Targeted pyrrolobenzodiazepine conjugates

Abstract

Conjugator-functionalized pyrrolobenzodiazepine (PBD) dimer compounds of Formula (I) are provided, wherein structures A and B are PBD and the conjugator is a group capable of forming a covalent linkage with an antibody. Antibody-drug conjugates comprising the conjugator-functionalized PBD dimer compounds are also disclosed.

Description

1. Cross-references to related applications

[0001] This application claims priority to PCT application No. PCT / CN2024 / 087468, filed on April 12, 2024, entitled “Targeted Pyrrolobenzodiazapine Conjugates,” which is hereby incorporated in its entirety by reference. 2. Reference to the electronic sequence list This application contains a sequence list, which has been submitted electronically in .XML format and is hereby incorporated herein by reference in its entirety. The .XML copy was created on March 13, 2025, named "F24W1548PR1.xml", and is 6 KB in size. The sequence list contained in this .XML file is an integral part of this specification and is hereby incorporated herein by reference in its entirety. 3. Technical Field This disclosure relates to targeted pyrrolobenzodiazepine (PBD) conjugates. 4. Background Technology Some pyrrolobenzodiazepines (PBDs) can recognize and bind to specific DNA sequences because their three-dimensional shape is isohelical with the minor groove of B-type DNA (Kohn, Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Various PBDs have been reported, differing in the number, type, and position of substituents, but all possess the following core structure: .

[0004] In ring B, there are imine (N═C), methamide (NH—CH(OH)), or methamide methyl ether (NH—CH(OMe)) groups at the N10-C11 positions. These groups are electrophilic centers responsible for alkylating DNA and conferring cytotoxic activity to PBD.

[0005] The bioactivity of PBD can be enhanced by linking two PBD units together via flexible alkylene links through their C8 / C′-hydroxy functional groups (Bose, DS et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, DE et al., J. Org. Chem., 61, 8141-8147 (1996)).

[0006] Due to the observed potency of PBD dimers, there is a need for PBD dimers that can be conjugated to target agents for use in targeted therapies. 5. Summary of the Invention This article describes compounds having the following general formula: , Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Structure A and structure B are independently PBD residues, for example having formula (IIa) or (IIb); The connector is a covalently connected part; and A conjugate is a conjugate group, such as those described herein.

[0008] This article provides compounds of formula (I): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A and structure B are each independently selected from one of the following formulas: (IIa) or (IIb); Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; -C(R 1 )- and -N(R 2 Between ) It can be a single bond or a double bond independently; when When it is a single bond, each R 1 Independently, it is H or OH, and each R 2 It is H; when When it is a double bond, each R 1 It is H, and each R 2 It does not exist; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7, or 8; and The sum of p and q is 2, 3, 4, 5, 6, 7 or 8.

[0009] This article also provides compounds of formula (V): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ;and Each Su is an independent sugar component.

[0010] This article also provides antibody-drug conjugates (ADCs) of formula (VI): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ; Each Su is an independent sugar component; Ab is a humanized, chimeric, or human antibody or its antigen-binding fragment; and x is approximately 1 to approximately 8. 6. Description of the attached drawings Figure 1 The percentage of cell killing in A375 cells by the payload compounds P1-1 and P1-2 described herein compared to the negative control, is shown, representing the percentage of cell killing in A375 cells by the payload compounds P1-1 and P1-2 as a percentage of the negative control, compared to the reference compounds Ref-1-1 and Ref-1-2.

[0012] Figure 2The percentage of cell killing in Calu-6 cells by the payload compounds P1-1 and P1-2 described herein compared to the negative control, is shown, representing the percentage of cell killing in Calu-6 cells by the payload compounds P1-1 and P1-2 as a percentage of the negative control, compared to the reference compounds Ref-1-1 and Ref-1-2.

[0013] Figure 3A The percentage of cell killing in NOMO-1 cells by the ADCs ADC3-B and ADC3-1 described herein compared to the negative control is shown.

[0014] Figure 3B The percentage of cell killing in K562 cells by the ADCs ADC3-B and ADC3-1 described herein compared to the negative control is shown.

[0015] Figure 4A The percentage of cell killing in NOMO-1 cells by the ADCs ADC3-A, ADC3-B, and ADC3-2 described herein compared to the negative control is shown.

[0016] Figure 4B The percentage of cell killing in K562 cells by the ADCs ADC3-A, ADC3-B, and ADC3-2 described herein compared to the negative control is shown. 7. Detailed Implementation This document provides pyrrolobenzodiazepine (PBD) dimers comprising two PBDs joined by a linker, the PBDs being identical or different. The PBD dimer can be conjugated to a conjugate via the linker, allowing the PBD dimer to be conjugated to a target agent (such as an antibody), for example, to form an antibody-drug conjugate (ADC). Each PBD in the PBD dimer can be further modified with cleavable groups (such as glycosidic groups). This document also provides ADCs comprising PBD dimers. ADCs can be used to treat diseases or conditions, such as cancer.

[0018] 7.1. Definition In this disclosure, unless otherwise indicated, the following terms have the following meanings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. If multiple definitions are provided for a term herein, those definitions shall prevail unless otherwise stated.

[0019] When a trade name is used in this document, unless the context otherwise indicates, reference to the trade name also refers to the product formulation, generic drug, and active pharmaceutical ingredient of the product under that trade name.

[0020] As used herein, the terms "conjugator" and "conjugating group" refer to a chemical moiety having a functional group capable of reacting with a corresponding functional group on a humanized, chimeric, or human antibody or its antigen-binding fragment to produce a covalently linked functional group. The functional group on the humanized, chimeric, or human antibody or its antigen-binding fragment can be a naturally occurring group (such as a thiol on a cysteine ​​residue), a synthetically incorporated natural group (such as a multihistidine sequence), or a non-natural group (such as an azide). A variety of conjugating groups and techniques for achieving conjugation are well known to those skilled in the art. Non-limiting examples of functional groups used for conjugating groups include alkynes (e.g., for conjugation with azides), maleimides (e.g., for conjugation with thiols), and o-phosphinoalkylbenzoates (e.g., for conjugation with azides). Examples of conjugated groups can also be found in International Publication No. WO 2023 / 125530 (wherein the compounds of formula (II) disclosed therein are referred to as “covalent linkers” or “L”), the entire contents of which are incorporated herein by reference.

[0021] As used herein, the term "amino acid" or "amino acid residue" refers to an organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups and a side chain (R group), said side chain being specific for each amino acid. Amino acids can be proteogenous or non-proteogenous. "Proteogenous" means that the amino acid is one of the twenty naturally occurring amino acids found in proteins. Proteogenous amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteogenous" means that the amino acid is not naturally found in proteins or is not directly produced by cellular mechanisms (e.g., it is a product of post-translational modification). Non-limiting examples of non-proteinogenic amino acids include γ-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylacetamide), hydroxyproline, β-alanine, ornithine, 4-azidophenylalanine, and citrulline.

[0022] The term "antibody" is used in the broadest sense and specifically encompasses intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting desired biological activity. An intact antibody has two main regions: a variable region and a constant region. The variable region binds to and interacts with the target antigen. The variable region includes a complementarity-determining region (CDR), which recognizes and binds to a specific binding site on a particular antigen. The constant region is recognized by and interacts with the immune system (see, for example, Janeway et al., 2001, Immuno. Biology, 5th edition, Garland Publishing, New York). Antibodies can belong to any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can be derived from any suitable species. In some embodiments, antibodies have a human or murine origin. Antibodies can be, for example, human, humanized, or chimeric.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of largely homogeneous antibodies, meaning that the individual antibodies constituting said population are identical, except for trace amounts of possibly naturally occurring mutations. Monoclonal antibodies exhibit high specificity against a single antigenic site. The modifier "monoclonal" should not be interpreted as requiring the antibody to be produced by any particular method.

[0024] A “complete antibody” is an antibody that contains an antigen-binding variable region and light chain constant domains (CL) and heavy chain constant domains CH1, CH2, CH3, and CH4, depending on the antibody class. The constant domains can be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0025] An "antibody fragment" comprises a portion of a complete antibody, including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, fragments generated from Fab expression libraries, or epitope-binding fragments of any of the above that are immune-specifically bound to a target antigen (e.g., cancer cell antigen, viral antigen, or microbial antigen).

[0026] An "antigen" is an entity that an antibody specifically binds to.

[0027] The terms "specific binding" and "specifically binds" refer to the fact that an antibody or antibody derivative binds to its corresponding target antigen in a highly selective manner, rather than to a multitude of other antigens. Typically, antibodies or antibody derivatives bind at least about 1 × 10⁻⁶. -7 M, 10 -8 M, 10 -9 M, 10 M, 10 -11 M or 10 -12 M binds with an affinity that is at least twice as strong as its affinity for a predetermined antigen as it binds to a nonspecific antigen other than the predetermined antigen or closely related antigens (e.g., BSA, casein).

[0028] The term "inhibit / inhibition of" means to reduce a measurable amount or to prevent it completely.

[0029] The term "therapeuticly effective amount" refers to the amount of a drug that is effective in treating a disease or symptom in a mammal. In the case of cancer, a therapeutically effective amount of a drug may, for example, reduce the number of cancer cells; reduce tumor size; inhibit (e.g., to some extent slow down or stop) the infiltration of cancer cells into surrounding organs; inhibit (e.g., to some extent slow down or stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. Insofar as the drug can inhibit growth and / or kill existing cancer cells, it may be cytoseptic and / or cytotoxic. For cancer therapies, efficacy may be measured, for example, by assessing time to progression (TTP) and / or determining response rate (RR).

[0030] The term "substantial" or "substantially" refers to the majority of a mixture or sample, i.e., >50% of the population, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0031] The terms "intracellularly cleaved" and "intracellular cleavage" refer to the metabolic process or reaction of a ligand-drug conjugate (e.g., an antibody-drug conjugate (ADC)) within a cell, whereby the covalent linkage (e.g., the bond between the payload and the conjugate or conjugated antibody) is disrupted, resulting in the dissociation of the free drug or another metabolite of the conjugate from the antibody within the cell. Therefore, the cleavage portion of a drug-linker-ligand conjugate is an intracellular metabolite.

[0032] The terms “cancer” and “cancerous” refer to or describe a physiological disorder or symptom in mammals that is typically characterized by an imbalance in cell growth. A “tumor” contains one or more cancer cells.

[0033] Examples of “patient” or “subject” include, but are not limited to, mammals such as humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, or cats, as well as birds or poultry. In one implementation, the patient is a human.

