Conjugation Reagents and Conjugates Thereof

Abstract

The present invention relates to an antibody conjugate and a method for producing the same. In particular, the present invention relates to a conjugate comprising an antibody, a linker and at least one active agent, i) a linker that connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) Each covalent bond between a linker and an antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

Description

[Technical Field]

[0001] The present invention relates to antibody conjugates and methods for making the same. More particularly, the present invention relates to obtaining an antibody, a linker, and an active agent, such as a drug or labeling moiety, to produce a conjugate. Furthermore, the present invention provides improved linkers for use in the conjugates and methods for introducing the linkers into the conjugates. More specifically, the conjugates can be antibody drug conjugates (ADCs). [Background technology]

[0002] Selective delivery of drugs to malignant cells without undesirable toxicity to healthy tissues is one of the major goals in the development of modern cancer therapeutics. Antibody-drug conjugates (ADCs) are a promising class of targeted therapeutics that aim to address this need (Walsh 2021, Beck 2014, Teicher 2012). By leveraging the exquisite specificity of antibodies for cell-surface antigens overexpressed by cancer cells, ADCs enable the selective delivery of highly cytotoxic payloads to tumor sites. The utility of this treatment strategy has been demonstrated recently by the Food and Drug Administration (FDA) approval of enfortumab vedotin (Padcev™), trastuzumab deruxtecan (Enhertu™), sacituzumab govitecan (Trodelvy™), and belantamab mafodotin (Blenrep™), increasing the number of ADCs on the market to 10 (Challita-Eid 2016, Modi 2020, Kaplon 2020, Syed 2020, and Hamadani 2021).

[0003] However, current methods for producing ADCs still suffer from various shortcomings. One common strategy for forming ADCs involves conjugation to surface-exposed lysine residues of the payload via an N-hydroxysuccinimide (NHS) ester motif. Another approach involves conjugation to antibody cysteine ​​residues. Human IgG1 antibodies contain four interchain disulfides that can be reduced to reveal eight nucleophilic cysteine ​​residues that can be conjugated to linkers containing electrophilic motifs, such as maleimides. Due to the high prevalence of reactive lysine (>50) and cysteine ​​(8) residues in antibodies, engineering at these residues suffers from an inherent lack of site selectivity, resulting in highly heterogeneous ADCs that vary in terms of conjugation site and the number of drug molecules attached to each antibody (drug-to-antibody ratio, DAR) (Kim 2014; Jackson 2016; Freedy 2016; Chudasama 2016). The conjugation site and DAR have been shown to have a significant effect on both the safety and efficacy of ADCs (Hamblett 2004; Shen 2012; Strop 2013). All currently FDA-approved ADCs are synthesized via lysine or cysteine ​​conjugation and exist as heterogeneous mixtures of conjugates with DARs ranging from 0 to 8 and up to 70 different conjugation sites, making their pharmacokinetic profiles unnecessarily complex and difficult to predict (Kim 2014; Agarwal 2015; Godwin 2017; Bross 2001; Hedrich 2018).

[0004] Furthermore, maleimide bioconjugation, one of the most commonly used cysteine ​​engineering strategies, has been shown to result in unstable linkages prone to retro-Michael addition under physiological conditions (Alley 2008; Lyon 2014). This instability can lead to premature dissociation of the payload from the antibody, potentially resulting in undesirable toxicity to healthy tissues.

