formula (B) at 0–37°C; and / or, the reaction time of the reduced ligand with the compound shown in formula (B) is 1–24 hours; and / or, the organic solvent is selected from any one or more of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitrile compounds, and alcohols; and / or, the buffer solution is selected from... Any one or more of the following groups: potassium dihydrogen phosphate-sodium hydroxide / sodium chloride-diethyltriaminepentaacetic acid buffer, disodium hydrogen phosphate-citric acid / sodium chloride-diethyltriaminepentaacetic acid buffer, histidine-sodium hydroxide / sodium chloride / diethyltriaminepentaacetic acid buffer, phosphate buffer solution / diethyltriaminepentaacetic acid buffer; and / or, the molar ratio of the reduced ligand to the compound shown in formula (B) is 1:1 to 1:20; and / or, the ligand includes any one of small molecule ligands, protein ligands, polypeptide ligands, carbohydrate ligands, nucleic acid ligands, antibody or antigen-binding fragments; and / or, The preparation method further includes a step of purifying the coupling product; wherein the purification step includes one or more of ion exchange chromatography, hydrophobic chromatography, reverse chromatography, or affinity chromatography.

24. A method for preparing the compound according to claim 3, characterized in that, The preparation method includes the following steps: Step 1: Contact the amino acid active ester of N1 with an amino protecting group with an amino acid to obtain intermediate M1; Step 2: In the presence of a condensing agent, the intermediate M1 obtained in the first step is contacted with p-aminobenzyl alcohol or p-aminobenzyl alcohol containing a tert-butoxycarbonyl protecting group to obtain intermediate M2. Step 3: Remove N1 from intermediate M2 obtained in step 2 to obtain intermediate M3; Step 4: The intermediate M3 obtained in step 3 is contacted with a compound containing a maleimide group to obtain intermediate M4; Step 5: The intermediate M4 obtained in step 4 is contacted with bis(4-nitrophenyl) carbonate to obtain intermediate M5; Step 6: The intermediate M5 obtained in step 5 is contacted with the bioactive molecule C to obtain intermediate M6 or the compound shown in formula (B); Step 7: Remove the tert-butyloxycarbonyl protecting group from intermediate M6 obtained in step 6 to obtain intermediate M7; Step 8: In the presence of a condensing agent, the intermediate M7 obtained in step 7 undergoes intramolecular condensation or condensation with the corresponding carboxylic acid to obtain the compound shown in formula (B).

25. The preparation method according to claim 24, characterized in that, In the first step, The molar ratio of the amino acid active ester with amino protecting group N1 to the amino acid is 1:1 to 1:1.2; The reaction temperature between the amino acid active ester of N1 with the amino protecting group and the amino acid is 0-37℃. The reaction time between the amino acid active ester of N1 with the amino protecting group and the amino acid is 0 to 24 hours. And / or, in the second step, The condensing agent is any one or more of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU). The molar ratio of intermediate M1 to p-aminobenzyl alcohol or p-aminobenzyl alcohol containing a tert-butyloxycarbonyl protecting group is 1:1 to 1:1.

5. The reaction temperature of intermediate M1 with p-aminobenzyl alcohol containing tert-butoxycarbonyl protecting group or p-aminobenzyl alcohol is 0-37°C. The reaction time between intermediate M1 and p-aminobenzyl alcohol containing a tert-butyloxycarbonyl protecting group is 0 to 24 hours. And / or, in the third step, The N1 in the intermediate M2 is removed at 0–37°C; The N1 in the intermediate M2 is removed in 0 to 4 hours; And / or, in the fourth step, The molar ratio of intermediate M3 to the compound containing maleimide groups is 1:1 to 1:1.5; The reaction temperature of the intermediate M3 with the compound containing the maleimide group is 0–37°C. The reaction time between the intermediate M3 and the compound containing the maleimide group is 0 to 24 hours; And / or, in the fifth step, The molar ratio of intermediate M4 to bis(4-nitrophenyl) carbonate is 1:1 to 1:1.5; The reaction temperature between the intermediate M4 and the bis(4-nitrophenyl) carbonate is 0–37°C. The reaction time between intermediate M4 and the bis(4-nitrophenyl) carbonate is 0 to 24 hours; And / or, in the sixth step, The molar ratio of intermediate M5 to bioactive molecule D is 1:1 to 1:1.5; The reaction temperature between the intermediate M5 and the bioactive molecule D is 0–37°C. The reaction time between the intermediate M5 and the bioactive molecule D is 0–24 hours; And / or, in the seventh step, The tert-butyloxycarbonyl protecting group of intermediate M6 is removed at 0–37°C; The tert-butyloxycarbonyl protecting group of intermediate M6 is removed in 0 to 4 hours; And / or, in the eighth step, The condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU). The reaction temperature at which the intermediate M7 undergoes intramolecular condensation or amide condensation is 0–37°C. The reaction time for the intermediate M7 to undergo intramolecular condensation or amide condensation is 0 to 4 hours.

26. A drug / drug composition, characterized in that, The drug / group of drugs comprises the compound according to any one of claims 1-4.

27. The pharmaceutical / pharmaceutical composition according to claim 26, characterized in that, The drug / drug composition may be used alone and / or in combination with one or more of the following therapeutic agents: chemotherapy, radiotherapy, immunotherapy, anti-autoimmune disease drugs, anti-infective drugs, or other antibody-drug conjugates.

28. The use of the compound according to any one of claims 1-21, or the preparation method according to any one of claims 22-25, or the drug / drug composition according to claim 26 or 27 in the preparation of a medicament for treating diseases related to abnormal cell activity, tumor diseases, autoimmune diseases, infectious diseases, and inflammatory diseases.

29. The application according to claim 28, characterized in that, The tumor was selected from tumors associated with the expression of one of the following targets: HER2, HER3, TROP2, B7H3, CDH6, EGFR, Nectin-4, TIM1, PSMA, EpCAM, MUC1, FGF2, c-MET, GFR, EphA2, ROR1, PD-L1, 5T4, NaPi2b, STEAP, BCMA, CEACAM5, SC-16, and Delt-like. protein3, Claudin18.2, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD70, CD73, CD74, CD79b, CD138, CD147, CD166 , CD223, MUC16, ASCT2, CD324, CD352, CD48a, CS1, FGFR2, FGFR3, ETBR, FGFR2, FLT3, LAMP-1, Ly6-E, NOTCH3, PRLR and RNF43.

30. The application according to claim 29, characterized in that, The tumors associated with target expression include tumors that highly express the target and / or tumors that are positive for the target.

31. The application according to claim 29, characterized in that, The tumors include solid tumors and hematologic tumors.

32. The application according to claim 31, characterized in that, The tumor is selected from one of the following groups: breast cancer, ovarian cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, multiple myeloma, prostate cancer, non-small cell lung cancer, small cell lung cancer, malignant melanoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, pancreatic cancer, prostate cancer, or any other tumor that grows and divides at an uncontrollable rate.

33. The application according to claim 28, characterized in that, The diseases associated with abnormal cell activity include hereditary hemoglobinopathies and muscular dystrophy; and / or, the autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, and psoriasis; and / or, the inflammatory diseases are enteritis.

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