[0034] Unless the context otherwise indicates, the term "treatment" refers to therapeutic treatment and preventative measures to prevent recurrence, wherein the goal is to suppress or slow (alleviate) unwanted physiological changes or conditions, such as the development or spread of cancer. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization of disease status (e.g., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonged survival compared to expected survival without treatment. Those requiring treatment include those who already have the disease or condition and those who are susceptible to it.

[0035] In the context of cancer, the term "treatment" includes any or all of the following: inhibiting the growth of tumor cells, cancer cells, or tumors; inhibiting the replication of tumor cells or cancer cells; reducing the overall tumor burden or the number of cancer cells; and improving one or more symptoms associated with the disease.

[0036] As used herein, in this specification, and in the appended claims, the indefinite article “a / an” and the definite article “the” include both plural and single indicators, unless the context clearly indicates otherwise.

[0037] As used herein and unless otherwise stated, the terms “about” and “approximately”, when used in conjunction with the amount or weight percentage of a component of a composition, mean an amount or weight percentage that is recognized by those skilled in the art to provide a pharmacological effect equivalent to that obtained from the specified amount or weight percentage. In some embodiments, the terms “about” and “approximately” as used herein cover an amount or weight percentage within 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percentage.

[0038] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base (including inorganic acids or bases and organic acids or bases).

[0039] As used herein and unless otherwise indicated, the term "solvent" means a compound or its salt, which also includes stoichiometric or non-stoichiometric amounts of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0040] As used herein and unless otherwise indicated, the term "hydrate" means a compound or its salt, which also includes stoichiometric or nonstoichiometric amounts of water bound together by noncovalent intermolecular forces.

[0041] As used herein and unless otherwise indicated, the term "prodrug" means a compound derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound. Examples of prodrugs include, but are not limited to, derivatives and metabolites of compounds, including biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs.

[0042] As used herein and unless otherwise indicated, the term "stereoisomer" refers to the atomic arrangement of a compound in three-dimensional space. Compounds that are called stereoisomers of each other have the same bonding structure but different arrangements in three-dimensional space. Stereoisomers may originate from chiral atoms, chiral centers, E / Z isomerism in alkenes, or rotationally locked bonds. Stereoisomers can be enantiomers, diastereomers, cis-trans isomers, trans-blocked isomers, epiomers, or end-group isomers.

[0043] As used herein and unless otherwise indicated, the term "steroisomerally pure" means that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereoisomerally pure compound having one chiral center will substantially free of its opposite enantiomers. A stereoisomerally pure compound having two chiral centers will substantially free of other diastereomers of the compound. A typical stereoisomerally pure compound contains more than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, more than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or more than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. The compounds may have a chiral center and may occur as racemic, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms are included within the embodiments disclosed herein, including mixtures thereof. The embodiments disclosed herein cover the use of pure stereoisomeric forms of such compounds, as well as the use of mixtures of those forms. For example, mixtures comprising equal or unequal amounts of enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers may be resolved by asymmetric synthesis or using standard techniques such as chiral columns or chiral resolving agents. See, for example, Jacques, J. et al. , Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH et al. Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions Page 268 (EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0044] It should also be noted that the compound may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In some embodiments, the compound is isolated into cis or trans isomers. In other embodiments, the compound is a mixture of cis and trans isomers.

[0045] "Tautomers" refer to the isomers of a compound that are in equilibrium with each other. The concentration of the isomers will depend on the environment in which the compound exists and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomers, which are referred to as tautomers of each other: .

[0046] It will be readily understood by those skilled in the art that various functional groups and other structures can exhibit tautomerism, and that all tautomers of the compound are within the scope of this disclosure.

[0047] It should also be noted that one or more atoms in the compound may contain atomic isotopes in non-natural proportions. For example, the compound may contain atomic isotopes such as tritium (…). 3 H), Iodine-125 ( 125 I), sulfur-35 ( 35 S) or carbon-14 ( 14 Radiolabeling can be performed using radioactive isotopes such as C, or deuterium (C). 2 H), carbon-13 ( 13 C) or nitrogen-15 ( 15 N) Isotope enrichment. As used herein, an "isotope" is an isotopically enriched compound. The term "isotope enrichment" means that the atoms have an isotopic composition other than the natural isotopic composition of the atoms. "Isotope enrichment" can also refer to a compound in which at least one atom has an isotopic composition different from the natural isotopic composition of the atoms. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer and inflammation treatment agents), research reagents (e.g., binding assay reagents), and diagnostic agents (e.g., in vivo imaging agents). All isotopic variations of the compounds described herein (whether radioactive or not) are intended to be covered within the scope of the embodiments provided herein. In some embodiments, isotopes of the compounds are provided, for example, isotopes enriched in deuterium, carbon-13, or nitrogen-15.

[0048] It should be noted that if there is an inconsistency between the described structure and the name of the structure, the described structure should be given higher weight.

[0049] When shown in the chemical structures described in this article, the symbol is drawn perpendicular to the other chemical bond. This refers to the connection point with a larger structure. As used in this article, it contains... The structure of a symbol will have only one connection point, and the larger structure it connects to will be clearly marked or defined, usually using unique variables. Symbol followed by other characters (e.g.) ) "will" refers to uniquely defined connection points, which will be explicitly stated in the context in which they are used.

[0050] As used herein, “sugar” or “glycosyl” or “sugar residue” or “sugar moiety” refers to a carbohydrate moiety that may comprise a 3-carbon (triose) unit, a 4-carbon (teuose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptaose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, “sugar” or “glycosyl” or “sugar residue” comprises furanose (e.g., furanose ribose, furanose fructose) or pyranose (e.g., pyranose glucose, pyranose galactose) or a combination thereof. In some cases, “sugar” or “glycosyl” or “sugar residue” comprises aldose or ketose, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, glucosamine, glucuronic acid, glucuronide, mannose, galactose, fructose, iduronic acid, and iduronide. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other “sugars” or “glycosyl groups” or “sugar residues” or “sugar moieties” include polysaccharides and / or oligosaccharides, including but not limited to amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, “sugars” or “glycosyl groups” or “sugar residues” or “sugar moieties” are amino-sugars. In some cases, “sugars” or “glycosyl groups” or “sugar residues” or “sugar moieties” are glucosamine residues (1-amino-1-deoxy-D-glucitol) that are linked via their amino groups to the remainder of the molecule, thereby forming an amide bond (i.e., glucosamide) with the remainder of the molecule. “Sugars” or “glycosyl groups” or “sugar residues” or “sugar moieties” can be natural or artificial sugars, or can be derived natural sugars. Derivatization of a sugar, sugar moiety, sugar residue, or sugar moiety can be, but is not limited to, removal of a hydroxyl group (deoxygenation), oxidation of the hydroxyl group to an aldehyde, ketone, or carboxylic acid, substitution with an amine, inversion of the chiral center, or acylation.

[0051] As used herein, the diagrammatic intent showing the substituent bonded to the cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) via inter-ring bonds indicates that, unless otherwise stated, the cyclic group may be substituted by the substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to the techniques described herein or known in the art to which this disclosure pertains.

[0052] The amino acid sequence of the antibody can be numbered using any known numbering scheme, including those described below: Kabat et al. (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme). Unless otherwise stated, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply the presence of differences in the sequence where no differences exist, and those skilled in the art can readily confirm the sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise stated, the “EU numbering scheme” is generally used when referring to residues in the constant region of the antibody heavy chain (e.g., as reported in Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md.).

[0053] As used in this article, the term "cytotoxic activity" refers to the activity that reduces or diminishes the cell viability of the tested cell line.

[0054] In the following claims and in the preceding description, unless the context requires otherwise due to the language of expression or necessary implication, the word “comprise” or variations thereof (such as “comprises / comprising”) are used to include the meaning of specifying the presence of the described feature in the various embodiments, rather than excluding the presence or addition of other features.

[0055] 7.2. Conjugated PBD dimer complex This article describes pyrrolobenzodiazepine (PBD) dimers covalently bonded to conjugated groups via covalently attached linkers. In the conjugated PBD dimers described herein, the conjugated groups are attached to the PBD dimer via linkers. The conjugated components can link the payload to the target group, thereby conferring selectivity to the complex in vivo. Such compounds themselves possess potent cytotoxic activity and can be used in antibody-drug conjugates, as described herein.

[0056] This article describes compounds having the following general formula: , Or its pharmaceutically acceptable salts, tautomers, solvates or stereoisomers, wherein: Structure A and structure B are independently PBD residues, for example having formula (IIa) or (IIb); The connector is a covalently connected part; and A conjugate is a conjugate group, such as those described herein.

[0057] This article describes the compound of formula (I): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A and structure B are each independently selected from one of the following formulas: (IIa) or (IIb); Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; -C(R 1 )- and -N(R 2 Between ) It can be a single bond or a double bond independently; when When it is a single bond, each R 1 Independently, it is H or OH, and each R 2 It is H; when When it is a double bond, each R 1 It is H, and each R 2 It does not exist; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7, or 8; and The sum of p and q is 2, 3, 4, 5, 6, 7 or 8.

[0058] In some implementations of formula (I), either or both of structure A and structure B are .

[0059] In some implementations of formula (I), either or both of structure A and structure B are .

[0060] In some implementations of equation (I), one of structure A and structure B is And the other one in structure A and structure B is .

[0061] In some implementations of equation (I), both p and q are 2.

[0062] In some embodiments of formula (I), the compound has the following formula: or .

[0063] In some embodiments of formula (I), the conjugate has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; and AA is an amino acid or its stereoisomer that exists independently each time it appears.

[0064] In some embodiments of formula (I), the conjugate has the following formula: .

[0065] In some embodiments of formula (I), the conjugate has the following formula: .

[0066] In some embodiments of formula (I), the compound has one of the following formulas: or , Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

[0067] 7.3. PBD dimer complexes functionalized with conjugates containing cleavable sugar moieties This paper also discloses conjugated PBD dimers in which each PBD is independently linked to a cleavable sugar moiety. These dimers have a prodrug design in which the cleavable sugar moiety is cleaved (e.g., by intracellular enzymes), thereby releasing the potent PBD dimer payload described herein. For example, at the sites indicated by dashed lines and arrows in the following structures, the action of β-GlcA releases the conjugated PBD dimer: .

[0068] This article also discloses compound (V): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ;and Each Su is an independent sugar component.