[0005] Several strategies have been reported to circumvent these issues by incorporating unnatural amino acids with bioorthogonal functionalities or by modifying antibody glycans (Fang 2014, Amant 2019, Amant 2019*, Walker 2019, Axup 2012, Zimmerman 2014). While these approaches have produced ADCs with high stability, defined attachment points, and precise DARs, the generation of such non-native antibody formats is generally laborious, low-yielding, and may pose the risk of immunogenicity. Therefore, the establishment of site-selective conjugation strategies using native antibodies is preferred. Disulfide-bridged linkers have emerged as an attractive class of reagents that enable the synthesis of homogeneous ADCs from native antibodies (Forte 2018). These linkers contain two cysteine ​​reactive groups that can react with reduced interchain disulfides in IgG molecules to cause covalent re-crosslinking of antibody chains. Dibromomaleimide (Behrens 2015, Smith 2010), pyridazinedione (Robinson 2017), bissulfone (Badescu 2014), and arylene-dipropiolonitrile (Koniev 2018) reagents have all been used in this context to successfully generate ADCs with demonstrated in vitro and in vivo activity. Recently, the development of disulfide-recrosslinked divinylpyrimidine (DVP) linkers has also been reported (Walsh 2019, Walsh 2020). The recrosslinking approach achieved significant improvements in homogeneity compared to stochastic cysteine ​​modifications, yielding a defined DAR of 4 for all synthesis batches. However, a major limitation of this approach is the formation of "half-antibody" species during bioconjugation, which is a result of non-native intrachain crosslinking of cysteine ​​residues in the hinge region of antibodies. Half-antibody formation has been observed with all types of disulfide cross-linkers (Forte 2018). The loss of covalent bonds between antibody heavy chains resulting from this non-native re-cross-linking is associated with reduced antibody stability, and therefore, the development of methods to prevent half-antibody formation is highly desirable (Bahou 2019).

Prior Technical Literature

Charter Documents

[0006] [Patent Document 1] US4816567 [Patent Document 2] WO2007 / 085930 [Patent Document 3] WO2017 / 186894の14からpage 86 [Patent Document 4] WO2019 / 011078

Non-licensed literature

[0007]

Non-patent document 1

Non-patent document 2

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Non-patent document 4

Non-patented document 5

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Non-patent document 8

[0008] The present invention aims to provide a disulfide bridge linker platform that addresses reported stability issues while still retaining the benefits of precise ratios between active agent and antibody and the ability to distribute the active agent. Additionally, the present invention aims to provide a disulfide bridge linker platform that addresses the issue of half-antibody formation and provides better control over the number of drug molecules attached to the antibody. [Means for solving the problem]

[0009] Thus, in a first aspect of the invention there is provided a conjugate comprising an antibody, a linker and at least one active agent, i) a linker connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) A conjugate is provided in which each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0010] The present invention provides linkers for use in antibody-drug conjugate (ADC) molecules that have utility in linking antibodies and active agents, such as antibodies and cytotoxins, to yield ADC molecules. The linkers provide an improved method for controlling the loading of active agents on antibodies. Thus, the present invention provides linkers for use in obtaining homogeneous ADCs with controlled drug-to-antibody ratios.

[0011] The linker may also provide an improved targeting payload for the active agent, thus improving the activity of the conjugate in which the active agent exerts biological activity, such as cytotoxicity. Additionally or alternatively, the linker may provide the conjugate with increased stability compared to currently known linker molecules for use in the conjugate. This may improve the tolerability of such conjugates.

[0012] The linker can be directly bonded to five to eight thiol groups (sulfur atoms) of cysteine ​​residues in the antibody. Thus, the linker connects to four reduced interchain disulfide bonds via one or both of the sulfur atoms associated with the four reduced interchain disulfide bonds. Thus, the linker serves to re-bridge one to four reduced interchain disulfide bonds in the antibody. Thus, the linker can be connected to an antibody (e.g., a human IgG1 antibody) via five to eight covalent bonds to the eight thiol groups of cysteine ​​residues involved in the interchain disulfide bonds. Specifically, the linker can be connected to an antibody (e.g., a human IgG1 antibody) via five to eight covalent bonds to the eight thiol groups of cysteine ​​residues involved in the interchain disulfide bonds in the hinge region of the antibody.

[0013] Preferably, the linker is directly bonded to 6 to 8, or more preferably 7 to 8, thiol groups (sulfur atoms) of cysteine ​​residues in the antibody.

[0014] Thus, the linker may re-bridge one to four of the reduced interchain disulfide bonds in an antibody (e.g., a human IgG1 antibody) via five to eight covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Preferably, the linker is directly bonded to six to eight, or more preferably seven to eight, thiol groups (sulfur atoms) of the cysteine ​​residues in the antibody. Thus, the linker re-bridges two to four, or more preferably three to four, of the reduced interchain disulfide bonds in an antibody (e.g., a human IgG1 antibody), preferably via six to eight (or more preferably seven to eight) covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Most preferably, the linker is directly bonded to the eight thiol groups (sulfur atoms) of the cysteine ​​residues in the antibody. Thus, the linker re-crosslinks all four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds.

