WO2025214466A1

WO2025214466A1 - Targeted pyrrolobenzodiazapine conjugates

Info

Publication number: WO2025214466A1
Application number: PCT/CN2025/088474
Authority: WO
Inventors: Charng-Sheng TSAI; Mei-Hsuan TSAI; Maomao HE; Zewei WANG; Xiaodong WEI; Xiaokun Yang
Original assignee: BeiGene Switzerland GmbH; Beigene Guangzhou Biologics Manufacturing Co Ltd
Current assignee: BeOne Medicines I GmbH; Beigene Guangzhou Biologics Manufacturing Co Ltd
Priority date: 2024-04-12
Filing date: 2025-04-11
Publication date: 2025-10-16
Anticipated expiration: 2026-10-12

Abstract

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

Description

TARGETED PYRROLOBENZODIAZAPINE CONJUGATES

1. CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of PCT Application No. PCT/CN2024/087468, filed April 12, 2024, entitled “Targeted Pyrrolobenzodiazapine Conjugates, ” which is hereby incorporated by reference in its entirety
2. REFERENCE TO ELECTRONIC SEQUENCE LISTING

The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said . XML copy, created on March 13, 2025, is named “F24W1548PR1. xml” and is 6 KB in size. The sequence listing contained in this . XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

3. FIELD

The present disclosure relates to targeted pyrrolobenzodiazepine (PBD) conjugates.

4. BACKGROUND

Some pyrrolobenzodiazepines (PBDs) have the ability to recognize and bind to specific sequences of DNA because their three-dimensional shape results in isohelicity with the minor groove of B-form DNA (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975) ; Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986) ) . Many PBDs have been reported, differing in the number, type, and position of substituents, though all with the following core structure:

In the B-ring there is an imine (N═C) , a carbinolamine (NH-CH (OH) ) , or a carbinolamine
methyl ether (NH-CH (OMe) ) at the N10-C11 position. This group is the electrophilic center responsible for alkylating DNA, and gives PBDs their cell-killing activity.

The biological activity of PBDs can be potentiated by joining two PBD units together through their C8/C′-hydroxyl functionalities via a flexible alkylene linker (Bose, D. S., et al., J. Am. Chem. Soc., 114, 4939-4941 (1992) ; Thurston, D.E., et al., J. Org. Chem., 61, 8141-8147 (1996) ) .

Due to the observed potency of PBD dimers, there exists a need for PBD dimers that can be conjugated to targeting agents for use in targeted therapy.
5. BRIEF SUMMARY

Described herein are compounds of the general structure:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein:
structure A and structure B are independently PBD residues, e.g., of formula (IIa) or (IIb) ; Linker is a covalent linking moiety; and
Conjugator is a conjugator group, e.g., as described herein.

Provided herein are compounds of Formula (I) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A and structure B is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is independently 1 or 2;
between -C (R¹) -and -N (R²) -is, independently, a single bond or a double bond;
whenis a single bond, each R¹ is independently H or OH, and each R² is H;
whenis a double bond, each R¹ is H, and each R² is absent;
structure C is a conjugator;
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.

Also provided herein are compounds of Formula (V) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is, independently, 1 or 2;
each R¹ is, independently, H or OH;
structure C is a conjugator;
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’ is, independently, and
each Su is independently a sugar moiety.

Also provided herein are antibody drug conjugates (ADCs) of Formula (VI) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is, independently, 1 or 2;
each R¹ is, independently, H or OH;
structure C is a conjugator;
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’ is, independently,
each Su is independently a sugar moiety;
Ab is a humanized, chimeric, or human antibody or an antigen binding fragment thereof; and
x is from about 1 to about 8.
6. BRIEF DESCRIPTION OF FIGURES

Figure 1 shows cellular killing by the payload compounds P1-1 and P1-2 described herein compared to reference compounds Ref-1-1 and Ref-1-2 as a percent of the negative control in A375 cells.

Figure 2 shows cellular killing by the payload compounds P1-1 and P1-2 described herein compared to reference compounds Ref-1-1 and Ref-1-2 as a percent of the negative control in Calu-6 cells.

Figure 3A shows cellular killing by the ADCs ADC3-B and ADC3-1 described herein compared to Isotype as a percent of the negative control in NOMO-1 cells.

Figure 3B shows cellular killing by the ADCs ADC3-B and ADC3-1 described herein compared to Isotype as a percent of the negative control in K562 cells.

Figure 4A shows cellular killing by the ADCs ADC3-A, ADC3-B, and ADC3-2 described herein compared to Isotype as a percent of the negative control in NOMO-1 cells.

Figure 4B shows cellular killing by the ADCs ADC3-A, ADC3-B, and ADC3-2 described herein compared to Isotype as a percent of the negative control in K562 cells.

7. DETAILED DESCRIPTION

Provided herein are pyrrolobenzodiazepine (PBD) dimers that include two PBDs, which may be the same or different, joined by a linker. The PBD dimers can be joined to a conjugator via the linker, which enables conjugation of the PBD dimers to a targeting agent, such as an antibody, e.g., to form an antibody drug conjugate (ADC) . Each PBD in the PBD dimer may be further modified with a cleavable group, such as a sugar-containing group. Also provided herein are ADCs comprising the PBD dimers. The ADCs may be used to treat a disease or disorder, such as cancer.
7.1. Definitions

In the present disclosure, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the event that there is a plurality of definitions for a term provided herein, these Definitions prevail unless stated otherwise.

When a trade name is used herein, reference to the trade name also refers to the product formulation, the generic drug, and the active pharmaceutical ingredient (s) of the trade name product, unless otherwise indicated by context.

As used herein, the term “conjugator, ” “conjugator group, ” or “conjugating group” refers to a chemical moiety having a functional group which is able to react with a corresponding functional group on a humanized, chimeric, or human antibody or an antigen binding fragment thereof, to create a covalent linkage. The functional group on the humanized, chimeric, or human antibody or an antigen binding fragment thereof may be a naturally occurring group (such as a thiol on a cysteine residue) , a synthetically-incorporated natural group (such as a poly-histidine sequence) , or a non-natural group (such as an azide) . Multiple conjugator groups and techniques for effecting conjugation are well known to those skilled in the art. Non-limiting examples of functional groups for use in conjugator groups include alkynes (e.g., for conjugation with azides) , maleimides (e.g., for conjugation with thiols) , and ortho-phosphino alkylbenzoates (e.g., for conjugation with azides) . Examples of conjugator groups can also be found in International Publication No. WO 2023/125530 (referred to therein as “covalent linkers” or “L” in the compound of Formula (II) disclosed therein) , the entirety of which is incorporated herein by reference.

As used herein, the term “amino acid” or “amino acid residue” refers to organic compounds that contain amine (-NH₂) and carboxyl (-COOH) functional groups, along with a side chain (R group) , which is specific to each amino acid. Amino acids may be proteinogenic or non-proteinogenic. By “proteinogenic, ” it is meant that the amino acid is one of the twenty naturally occurring amino acids found in proteins. The proteinogenic 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. By “non-proteinogenic, ” it is meant that either the amino acid is not found naturally in protein, or is not directly produced by cellular machinery (e.g., is the product of post-translational modification) . Non-limiting examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA) , taurine (2-aminoethanesulfonic acid) , theanine (L-γ-glutamylethylamide) , hydroxyproline, beta-alanine, ornithine, 4-azidophenylalanine, and citrulline.

The term “antibody” herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) , and antibody fragments that exhibit the desired biological activity. An intact antibody has primarily two regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region includes a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g., Janeway et al., 2001, Immuno. Biology, 5th Ed., Garland Publishing, New York) . An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass. The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. An antibody can be, for example, human, humanized, or chimeric.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.

An “intact antibody” is one that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3, and CH4, as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

An “antibody fragment” comprises a portion of an intact antibody, comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab’, F (ab’) 2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment (s) , a fragment (s) produced by a Fab expression library, or an epitope-binding fragment of any of the above which immunospecifically binds to a target antigen (e.g., a cancer cell antigen, a viral antigen or a microbial antigen) .

An “antigen” is an entity to which an antibody specifically binds.

The terms “specific binding” and “specifically binds” mean that an antibody or antibody derivative will bind, in a highly selective manner to its corresponding target antigen and not with the multitude of other antigens. Typically, the antibody or antibody derivative binds with an affinity of at least about 1×10^-7 M, 10^-8 M, 10^-9 M, 10 M, 10^-11 M, or 10^-12 M and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen.

The term “inhibit” or “inhibition of” means to reduce by a measurable amount, or to prevent entirely.

The term “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of a drug may, for example, reduce the number of cancer cells; reduce the tumor size; inhibit (e.g., slow to some extent or stop) cancer cell infiltration into peripheral organs; inhibit (e.g., slow to some extent or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may inhibit growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR) .

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

The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on a ligand drug conjugate (e.g., an antibody drug conjugate (ADC) ) , whereby the covalent attachment, e.g., the bond between the payload and the conjugator or conjugated antibody, is broken, resulting in the free drug, or another metabolite of the conjugate dissociated from the antibody inside the cell. The cleaved moieties of the drug-linker-ligand conjugate are thus intracellular metabolites.

The terms “cancer” and “cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells.

Examples of a “patient” or “subject” include, but are not limited to, mammals such as a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, or cat, and birds or fowl. In an embodiment, the patient is a human.