[0069] In some implementations of equation (V), either or both of structure A' and structure B' are .

[0070] In some implementations of equation (V), either or both of structure A' and structure B' are .

[0071] In some implementations of equation (V), one of structure A' and structure B' is And the other one of structure A' and structure B' is .

[0072] In some implementations of equation (V), both p and q are 2.

[0073] In some embodiments of formula (V), the compound has the following formula: or .

[0074] In some embodiments of formula (V), the conjugate has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; and AA is an amino acid or its stereoisomer that exists independently each time it appears.

[0075] In some embodiments of formula (V), the conjugate has the following formula: .

[0076] In some embodiments of formula (V), the conjugate has the following formula: .

[0077] In some embodiments of formula (V), each sugar component is independent. , , or or its stereoisomers; and Each m is independently 0 or 1.

[0078] In some embodiments of formula (V), the sugar component is And m is 0.

[0079] In some implementations of formula (V), each R 2’ Independently or .

[0080] In some implementations of formula (V), each R 2’ Independently or .

[0081] In some embodiments of formula (V), the compound has one of the following formulas: , or , Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

[0082] 7.4. Cleavage-functionalized PBD dimer antibody-drug conjugates This document also discloses antibody-drug conjugates (ADCs), including humanized, chimeric, or human antibodies or antigen-binding fragments thereof, which are directly or indirectly covalently linked to the PBD dimer described herein.

[0083] This article also describes antibody-drug conjugates of formula (VI): , Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ; Each Su is an independent sugar component; Ab is a humanized, chimeric, or human antibody or its antigen-binding fragment; and x is approximately 1 to approximately 8.

[0084] In some embodiments of formula (VI), either or both of structure A' and structure B' are .

[0085] In some embodiments of formula (VI), either or both of structure A' and structure B' are .

[0086] In some implementations of formula (VI), one of structure A' and structure B' is And the other one of structure A' and structure B' is .

[0087] In some implementations of equation (VI), both p and q are 2.

[0088] In some implementations of equation (VI), the ADC has the following formula: or .

[0089] In some embodiments of formula (VI), the ligand has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; Each time AA appears, it is an independently naturally occurring amino acid or its stereoisomer; It is the connection point with Ab.

[0090] In some embodiments of formula (VI), the ligand has the following formula: .

[0091] In some embodiments of formula (VI), the ligand has the following formula: .

[0092] In some embodiments of formula (VI), the sugar component is , , or or its stereoisomers; and Each m is independently 0 or 1.

[0093] In some embodiments of formula (VI), the sugar component is And m is 0.

[0094] In some implementations of formula (VI), each R 2’ Independently or .

[0095] In some implementations of equation (VI), the ADC has one of the following equations: , or , Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

[0096] In some implementations of formula (VI), x is approximately 2.

[0097] 7.5. Method or process for preparing the conjugate This document provides a method for preparing an ADC, wherein a humanized, chimeric, or human antibody or its antigen-binding fragment is contacted with a PBD dimer described herein under conditions suitable for forming a bond between the antibody or its antigen-binding fragment and the PBD dimer. The reaction conditions can be any suitable reaction conditions known in the art. The following examples provide instances of conditions used for such reactions.

[0098] 7.6. Pharmaceutical Compositions This document also provides compositions comprising the ADCs described herein, including pharmaceutical compositions. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise pharmaceutically acceptable excipients.

[0099] The pharmaceutical compositions according to this disclosure can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an antibody-drug conjugate having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residuals) This includes polypeptides (based on hydroxyl groups); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein also include interstitial drug dispersants such as neutral-active soluble hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans (such as chondroitinase).

[0100] Exemplary lyophilized formulations are described in U.S. Patent No. 6,267,958. Aqueous formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, the latter comprising histidine-acetate buffer.

[0101] The pharmaceutical composition can be formulated for administration by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if local treatment is required). Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.

[0102] 7.7. How to use In some implementations, this document describes a method for treating a disease or condition (e.g., cancer) in a subject (e.g., a patient) in need, the method comprising administering a therapeutically effective amount of the ADC disclosed herein to the patient.

[0103] The ADCs disclosed herein can be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if local treatment is required). Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be carried out via any suitable route, such as by injection (e.g., intravenous or subcutaneous), depending in part on whether the administration is acute or prolonged. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0104] The ADC disclosed herein can be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to medical practitioners.

[0105] 8. Examples List of abbreviations

[0106] UPLC analysis method Method A: Mobile phase A: Water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.2 min, 10% - 95% B for 5.8 min, 95% B for 0.5 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 µm.

[0107] Method B: Mobile phase A: water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.5 min, 10% - 90% B for 2.5 min, 90% B for 0.2 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 µm.

[0108] Method C: Mobile phase A: water containing 0.1% FA, B: MeCN; Gradient: 10% B for 0.2 min, 10% - 90% B for 1.3 min, 90% B for 0.3 min; Flow rate: 0.6 mL / min; Column: ACQUITY UPLC® BEH C18 1.7 µm.

[0109] Example 1: Synthesis of intermediate compounds Synthetic Int-2 Step 1: ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)carbamate (Int-2b) EEDQ (4.77 g, 19.3 mmol) was added to a solution of Int-2a (5 g, 18.4 mmol) and (4-aminophenyl)methanol (2.37 g, 19.3 mmol) in 100 mL of anhydrous THF. The mixture was stirred at room temperature for 40 hours. The mixture was concentrated. The residue was slurried with MTBE (30 V) and stirred for 2 hours. The solid was separated by filtration under vacuum for 3 hours to give Int-2b (5.16 g, 74% yield).

[0110] MS (ESI) m / z: 378.4 [M+H] + .

[0111] Step 2: (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropyl-2-yl)-3-methylbutyramide (Int-2c) Pd(PPh3)4 (76.6 mg, 0.066 mmol) was added to a solution of Int-2b (500 mg, 1.33 mmol) in CH2Cl2 (10 mL) and pyrrolidine (270.8 μL, 3.31 mmol) at room temperature under N2. The reaction mixture was stirred at room temperature for 0.5 h. The reactants were concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 92 / 8) to give Int-2c (370 mg, 95% yield) as a white solid.

[0112] MS (ESI) m / z: 294.3 [M+H] + .

[0113] Step 3: ((17S,20S)-21-((4-(hydroxymethyl)phenyl)amino)-17-isopropyl-20-methyl-15,18,21-trioxo-3,6,9,12-tetraoxa-16,19-diazaeicosyl)carbamate (9H-fluorene-9-yl)methyl ester (Int-2) DIEA (326 mg, 2.52 mmol) was added to a solution of Int-2c (370 mg, 1.26 mmol) and HATU (575.8 mg, 1.51 mmol) in 4 mL of anhydrous DMF. The mixture was stirred at room temperature for 10 min. Then, a solution of Int-2d (645 mg, 1.33 mmol) in DMF (4 mL) was added to the mixture. The reaction mixture was stirred for 1 h. The mixture was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 95 / 5) to give Int-2 (680 mg, 71% yield) as a light brown solid.

[0114] MS (ESI) m / z: 763.4 [M+H] + .

[0115] Synthetic Int-5 Step 1: Bis(2-hydroxyethyl)carbamate (1-5b) AllocCl (361.45 mg, 3 mmol) and K₂CO₃ (1036 mg, 7.5 mmol) were added to a solution of 1-5a (423 mg, 3 mmol) in THF (3.1 mL) and water (5.7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 hours. TLC (petroleum ether:EtOAc = 1:1, v / v) showed that the reaction was complete. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over Na₂SO₄ and filtered. The solvent was evaporated and the crude product 1-5b (567 mg) was used for the next step without further treatment and purification.

[0116] Step 2: Bis(4-methylbenzenesulfonic acid)(((allyloxy)carbonyl)azadiyl)bis(ethane-2,1-diyl) ester (Int-5) TsCl (1.7 g, 9 mmol) and triethylamine (1.67 mL, 12 mmol) were added to a solution of 1-5b (567 mg, 3 mmol) in CH2Cl2 (7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 h. TLC (petroleum ether:EtOAc = 3:1, v / v) showed that the reaction was complete. The reactants were poured into water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The solvent was evaporated and purified by silica gel column chromatography (eluent: petroleum ether EtOAc = 100 / 0 to 30 / 70) to give Int-5 as a colorless oil (1.2 g, 73.08% yield).

[0117] Synthetic Int-7 Step 1: ((17S,20S)-17-isopropyl-20-methyl-21-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-15,18,21-trioxo-3,6,9,12-tetraoxa-16,19-diazatecodecyl)carbamate (9H-fluorene-9-yl)methyl ester (Int-7) DIEA (233 μL, 1.31 mmol) was added to a mixture of Int-2 (500 mg, 0.656 mmol), bis(4-nitrophenyl) carbonate (300 mg, 0.981 mmol), and DMF (10 mL). The mixture was stirred overnight at room temperature. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 0 / 100) to give Int-7 (386 mg, 63.5% yield) as a white solid.

[0118] MS (ESI) m / z: 928.7 [M+H] + .

[0119] Synthetic Int-8 Step 1: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (Int-8c) Int-8b (1.14 g, 2.87 mmol) and 4 Å molecular sieve (4.00 g) were added to a solution of compound Int-8a (1.00 g, 2.41 mmol) in CH2Cl2 (30 mL). The mixture was stirred at room temperature for 30 min. The mixture was then cooled to -20 °C. Next, AgOTf (0.74 g, 2.87 mmol) was added to the mixture in fractions at -20 °C. The mixture was stirred at -20 °C for 4 h. The mixture was filtered, and the filtrate was diluted with EtOAc (150 mL) and washed with saturated NaHCO3 (3 x 100 mL) and brine (3 x 100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography (eluting with petroleum ether / EtOAc = 0%–40%) to give the crude product. The crude product was further purified with isopropanol to give compound Int-8c (340 mg, 19.3% yield) as a white solid.

[0120] MS (ESI) m / z: 734.4 [M+H] + .

[0121] Step 2: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-2-amino-3-(benzyloxy)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (Int-8d) Et₂NH (125 mg, 1.70 mmol) was added to a solution of compound Int-8c (250 mg, 0.34 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 30 min. The mixture was concentrated under high vacuum and co-evaporated with toluene (3 x 3 mL) to give compound Int-8d (174 mg, crude substance) as a brown oil, which was used directly in the next step without further purification.

[0122] MS (ESI) m / z: 512.3 [M+H] + .