[0015] In a second aspect of the present invention, a conjugation reagent capable of reacting with an antibody is provided, said conjugation reagent comprising a linker, at least one active agent, and eight functional groups capable of reacting with five to eight sulfur atoms in the antibody.

[0016] Thus, preferably, the conjugation reagent of the present invention has formula (II) shown below: (DL 1 -FG') n -L-(Z)8 (Formula II) (In the formula, FG' is a functional linking moiety; n is an integer selected from 0 to 20; L and L 1 is independently a linker, Z is a functional group capable of reacting with a sulfur atom from a cysteine ​​moiety of an antibody; D is an active agent), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0017] This conjugation reagent is capable of reacting with an antibody to form the conjugate of the first aspect of the invention.

[0018] Preferably, the conjugation reagent comprises a linker, at least one active agent, and eight functional groups capable of reacting with six to eight, more preferably seven to eight, and most preferably eight sulfur atoms in the antibody.

[0019] In a third aspect of the invention, there is provided an intermediate conjugate comprising an antibody, a linker, and at least one functional group capable of reacting with another moiety to form a functional linking moiety, i) a linker connects at least one functional group to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) An intermediate conjugate is provided in which each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0020] Thus, the intermediate conjugate can be reacted with at least one active agent to form the conjugate of the first aspect of the present invention. This reaction between the intermediate conjugate and the active agent can occur via reaction of at least one functional group on the intermediate conjugate and another functional moiety on the active agent. Thus, a functional linking moiety is formed that links the intermediate conjugate to the active agent.

[0021] In a fourth aspect of the invention, there is provided a compound capable of reacting with both an antibody and at least one active agent, said compound comprising a linker, eight functional groups capable of reacting with five to eight sulfur atoms in the antibody, and at least one functional group capable of reacting with another moiety to form a functional linking moiety, which serves as a means by which at least one active agent can be attached to the compound.

[0022] Thus, preferably, the compounds of the present invention have formula (I) as shown below: (FG) n -L-(Z)8 (Formula I) (In the formula, FG is a functional group that is capable of reacting with another moiety to form a functional linking moiety; n is an integer selected from 0 to 20; L is a linker, Z is a functional group capable of reacting with a sulfur atom from a cysteine ​​moiety of an antibody), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0023] The compound of the fourth aspect is a precursor to the conjugation reagent of the second aspect of the invention. The compound of the fourth aspect is also a precursor to the intermediate conjugate of the third aspect of the invention. The conjugation reagent can be formed by reacting a compound of formula (I) with an active agent, which can have a linker group already attached. The intermediate conjugate can be formed by reacting a compound of formula (I) with an antibody (an antibody that has had its interchain disulfide bonds reduced).

[0024] In a fifth aspect of the invention there is provided a pharmaceutical composition comprising the conjugate of the first aspect of the invention, wherein the active agent is a drug and a carrier, excipient or diluent. The fifth aspect also provides the conjugate of the first aspect of the invention, wherein the active agent is a drug for use in a method of treatment.

[0025] It will be appreciated that the pharmaceutical composition according to the fifth aspect of the invention may comprise a mixture of conjugates of the first aspect of the invention. That is, it will be appreciated that the pharmaceutical composition may comprise a mixture of conjugates in which the linker is attached to the antibody via 5, 6, 7 or 8 independent covalent bonds. It will also be appreciated that the pharmaceutical composition may comprise conjugates in which the linker is attached to the antibody via 1 to 4 independent covalent bonds.

[0026] Preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 65% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. More preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 70% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. Even more preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 75% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. Even more preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 80% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. Even more preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 85% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. Even more preferably, the pharmaceutical composition comprises a mixture of conjugates, wherein at least 90% of the conjugates comprise a linker attached to the antibody via eight independent covalent bonds. Even more preferably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 95% of the conjugates consist of a linker attached to the antibody via eight independent covalent bonds. Most preferably, the pharmaceutical composition comprises a mixture of conjugates wherein at least 98% of the conjugates consist of a linker attached to the antibody via eight independent covalent bonds.