The terms “treat” or “treatment, ” unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder.

In the context of cancer, the term “treating” includes any or all of inhibiting growth of tumor cells, cancer cells, or of a tumor, inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

As used herein, and in the specification and the accompanying claims, the indefinite articles “a” and “an” and the definite article “the” include the plural as well as single referents, unless the context clearly indicates otherwise.

As used herein, and unless otherwise specified, the terms “about” and “approximately, ” when used in connection with amounts, or weight percentage of ingredients of a composition, mean an amount or weight percent that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified amount or weight percent. In certain embodiments, the terms “about” and “approximately, ” when used in this context, contemplate an amount or weight percent within 30%, within 20%, within 15%, within 10%, or within 5%, of the specified amount or weight percent.

As used herein, the term “pharmaceutically acceptable salt (s) ” refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base including an inorganic acid or base and an organic acid or base.

As used herein and unless otherwise indicated, the term “solvate” means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

As used herein and unless otherwise indicated, the term “hydrate” means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

As used herein and unless otherwise indicated, the term “prodrug” means a compound derivative that can hydrolyze, oxidize, or otherwise react 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 a compound that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.

As used herein and unless otherwise indicated, the term “stereoisomer” refers to a compound’s arrangement of atoms in three-dimensional space. Compounds which are said to be stereoisomers of each other have the same bonding structure but differ in the arrangement of the structure in three-dimensional space. Stereoisomers may arise from chiral atoms, chiral centers, E/Z-isomerism in alkenes, or rotationally locked bonds. Stereoisomers may be enantiomers, diastereomers, cis-trans isomers, atropisomers, epimers, or anomers.

As used herein and unless otherwise indicated, the term “stereomerically pure” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80%by weight of one stereoisomer of the compound and less than about 20%by weight of other stereoisomers of the compound, greater than about 90%by weight of one stereoisomer of the compound and less than about 10%by weight of the other stereoisomers of the compound, greater than about 95%by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater than about 97%by weight of one stereoisomer of the compound and less than about 3%by weight of the other stereoisomers of the compound. The compounds can have chiral centers and can occur as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms are included within the embodiments disclosed herein, including mixtures thereof. The use of stereomerically pure forms of such compounds, as well as the use of mixtures of those forms, are encompassed by the embodiments disclosed herein. For example, mixtures comprising equal or unequal amounts of the enantiomers of a particular compound may be used in methods and compositions disclosed herein. These isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, e.g., Jacques, J., et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981) ; Wilen, S.H., et al., Tetrahedron 33: 2725 (1977) ; Eliel, E.L., Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962) ; and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972) .

It should also be noted that the compounds can include E and Z isomers, or a mixture thereof, and cis and trans isomers, or a mixture thereof. In certain embodiments, the compounds are isolated as either the cis or trans isomer. In other embodiments, the compounds are a mixture of the cis and trans isomers.

“Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in an aqueous solution, pyrazoles may exhibit the following isomeric forms, which are referred to as tautomers of each other:

As readily understood by one skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism and all tautomers of the compounds are within the scope of the present disclosure.

It should also be noted the compounds can contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H) , iodine-125 (¹²⁵I) , sulfur-35 (³⁵S) , or carbon-14 (¹⁴C) , or may be isotopically enriched, such as with deuterium (²H) , carbon-13 (¹³C) , or nitrogen-15 (¹⁵N) . As used herein, an “isotopologue” is an isotopically enriched compound. The term “isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. The term “isotopic composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer and inflammation therapeutic 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 encompassed within the scope of the embodiments provided herein. In some embodiments, there are provided isotopologues of the compounds, for example, the isotopologues are deuterium, carbon-13, or nitrogen-15 enriched compounds.

It should be noted that if there is a discrepancy between a depicted structure and a name for that structure, the depicted structure is to be accorded more weight.

When shown in chemical structures herein, the symbol drawn perpendicular to another chemical bond refers to a point of attachment to a larger structure. As used herein, the structures containing the symbol will only have one point of attachment, and the larger structure to which it is attached will have this point clearly labeled or defined, typically with a unique variable. The symbol followed by other characters (for example ***) will be in reference to uniquely defined points of attachment as will be made clear in the context in which they are used.

As used herein, “sugar” or “sugar group” or “sugar residue” or “sugar moiety” refers to a carbohydrate moiety which may comprise 3-carbon (triose) units, 4-carbon (tetrose) units, 5-carbon (pentose) units, 6-carbon (hexose) units, 7-carbon (heptose) units, or combinations thereof, and may be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, an oligosaccharide, or any other polysaccharide. In some instances, a “sugar” or “sugar group” or “sugar residue” comprises furanoses (e.g., ribofuranose, fructofuranose) or pyranoses (e.g., glucopyranose, galactopyranose) , or a combination thereof. In some instances, a “sugar” or “sugar group” or “sugar residue” comprises aldoses or ketoses, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, glucosamine, glucuronic acid, glucuronamide, mannose, galactose, fructose, iduronic acid, iduronamide. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other “sugars” or “sugar groups” or “sugar residues” or “sugar moiety” include polysaccharides and/or oligosaccharides, including, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose. In some instances, a “sugar” or “sugar group” or “sugar residue” or “sugar moiety” is an amino-sugar. In some instances, a “sugar” or “sugar group” or “sugar residue” or “sugar moiety” is a glucamine residue (1-amino-1-deoxy-D-glucitol) linked to the rest of molecule via its amino group to form an amide linkage with the rest of the molecule (i.e., a glucamide) . A “sugar” or “sugar group” or “sugar residue” or “sugar moiety” may be a natural or artificial sugar or may be a derivatized natural sugar. A derivatization of a “sugar” or “sugar group” or “sugar residue” or “sugar moiety” may be, but is not limited to, removal of a hydroxyl (deoxygenation) , oxidizing of a hydroxy to an aldehyde, ketone, or carboxylic acid, substitution with an amine, inversion of a chiral center, or acylation.

As used herein, illustrations showing substituents bonded to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through a bond between ring atoms are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains.

The amino acid sequence of an antibody can be numbered using any known numbering schemes, including those described by 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 specified, the numbering scheme used herein is the Kabat numbering scheme. However, selection of a numbering scheme is not intended to imply differences in sequences where they do not exist, and one of skill in the art can readily confirm a sequence position by examining the amino acid sequence of one or more antibodies. Unless stated otherwise, the “EU numbering scheme” is generally used when referring to a residue in an antibody heavy chain constant region (e.g., as reported in Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ) .

As used herein, the term “cell-killing activity” refers to the activity that decreases or reduces the cell viability of the tested cell line.

In the claims that follow and in the preceding description, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments.
7.2. Conjugator-Functionalized PBD Dimer Complexes

Described herein are pyrrolobenzodiazepine (PBD) dimers covalently joined by a linker covalently joined to a conjugator group. In the conjugator-functionalized PBD dimers described herein, the conjugator group is joined to the PBD dimer via the linker. The conjugator component enables linking of the payload to a targeting group that gives selectivity of the complex in vivo. Such compounds have potent cell-killing effect on their own and can be used in antibody-drug conjugates, as described herein.

Described herein are compounds of Formula (I) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A and structure B is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is independently 1 or 2;
between -C (R¹) -and -N (R²) -is, independently, a single bond or a double bond;
whenis a single bond, each R¹ is independently H or OH, and each R² is H;
whenis a double bond, each R¹ is H, and each R² is absent;
structure C is a conjugator;
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.

In some embodiments of Formula (I) , either or both of structure A and structure B is

In some embodiments of Formula (I) , one of structure A and structure B is and the other of structure A and structure B is

In some embodiments of Formula (I) , p and q are both 2.

In some embodiments of Formula (I) , a compound has the following formula:

In some embodiments of Formula (I) , the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;
s is 1, 2, or 3; and
each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer
thereof.

In some embodiments of Formula (I) , the conjugator has the following formula:

In some embodiments of Formula (I) , a compound has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.
7.3. Conjugator-Functionalized PBD Dimer Complexes Containing Cleavable Sugar
Moieties
[0192] Also disclosed herein are conjugator-functionalized PBD dimers wherein each PBD is
independently linked to a cleavable sugar moiety. Such dimers have a prodrug design in that the cleavable sugar moieties are cleaved, such as by intracellular enzymes, to release the potent PBD dimer payloads described herein. For example, a conjugator-functionalized PBD dimer is released by action of β-GlcA at sites indicated with dotted lines and arrows in the structure below:

Also disclosed herein are compounds of Formula (V) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is, independently, 1 or 2;
each R¹ is, independently, H or OH;
structure C is a conjugator;
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’ is, independently, and
each Su is independently a sugar moiety.

In some embodiments of Formula (V) , either or both of structure A’ and structure B’ is

In some embodiments of Formula (V) , one of structure A’ and structure B’ is and the other of structure A’ and structure B’ is

In some embodiments of Formula (V) , p and q are both 2.

In some embodiments of Formula (V) , a compound has the following formula:

In some embodiments of Formula (V) , the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;
s is 1, 2, or 3; and
each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer
thereof.

In some embodiments of Formula (V) , the conjugator has the following formula:

In some embodiments of Formula (V) , each sugar moiety is independently
or a stereoisomer
thereof; and
each m is independently 0 or 1.

In some embodiments of Formula (V) , the sugar moiety isand m is 0.