[0123] Step 3: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-3-(benzyloxy)-2-((S)-2-(((benzyloxy)carbonyl)amino)-3-methylbutamido)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (Int-8f) Compounds Int-8e (103 mg, 0.41 mmol), HATU (168 mg, 0.44 mmol), and DIEA (88 mg, 0.68 mmol) were added to a solution of compound Int-8d (174 mg, 0.34 mmol) in DMF (4 mL). The mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed with saturated NaHCO3 solution (3 x 30 mL) and brine (3 x 30 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated to give the residue. The residue was purified by rapid column chromatography (eluting with CH2Cl2 / EtOAc = 100 / 0~70 / 30). Compound Int-8f (230 mg, 91% yield) was given as a white solid.

[0124] MS (ESI) m / z: 745.4 [M+H] + .

[0125] Step 4: N-(L-valine)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (Int-8g) Pd / C (10%, 23 mg) was added to a solution of compound Int-8f (230 mg, 0.31 mmol) in MeOH (3 mL). The mixture was stirred at room temperature under H2 (15 psi) for 1 hour. H2O (3 mL) was added to the mixture, filtered, and concentrated to give crude product Int-8g (161 mg, crude substance) as a white solid, which was used directly without further purification.

[0126] MS (ESI) m / z: 521.3 [M+H] + .

[0127] Step 5: N-(acetyl-L-valine)-O-((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)-L-serine (Int-8h) At 0 °C, Ac₂O (316 mg, 3.09 mmol) was added to a solution of compound Int-8 g (161 mg, 0.31 mmol) in AcOH (3 mL). The mixture was stirred at room temperature for 1 hour. The mixture was concentrated and analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). Purification was performed using a mobile phase of 150 mm; A-water (0.1% TFA): B-acetonitrile; flow rate: 20 mL / min. The compound Int-8h (145 mg, 83.3% yield) was obtained as a white solid.

[0128] MS (ESI) m / z: 563.4 [M+H] + .

[0129] Step 6: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-acetamido-3-methylbutamido)-3-((4-(hydroxymethyl)phenyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (Int-8) Int-8h (200 mg, 0.36 mmol) and 4-aminobenzyl alcohol (46 mg, 0.37 mmol) were mixed in anhydrous CH2Cl2 (1 mL). EEDQ (131.9 mg, 0.53 mmol) was added to the mixture. The mixture was stirred at room temperature under a nitrogen atmosphere for 12 hours. The solvent was removed under vacuum to obtain the crude product, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 10 / 90) to obtain Int-8 (80 mg, 34%) as a pale yellow solid.

[0130] MS (ESI) m / z: 668.0 [M+H] + .

[0131] Example 2: Synthetic Payload Example P1-1 (1-21) Step 1: (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidone-1-yl)(4-hydroxy-5-methoxy-2-nitrophenyl) ketone (1-21b) t-BuOK (6.95 g, 61.98 mmol) was added to a mixture of MePh3PBr (24.6 g, 68.86 mmol) in THF (100 mL) at 0 °C under N2. The mixture was stirred at 0 °C for 2 h, and then a solution of 1-21a (4 g, 6.89 mmol) in THF (30 mL) was added dropwise to the mixture and stirred at 0 °C for 16 h. The mixture was neutralized with citric acid and extracted with EtOAc (3 x 100 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give 1-21b (1.1 g, 37.8% yield) as a yellow solid.

[0132] MS (ESI) m / z: 423.2 [M+H] + .

[0133] Step 2: Bis(2-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)ethyl)carbamate (1-21c) K₂CO₃ (55.5 mg, 0.4 mmol) was added to a solution of 1-13c (100 mg, 0.2 mmol) and 1-21b (186.83 mg, 0.44 mmol) in DMSO (3 mL). The mixture was stirred at 50 °C for 16 hours. The reaction was quenched with H₂O and extracted with EtOAc (30 mL). The organic phase was dried over Na₂SO₄, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography (eluent: hexane / EtOAc = 100 / 0 to 0 / 100) to give 1-21c (108 mg, 53.83% yield) as a yellow solid.

[0134] MS (ESI) m / z: 998.5 [M+H] + .

[0135] Step 3: Bis(2-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (1-21d) Zn powder (28.24 mg, 4.08 mmol) was added to a mixture of EtOH (3 mL), AcOH (0.2 mL), and H2O (0.2 mL) and stirred at room temperature for 10 min. Then, a solution of 1-21d (108 mg, 0.11 mmol) in EtOH (2 mL) was added and stirred at room temperature for 1 hour. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated to obtain a crude product. This crude product was purified by silica gel chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to obtain a yellow oily product of 1-21d (73 mg, 72.2% yield).

[0136] MS (ESI) m / z: 938.6 [M+H] + .

[0137] Step 4: Bis(2-(5-(((allyloxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (1-21e) Alloc-Cl (53 μL, 0.49 mmol) and pyridine (24.73 μL, 0.311 mmol) were added to a solution of 1-21d (73 mg, 0.077 mmol) in anhydrous CH2Cl2 (2 mL) at -10 °C under a nitrogen atmosphere. The mixture was stirred at -10 °C for 1 hour. The solution was added to water (10 mL) and extracted with CH2Cl2 (3 x 5 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by rapid column chromatography (eluent: petroleum ether / EtOAc = 1 / 3) to give 1-21e (75 mg, 87.13% yield) as a grayish-white solid.

[0138] MS (ESI) m / z: 1106.6 [M+H] + .

[0139] Step 5: Bis(2-(5-((((allyloxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (1-21f) p-Toluenesulfonic acid hydrate (25.79 mg, 0.13 mmol) was added to a solution of 1-21e (75 mg, 0.067 mmol) in THF (2 mL) and water (0.1 mL). The reaction mixture was stirred at room temperature for 1 hour. The mixture was directly concentrated and purified by rapid column chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give 1-21f (52 mg, 87.38% yield) as a yellow solid.

[0140] MS (ESI) m / z: 878.4 [M+H] + .

[0141] Step 6: 8,8'-(((((allyloxy)carbonyl)azadiyl)bis(ethane-2,1-diyl))bis(oxy))(11aS,11a'S)-bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid)1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-carboxylic acid diallyl ester (1-21g) DMP (55.27 mg, 0.13 mmol) was added to a solution of 1-21f (52 mg, 0.059 mmol) in CH2Cl2 (2 mL) at 0 °C. The reaction mixture was then heated to room temperature and stirred for 4 hours. The reaction mixture was filtered, and the filtrate was quenched with a saturated aqueous sodium thiosulfate solution (5 mL). Subsequently, a saturated aqueous NaHCO3 solution (5 mL) and H2O (10 mL) were slowly added. The mixture was extracted with dichloromethane (3 x 5 mL), and the organic layer was washed with brine (5 mL). The solution was dried over Na2SO4, filtered, and concentrated to give a crude product. The crude product was purified by rapid column chromatography (eluent: CH2Cl2 / MeOH = 20 / 1) to give 1-21g (32 mg, 61.82% yield) as a white solid.

[0142] MS (ESI) m / z: 874.4 [M+H] + .

[0143] Step 7: (11aS,11a'S)-8,8'-((azadiylbis(ethane-2,1-diyl))bis(oxy))bis(7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one) (1-21) Pd(PPh3)4 (2.12 mg, 0.0018 mmol) and pyrrolidine (6 μL, 0.073 mmol) were added to a solution of 1-21 g (32 mg, 0.036 mmol) and CH2Cl2 (2 mL), and the mixture was stirred at room temperature under N2 for 15 min. The reactants were neutralized with AcOH and concentrated to give the crude product, which was then analyzed by preparative HPLC (column: XBridge Prep C18 OBD 5 μm 19). The mobile phase was 150 mL / min; the flow rate was 20 mL / min. The solution was purified to give 1-21 (9.7 mg, 45.23% yield) as a white solid.

[0144] MS (ESI) m / z: 586.3 [M+H] + .

[0145] Example P1-2 (1-13) Step 1: Bis(2-hydroxyethyl)carbamate (1-13b) AllocCl (361.45 mg, 3 mmol) and K₂CO₃ (1036 mg, 7.5 mmol) were added to a solution of 1-13a (423 mg, 3 mmol) in THF (3.1 mL) and water (5.7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 hours. TLC (petroleum ether:EtOAc = 1:1, v / v) showed that the reaction was complete. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over Na₂SO₄ and filtered. The solvent was evaporated and the crude product 1-13b (567 mg) was used in the next step without further treatment or purification.

[0146] Step 2: bis(4-methylbenzenesulfonic acid)(((allyloxy)carbonyl)azadiyl)bis(ethane-2,1-diyl) ester (1-13c) TsCl (1.7 g, 9 mmol) and triethylamine (1.67 mL, 12 mmol) were added to a solution of 1-13b (567 mg, 3 mmol) in CH2Cl2 (7 mL) at 0 °C. The mixture was stirred at 20 °C for 16 hours. TLC (petroleum ether:EtOAc = 3:1, v / v) showed that the reaction was complete. The reactants were poured into water (20 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4 and filtered. The solvent was evaporated and purified by silica gel column chromatography (eluent: petroleum ether EtOAc = 100 / 0 to 30 / 70) to give 1-13c (1.2 g, 73.08% yield) as a colorless oil.

[0147] Step 3: 8,8''-(((((allyloxy)carbonyl)azadiyl)bis(ethane-2,1-diyl))bis(oxy))(11aS,11a''S)-bis(11-hydroxy-7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10(5H)-formic acid) diallyl ester (1-13d) K₂CO₃ (41.70 mg, 0.30 mmol) was added to a solution of 1-13c (50 mg, 0.10 mmol) and 1-5b (75.25 mg, 0.20 mmol) in DMSO (2 mL) at room temperature. The mixture was stirred at 50 °C for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was diluted with EtOAc (10 mL) and washed with brine (3 x 8 mL). The organic layer was dried over Na₂SO₄ and concentrated to give the residue, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 10 / 90) to give 1-13d (38 mg, 49.64% yield) as a colorless oil.

[0148] MS (ESI) m / z: 902.3 [M+H] + .

[0149] Step 4: (11aS,11a''S)-8,8''-((azadiylbis(ethane-2,1-diyl))bis(oxy))bis(7-methoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazadi-2,1'-cyclopropane]-5-one) (1-13) Pd(PPh3)4 (5.77 mg, 0.005 mmol) and pyrrolidine (12 μL, 0.12 mmol) were added to a solution of 1-13d (45 mg, 0.05 mmol) and CH2Cl2 (3 mL) at room temperature. The mixture was stirred at 20 °C for 30 min. The reaction was complete as indicated by LCMS. The solvent was evaporated and the residue was purified by preparative HPLC (0.01% FA) to give product 1-13 (12 mg, 35.27% yield) as a white solid.