[0027] Thus, preferably, at least 65% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. More preferably, at least 70% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Even more preferably, at least 75% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Even more preferably, at least 85% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Even more preferably, at least 90% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds. Even more preferably, at least 95% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds.Most preferably, at least 98% of the conjugates present in the pharmaceutical composition are conjugates of the first aspect of the invention, in which the linker re-bridges four of the reduced interchain disulfide bonds in the antibody (e.g., a human IgG1 antibody) via eight covalent bonds to each of the eight thiol groups (sulfur atoms) of the cysteine ​​residues involved in the interchain disulfide bonds.

[0028] In a sixth aspect of the present invention, there is provided the use of a conjugate of the first aspect of the present invention in the manufacture of a medicament for treating a proliferative disease, wherein the active agent is a cytotoxic drug. The sixth aspect also provides a conjugate of the first aspect of the present invention for use in treating a proliferative disease, wherein the active agent is a cytotoxic drug. The sixth aspect also provides a method of treating a proliferative disease, comprising administering to a patient in need thereof a therapeutically effective amount of a conjugate of the first aspect of the present invention, wherein the active agent is a cytotoxic drug. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition Throughout this description and the claims, the words "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude other moieties, additives, ingredients, integers, or steps. Throughout this description and the claims, the singular includes the plural unless the context clearly dictates otherwise. In particular, where the indefinite article is used, the specification is to be understood to contemplate the plural as well as the singular unless the context clearly dictates otherwise.

[0030] It should be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention may be applicable to any other aspect, embodiment, or example described herein, to the extent not inconsistent therewith. All features disclosed herein (including any accompanying claims, abstract, and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention extends to any novel, or any novel combination of, features disclosed herein (including any accompanying claims, abstract, and drawings), or any novel, or any novel combination of steps of any method or process so disclosed.

[0031] The terms "specifically bind" and "specifically binding" refer to the binding of an antibody to a predetermined molecule (e.g., an antigen). Typically, an antibody binds to at least about 1 x 10 7 M -1 and binds to a given molecule with an affinity that is at least two-fold higher than the affinity for binding to a nonspecific molecule other than the given molecule or a closely related molecule (e.g., BSA, casein).

[0032] antibody The term "antibody" herein is used broadly to specifically cover monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies, and antibody fragments, so long as they exhibit the desired biological activity (Miller et al. (2003), Jour. of Immunology 170:4854-4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th ed., Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, which are captured by CDRs in multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules or portions thereof that contain an antigen-binding site that immunospecifically binds to an antigen of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. Immunoglobulins 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 of immunoglobulin molecule. Immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

[0033] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab'), and scFv fragments; bispecific antibodies; linear antibodies; fragments produced by an Fab expression library; anti-idiotypic (anti-Id) antibodies; CDRs (complementarity-determining regions); and epitope-binding fragments of any of the above that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for minor, naturally occurring mutations that may be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597, or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

[0035] The monoclonal antibodies herein specifically include chimeric antibodies, humanized antibodies and human antibodies.

[0036] Examples of cell binding agents include those agents described for use in WO2007 / 085930, which is incorporated herein.

[0037] Tumor-associated antigens and syngeneic antibodies for use in embodiments of the present invention are listed below and are also described in more detail on pages 14 to 86 of WO2017 / 186894, which is incorporated herein by reference.

[0038] activator The active agent can be a drug, a labeling moiety, or a targeting moiety. Preferably, the active agent is a drug or a labeling agent. The active agent must include a functional group that can be linked to a linker. Such groups can include, for example, an amino group, an imine group, or a hydroxy group. In situations where more than one active agent is present, it should be understood that two or more of the same or different active agents can be present. For example, in situations where two or more active agents are present, the two or more active agents can be independently selected from a drug, a labeling moiety, or a targeting moiety. It is also understood that in situations where more than one drug, a labeling moiety, or a targeting moiety is present, each drug, labeling moiety, and targeting moiety can be the same or different.

[0039] drugs The drug may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the drug is preferably a cytotoxin.

[0040] Preferably, the cytotoxin is a biologically active cytotoxic agent, which may be selected from the group including auristatins, maytansinoids, tubulysins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (particularly pyrrolobenzodiazepine dimers), camptothecin analogs, and doxorubicin.