In some embodiments of Formula (V) , each R^2’ is, independently,

In some embodiments of Formula (V) , a compound has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.
7.4. Cleavable-Functionalized PBD Dimer Antibody-Drug Conjugates

Also disclosed herein are antibody-drug conjugates (ADCs) including a humanized, chimeric, or human antibody or an antigen binding fragment thereof covalently linked, directly or indirectly, to a PBD dimer described herein.

Also described herein are antibody-drug conjugates of Formula (VI) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,
wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;
each m and n is, independently, 1 or 2;
each R¹ is, independently, H or OH;
structure C is a conjugator;
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’ is, independently,
each Su is independently a sugar moiety;
Ab is a humanized, chimeric, or human antibody or an antigen binding fragment thereof; and
x is from about 1 to about 8.

In some embodiments of Formula (VI) , either or both of structure A’ and structure B’ is

In some embodiments of Formula (VI) , one of structure A’ and structure B’ is and the other of structure A’ and structure B’ is

In some embodiments of Formula (VI) , p and q are both 2.

In some embodiments of Formula (VI) , the ADC has the following formula:

In some embodiments of Formula (VI) , the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;
s is 1, 2, or 3;
each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer
thereof;
***is the point of attachment to Ab.

In some embodiments of Formula (VI) , the conjugator has the following formula

In some embodiments of Formula (VI) , the conjugator has the following formula:

In some embodiments of Formula (VI) , the sugar moiety is or a stereoisomer thereof; and each m is independently 0 or 1.

In some embodiments of Formula (VI) , the sugar moiety isand m is 0.

In some embodiments of Formula (VI) , each R^2’ is, independently,

In some embodiments of Formula (VI) , the ADC has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.

In some embodiments of Formula (VI) , x is about 2.
7.5. Methods or Processes of Making the Conjugates

Provided herein are methods of preparing an ADC by contacting a humanized, chimeric, or human antibody or an antigen binding fragment thereof with a PBD dimer described herein under conditions suitable for forming a bond between the antibody or an antigen binding fragment thereof and the PBD dimer. The reaction conditions may be any suitable reaction conditions known in the art. Examples of conditions for such reactions are provided in the Examples below.
7.6. Pharmaceutical Compositions

Also provided herein are compositions, including pharmaceutical compositions, comprising an ADC set forth herein. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise a pharmaceutically acceptable excipient.

Pharmaceutical compositions in accordance with the present disclosure can be prepared by mixing an antibody drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) , in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as polyethylene glycol (PEG) . Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP) , for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (Baxter International, Inc. ) . Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Nos. US 7, 871, 607 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized formulations are described in US Patent No. 6, 267, 958. Aqueous formulations include those described in US Patent No. 6, 171, 586 and WO2006/044908, the latter formulations including a histidine-acetate buffer.

Pharmaceutical compositions can be formulated for administration by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
7.7. Methods of Using

In some embodiments, set forth herein is a method of treating a disease or disorder (e.g., a cancer) in a subject (e.g., patient) in need thereof, comprising administering to the patient a therapeutically effective amount of an ADC disclosed herein.

The ADCs disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is acute or chronic. Various dosing schedules, including but not limited to, single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

ADCs of the disclosure can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
8. EXAMPLES

List of Abbreviations

UPLC analysis methods

Method A: Mobile phase A: 0.1%FA in water, B: MeCN; Gradient: 10%B maintain 0.2 min, 10%-95%B, 5.8 min, 95%B maintain 0.5 min; Flow rate: 0.6 mL/min; Column: ACQUITYBEH C18 1.7μm.

Method B: Mobile phase A: 0.1%FA in water, B: MeCN; Gradient: 10%B maintain 0.5 min, 10%-90%B, 2.5 min, 90%B maintain 0.2 min; Flow rate: 0.6 mL/min; Column: ACQUITYBEH C18 1.7μm.

Method C: Mobile phase A: 0.1%FA in water, B: MeCN; Gradient: 10%B maintain 0.2 min, 10%-90%B, 1.3 min, 90%B maintain 0.3 min; Flow rate: 0.6 mL/min; Column: ACQUITYBEH C18 1.7μm.
Example 1: Synthesis of Intermediate Compounds
Synthesis of Int-2

Step 1: allyl ( (S) -1- ( ( (S) -1- ( (4- (hydroxymethyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-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 dry 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 isolated by filtration under a vacuum for 3 hours to give Int-2b (5.16 g, 74%yield) .

MS (ESI) m/z: 378.4 [M+H] ⁺.

Step 2: (S) -2-amino-N- ( (S) -1- ( (4- (hydroxymethyl) phenyl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (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 allowed to stir for 0.5 hours at room temperature. The reaction was concentrated and purified by silica gel column chromatography (CH₂Cl₂/MeOH = 92/8) to give Int-2c (370 mg, 95%yield) as a white solid.

MS (ESI) m/z: 294.3 [M+H] ⁺.

Step 3: (9H-fluoren-9-yl) methyl ( (17S, 20S) -21- ( (4- (hydroxymethyl) phenyl) amino) -17-isopropyl-20-methyl-15, 18, 21-trioxo-3, 6, 9, 12-tetraoxa-16, 19-diazahenicosyl) carbamate (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 dry 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 was stirred for 1 hour. 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.

MS (ESI) m/z: 763.4 [M+H] ⁺.
Synthesis of Int-5

Step 1: allyl bis (2-hydroxyethyl) carbamate (1-5b)

To a solution of 1-5a (423 mg, 3 mmol) in THF (3.1 mL) and water (5.7 mL) was added AllocCl (361.45 mg, 3 mmol) and K₂CO₃ (1036 mg, 7.5 mmol) at 0 ℃. The mixture was stirred at 20 ℃ for 16 hours. TLC (petroleum ether: EtOAc=1: 1, v/v) showed the reaction was completed. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (3x20 mL) . The combined organic layer was dried over Na₂SO₄ and filtered. Solvent was evaporated and the crude product 1-5b (567 mg) was used in the next step without further workup and purification.

Step 2: ( ( (allyloxy) carbonyl) azanediyl) bis (ethane-2, 1-diyl) bis (4-methylbenzenesulfonate) (Int-5)

To a solution of 1-5b (567 mg, 3 mmol) in CH₂Cl₂ (7 mL) was added TsCl (1.7 g, 9 mmol) and triethylamine (1.67 mL, 12 mmol) at 0 ℃. The mixture was stirred at 20 ℃ for 16 hours. TLC (petroleum ether: EtOAc=3: 1, v/v) showed the reaction was completed. The reaction was poured into water (20 mL) and extracted with EtOAc (3x30 mL) . The combined organic layer was dried over Na₂SO₄ and filtered. Solvent was evaporated and the residue was purified by silica column gel chromatography (eluent: petroleum ether/EtOAc = 100/0 to 30/70) to afford Int-5 (1.2 g, 73.08%yield) as a colorless oil.
Synthesis of Int-7

Step 1: (9H-fluoren-9-yl) methyl ( (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-diazahenicosyl) carbamate (Int-7)

To a mixture of Int-2 (500 mg, 0.656 mmol) , bis (4-nitrophenyl) carbonate (300 mg, 0.981 mmol) , and DMF (10 mL) was added DIEA (233 μL, 1.31 mmol) . The mixture was stirred at room temperature overnight. The mixture was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (petroleum ether/EtOAc = 0/100) to afford Int-7 (386 mg, 63.5%yield) as a white solid.

MS (ESI) m/z: 928.7 [M+H] ⁺.
Synthesis of Int-8

Step 1: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -2- ( ( ( (9H-fluoren-9-yl) methoxy) carbonyl) amino) -3- (benzyloxy) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (Int-8c)

To a solution of compound Int-8a (1.00 g, 2.41 mmol) in CH₂Cl₂ (30 mL) was added Int-8b (1.14 g, 2.87mmol) andmolecular sieves (4.00 g) . The mixture was stirred at room temperature for 30 min. The mixture was cooled to -20 ℃. Then AgOTf (0.74 g, 2.87 mmol) was added into the mixture in portions at -20 ℃. The mixture was stirred at -20 ℃ for 4 hours. The mixture was filtered, and the filtrate was diluted with EtOAc (150 mL) , washed with sat. NaHCO₃ (3x100 mL) , brine (3x100 mL) . The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated. The residue was purified by flash column chromatography (eluted 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.

MS (ESI) m/z: 734.4 [M+H] ⁺.

Step 2: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -2-amino-3- (benzyloxy) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (Int-8d)

To a solution of compound Int-8c (250 mg, 0.34 mmol) in DMF (1 mL) was added Et₂NH (125 mg, 1.70 mmol) . The mixture was stirred at room temperature for 30 min. The mixture was concentrated under high vacuum, and co-evaporated with toluene (3x3 mL) to give compound Int-8d (174 mg, crude) as a brown oil, which was used directly in the next step without further purification.

MS (ESI) m/z: 512.3 [M+H] ⁺.

Step 3: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -3- (benzyloxy) -2- ( (S) -2- ( ( (benzyloxy) carbonyl) amino) -3-methylbutanamido) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (Int-8f)

To a solution of compound Int-8d (174 mg, 0.34 mmol) in DMF (4 mL) were added compound Int-8e (103 mg, 0.41 mmol) , HATU (168 mg, 0.44 mmol) and DIEA (88 mg, 0.68 mmol) . The mixture was stirred at room temperature for 4 hours. The reaction was diluted with EtOAc (50 mL) , washed with sat. NaHCO₃ solution (3x30 mL) , brine (3x30 m) . The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to give a residue. The residue was purified by flash column chromatography (eluted with CH₂Cl₂ /EtOAc = 100/0～70/30) . Compound Int-8f (230 mg, 91%yield) was obtained as a white solid.