[0150] MS (ESI) m / z: 614.3 [M+H] + .

[0151] 1 H NMR (400 MHz, CDCl3) δ 7.79 (2 H, d, J = 4.4), 7.51 (2 H, s), 6.84 (2H, d, J = 2.2), 4.30 – 4.13 (4 H, m), 3.93 (6 H, d, J = 0.9), 3.87 – 3.81 (2 H,m), 3.67 (2 H, d, J = 11.7), 3.49 (2 H, d, J = 11.7), 3.26 – 3.17 (4 H, m), 2.52(2 H, dd, J = 13.0, 8.1), 2.00 (2 H, dd, J = 13.0, 2.6), 0.73 (8 H, ddd, J = 11.3, 8.3, 4.5).

[0152] Example 3: Synthesis of the reference compound Table 1 below summarizes the reference connector-payload compounds.

[0153] Example Ref-2-1 Ref-2-1a is synthesized according to the procedure described in US2015283262A1 and Org. Process Res. Dev. 2018, 22, 1241-1256.

[0154] Step 1 (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][ [1,4]diaza-10(5H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl ester (Ref-2-1) HATU (5.0 mg, 0.01 mmol) and DIEA (4 μL, 0.02 mmol) were added to a solution of Int-1 (5.3 mg, 0.01 mmol, prepared according to the procedures described in WO2022120132 A1 and WO2022079211 A1) in 0.5 mL of DMF at 0 °C. The mixture was stirred at room temperature for 15 min. 0.5 mL of DMF containing Ref-2-1a (10 mg, 0.01 mmol) was added to the mixture at 0 °C, and the reaction mixture was stirred at room temperature for 2 h. Preparative HPLC was performed (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified using a mobile phase of 150 mm (A-water (formic acid-free):B-acetonitrile; flow rate: 20 mL / min). The fractions were lyophilized to give Ref-2-1 (2.5 mg, 17.1% yield) as a white solid.

[0155] MS (ESI) m / z: 1345.9 [M+H] + .

[0156] Example Ref-2-2 Int-3 is synthesized according to the procedure described in WO 2017059289 A1 and Org. Process Res. Dev. 2022, 26, 2155-2175.

[0157] Step 1: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamate (9H-fluorene-9-yl)methyl tert-butyl ester (Ref-2-2a) Triphosgene (24.9 mg, 0.084 mmol) was added to a solution of Int-3 (200 mg, 0.1210 mmol) and 200 mg 4Å MS in anhydrous THF (2.5 mL), followed by the addition of Et3N (64 μL, 0.462 mmol) at 0 °C under N2. The mixture was stirred at 0 °C under N2 for 10 min. Isocyanate formation was monitored by LCMS analysis after quenching with MeOH. A solution of Int-2 (176 mg, 0.231 mmol), dibutyltin dilaurate (13.3 mg, 0.021 mmol), and Et3N (43.7 μL, 0.315 mmol) in anhydrous THF (2.5 mL) was added to the mixture. The mixture was stirred at room temperature for 3 h. The mixture was then filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 4 / 96) to give Ref-2-2a (304 mg, 83% yield) as a white solid.

[0158] MS (ESI) m / z: 1742.1 [M+H] + .

[0159] Step 2: (5-((5-(5-((((4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)tert-butyl carbamate (Ref-2-2b) p-Toluenesulfonic acid hydrate (40 mg, 0.21 mmol) was added to a solution of Ref-2-2a (304 mg, 0.17 mmol) in THF (3 mL) and water (0.15 mL). The reaction mixture was stirred at 22 °C for 4 hours. The mixture was diluted with EtOAc (20 mL) and washed with water, saturated NaHCO3, and brine. The organic phase was concentrated and purified by silica gel column chromatography (CH2Cl2 / MeOH = 5 / 95) to give Ref-2-2b (213 mg, 81% yield) as a white solid.

[0160] MS (ESI) m / z: 1513.9 [M+H] + .

[0161] Step 3: (11S,11aS)-8-((5-(((11S,11aS)-10-(tert-butyloxycarbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2 ,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecosano-25-amido)benzyl ester (Ref-2-2c) DMP (58.9 mg, 0.139 mmol) was slowly added fractionally to a solution of Ref-2-2b (100 mg) in anhydrous CH2Cl2 (2 mL) at 0 °C. The reaction mixture was then heated to room temperature and stirred overnight. The reaction mixture was quenched with saturated Na2S2O3, followed by the addition of saturated NaHCO3 and water. The layers were separated and the organic layer was washed with saturated Na2S2O3, saturated NaHCO3, and brine, and dried over Na2SO4. The crude product was purified by silica gel column chromatography (CH2Cl2 / MeOH = 5 / 95) to give Ref-2-2c (80 mg, 80% yield) as a white solid.

[0162] MS (ESI) m / z: 1509.8 [M+H] + .

[0163] Step 4: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H -Benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((21S,24S)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecosano-25-amido)benzyl ester (Ref-2-2d) Ref-2-2c (20 mg, 0.013 mmol) was cooled to -3 °C. Separately, a solution of 95% TFA in H₂O (0.5 mL) was cooled to -3 °C and then added to Ref-2-2c. The reaction mixture was stirred at -3 °C for 30 min, then poured onto a 1:1 solution (16 mL) of CHCl₃: saturated NaHCO₃ at 0 °C. The organic layer was separated, dried over Na₂SO₄, filtered, and then removed under vacuum. The crude material was used directly in the next step without further purification.

[0164] MS (ESI) m / z: 1391.7 [M+H] + .

[0165] Step 5: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,1 1a-Tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid 4-((17S,20S)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diaza-eicosano-21-amido)benzyl ester (Ref-2-2e) Et₂NH (14 μL, 0.129 mmol) was added to a solution of crude Ref-2-2d (18 mg, 0.013 mmol) in 0.5 mL DMF. The mixture was stirred at room temperature for 0.5 hours. After the reaction was complete, the sample was analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified to give Ref-2-2e (5 mg, 33% yield) using a mobile phase of 150 mm (A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min).

[0166] MS (ESI) m / z: 1169.7 [M+H] + .

[0167] Step 6: (11S,11aS)-11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[ 1,2-a][1,4]diaza-10(5H)-formic acid 4-((21S,24S)-1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl ester (Ref-2-2) DIEA (8.6 μL, 0.05 mmol) was added to Ref-2-2e (14.5 mg, 0.012 mmol) and Int 4 (10.8 mg, 0.037 mmol, according to...) J. Am. Chem. Soc. Prepared according to the procedure described in 2020, 142, 20, 9285-9301) in 1 mL of DMF solution. The mixture was stirred at room temperature for 20 min. The solution was then analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). The mixture was purified to give Ref-2-2 (7.9 mg, 46% yield) as a light gray solid. 150 mm; mobile phase: A-water (without formic acid): B-acetonitrile; flow rate: 20 mL / min)

[0168] MS (ESI) m / z: 1345.7 [M+H] + .

[0169] Example 4: Synthesis of test compounds Table 1 below summarizes the test connector-payload compounds.

[0170] Example 2-1 Step 1: (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidone-1-yl)(4-hydroxy-5-methoxy-2-nitrophenyl) ketone (2-1b) t-BuOK (6.95 g, 61.98 mmol) was added to a mixture of MePh3PBr (24.6 g, 68.86 mmol) in THF (100 mL) at 0 °C under N2. The mixture was stirred at 0 °C for 2 h, and then a solution of 2-1a (4 g, 6.89 mmol) in THF (30 mL) was added dropwise to the mixture and stirred at 0 °C for 16 h. The mixture was neutralized with citric acid and extracted with EtOAc (3 x 100 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product, which was purified by silica gel column chromatography (eluent: hexane / EtOAc = 100 / 0 to 25 / 75) to give 2-1b (1.1 g, 37.8% yield) as a yellow solid.

[0171] MS (ESI) m / z: 423.2 [M+H] + .

[0172] Step 2: Bis(2-(4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxy-5-nitrophenoxy)ethyl)carbamate (2-1c) Int-5 (400 mg, 0.8 mmol) and 2-1b (747.5 mg, 1.77 mmol) were mixed in anhydrous DMSO (2 mL). K2CO3 (333.6 mg, 2.42 mmol) was added to the mixture. The mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction solution was then diluted with EtOAc (10 mL) and washed with H2O (10 mL) and brine (5 mL). The organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 25 / 75) to give 2-1c (250 mg, 31.1% yield) as a pale yellow solid.

[0173] MS (ESI) m / z: 998.7 [M+H] + .

[0174] Step 3: Bis(2-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (2-1d) Zn powder (618.7 mg, 9.5 mmol) was added to a solution of EtOH (3 mL), AcOH (200 μL), and H₂O (200 μL). The mixture was stirred at room temperature for 10 min. A solution of 2-1c (250 mg, 0.26 mmol) in EtOH (2 mL) was added to the reaction mixture. The mixture was stirred at room temperature for 1 hour. The mixture was filtered to remove excess Zn, and the filtrate was concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (MeOH / CH₂Cl₂ = 5 / 95) to give 2-1d (220 mg, 92% yield) as a yellow oil.

[0175] MS (ESI) m / z: 938.8 [M+H] + .

[0176] Step 4: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-((2-(5-((((4-(((2S,3R,4R,5S,6S)-5-acetoxy-3,4-diacetyl-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy) Methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)((allyloxy)carbonyl)amino)ethoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (2-1e) 2-1d (220 mg, 0.24 mmol) was dissolved in anhydrous THF (2 mL), and 4 Å molecular sieve (200 mg) was added. The mixture was cooled to 0 °C, and then triphosgene (50.2 mg, 0.17 mmol) was added, followed by triethylamine (145 μL, 1.03 mmol). The mixture was stirred at 0 °C under a nitrogen atmosphere for 10 min. Int-6 (238 mg, 0.54 mmol), triethylamine (99 μL, 0.7 mmol), and dibutyltin dilaurate (55.6 μL, 0.09 mmol) were mixed in anhydrous THF (1 mL), and this solution was then gradually added to the above reaction solution at 0 °C. The reaction solution was heated to room temperature and stirred for 2 hours. The reaction solution was concentrated and then purified by column chromatography (MeOH / CH2Cl2 = 5 / 95) to obtain 2-1e (430 mg, 98% yield) as a pale yellow solid.