[0041] However, the cytotoxin may also or alternatively be selected from other known cytotoxins, including ricin subunits and other peptide-based cytotoxic agents, although such agents are less commonly available in the art.

[0042] Sign part The labeling moiety can be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), indocicarbocyanine (Cy5), indocarbocyanine (Cy3), and those known under the trade names Alexa Fluor (e.g., 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (e.g., 405, 488, 550, 650, 680, 755, 800).

[0043] The labeling moiety can also be a biotin tag, which is derived from biotin.

[0044] The labeling moiety can also be a radioisotope or a radioisotope-containing moiety. Preferably, the labeling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [F]fludeoxyglucose (F)(FDG)-glucose analogs, [C]acetate, [C]methionine, [C]choline, copper 64 Cu dotatate, [18F]EF5, [18F]fluciclovine, [18F]fluorocholine, [18F]fluoroethyl-L-tyrosine, [18F]fluoromisonidazole, [18F]fluorothymidine F-18, 64 [68Ga]DOTA-pseudopeptide, [68Ga]DOTA-TATE, and [68Ga]prostate-specific membrane antigen (PSMA).

[0045] targeting part The targeting moiety is a moiety that helps direct the conjugate to a specific site of occurrence in the body. It is recognized that any suitable targeting moiety can be used. Preferably, the targeting moiety is a carbohydrate, such as alpha-D-galacto-hexopyranosyl-(1->3)-2-acetamido-2-deoxy-D-galacto-hexopyranose (Gal-alpha1,3-GalNAc).

[0046] Conjugates of the Invention According to one aspect of the invention, there is provided a conjugate comprising an antibody, a linker, and at least one active agent, i) a linker connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) A conjugate is provided in which each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0047] Preferably, each covalent bond between a linker and an antibody is formed from the reaction between a sulfur atom in the antibody and an electrophilic functional group in the linker.

[0048] In certain embodiments, the conjugate has formula (III) shown below: [ka] (In the formula, Z A is a functional linking group, Pep indicates the position at which the moiety is attached, directly or indirectly, to the antibody so that the re-cross-linking moiety re-cross-links reduced interchain disulfide bonds in the antibody; [ka] indicates the position where the moiety is attached to the linker).

[0049] Preferably, the conjugate comprises two to four re-bridging moieties of formula (III). More preferably, the conjugate comprises three to four re-bridging moieties of formula (III). Most preferably, the conjugate comprises four re-bridging moieties of formula (III).

[0050] Functional linking group Z A It is understood that the functional linking group Z can be any functional group that is capable of linking the antibody to the linker. A is therefore a functional grouping capable of forming a covalent attachment to a sulfur atom present in an antibody. Preferably, the sulfur atom is a sulfur atom from a cysteine ​​residue in the antibody, more preferably a sulfur atom from a cysteine ​​residue from a reduced interchain disulfide bond of the antibody.

[0051] Functional linking groups capable of linking an antibody to a linker are thus well known in the art. Therefore, one skilled in the art can select a suitable functional linking group for use in the present invention. Suitable functional linking groups Z A Examples include, for example, those described in Xu 2021, Stieger 2021, Walsh 2019, Walsh 2020, Badescu 2014, Koniev 2018, Robinson 2017, Behrens 2015 and WO2019 / 011078.

[0052] In one embodiment, Z A is selected from one of the functional linking groups shown below, such that the conjugate is independently selected from the following group: [ka] [ka] (In the formula, A 1 , A 2 and A 3 One or two of these are N, and the other A 1 , A2 and A 3 One or two of are CH, or A 1 , A 2 and A 3 All three are N or C, a and b are integers selected from 0 or 1; X is N, NR N , O and S; R N is H or C 1~2 is alkyl, R 1 and R 2 independently, C 1~ selected from C6 alkylene and C1 to C6 alkylene containing O in the skeleton, R 3 is selected from hydrogen or C1-C4 alkyl, Y 1 and Y 2 is independently absent, or O, NR 4 , C(=O), C(=O)NR 4 Or NR 5 C(=O), p and q are independently integers selected from 0 or 1; Q is CR 6 , N or aryl; R 4 , R 5 and R 6 are independently selected from hydrogen and C1-C4 alkyl; Pep indicates the position at which the moiety is linked, directly or indirectly, to the antibody; [ka] indicates the position at which the moiety is attached to the linker), and

[0053] In one embodiment, A 1 , A 2 and A 3 Two of them are N (for example, A 1 and A2 is N), and A 1 , A 2 and A 3 Others are CH (e.g., A 3 is CH). Preferably, in this embodiment, the integers a and b are 1.