MS (ESI) m/z: 745.4 [M+H] ⁺.

Step 4: N- (L-valyl) -O- ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) -L-serine (Int-8g)

To a solution of compound Int-8f (230 mg, 0.31 mmol) in MeOH (3 mL) was added Pd/C (10%, 23 mg) . The mixture was stirred at room temperature under H₂ (15 psi) for 1 hour. H₂O (3 mL) was added into the mixture, filtered, and concentrated to give crude product Int-8g (161 mg, crude) as a white solid, which was used directly without further purification.

MS (ESI) m/z: 521.3 [M+H] ⁺.

Step 5: N- (acetyl-L-valyl) -O- ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) -L-serine (Int-8h)

To a solution of compound Int-8g (161 mg, 0.31 mmol) in AcOH (3 mL) was added Ac₂O (316 mg, 3.09 mmol) at 0 ℃. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated and purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (0.1%TFA) : B-acetonitrile; Flow rate: 20 mL/min) . Compound Int-8h (145 mg, 83.3%yield) was obtained as a white solid.

MS (ESI) m/z: 563.4 [M+H] ⁺.

Step 6 (2R, 3R, 4S, 5S, 6S) -2- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( (4- (hydroxymethyl) phenyl) amino) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (Int-8)

Int-8h (200 mg, 0.36 mmol) and 4-aminobenzyl alcohol (46 mg, 0.37 mmol) were mixed in dry CH₂Cl₂ (1 mL) . To the mixture was added EEDQ (131.9 mg, 0.53 mmol) . The mixture was stirred at room temperature for 12 hours under a nitrogen atmosphere. The solvent was removed under vacuum to give a crude product, which was purified by silica gel column chromatography (MeOH/CH₂Cl₂= 10/90) to obtain Int-8 as a light-yellow solid (80 mg, 34%) .

MS (ESI) m/z: 668.0 [M+H] ⁺.
Example 2: Synthesis of Payloads
Example P1-1 (1-21)

Step 1: (S) - (2- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methylenepyrrolidin-1-yl) (4-hydroxy-5-methoxy-2-nitrophenyl) methanone (1-21b)

A mixture of MePh₃PBr (24.6 g, 68.86 mmol) in THF (100 mL) was added t-BuOK (6.95 g, 61.98 mmol) at 0 ℃ under N₂. The mixture was stirred at 0 ℃ for 2 h, 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 ℃ for 16 hours. The mixture was neutralized with citric acid and extracted with EtOAc (3x100 mL) . The organic phase was dried over Na₂SO₄, filtered, and concentrated to give crude product, which was purified by silica column gel chromatography (eluent: hexane/EtOAc = 100/0 to 25/75) to give 1-21b (1.1 g, 37.8%yield) as a yellow solid.

MS (ESI) m/z: 423.2 [M+H] ⁺.

Step 2: Allylbis (2- (4- ( (S) -2- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methylenepyrrolidine-1-carbonyl) -2-methoxy-5-nitrophenoxy) ethyl) carbamate (1-21c)

To a solution of 1-13c (100 mg, 0.2 mmol) and 1-21b (186.83 mg, 0.44 mmol) in DMSO (3 mL) was added K₂CO₃ (55.5 mg, 0.4 mmol) . The mixture was stirred at 50 ℃ 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 crude product, which was purified by silica column gel chromatography (eluent: hexane/EtOAc = 100/0 to 0/100) to give 1-21c (108 mg, 53.83%yield) as a yellow solid.

MS (ESI) m/z: 998.5 [M+H] ⁺.

Step 3: Allylbis (2- (5-amino-4- ( (S) -2- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methylenepyrrolidine-1-carbonyl) -2-methoxyphenoxy) ethyl) carbamate (1-21d)

A mixture of Zn powder (28.24 mg, 4.08 mmol) in EtOH (3 mL) , AcOH (0.2 mL) and H₂O (0.2 mL) was stirred at room temperature for 10 min, then a solution of 1-21c (108 mg, 0.11 mmol) in EtOH (2 mL) was added and stirred at room temperature for 1 hour. The reaction was filtered through celite, and the filtrate was concentrated to give crude product, which was purified by silica gel chromatography (eluent: CH₂Cl₂/MeOH = 20/1) to obtain 1-21d (73 mg, 72.2 %yield) as a yellow oil.

MS (ESI) m/z: 938.6 [M+H] ⁺.

Step 4: Allylbis (2- (5- ( ( (allyloxy) carbonyl) amino) -4- ( (S) -2- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methylenepyrrolidine-1-carbonyl) -2-methoxyphenoxy) ethyl) carbamate (1-21e)

To a solution of 1-21d (73 mg, 0.077 mmol) in dry CH₂Cl₂ (2 mL) was added Alloc-Cl (53 μL, 0.49 mmol) and pyridine (24.73 μL, 0.311 mmol) under N₂ atmosphere at -10 ℃. The mixture was stirred at -10 ℃ for 1 hour. The solution was added into water (10 mL) , and extracted with CH₂Cl₂ (3x5 mL) . The organic phase was dried over Na₂SO₄, filtered and concentrated to give crude product, which was purified by flash column chromatography (eluent: petroleum ether/EtOAc = 1/3) to give 1-21e (75 mg, 87.13%yield) as an off-white solid.

MS (ESI) m/z: 1106.6 [M+H] ⁺.

Step 5: allylbis (2- (5- ( ( (allyloxy) carbonyl) amino) -4- ( (S) -2- (hydroxymethyl) -4-methylenepyrrolidine-1-carbonyl) -2-methoxyphenoxy) ethyl) carbamate (1-21f)

Para-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 flash column chromatography (eluent: CH₂Cl₂/MeOH = 20/1) to give 1-21f (52 mg, 87.38%yield) as a yellow solid.

MS (ESI) m/z: 878.4 [M+H] ⁺.

Step 6: diallyl8, 8'- ( ( ( ( (allyloxy) carbonyl) azanediyl) 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] diazepine-10 (5H) -carboxylate) 1H, 3H-spiro [benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-2, 1'-cyclopropane] -10 (5H) -carboxylate (1-21g)

DMP (55.27 mg, 0.13 mmol) was added at 0 ℃ to a solution of 1-21f (52 mg, 0.059 mmol) in CH₂Cl₂ (2 mL) , the reaction was then warmed to room temperature and stirred for 4 hours. The reaction mixture was filtered, and the filtrate was quenched with sat. aq. sodium thiosulfate solution (5 mL) , followed by a slow addition of sat. aq. NaHCO₃ (5 mL) and H₂O (10 mL) . The mixture was extracted with dichloromethane (3x5 mL) , and the organic layer was washed with brine (5 mL) , dried over Na₂SO₄, filtered, and concentrated to give crude product, which was purified by flash column chromatography (eluent: CH₂Cl₂/MeOH = 20/1) to give 1-21g (32 mg 61.82%yield) as a white solid.

MS (ESI) m/z: 874.4 [M+H] ⁺.

Step 7: (11aS, 11a'S) -8, 8'- ( (azanediylbis (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] diazepin-5-one) (1-21)

To a solution of 1-21g (32 mg, 0.036 mmol) and CH₂Cl₂ (2 mL) was added Pd (PPh₃) ₄ (2.12 mg, 0.0018 mmol) and pyrrolidine (6 μL, 0.073 mmol) and stirred at room temperature for 15 min under N₂. The reaction was neutralized with AcOH and concentrated to give the crude product, which was purified by prep-HPLC (column: XBridge Prep C18 OBD 5μm 19*150 mm) ; mobile phase: A-water (0.01%formic acid) : B-acetonitrile; flow rate: 20 mL/min) to give 1-21 (9.7 mg, 45.23%yield) as a white solid.

MS (ESI) m/z: 586.3 [M+H] ⁺.
Example P1-2 (1-13)

Step 1: allyl bis (2-hydroxyethyl) carbamate (1-13b)

To a solution of 1-13a (423 mg, 3 mmol) in THF (3.1 mL) and water (5.7 mL) were added AllocCl (361.45 mg, 3 mmol) and K₂CO₃ (1036 mg, 7.5 mmol) at 0 ℃. The mixture was stirred at 20 ℃ for 16 hours. TLC (petroleum ether: EtOAc=1: 1, v/v) showed the reaction was completed. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (3x20 mL) . The combined organic layer was dried over Na₂SO₄ and filtered. Solvent was evaporated and the crude product 1-13b (567 mg) was used in the next step without further workup or purification.

Step 2: ( ( (allyloxy) carbonyl) azanediyl) bis (ethane-2, 1-diyl) bis (4-methylbenzenesulfonate) (1-13c)

To a solution of 1-13b (567 mg, 3 mmol) in CH₂Cl₂ (7 mL) was added TsCl (1.7 g, 9 mmol) and triethylamine (1.67 mL, 12 mmol) at 0 ℃. The mixture was stirred at 20 ℃ for 16 hours. TLC (petroluem ether: EtOAc=3: 1, v/v) showed the reaction was completed. The reaction was poured into water (20 mL) and extracted with EtOAc (3x30 mL) . The combined organic layer was dried over Na₂SO₄ and filtered. Solvent was evaporated and the residue was purified by silica column gel chromatography (eluent: petroleum ether/EtOAc = 100/0 to 30/70) to afford 1-13c (1.2 g, 73.08%yield) as a colorless oil.