[0177] MS (ESI) m / z: 1871.4 [M+H] + .

[0178] Step 5: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-((2-(5-((((4-(((2S,3R,4R,5S,6S)-5-acetoxy-3,4-diacetyl-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilane) 4-Methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-1f) Pd(PPh3)4 (5 mg, catalytic amount) and pyrrolidine (48 μL, 0.58 mmol) were added to a solution of 2-1e (430 mg, 0.23 mmol) in 3 mL of CH2Cl2 at 0 °C. The reaction solution was stirred at room temperature for 1 hour. The organic solvent was removed by evaporation. The crude product was purified by silica gel column chromatography (MeOH / CH2Cl2 = 5 / 95) to give 2-1f (350 mg, 85.2%) as a white solid.

[0179] MS (ESI) m / z: 1787.4 [M+H] + .

[0180] Step 6: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(5-((((4-(((2S,3R,4R,5S,6S)-5-acetoxy-3,4-diacetyl-6-(methoxycarbonyl)) Tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-1g) DIEA (102 mg, 0.59 mmol) was added to a solution of 2-1f (350 mg, 0.2 mmol), Int-7 (185.4 mg, 0.2 mmol), HOBt (8 mg, 0.059 mmol), and DMF (5 mL). The mixture was stirred at room temperature for 5 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 15 / 85) to give 2-1 g (240 mg, 47.6% yield) as a white solid.

[0181] MS (ESI) m / z: 2575.8 [M+H] + .

[0182] Step 7: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(5-((((4-(((2S,3R,4R,5S,6S)-5-acetoxy-3, 4-Diacetyl-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-1h) 2-1 g (240 mg, 0.09 mmol) of the compound was dissolved in THF / H2O (2 mL / 40 μL, 50 / 1), and TsOH was added. . H2O (35 mg, 0.18 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The solvent was then removed by evaporation, and the residue was purified by column chromatography (MeOH / CH2Cl2 = 7 / 93) to give a white solid of 2–1 h (135 mg, 61.7% yield).

[0183] MS (ESI) m / z: 2347.1 [M+H] + .

[0184] Step 8: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((11S,11aS)-8-(2-((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(((11S,11aS)-10-(((4-(((2S,3R,4R,5S,6S)-5-acetoxy-3,4-diacetyl-6-(methoxycarbonyl)tetrahydro-2H-pyridine) 11-H-(2-yl)oxy)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)ethyl)amino)ethoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-1i) Des Martin periodane (61 mg, 0.14 mmol) was added to a solution of 2-1h (135 mg, 0.06 mmol) in CH2Cl2 (1 mL) at room temperature, and the mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction solution was then diluted with CH2Cl2 (10 mL) and washed with saturated Na2S2O3 (5 mL) and brine (5 mL). The organic phase was collected, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (MeOH / CH2Cl2 = 8 / 92) to give 2-1i as a yellow solid (105 mg, 77.8% yield).

[0185] MS (ESI) m / z: 2343.3 [M+H] + .

[0186] Step 9: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((((11S,11aS,11'S,11a'S)-8,8'-((((((4-((17R,20R)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosode-21-amido)benzyl)oxy)carbonyl )azadiyl)bis(ethane-2,1-diyl))bis(oxy))bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazadi-8,10-diyl-10-carbonyl))bis(oxy))bis(methylene))bis(4,1-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-1j) 2-1i (105 mg, 0.045 mmol) was added to a solution of saturated LiBr (2 mL) in CH3CN / H2O (10 / 1, v / v) and triethylamine (0.5 mmol, 62 μL). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 2 hours. The solvent was removed by evaporation to give a crude product as a yellow oil, which was used directly in the next step without further purification.

[0187] Diethylamine (0.45 mmol, 46.2 μL) was added to 1 mL of DMF containing the crude product described above at 0 °C. The reaction solution was stirred at 0 °C for 0.5 h. The reaction solution was neutralized with AcOH and then analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). 1250 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) purification, after lyophilization, provided 2-1j (26 mg, 31.7% yield) as a white solid.

[0188] MS (ESI) m / z: 1841.2 [M+H] + .

[0189] Step 10: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((((11S,11aS,11'S,11a'S)-8,8'-((((((4-((21R,24R)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazadi Pentadecane-25-amido)benzyl)oxy)carbonyl)azadiyl)bis(ethane-2,1-diyl))bis(oxy))bis(11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazadi-8,10-diyl-10-carbonyl))bis(oxy))bis(methylene))bis(4,1-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-1) Int-4 (4.1 mg, 0.014 mmol) and DIPEA (6.4 μL, 0.035 mmol) were added to a solution of 2-1j (13 mg, 0.007 mmol) in 0.5 mL of DMF at 0 °C. The reaction solution was stirred at 0 °C for 1 hour. The reaction solution was neutralized with AcOH and then analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). 150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) purification, after lyophilization, provides 2-1 as a white solid (4.4 mg, 30.9% yield).

[0190] MS (ESI) m / z: 2017.0 [M+H] + .

[0191] Example 2-2 Step 1: Bis(2-(5-amino-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)carbamate (2-2b) K₂CO₃ (422 mg, 3.06 mmol) was added to a solution of Int-5 (505 mg, 1.02 mmol), 2-2a (826 mg, 2.03 mmol) (prepared according to a similar procedure as described in CN111164208A), and DMSO (10 mL). The mixture was stirred overnight at 50 °C. After the reaction was complete, the mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic matter was washed with brine (20 mL), dried over Na₂SO₄, filtered, and the filtrate was concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 10 / 90) to give 2-2b (200 mg, 20.3% yield) as a yellow solid.

[0192] MS (ESI) m / z: 966.9 [M+H] + .

[0193] Step 2: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-(((allyloxy)carbonyl)(2-(4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxy) Carbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)ethyl)amino)ethoxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (2-2c).

[0194] Compound 2-2c (267 mg, 61.7% yield) was synthesized according to the synthetic procedure in step 4 of Example 2-1.

[0195] MS (ESI) m / z: 1899.0 [M+H] + .

[0196] Step 3: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-((2-(4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetra- Hydrogen-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)ethyl)amino)ethoxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2d) Compound 2-2d (320 mg) was synthesized according to the synthetic procedure in step 5 of Example 2-1.

[0197] MS (ESI) m / z: 1815.4 [M+H] + .

[0198] Step 4: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methyl oxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)ethyl)amino)ethoxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2e) DIEA (180 mg, 0.618 mmol) was added to a solution of 2-2d (374 mg, 0.206 mmol), Int-7 (191 mg, 0.206 mmol), HOBt (8 mg, 0.059 mmol), and DMF (5 mL). The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (petroleum ether / EtOAc = 10 / 90) to give 2-2e (300 mg, 25.6% yield) as a yellow solid.

[0199] MS (ESI) m / z: 2603.7 [M+H] + .

[0200] Step 5: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(4-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methyl oxy-5-((((4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)phenoxy)ethyl)amino)ethoxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2f) Compound 2-2f (115 mg, 42.1% yield) was synthesized according to the synthetic procedure in step 7 of Example 2-1.

[0201] MS (ESI) m / z: 2375.6 [M+H] + .

[0202] Step 6: Triacetic acid (2S,3R,4S,5S,6S)-2-(4-((((11S,11aS)-8-(2-((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(((11S,11aS)-10-(((4-(((2S,3R,4R,5S,6S)-4,5-diacetoxy-3-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl) )oxy)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)ethyl)amino)ethoxy)-11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-2g) 2-2 g (120 mg, crude substance) of compound were synthesized according to the synthesis procedure of step 8 in Example 2-1.

[0203] MS (ESI) m / z: 2371.2 [M+H] + .

[0204] Step 7: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((((11S,11aS,11''S,11a''S)-8,8''-((((((4-((17R,20R)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosode-21-amido)benzyl)oxy Carbonyl) Azadiyl) bis(ethane-2,1-diyl) bis(oxy) bis(11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazadi-2,1'-cyclopropane]-10-carbonyl) bis(oxy) bis(methylene) bis(4,1-phenylene) bis(oxy) bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-2h) The compound was synthesized for 2-2 hours (24 mg, 25.3% yield) according to the synthetic procedure of step 9 in Example 2-1.

[0205] MS (ESI) m / z: 1868.5 [M+H] + .

[0206] Step 8: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6S,6'S)-6,6'-((((((11S,11aS,11''S,11a''S)-8,8''-((((((4-((21R,24R)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23 ... azapentadecane-25-amido)benzyl)oxy)carbonyl)azadiyl)bis(ethane-2,1-diyl))bis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl))bis(oxy))bis(methylene))bis(4,1-phenylene))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-2) Compound 2-2 (11.4 mg, 43.2% yield) was synthesized according to the synthetic procedure of step 10 in Example 2-1.

[0207] MS (ESI) m / z: 2045.4 [M+H] + .

[0208] Example 2-3 Step 1: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-acetamido-3-methylbutamido)-3-((4-((((5-(2-((2-(5-((((4-((S)-2-((S)-2-acetamido-3-methylbutamido)-3-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)propamido)benzyl)oxy)carbonyl)amino)-4-( (S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)((allyloxy)carbonyl)amino)ethoxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (2-3a) Triphosgene (19 mg, 0.065 mmol) and TEA (50 μL, 0.36 mmol) were added to a mixture of 2-2b (78 mg, 0.081 mmol) and anhydrous THF (3 mL) at 0 °C. The mixture was stirred at 0 °C under N2 for 10 min, followed by the addition of a mixture of Int-8 (135 mg, 0.202 mmol), TEA (34 μL, 0.24 mmol), DBTDL (10 mg, 0.016 mmol), and THF (2 mL). The mixture was stirred overnight at room temperature under N2. The mixture was filtered and the filtrate was concentrated under vacuum to obtain the residue, which was purified by silica gel column chromatography (dichloromethane / methanol = 98 / 2) to give 2-3a (141 mg, 74.2% yield) as a yellow solid.

[0209] MS (ESI) m / z: 2352.5 [M+H] + .