[0054] In other embodiments, A 1 , A 2 and A 3 All three of are N. Preferably, in this embodiment, the integers a and b are 1.

[0055] In some embodiments, A 1 , A 2 and A 3 One of the is N (e.g., A 3 is N), and A 1 , A 2 and A 3 The other two are CH (e.g., A 1 and A 2 is CH). Preferably, in this embodiment, the integers a and b are 0.

[0056] Preferably, X is NH or O. Most preferably, X is NH.

[0057] In one embodiment, R 1 and R 2 independently, C 1~ C alkylene. Preferably, R 1 and R 2 independently, C 1~ C4 alkylene.

[0058] In some embodiments, R 3 is a C1-C4 alkyl (for example, a methyl group).

[0059] In some embodiments, Y 1 and Y 2 is not independently present, or O, NR 4 and C(=O). Preferably, Y 1and Y 2 does not exist independently.

[0060] Preferably, Q is CR 6 or N. In some embodiments, Q is CR 6 In other embodiments, Q is N.

[0061] In some embodiments, R 4 , R 5 and R 6 are independently selected from hydrogen and methyl. 4 , R 5 and R 6 are each hydrogen.

[0062] In certain embodiments, the conjugates independently comprise: [ka] (In the formula, A 1 , A 2 , A 3 , X and Pep each comprise 1 to 4 (preferably 2 to 4, more preferably 3 to 4, and most preferably 4) re-bridging moieties selected from the group:

[0063] In other embodiments, the conjugate has the general formula (IIIa) shown below: [ka] (In the formula, A 1 , A 2 , A 3 , X, Pep and [ka] each of which contains 1 to 4 (preferably 2 to 4, more preferably 3 to 4, and most preferably 4) re-crosslinking moieties (as defined herein).

[0064] In some embodiments, A1 and A 2 is N.

[0065] In other embodiments, A 1 and A 3 is N.

[0066] In some embodiments, X is NR N , O and S; R N is H or C 1~2 In these embodiments, only a single active agent may be attached to X via the linker.

[0067] In some of these embodiments, X is NR N X can be NH, NCH3 or NCH2CH3.

[0068] In other of these embodiments, X is O.

[0069] In other of these embodiments, X is S.

[0070] In some embodiments, X is N. In these embodiments, X is: [ka] (In the formula, A 1 , A 2 , A 3 , Pep and [ka] Each of may be attached to the linker via two covalent bonds shown in (as defined herein).

[0071] Linker (L) A linker is a group (functional group) that connects an antibody to an active agent. The linker is attached to the antibody via 5 to 8 covalent bonds (preferably via 6 to 8, more preferably via 7 to 8, and most preferably via 8) formed between a sulfur atom in the antibody and a functional group (e.g., an electrophilic functional group) in the linker. The sulfur atom in the antibody can be a sulfur atom in a cysteine ​​residue in the antibody, for example, a sulfur atom from a cysteine ​​residue in the antibody, which is involved in the interchain disulfide linkage of the antibody (i.e., the sulfur atom from the cysteine ​​residue becomes available following reduction of the interchain disulfide linkage of the antibody).

[0072] The linker is therefore a functional linking group (Z A The linker can thus be attached to the antibody via a functional linking group (Z A ) can be connected to at least one active agent. The linker can be connected to at least one active agent via a functional linking moiety (FG'), as defined herein below.

[0073] It is understood that the linker can be any group (functional group) that is capable of connecting at least one active agent to the antibody.

[0074] In certain embodiments, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 10 to 60 atoms. Preferably, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 10 to 50 atoms. More preferably, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 10 to 40 atoms. Even more preferably, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 12 to 40 atoms. Even more preferably, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 15 to 40 atoms. Most preferably, the linker is such that at least two of the re-bridging moieties (i.e., groups of Formula (III)) are separated by 16 to 38 atoms.