Step 3: diallyl 8, 8” - ( ( ( ( (allyloxy) carbonyl) azanediyl) 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] diazepine-2, 1'-cyclopropane] -10 (5H) -carboxylate) (1-13d)

To a solution of 1-13c (50 mg, 0.10 mmol) and 1-5b (75.25 mg, 0.20 mmol) in DMSO (2 mL) was added K₂CO₃ (41.70 mg, 0.30 mmol) at room temperature The mixture was stirred at 50 ℃ for 16 hours. LCMS showed the reaction was completed. The reaction mixture was diluted with EtOAc (10 mL) and washed with brine (3x8 mL) . The organic layer was dried over Na₂SO₄ and concentrated to give the residue which was purified by silica column gel chromatography (eluent: petroleum ether/EtOAc = 100/0 to 10/90) to afford 1-13d (38 mg, 49.64%yield) as a colorless oil.

MS (ESI) m/z: 902.3 [M+H] ⁺.

Step 4: (11aS, 11a”S) -8, 8”- ( (azanediylbis (ethane-2, 1-diyl) ) bis (oxy) ) bis (7-methoxy-1, 11a-dihydro-3H, 5H-spiro [benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-2, 1'-cyclopropan] -5-one) (1-13)

To a solution of 1-13d (45 mg, 0.05 mmol) and CH₂Cl₂ (3 mL) were added Pd (PPh₃) ₄ (5.77 mg, 0.005 mmol) and pyrrolidine (12 μL, 0.12 mmol) at room temperature The mixture was stirred at 20 ℃ for 30 min. LCMS showed the reaction was completed. Solvent was evaporated and the residue was purified by prep-HPLC (0.01%FA) to give product 1-13 (12 mg, 35.27%yield) as a white solid.

MS (ESI) m/z: 614.3 [M+H] ⁺.

¹H NMR (400 MHz, CDCl₃) δ 7.79 (2 H, d, J = 4.4) , 7.51 (2 H, s) , 6.84 (2 H, 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) .
Example 3: Synthesis of Reference Compounds

Reference linker-payload compounds are summarized below in Table 1.
Example Ref-2-1

Ref-2-1a was synthesized according to the procedures described in US2015283262A1 and Org. Process Res. Dev. 2018, 22, 1241-1256.

Step 1 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-triazapentacosan-25-amido) benzyl (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] diazepin-8-yl) oxy) pentyl) oxy) -2-methyl-5-oxo-11, 11a-dihydro-1H-benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-10 (5H) -carboxylate (Ref-2-1)

To a solution of Int-1 (5.3 mg, 0.01 mmol, prepared according to procedures described in WO2022120132 A1 and WO2022079211 A1) in 0.5 mL DMF were added HATU (5.0 mg, 0.01 mmol) and DIEA (4 μL, 0.02 mmol) at 0 ℃. The mixture was stirred at room temperature for 15 min. Ref-2-1a (10 mg, 0.01 mmol) in 0.5 mL DMF was added to the mixture at 0 ℃ and the reaction was stirred at room temperature for 2 hours. The mixture was purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (no formic acid) : B-acetonitrile; Flow rate: 20 mL/min) . The fraction was lyophilized to give Ref-2-1 (2.5 mg, 17.1%yield) as a white solid.

MS (ESI) m/z: 1345.9 [M+H] ⁺.
Example Ref-2-2

Int-3 was synthesized according to the procedures described in WO 2017059289 A1 and Org. Process Res. Dev. 2022, 26, 2155-2175.

Step 1: (9H-fluoren-9-yl) methyl tert-butyl (5- ( (5- (5- ( ( ( (4- ( (21S, 24S) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-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 (Ref-2-2a)

To a solution of Int-3 (200 mg, 0.1210 mmol) and 200 mg MS in dry THF (2.5 mL) was added triphosgene (24.9 mg, 0.084 mmol) , then Et₃N (64 μL, 0.462 mmol) was added at 0 ℃ under N₂. The mixture was stirred at 0 ℃ for 10 min under N₂. Formation of the isocyanate was monitored by LCMS analysis by quenching with MeOH. A solution of Int-2 (176 mg, 0.231 mmol) , dibutyltin dilaurate (13.3 mg, 0.021 mmol) , Et₃N (43.7 μL, 0.315 mmol) in dry THF (2.5 mL) was added to the mixture. The mixture was stirred for 3 hours at room temperature. 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.

MS (ESI) m/z: 1742.1 [M+H] ⁺.

Step 2: tert-butyl (5- ( (5- (5- ( ( ( (4- ( (21S, 24S) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-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) carbamate (Ref-2-2b)

para-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 allowed to stir for 4 hours at 22 ℃. The mixture was diluted with EtOAc (20 mL) , washed with water, sat. 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.

MS (ESI) m/z: 1513.9 [M+H] ⁺.

Step 3: 4- ( (21S, 24S) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl (11S, 11aS) -8- ( (5- ( ( (11S, 11aS) -10- (tert-butoxycarbonyl) -11-hydroxy-7-methoxy-2-methylene-5-oxo-2, 3, 5, 10, 11, 11a-hexahydro-1H-benzo [e] pyrrolo [1, 2-a] [1, 4] diazepin-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] diazepine-10 (5H) -carboxylate (Ref-2-2c)

DMP (58.9 mg, 0.139 mmol) was added slowly, portion-wise to the solution of Ref-2-2b (100 mg) in dry CH₂Cl₂ (2 mL) at 0 ℃. The reaction was then warmed to room temperature and stirred overnight. The reaction was quenched with sat. Na₂S₂O₃, followed by addition of sat. NaHCO₃ and water. The layers were separated and the organic layer was washed with sat. Na₂S₂O₃, sat. NaHCO₃ and brine, 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.

MS (ESI) m/z: 1509.8 [M+H] ⁺.

Step 4: 4- ( (21S, 24S) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl (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] diazepin-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11a-tetrahydro-1H-benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-10 (5H) -carboxylate (Ref-2-2d)

Ref-2-2c (20 mg, 0.013 mmol) was cooled to -3 ℃. Separately, a solution of 95%TFA in H₂O (0.5 mL) was cooled to -3 ℃ before adding to Ref-2-2c. The reaction mixture was stirred at -3 ℃ for 30 min before pouring onto a 1: 1 solution of CHCl₃ : sat. NaHCO₃ (16 mL) at 0 ℃. The organic layer was separated, dried on Na₂SO₄ before being filtered and then removed in vacuo. The crude material was used directly in the next step without further purification.

MS (ESI) m/z: 1391.7 [M+H] ⁺.

Step 5: 4- ( (17S, 20S) -1-amino-17-isopropyl-20-methyl-15, 18-dioxo-3, 6, 9, 12-tetraoxa-16, 19-diazahenicosan-21-amido) benzyl (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] diazepin-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11a-tetrahydro-1H-benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-10 (5H) -carboxylate (Ref-2-2e)

To a solution of crude Ref-2-2d (18 mg, 0.013 mmol) in 0.5 mL DMF was added Et₂NH (14 μL, 0.129 mmol) . The mixture was stirred at room temperature for 0.5 hours. After the reaction was completed, the mixture was purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (0.1%formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to give Ref-2-2e (5 mg, 33%yield) .

MS (ESI) m/z: 1169.7 [M+H] ⁺.

Step 6: 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-triazapentacosan-25-amido) benzyl (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] diazepin-8-yl) oxy) pentyl) oxy) -2-methylene-5-oxo-2, 3, 11, 11a-tetrahydro-1H-benzo [e] pyrrolo [1, 2-a] [1, 4] diazepine-10 (5H) -carboxylate (Ref-2-2)

DIEA (8.6 μL, 0.05 mmol) was added to the solution of Ref-2-2e (14.5 mg, 0.012 mmol) and Int 4 (10.8 mg, 0.037 mmol, prepared according to procedures described in J. Am. Chem. Soc. 2020, 142, 20, 9285–9301) in 1 mL DMF. The mixture was stirred at room temperature for 20 min. The mixture was purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (no formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to give Ref-2-2 (7.9 mg, 46%yield) as a light gray solid.

MS (ESI) m/z: 1345.7 [M+H] ⁺.
Example 4: Synthesis of Test Compounds

Test linker-payload compounds are summarized below in Table 1.
Example 2-1

Step 1: (S) - (2- ( ( (tert-butyldimethylsilyl) oxy) methyl) -4-methylenepyrrolidin-1-yl) (4-hydroxy-5-methoxy-2-nitrophenyl) methanone (2-1b)

To a mixture of MePh₃PBr (24.6 g, 68.86 mmol) in THF (100 mL) was added t-BuOK (6.95 g, 61.98 mmol) at 0 ℃ under N₂. The mixture was stirred at 0 ℃ for 2 h, 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 ℃ for 16 hours. The mixture was neutralized with citric acid and extracted with EtOAc (3x100 mL) . The organic phase was dried over Na₂SO₄, filtered, and concentrated to give crude product, which was purified by silica column gel chromatography (eluent: hexane/EtOAc = 100/0 to 25/75) to give 2-1b (1.1 g, 37.8%yield) as a yellow solid.