[0210] Step 2: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-2-((S)-2-acetamido-3-methylbutamido)-3-((4-((((5-(2-((2-(5-((((4-((S)-2-((S)-2-acetamido-3-methylbutamido)-3-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)propamido)benzyl)oxy)carbonyl)amino )-4-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-trimethyl ester (2-3b) Add 5,5-dimethylcyclohexane-1,3-dione (21 mg, 0.15 mmol) and Pd(PPh3)4 (7 mg, 0.006 mmol) to a mixture of 2-3a (141 mg, 0.06 mmol) and THF (2 mL). Stir the mixture at room temperature under N2 for 2 hours. Filter the mixture. Concentrate the filtrate under vacuum to obtain the residue, which is purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give 2-3b (80 mg, 58.8% yield) as a yellow solid.

[0211] MS (ESI) m / z: 2269.3 [M+H] + .

[0212] Step 3: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-3-((4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(5-((((4-(((S)-2-((S)-2-acetamido-3-methylbutamido)-3-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy) -6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)propamido)benzyl)oxy)carbonyl)amino)-4-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-2-((S)-2-acetamido-3-methylbutamido)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-3c) DIPEA (5 μL, 0.03 mmol) was added to a solution of 2-3b (20 mg, 0.0088 mmol), Int-7 (826 mg, 2.03 mmol), HOBt (4 mg, 0.004 mmol), and DMF (1 mL). The mixture was stirred overnight at room temperature. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give 2-3c (26 mg, 96.7% yield) as a yellow solid.

[0213] MS (ESI) m / z: 1529.4 [1 / 2M+H] + .

[0214] Step 4: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-3-((4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(5-((((4-(((S)-2-((S)-2-acetamido-3-methylbutamido)-3-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy) -6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)propamido)benzyl)oxy)carbonyl)amino)-4-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-2-((S)-2-acetamido-3-methylbutamido)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-3d) PTSA (3.4 mg, 0.018 mmol) was added to a mixture of 2-3c (27 mg, 0.0088 mmol) and THF (1 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under vacuum to obtain a residue, which was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give 2-3d (15 mg, 60.0% yield) as a yellow solid.

[0215] MS (ESI) m / z: 2830.1 [M+H] + .

[0216] Step 5: Triacetic acid (2R,3R,4S,5S,6S)-2-((S)-3-((4-((((5-(2-(((((4-((21R,24R)-1-(9H-fluorene-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy)carbonyl)(2-(5-((((4-(((S)-2-((S)-2-acetamido-3-methylbutamido)-3-(((2R,3R,4S,5S,6S)-3,4,5-triacetoxy) -6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)propamido)benzyl)oxy)carbonyl)amino)-4-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-2-methoxyphenoxy)ethyl)amino)ethoxy)-2-((S)-6-(hydroxymethyl)-5-azaspiro[2.4]heptane-5-carbonyl)-4-methoxyphenyl)carbamoyl)oxy)methyl)phenyl)amino)-2-((S)-2-acetamido-3-methylbutamido)-3-oxopropoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl ester (2-3e) BAIB (23 mg, 0.071 mmol) and TEMPO (1.1 mg, 0.0071 mmol) were added to a mixture of 2-3d (67 mg, 0.024 mmol) and dichloromethane (2 mL). The mixture was stirred at room temperature for 4 hours. The mixture was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5) to give 2-3e as a yellow solid (40 mg, 95.7% yield).

[0217] MS (ESI) m / z: 2826.1 [M+H] + .

[0218] Step 6: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6R,6'R)-6,6'-(((2S,2'S)-(((((11S,11aS,11''S,11a''S)-8,8''-(((((4-((17R,20R)-1-amino-17-isopropyl-20-methyl-15,18-dioxo-3,6,9,12-tetraoxa-16,19-diazaeicosode-21-amido)benzyl)oxy)carbonyl)azadiyl)bis(ethane-2,1-di) bis(11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-10-carbonyl) bis(methylene) bis(4,1-phenylene) bis(azadiyl) bis(2-((S)-2-acetamido-3-methylbutamido)-3-oxopropane-3,1-diyl) bis(oxy) bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-3f) DEA (7 μL, 0.07 mmol) was added to a solution of 2-3e (10 mg, 0.0035 mmol) and DMF (1 mL). The mixture was stirred at room temperature for 1 hour and concentrated under vacuum to obtain a residue. The residue was dissolved in saturated LiBr (1 mL, acetonitrile / water = 10:1), and then TEA (5 μL, 0.04 mmol) was added to the mixture. The mixture was stirred at room temperature for 30 min. The mixture was adjusted to pH 7 using AcOH and filtered, and then analyzed by preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). 150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) purified filtrate, providing 2-3 f (2 mg, 25.0% yield) as a white solid.

[0219] MS (ESI) m / z: 2322.7 [M+H] + .

[0220] Step 7: (2S,2'S,3S,3'S,4S,4'S,5R,5'R,6R,6'R)-6,6'-(((2S,2'S)-(((((11S,11aS,11''S,11a''S)-8,8''-(((((4-((21R,24R)-1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-21-isopropyl-24-methyl-3,19,22-trioxo-2,7,10,13,16-pentaoxa-4,20,23-triazapecopentane-25-amido)benzyl)oxy )carbonyl)azadiyl)bis(ethane-2,1-diyl))bis(oxy))bis(11-hydroxy-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diazadi-2,1'-cyclopropane]-10-carbonyl))bis(oxy))bis(methylene))bis(4,1-phenylene))bis(azadiyl))bis(2-((S)-2-acetamido-3-methylbutamido)-3-oxopropane-3,1-diyl))bis(oxy))bis(3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-3) Add DIPEA (2 μL, 0.01 mmol) to a mixture of 2-3f (5 mg, 0.0022 mmol), Int-4 (1.2 mg, 0.0043 mmol), and DMF (1 mL). Stir the mixture at room temperature for 30 min. Adjust the pH to 7 using AcOH, filter, and perform preparative HPLC (method: column: XBridge Prep C18 OBD 5 μm 19). 150 mm; mobile phase: A-water (0.1% formic acid): B-acetonitrile; flow rate: 20 mL / min) purified filtrate, providing 2-3 (3 mg, 54.5% yield) as white solid.

[0221] MS (ESI) m / z: 2500.1 [M+H] + .

[0222] Table 1: Joint effective load structure and corresponding released free effective load

[0223] Example 5: Preparation and Characterization of Antibody-Drug Conjugates (ADCs) Drug: Antibody Ratio (DAR) 2 ADC Preparation. Anti-CD74 antibody mAb1 SP7219 (0.5-25 mg / mL, 50 mM Tris-HCl buffer, pH 7.0-8.5) in reaction buffer was incubated with 1 / 2000-1 / 500 w / w (EndoS2 / mAb weight ratio) endoS2 at reaction temperature (0-40℃) for 1-24 hours to hydrolyze the glycoform present at the Fc N-glycosylation site. 2-40 eq. UDP-GalNAz (20 mM) and 0.1 w / w%-10 w / w% (GalT / mAb weight ratio) β1,4-GalT were added to the reaction mixture and incubated at reaction temperature (0-40℃) in reaction buffer (50 mM Tris-HCl buffer, pH 7.0-8.5, 20 mM MnCl2) for 8-24 hours. The reaction mixture was purified with protein A resin to obtain mAb1-GalNAz (N-azidoacetylgalactosamine modified).

[0224] Organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and adapter-payload stock solution (10-25 eq, 10 mM stock solution in organic solvent) are incrementally added to reaction buffer (PBS buffer pH 7.0-8.5) containing mAb1-GalNAz (1-20 mg / mL) for 0.5-24 hours at 0-25°C. The solution is then submitted for buffer exchange (rotary desalting column, ultrafiltration, and dialysis) to storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween 20, 60, 80).

[0225] ADC Characterization. All ADCs were characterized using the following analytical methods. The DAR of the ADCs was determined using either the LCMS method or the HIC method.

[0226] DAR confirmed LCMS method. LC-MS analysis was performed under the following measurement conditions: LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac™ RP, 2.1 50 mm, 4 μm, 1,500 Å, Thermo Scientific™ Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) aqueous solution Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient program: 25% B-25% B (0 min-2 min), 25% B-50% B (2 min-18 min), 50% B-90% B (18 min-18.1 min), 90% B-90% B (18.1 min-20 min), 90% B-25% B (20 min-20.1 min), 25% B-25% B (20.1 min-25 min) Injected sample volume: 1 μg MS parameters: Full and denatured MS data were acquired in HMR mode with R=15k, and deconvolution was performed using the ReSpect™ algorithm and sliding window integration in ThermoScientific™ BioPharma Finder™ 4.0 software.

[0227] HIC method. HPLC analysis was performed under the following measurement conditions: HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID×3.5 cm, 2.5 μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient program: 0% B - 0% B (0 min - 2 min), 0% B - 100% B (2 min - 15 min), 100% B - 100% B (15 min - 16 min), 100% B - 0% B (16 min - 17 min), 0% B - 0% B (17 min - 20 min) Injected sample volume: 20 μg ADC purity SEC method. HPLC analysis was performed under the following measurement conditions: HPLC system: Waters H-Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7 μm 4.6x150 mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropanol, pH 7.0 Gradient program: 10 min isocratic elution at a flow rate of 0.3 mL / min Injected sample volume: 20 μg The SEC purity of the ADCs is all >95%.

[0228] ADC hydrophobicity assessment ADCs with higher hydrophobicity exhibit later retention times in hydrophobic interaction column (HIC) chromatography. This comparison used the DAR2 peak of an example ADC.

[0229] HIC Method 1. HPLC analysis was performed under the following measurement conditions: HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID×3.5 cm, 2.5 μm butyl nonporous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (v / v) isopropanol, pH 7.0 Gradient program: 0% B - 0% B (0 min - 2 min), 0% B - 100% B (2 min - 15 min), 100% B - 100% B (15 min - 16 min), 100% B - 0% B (16 min - 17 min), 0% B - 0% B (17 min - 20 min) Injected sample volume: 20 μg HIC Method 2. HPLC analysis was performed under the following measurement conditions: HPLC System: Waters ACQUITY ARC HPLC System Detector: Measurement wavelength: 280 nm Column: MABPac HIC-10, 5 μm, 4.6×10 mm (Thermo) Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20% B - 20% B (0 min - 1 min), 0% B - 0% B (1 min - 35 min), 20% B - 20% B (35 min - 40 min) Flow rate: 0.5 mL / min Sample preparation: Dilute the sample to 0.5 mg / mL with the initial mobile phase.