MS (ESI) m/z: 423.2 [M+H] ⁺.

Step 2: Allylbis (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 dry DMSO (2 mL) . To the mixture was added K₂CO₃ (333.6 mg, 2.42 mmol) . The mixture was stirred at room temperature overnight under a nitrogen atmosphere. The reaction solution was then diluted with EtOAc (10 mL) , washed with H₂O (10 mL) , and brine (5 mL) . The organic phase was collected and dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum to give a residue which was purified by silica gel column chromatography (Petroleum ether/EtOAc = 25/75) to obtain 2-1c as a light-yellow solid (250 mg, yield 31.1%) .

MS (ESI) m/z: 998.7 [M+H] ⁺.

Step 3: allyl 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) were added to the reaction mixture. The mixture was stirred at room temperature for 1 hour. The mixture was filtrated to remove the excess amount of Zn, the filter was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (MeOH/CH₂Cl₂= 5/95) to afford 2-1d (220 mg, 92%yield) as a yellow oil.

MS (ESI) m/z: 938.8 [M+H] ⁺.

Step 4: (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-triyl triacetate (2-1e)

2-1d (220 mg, 0.24 mmol) was dissolved in dry THF (2 mL) , molecular sieves (200 mg) was added. The mixture was cooled to 0 ℃, then triphosgene (50.2 mg, 0.17 mmol) was added, followed by addition of triethylamine (145 μL, 1.03 mmol) . The mixture was stirred at 0 ℃ for 10 min under a nitrogen atmosphere. Int-6 (238 mg, 0.54 mmol) , triethylamine (99 μL, 0.7 mmol) and dibutyltin dilaurate (55.6 μL, 0.09 mmol) was mixed in dry THF (1 mL) , and then this solution was gradually added to the above reaction solution at 0 ℃. The reaction solution was warmed 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 as a light-yellow solid (430 mg, 98%yield) .

MS (ESI) m/z: 1871.4 [M+H] ⁺.

Step 5: (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) 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 triacetate (2-1f)

To a solution of 2-1e (430 mg, 0.23 mmol) in 3 mL CH₂Cl₂ was added Pd (PPh₃) ₄ (5 mg, cat. ) and pyrrolidine (48 μL, 0.58 mmol) at 0 ℃. 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 obtain 2-1f as a white solid (350 mg, 85.2%) .

MS (ESI) m/z: 1787.4 [M+H] ⁺.

Step 6: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-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 triacetate (2-1g)

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) were added DIEA (102 mg, 0.59 mmol) . The mixture was stirred at room temperature for 5 hours. The mixture was concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography (MeOH/CH₂Cl₂= 15/85) to afford 2-1g (240 mg, 47.6%yield) as a white solid.

MS (ESI) m/z: 2575.8 [M+H] ⁺.

Step 7: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-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 triacetate (2-1h)

Compound 2-1g (240 mg, 0.09 mmol) was dissolved in THF/H₂O (2 mL/40 μL, 50/1) , TsOH^. 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, the residue was purified by column chromatography (MeOH/CH₂Cl₂= 7/93) to obtain 2-1h as a white solid (135 mg, 61.7%yield) .

MS (ESI) m/z: 2347.1 [M+H] ⁺.

Step 8: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (11S, 11aS) -8- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) carbonyl) (2- ( ( (11S, 11aS) -10- ( ( (4- ( ( (2S, 3R, 4R, 5S, 6S) -5-acetoxy-3, 4-diacetyl-6- (methoxycarbonyl) tetrahydro-2H-pyran-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] diazepin-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] diazepine-10-carbonyl) oxy) methyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2-1i)

To a solution of 2-1h (135 mg, 0.06 mmol) in CH₂Cl₂ (1 mL) was added Dess-Martin periodinane (61 mg, 0.14 mmol) at room temperature, and the mixture was stirred at room temperature for 1 hour under a nitrogen atmosphere. The reaction solution was then diluted with CH₂Cl₂ (10 mL) , washed with sat. Na₂S₂O₃ (5 mL) and brine (5 mL) . The organic phase was collected and dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum to give a residue which was purified by silica gel column chromatography (MeOH/CH₂Cl₂= 8/92) to obtain 2-1i as a yellow solid (105 mg, 77.8%yield) .

MS (ESI) m/z: 2343.3 [M+H] ⁺.

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-diazahenicosan-21-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-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)

To a solution of sat. LiBr (2 mL) in CH₃CN/H₂O (10/1, v/v) and triethylamine (0.5 mmol, 62 μL) was added 2-1i (105 mg, 0.045 mmol) . The reaction mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. The solvent was removed by evaporation to give crude product as a yellow oil, which was directly used in the next step without further purification.

To the above crude product in 1 mL DMF was added diethylamine (0.45 mmol, 46.2 μL) at 0 ℃. The reaction solution was stirred at 0 ℃ for 0.5 hours. The reaction solution was neutralized with AcOH, and then purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*1250 mm; Mobile phase: A-water (0.1%formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to provide 2-1j as a white solid after lyophilization (26 mg, 31.7%yield) .

MS (ESI) m/z: 1841.2 [M+H] ⁺.

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-triazapentacosan-25-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-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)

To a solution of 2-1j (13 mg, 0.007 mmol) in 0.5 mL DMF was added Int-4 (4.1 mg, 0.014 mmol) and DIPEA (6.4 μL, 0.035 mmol) at 0 ℃. The reaction solution was stirred at 0 ℃for 1 hour. The reaction solution was neutralized with AcOH, and then purified by prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (0.1%formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to provide 2-1 as a white solid after
lyophilization (4.4 mg, 30.9%yield) .

MS (ESI) m/z: 2017.0 [M+H] ⁺.
Example 2-2

Step 1: allyl bis (2- (5-amino-4- ( (S) -6- ( ( (tert-butyldimethylsilyl) oxy) methyl) -5-azaspiro [2.4] heptane-5-carbonyl) -2-methoxyphenoxy) ethyl) carbamate (2-2b)

To a solution of Int-5 (505 mg, 1.02 mmol) , 2-2a (826 mg, 2.03 mmol) (prepared according to a similar procedure described in CN111164208A) and DMSO (10 mL) were added K₂CO₃ (422 mg, 3.06 mmol) . The mixture stirred at 50 ℃ overnight. After the reaction was completed, the mixture was diluted with water (20 mL) , extracted with EtOAc (3x20 mL) . The combined organics were washed with brine (20 mL) , dried over Na₂SO₄, filtered and the filtrate was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (Petroleum ether/EtOAc = 10/90) to afford 2-2b (200 mg, 20.3%yield) as a yellow solid.

MS (ESI) m/z: 966.9 [M+H] ⁺.

Step 2: (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- (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 triacetate (2-2c) .

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

MS (ESI) m/z: 1899.0 [M+H] ⁺.

Step 3: (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) 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 triacetate (2-2d)

Compound 2-2d (320 mg) was synthesized according to the synthetic procedure of step 5 of example 2-1.

MS (ESI) m/z: 1815.4 [M+H] ⁺.

Step 4: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) 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- (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 triacetate (2-2e)

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) was added DIEA (180 mg, 0.618 mmol) . The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (Petroleum ether/EtOAc = 10/90) to afford 2-2e (300 mg, 25.6%yield) as a yellow solid.

MS (ESI) m/z: 2603.7 [M+H] ⁺.

Step 5: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) carbonyl) (2- (4- ( (S) -6- (hydroxymethyl) -5-azaspiro [2.4] heptane-5-carbonyl) -2-methoxy-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 triacetate (2-2f)

Compound 2-2f (115 mg, 42.1%yield) was synthesized according to the synthetic procedure of step 7 of example 2-1.

MS (ESI) m/z: 2375.6 [M+H] ⁺.

Step 6: (2S, 3R, 4S, 5S, 6S) -2- (4- ( ( ( (11S, 11aS) -8- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-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] diazepine-2, 1'-cyclopropan] -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] diazepine-2, 1'-cyclopropane] -10-carbonyl) oxy) methyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2-2g)

Compound 2-2g (120 mg, crude) was synthesized according to the synthetic procedure of step 8 of example 2-1.

MS (ESI) m/z: 2371.2 [M+H] ⁺.

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-diazahenicosan-21-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-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)

Compound 2-2h (24 mg, 25.3%yield) was synthesized according to the synthetic procedure of step 9 of example 2-1.

MS (ESI) m/z: 1868.5 [M+H] ⁺.

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-triazapentacosan-25-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-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 of example 2-1.

MS (ESI) m/z: 2045.4 [M+H] ⁺.
Example 2-3

Step 1: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( (4- ( ( ( (5- (2- ( (2- (5- ( ( ( (4- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (mehoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) propanamido) 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-triyl triacetate (2-3a)

To a 0 ℃ mixture of 2-2b (78 mg, 0.081 mmol) and anhydrous THF (3 mL) were added triphosgene (19 mg, 0.065 mmol) and TEA (50 μL, 0.36 mmol) . The mixture was stirred at 0 ℃for 10 mins under N₂, before 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) was added. The mixture was stirred at room temperature overnight under N₂. The mixture was filtrated and the filtrate was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (dichloromethane/methanol = 98/2) to afford 2-3a (141 mg, 74.2%yield) as a yellow solid.

MS (ESI) m/z: 2352.5 [M+H] ⁺.

Step 2: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( (4- ( ( ( (5- (2- ( (2- (5- ( ( ( (4- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) propanamido) 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-triyl triacetate (2-3b)

To a mixture of 2-3a (141 mg, 0.06 mmol) and THF (2 mL) was added 5, 5-dimethylcyclohexane-1, 3-dione (21 mg, 0.15 mmol) and Pd (PPh₃) ₄ (7 mg, 0.006 mmol) . The mixture was stirred at room temperature for 2 hours under N₂. The mixture was filtrated. The filter was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (dichloromethane/methanol = 95/5) to afford 2-3b (80 mg, 58.8%yield) as a yellow solid.

MS (ESI) m/z: 2269.3 [M+H] ⁺.

Step 3: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -3- ( (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) carbonyl) (2- (5- ( ( ( (4- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) propanamido) 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-methylbutanamido) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2-3c)

To a solution of 2-3b (20 mg, 0.0088 mmol) , Int-7 (826 mg, 2.03 mmol) , HOBt (4 mg, 0.004) and DMF (1 mL) was added DIPEA (5 μL, 0.03 mmol) . The mixture was stirred at room temperature overnight. The mixture was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (dichloromethane/methanol = 95/5) to afford 2-3c (26 mg, 96.7%yield) as a yellow solid.

MS (ESI) m/z: 1529.4 [1/2M+H] ⁺.

Step 4: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -3- ( (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) carbonyl) (2- (5- ( ( ( (4- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) propanamido) 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-methylbutanamido) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2-3d)

To a mixture of 2-3c (27 mg, 0.0088 mmol) and THF (1 mL) was added PTSA (3.4 mg, 0.018 mmol) . The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under vacuum to give a residue which was purified by silica gel column chromatography (dichloromethane/methanol = 95/5) to afford 2-3d (15 mg, 60.0%yield) as a yellow solid.

MS (ESI) m/z: 2830.1 [M+H] ⁺.

Step 5: (2R, 3R, 4S, 5S, 6S) -2- ( (S) -3- ( (4- ( ( ( (5- (2- ( ( ( (4- ( (21R, 24R) -1- (9H-fluoren-9-yl) -21-isopropyl-24-methyl-3, 19, 22-trioxo-2, 7, 10, 13, 16-pentaoxa-4, 20, 23-triazapentacosan-25-amido) benzyl) oxy) carbonyl) (2- (5- ( ( ( (4- ( (S) -2- ( (S) -2-acetamido-3-methylbutanamido) -3- ( ( (2R, 3R, 4S, 5S, 6S) -3, 4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) propanamido) 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-methylbutanamido) -3-oxopropoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2-3e)

To a mixture of 2-3d (67 mg, 0.024 mmol) and dichloromethane (2 mL) were added BAIB (23 mg, 0.071 mmol) and TEMPO (1.1 mg, 0.0071 mmol) . The mixture was stirred at room temperature for 4 hours. The mixture was purified by silica gel column chromatography (dichloromethane/methanol = 95/5) to afford 2-3e (40 mg, 95.7%yield) as a yellow solid.

MS (ESI) m/z: 2826.1 [M+H] ⁺.

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-diazahenicosan-21-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-2, 1'-cyclopropane] -10-carbonyl) ) bis (oxy) ) bis (methylene) ) bis (4, 1-phenylene) ) bis (azanediyl) ) bis (2- ( (S) -2-acetamido-3-methylbutanamido) -3-oxopropane-3, 1-diyl) ) bis (oxy) ) bis (3, 4, 5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-3f)

To a solution of 2-3e (10 mg, 0.0035 mmol) and DMF (1 mL) was added DEA (7 μL, 0.07 mmol) . The mixture was stirred at room temperature for 1 hour and concentrated under vacuum to give a residue. The residue was dissolved in sat. LiBr (1 mL, acetonitrile/water=10: 1) , then TEA (5 μL, 0.04 mmol) was added to the mixture. The mixture was stirred at room temperature for 30 mins. The mixture was adjusted to pH=7 using AcOH and filtrated and the filter purified using prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (0.1%formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to provide 2-3f (2 mg, 25.0%yield) as a white solid.

MS (ESI) m/z: 2322.7 [M+H] ⁺.

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-triazapentacosan-25-amido) benzyl) oxy) carbonyl) azanediyl) 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] diazepine-2, 1'-cyclopropane] -10-carbonyl) ) bis (oxy) ) bis (methylene) ) bis (4, 1-phenylene) ) bis (azanediyl) ) bis (2- ( (S) -2-acetamido-3-methylbutanamido) -3-oxopropane-3, 1-diyl) ) bis (oxy) ) bis (3, 4, 5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid) (2-3)

To a mixture of 2-3f (5 mg, 0.0022 mmol) , Int-4 (1.2 mg, 0.0043 mmol) and DMF (1 mL) was added DIPEA (2 μL, 0.01 mmol) . The mixture was stirred at room temperature for 30 mins. The mixture was adjusted to pH=7 using AcOH and filtrated and the filter purified using prep-HPLC (Method: column: XBridge Prep C18 OBD 5μm 19*150 mm; Mobile phase: A-water (0.1%formic acid) : B-acetonitrile; Flow rate: 20 mL/min) to provide 2-3 (3 mg, 54.5%yield) as a white solid.

MS (ESI) m/z: 2500.1 [M+H] ⁺.

Table 1: Linker payload structures and the corresponding released free payload

Example 5: Antibody Drug Conjugate (ADC) Preparation and Characterization

Drug-to-antibody ratio (DAR) 2 ADC preparation. Anti-CD74 antibody mAb1 SP7219 in reaction buffer (with concentration 0.5-25 mg/mL, 50 mM tris-HCl buffer pH 7.0-8.5) was incubated with 1/2000-1/500 w/w (EndoS2/mAb weight ratio) endoS2 under reaction temperature (0-40 ℃) for 1-24 hours to hydrolyze glycoforms present at Fc N-glycosylation sites. 2-40 eq. UDP-GalNAz (20 mM) and 0.1 w/w%-10 w/w% (GalT/mAb weight ratio) β1, 4-GalT were added into the reaction mixture and incubated in reaction buffer (50 mM Tris-HCl buffer pH 7.0-8.5, 20 mM MnCl₂) for 8-24 hours at reaction temperature (0-40 ℃) . The reaction mixture was purified with protein A resin to give the mAb1-GalNAz (N-azidoacetylgalactosamine modification) .

Organic solvent (e.g.: DMSO, DMF, DMA, PG, acetonitrile, 0-25%v/v) and linker-payload stock (10-25 eq, 10 mM stock in organic solvent) were added stepwise in reaction buffer (PBS buffer pH 7.0-8.5) with mAb1-GalNAz (1-20mg/mL) under 0-25 ℃ for 0.5-24 hours. The solution was submitted to buffer exchange (spin desalting column, ultrafiltration, and dialysis) into storage buffer (for example: pH 5.5-6.5 histidine acetate buffer, with optional additive such as sucrose, trehalose, tween 20, 60, 80) .

ADC characterization. All ADCs were characterized via the following analytical methods. DAR of the ADCs were determined by the following LCMS method or HIC method.

DAR determination
LCMS method. LC-MS analysis was carried out under the following measurement
conditions:
LC-MS system: Vanquish Flex UHPLC and Orbitrap Exploris 240 Mass Spectrometer
Column: MAbPac^TM RP, 2.1*50mm, 4μm, Thermo Scientific^TM
Column temperature: 80 oC
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 amount: 1 μg
MS paramaters: Intact and denaturing MS data were acquired in HMR mode at setting of
R=15k and deconvolved using the ReSpect^TM algorithm and Sliding Window integration in Thermo Scientific^TM BioPharma Finder^TM 4.0 software.
HIC method. HPLC analysis was carried out 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 amount: 20 μg

ADC purity
SEC method. HPLC analysis was carried out 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 4.6x150mm, Waters
Column temperature: room temperature
Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15%isopropyl
alcohol, pH 7.0
Gradient program: under 10 min isocratic elutions with the flow rate of 0.3 mL/min
Injected sample amount: 20 μg
SEC purity of ADCs were all > 95 %purity.

ADC hydrophobicity evaluation
ADCs with a higher hydrophobic property appear with later retention times from
hydrophobicity interaction column (HIC) chromatography. The DAR2 peak of the example ADCs was used for this comparison.
HIC Method 1. HPLC analysis was carried out 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 amount: 20 μg
HIC Method 2. HPLC analysis was carried out 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: The sample was diluted with initial mobile phase to 0.5 mg/mL.

Table 2: ADCs (Ab in ADC3-A, ADC3-B, ADC3-1, ADC3-2, and ADC3-3 is SP7219; Ab in Isotype ADC is CB6)

Antibody sequences
SP7219 wt sequence (anti-CD74 antibody)

Light Chain Sequence (SEQ ID NO: 1)

Heavy Chain Sequence (SEQ ID NO: 2)

CB6 sequence (Isotype antibody)

Light Chain sequence (SEQ ID NO: 3)

Heavy Chain sequence (SEQ ID NO: 4)

Example 6: Payload Direct Killing in A375 and Calu-6 cancer lines
Cell lines

A375 (ATCC, CRL-1619) . A-375 is a cell line exhibiting epithelial morphology that was isolated from the skin of a 54-year-old female patient with malignant melanoma, and A375 was purchased from ATCC. The base medium for A375 is DMEM, high glucose, with GlutaMAX^TM Supplement (Gibco, 10566024) . To make the complete growth medium, fetal bovine serum to a final concentration of 10% (Gibco, 10099-141C) was added to the base medium. The cell line was grown in a humidified 5%CO₂ atmosphere at 37 ℃, and was regularly tested for the presence of mycoplasma with MycoAlert^TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710) .