[0230] Table 2: ADCs (The Ab in ADC3-A, ADC3-B, ADC3-1, ADC3-2, and ADC3-3 is SP7219; the Ab in the same type of ADC is CB6)

[0231] antibody sequence SP7219 wt sequence (anti-CD74 antibody) Light chain sequence (SEQ ID NO: 1) DIQMTQSPSSVSASVGDRVTITCRASQGIGSWLAWYQQKPGKAPKLLIYAADRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHTYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFNFSDYGMHWVRQAPGKGLEWVAVIWYDGSISYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGTVEHGAVYGTDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CB6 sequence (isotype antibody) Light chain sequence (SEQ ID NO: 3) DIVMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPEYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKGLEWVSVIYSGGSTFYADSVKGRFTISRDNSMNTLFLQMNSLRAEDTAVYYCARVLPMYGDYLDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPPAAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 6: Direct killing effect of payload in A375 and Calu-6 cancer cell lines cell lines A375 (ATCC, CRL-1619). A375 is a cell line exhibiting epithelial morphology isolated from the skin of a 54-year-old female patient with malignant melanoma, and was purchased from ATCC. The basal medium for A375 was DMEM, high glucose, and GlutaMAX™ supplement (Gibco, 10566024). To prepare complete growth medium, fetal bovine serum was added to the basal medium at a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0232] Calu-6 (ATCC, HTB-56). Calu-6 was purchased from ATCC and was an epithelial cell line isolated from a 61-year-old white female patient with anaplastic carcinoma. Calu-6 was basaled in Eagle's minimum essential medium (ATCC, 30-2003). To prepare complete growth medium, fetal bovine serum was added to the basal medium at a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was periodically tested using a MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0233] Methods. The direct killing effect of the payload was evaluated in the A375 and Calu-6 cancer cell lines. Cells were seeded (1E3 / well for A375 or 2E3 / well for Calu-6) into 96-well plates (Greiner: 655090), 100 µl / well, and incubated overnight at 37°C and 5% CO2. Fresh growth medium containing different concentrations of the payload compound was added, 50 µl / well, and incubated for 6 days at 37°C and 5% CO2. Cell viability was assessed using Cell Titer-Glo (Promega, G7573), 70 µl / well. The plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal. The plates were analyzed using a microplate reader.

[0234] The data is summarized in Table 3 and Figure 1 and Figure 2 The results showed that, compared with the reference payloads Ref-1-1 and Ref-1-2, payloads P1-1 and P1-2 exhibited less direct cell killing in A375 and Calu-6 cell lines. The data indicate that payloads P1-1 and P1-2 alone were less toxic to cancer cells than the reference payloads.

[0235] Table 3: Cell-killing effect of payload on A375 and Calu-6 cells

[0236] Example 7: Direct killing of NOMO-1 and K562 cancer cell lines by ADC cell lines NOMO-1 (JCRB, IFO50474). NOMO-1 is a cell line exhibiting hematopoietic lymphocyte morphology, and NOMO-1 was purchased from JCRB. The basal medium was RPMI-1640 medium prepared by ATCC, ATCC 30-2001. To prepare complete growth medium, fetal bovine serum was added to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was tested periodically using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0237] K562 (ATCC, CCL-243). K562 is a cell line exhibiting lymphoblastic morphology, and K562 was purchased from ATCC. The basal medium for K562 was Iscove modified Dulbecco medium prepared by ATCC, catalog number 30-2005. To prepare complete growth medium, fetal bovine serum was added to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humid 5% CO2 atmosphere, and the presence of mycoplasma was tested periodically using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0238] Table 4: CD74 expression levels

[0239] Methods. NOMO-1 (6E3 / well) or K562 (3E3 / well) cells were seeded at 100 µl / well into 96-well plates (Greiner: 655090). 100 µg / ml Fc inhibitor was added to the NOMO-1 cells. The cells were incubated at 37°C and 5% CO2 for 2 h.

[0240] Add fresh growth medium containing different concentrations of ADC, 50 µl / well. Incubate cells at 37°C and 5% CO2 for 6 days.

[0241] Cell viability was assessed using Cell Titer-Glo (Promega, G7573). Cell Titer-Glo® reagent was added at 70 µL / well. The plates were incubated at room temperature for 10 minutes to stabilize the luminescence signal. The plates were then analyzed using a microplate reader.

[0242] The data is presented in Tables 5 and 6, and Figures 3A-4BThe results showed that ADC3-1 and ADC3-2 (each with a prodrug design) exhibited similar killing efficacy in NOMO-1 cells (CD74-positive cell line) compared to reference ADC3-A and ADC3-B. In K562 cells (CD74-negative cell line), ADC3-1 and ADC3-2 did not show killing activity, while ADC3-A and ADC3-B showed similar non-specific killing activity compared to their counterparts. The current data are presented in conjunction with Table 3 and... Figure 1-2 The direct killing results of the payload shown indicate that PBD dimer-based ADCs with prodrug design can minimize non-specific killing of antigen-negative cells while maintaining activity against antigen-positive cells, and that any payload released prematurely in systemic circulation may cause less damage to healthy tissues.

[0243] Table 5: Cell killing effect of ADCs on NOMO-1 and K562 cells

[0244] Table 6: Cell killing effect of ADCs on NOMO-1 and K562 cells

[0245] Although the foregoing disclosure has been presented in considerable detail through illustration and examples for the purpose of clear understanding, it will be apparent to those skilled in the art that some minor changes and modifications will be made. Therefore, the descriptions and embodiments described should not be considered limiting.

[0246] It should be understood that if any prior art publications are mentioned herein, such mentions do not constitute an acknowledgment that such publications constitute part of the general knowledge of the field in any country.

[0247] All non-patent publications, patents, patent applications, and published patent applications cited in this document are hereby incorporated herein in their entirety by way of citation.

Claims

1. A compound of formula (I): ， Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A and structure B are each independently selected from one of the following formulas: (IIa) or (IIb); Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; -C(R 1 )- and -N(R 2 Between ) It can be a single bond or a double bond independently; when When it is a single bond, each R 1 Independently, it is H or OH, and each R 2 It is H; when When it is a double bond, each R 1 It is H, and each R 2 It does not exist; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7, or 8; and The sum of p and q is 2, 3, 4, 5, 6, 7 or 8.

2. The compound of claim 1, wherein either or both of structure A and structure B are .

3. The compound of claim 1, wherein either or both of structure A and structure B are .

4. The compound of claim 1, wherein one of structure A and structure B is And the other one in structure A and structure B is .

5. The compound according to any one of claims 1 to 4, wherein both p and q are 2.

6. The compound according to any one of claims 1 to 3 and 5, wherein the compound has the following formula: or .

7. The compound according to any one of claims 1 to 6, wherein the conjugate has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; and AA is an amino acid or its stereoisomer that exists independently each time it appears.

8. The compound according to any one of claims 1 to 7, wherein the conjugate has the following formula: 。 9. The compound according to any one of claims 1 to 8, wherein the conjugate has the following formula: 。 10. The compound according to any one of claims 1 to 9, wherein the compound has one of the following formulas: or , Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

11. A compound of formula (V): ， Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ;and Each Su is an independent sugar component.

12. The compound of claim 11, wherein either or both of structure A' and structure B' are .

13. The compound of claim 11, wherein either or both of structure A' and structure B' are .

14. The compound according to any one of claims 11-13, wherein one of structure A' and structure B' is And the other one of structure A' and structure B' is .

15. The compound according to any one of claims 11 to 14, wherein both p and q are 2.

16. The compound according to any one of claims 11 to 13 and 15, wherein the compound has the following formula: or .

17. The compound according to any one of claims 10 to 15, wherein the conjugate has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; and AA is an amino acid or its stereoisomer that exists independently each time it appears.

18. The compound of any one of claims 11 to 17, wherein the conjugate has the following formula: 。 19. The compound according to any one of claims 11 to 18, wherein the conjugate has the following formula: 。 20. The compound according to any one of claims 11 to 19, wherein... Each sugar component is independent , , or or its stereoisomers; and Each m is independently 0 or 1.

21. The compound of claim 20, wherein the sugar moiety is And m is 0.

22. The compound according to any one of claims 11 to 21, wherein each R 2’ Independently or .

23. The compound of claim 22, wherein each R 2’ Independently or .

24. The compound of any one of claims 11 or 15 to 23, wherein the compound has one of the following formulas: , or , Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

25. An antibody-drug conjugate (ADC) of formula (VI): ， Or its pharmaceutically acceptable salts, tautomers, solvates, or stereoisomers. Structure A' and structure B' are each independently selected from one of the following formulas: (IIa') or (IIb'), Ring D is a cyclopropyl ring or a cyclobutyl ring; Each m and n is independently 1 or 2; Each R 1 It can be H or OH independently; Structure C is a fusible linker; p and q are independently 1, 2, 3, 4, 5, 6, 7 or 8; The sum of p and q is 2, 3, 4, 5, 6, 7 or 8; Each R 2’ Independently or ; Each Su is an independent sugar component; Ab is a humanized, chimeric, or human antibody or its antigen-binding fragment; and x is approximately 1 to approximately 8.

26. The ADC of claim 25, wherein either or both of structure A' and structure B' are .

27. The ADC of claim 25, wherein either or both of structure A' and structure B' are .

28. The ADC of claim 25, wherein one of structure A' and structure B' is And the other one of structure A' and structure B' is .

29. The ADC of any one of claims 25 to 28, wherein both p and q are 2.

30. The ADC of any one of claims 25 to 27 and 29, wherein the ADC has the following formula: or .

31. The ADC of any one of claims 25 to 30, wherein the conjugate has the following formula: r is 1, 2, 3, 4, 5, or 6; s is 1, 2, or 3; Each time AA appears, it is an independently naturally occurring amino acid or its stereoisomer; It is the connection point with Ab.

32. The ADC as claimed in any one of claims 25 to 31, wherein The fascicle has the following formula: .

33. The ADC of any one of claims 25 to 32, wherein the conjugate has the following formula: 。 34. The ADC of any one of claims 25 to 33, wherein The sugar portion is , , or or its stereoisomers; and Each m is independently 0 or 1.

35. The ADC of any one of claims 25-34, wherein the sugar moiety is And m is 0.

36. The ADC of any one of claims 25 to 35, wherein each R 2’ Independently or .

37. The ADC of any one of claims 25 to 36, wherein each R 2’ Independently or .

38. The ADC of any one of claims 25 or 29 to 37, wherein the ADC has one of the following formulas: 、 or ， Or its pharmaceutically acceptable salt, tautomer, solvate or stereoisomer.

39. The ADC of any one of claims 25 to 38, wherein x is about 2.

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