Calu-6 (ATCC, HTB-56) . Calu-6, purchased from ATCC, is a cell line exhibiting epithelial morphology that was isolated from a 61-year-old white female patient with anaplastic carcinoma, and Calu-6 was purchased from ATCC. The base medium for Calu-6 is Eagle's Minimum Essential Medium (ATCC, 30-2003) . To make the complete growth medium, fetal bovine serum to a final concentration of 10% (Gibco, 10099-141C) was added to the base medium. The cell line was grown in a humidified 5%CO₂ atmosphere at 37 ℃, and was regularly tested for the presence of mycoplasma with MycoAlert^TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710) .

Method. Payload direct killing was assessed in A375 and Calu-6 cancer lines. Cells were seeded (A375 at 1E3/well or Calu-6 at 2E3/well) into 96-well plates (Greiner: 655090) , 100 μl/well, and incubated at 37 ℃, 5%CO₂, overnight. Fresh growth medium was added containing varying concentrations of the payload compounds, 50 μl/well, and incubated at 37 ℃, 5%CO₂, for 6 days. The cell viability was detected by Cell Titer-Glo (Promega, G7573) , 70 μl/well. The plates were allowed to incubate at room temperature for 10 minutes to stabilize the luminescent signal. The plates were analyzed with a Microplate Reader.

Data are summarized in Table 3 and Figures 1 and 2. The results demonstrate that payloads P1-1 and P1-2 showed less direct cellular killing in A375 and Calu-6 cell lines compared to reference payloads Ref-1-1 and Ref-1-2. The data indicate that payloads P1-1 and P1-2 alone are less toxic to cancer cells than are the reference payloads.

Table 3: Payload cellular killing of A375 and Calu-6 cells

Example 7: ADC direct killing in NOMO-1 and K562 cancer lines
Cell lines

NOMO-1 (JCRB, IFO50474) . NOMO-1 is a cell line exhibiting hemo-lymphocytic morphology, and NOMO-1 was purchased from JCRB. The base medium is ATCC-formulated RPMI-1640 Medium, ATCC 30-2001. To make the complete growth medium, fetal bovine serum was added to a final concentration of 10% (Gibco, 10099-141C) . The cell line was grown in a humidified 5%CO₂ atmosphere at 37 ℃, and was regularly tested for the presence of mycoplasma with MycoAlert^TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710) .

K562 (ATCC, CCL-243) . K562 is a cell line exhibiting lymphoblast morphology, and K562 was purchased from ATCC. The base medium for K562 is ATCC-formulated Iscove's Modified Dulbecco's Medium, Catalog No. 30-2005. To make the complete growth medium, fetal bovine serum was added to a final concentration of 10% (Gibco, 10099-141C) . The cell line was grown in a humidified 5%CO₂ atmosphere at 37 ℃, and was regularly tested for the presence of mycoplasma with MycoAlert^TM PLUS Mycoplasma Detection Kit (Lonza, LT07-710) .

Table 4: CD74 expression level

Method. NOMO-1 (6E3/well) or K562 (3E3/well) cells were seeded into 96-well plates (Greiner: 655090) at 100 μl/well. 100 μg/ml Fc blocker was added to NOMO-1 cells. Cells were incubated at 37 ℃, 5%CO₂, for 2 hrs.

Fresh growth-medium containing varying concentrations of ADC was added at 50 μl/well. Cells were incubated at 37℃, 5%CO₂, for 6 days.

Cell viability was detected by Cell Titer-Glo (Promega, G7573) . Reagent was added at 70 μL/well. Plates were incubated at room temperature for 10 minutes to stabilize the luminescent signal. Then the plates were analyzed with a microplate reader.

Data are presented in Tables 5 and 6 and Figures 3A-4B. The results demonstrate that ADC3-1 and ADC3-2, each having a prodrug design, exhibited similar killing potency compared to reference ADC3-A and ADC3-B in NOMO-1 cells (CD74 positive cell line) . In K562 cells (CD74 negative cell line) , ADC3-1 and ADC3-2 showed no killing, whereas ADC3-A and ADC3-B showed similar non-specific killing compared to Isotype ADC. The present data, in combination with the payload direct killing results shown in Table 3 and Figures 1-2, indicate that a PBD dimer-based ADC with a prodrug design could minimize non-specific killing in antigen negative cells while remaining active in antigen positive cells, and any premature release of payload in the systemic circulation might cause less harm to healthy tissue.

Table 5: ADC cellular killing of NOMO-1 and K562 cells

Table 6: ADC cellular killing of NOMO-1 and K562 cells

Although the foregoing disclosure has been presented in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country.

The disclosures of all non-patent publications, patents, patent applications, and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entireties.

Claims

A compound of Formula (I) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof, wherein each of structure A and structure B is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;

each m and n is independently 1 or 2;

between -C (R¹) -and -N (R²) -is, independently, a single bond or a double bond;

whenis a single bond, each R¹ is independently H or OH, and each R² is H;

whenis a double bond, each R¹ is H, and each R² is absent;

structure C is a conjugator;

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.
The compound of claim 1, wherein either or both of structure A and structure B is
The compound of claim 1, wherein either or both of structure A and structure B is
The compound of claim 1, wherein one of structure A and structure B isand the other of structure A and structure B is
The compound of any one of claims 1 to 4, wherein p and q are both 2.
The compound of any one of claims 1 to 3 and 5, wherein the compound has the following formula:
The compound of any one of claims 1 to 6, wherein the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;

s is 1, 2, or 3; and

each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer thereof.
The compound of any one of claims 1 to 7, wherein the conjugator has the following formula:
The compound of any one of claims 1 to 8, wherein the conjugator has the following formula:
The compound of any one of claims 1 or 5 to 9, wherein the compound has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.
A compound of Formula (V) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,

wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;

each m and n is, independently, 1 or 2;

each R¹ is, independently, H or OH;

structure C is a conjugator;

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’ is, independently, and

each Su is independently a sugar moiety.
The compound of claim 11, wherein either or both of structure A’ and structure B’ is
The compound of claim 11, wherein either or both of structure A’ and structure B’ is
The compound of any one of claims 11-13, wherein one of structure A’ and structure B’ isand the other of structure A’ and structure B’ is
The compound of any one of claims 11 to 14, wherein p and q are both 2.
The compound of any one of claims 11 to 13 and 15, wherein the compound has the following formula:
The compound of any one of claims 10 to 15, wherein the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;

s is 1, 2, or 3; and

each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer thereof.
The compound of any one of claims 11 to 17, wherein the conjugator has the following formula:
The compound of any one of claims 11 to 18, wherein the conjugator has the following formula:
The compound of any one of claims 11 to 19, wherein

each sugar moiety is independently or a stereoisomer thereof; and

each m is independently 0 or 1.
The compound of claim 20, wherein the sugar moiety isand m is 0.
The compound of any one of claims 11 to 21, wherein each R^2’ is, independently,
The compound of claim 22, wherein each R^2’ is, independently,
The compound of any one of claims 11 or 15 to 23, wherein the compound has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.
An antibody drug conjugate (ADC) of Formula (VI) :

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof,

wherein each of structure A’ and structure B’ is independently selected from one of the following formulas:

ring D is a cyclopropyl ring or a cyclobutyl ring;

each m and n is, independently, 1 or 2;

each R¹ is, independently, H or OH;

structure C is a conjugator;

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’ is, independently,

each Su is independently a sugar moiety;

Ab is a humanized, chimeric, or human antibody or an antigen binding fragment thereof; and

x is from about 1 to about 8.
The ADC of claim 25, wherein either or both of structure A’ and structure B’ is
The ADC of claim 25, wherein either or both of structure A’ and structure B’ is
The ADC of claim 25, wherein one of structure A’ and structure B’ isand the other of structure A’ and structure B’ is
The ADC of any one of claims 25 to 28, wherein p and q are both 2.
The ADC of any one of claims 25 to 27 and 29, wherein the ADC has the following formula:
The ADC of any one of claims 25 to 30, wherein the conjugator has the following formula:

r is 1, 2, 3, 4, 5, or 6;

s is 1, 2, or 3;

each occurrence of AA is independently a naturally occurring amino acid, or a stereoisomer thereof;

is the point of attachment to Ab.
The ADC of any one of claims 25 to 31, wherein

the conjugator has the following formula
The ADC of any one of claims 25 to 32, wherein the conjugator has the following formula:
The ADC of any one of claims 25 to 33, wherein

the sugar moiety is or a stereoisomer thereof; and

each m is independently 0 or 1.
The ADC of any one of claims 25-34, wherein the sugar moiety isand m is 0.
The ADC of any one of claims 25 to 35, wherein each R^2’ is, independently,
The ADC of any one of claims 25 to 36, wherein each R^2’ is, independently,
The ADC of any one of claims 25 or 29 to 37, wherein the ADC has one of the following formulas:

or a pharmaceutically acceptable salt, tautomer, solvate, or stereoisomer thereof.
The ADC of any one of claims 25 to 38, wherein x is about 2.