[0075] In some embodiments, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue, an arylene-containing moiety, a heteroarylene-containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety, and combinations thereof. Preferably, the linker comprises 1 to 20 of the above-mentioned groups. More preferably, the linker comprises 1 to 15 of the above-mentioned groups. Even more preferably, the linker comprises 1 to 10 of the above-mentioned groups.

[0076] Preferably, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue, and combinations thereof.

[0077] An alkylenediamine moiety is understood to be a functional group containing or derived from an alkylenediamine moiety, such as ethylenediamine.

[0078] A polyethylene glycol moiety is understood to be a functional group that includes one or more alkylene glycol moieties, e.g., one or more ethylene glycol moieties. In some embodiments, the linker includes 1 to 10 alkylene glycol moieties (e.g., ethylene glycol moieties).

[0079] It is understood that the amino acid residue comprises both natural and non-natural amino acid residues. Preferably, the amino acid residue is a natural amino acid residue. The amino acid residue may also comprise two or more amino acids, such as dipeptide and tripeptide moieties. Preferred amino acid residues include Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp residues.

[0080] An arylene-containing moiety is understood to be a functional group containing one or more arylene groups. Preferably, the arylene is a 6-membered arylene, such as phenylene. In one embodiment, the arylene-containing moiety is a functional group containing a 1,3,5-benzyl group.

[0081] It is understood that a heteroarylene-containing moiety is a functional group containing one or more heteroarylene groups. Preferably, the heteroarylene is a functional group containing a 6-membered heteroaryl ring, such as a triazine. In some embodiments, the arylene-containing moiety is a functional group containing a 1,3,5 triazine group.

[0082] It is understood that a heterocyclyl-containing moiety is a functional group containing one or more heterocyclic groups. Preferably, the heterocycle is a 6-membered heterocycle, such as a triazinane. In one embodiment, the heterocyclyl-containing moiety is a functional group containing a 1,3,5 triazinane group.

[0083] A cycloalkyl-containing moiety is understood to be a functional group containing one or more cycloalkyl groups. Preferably, the cycloalkyl is a 6-membered cycloalkyl, such as cyclohexane. In one embodiment, the cycloalkyl-containing moiety is a functional group containing 1,3,5 cyclohexane.

[0084] Preferred conjugates In certain embodiments, the conjugate has formula (IIIA) shown below: [ka] (In the formula, each Z 1 is a re-bridging moiety of formula (III) as defined hereinabove, wherein the re-bridging moiety is attached to the antibody at the position indicated in formula (III); Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=O)NR 7 , C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene; v is an integer selected from 0, 1, or 2; Each Q 2 are, independently, N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 is independently absent or is of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently bonded, C1 to C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is of formula IVc or formula IVe: [ka] is the basis of W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amide, O or N in the skeleton; Ring A is a 6-membered aryl, 6-membered heteroaryl, 6-membered heterocyclyl, or 6-membered cycloalkyl; each X is independently selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16-, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently bonded, C1 to C 12 Alkylene and C1-C containing O in the skeleton 12 alkylene; m is an integer selected from 1 or 2; W 1C is the formula W C1 or formula W C2 : [ka] is selected from W Q1 is the formula W C3 : [ka] is the basis of Q W1 does not exist, or C1 to C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2 is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; X W1 is selected from O or NH, X W2 is hydrogen, C1-C4 alkyl, OR x1 and NR x1 R x2 Selected from R x1 and R x2 are independently selected from hydrogen and C1-C4 alkyl; [ka] is XW1 Formula W Q2 indicates the position of the bond to the base of [ka] is W Q1 is the formula R 24 indicates the position of the bond to the base of W Q2 is the formula W C4 : [ka] is the basis of Q W1A does not exist, or C1~C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2A is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; L Q1 is C1~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; X W3 is selected from O or NH, [ka] is X W4 But, Q W2A via the substituents, the formula W Q2 indicates the position of attachment to another group of XW 4 is selected from the following groups: hydrogen or -C(=O)R X3 Point to the location attached to one of the R x3 is selected from hydrogen and C1-C4 alkyl; [ka] Q W2A But X W1 via the group W Q1indicates the position of the bond to the base of X W4 is selected from O or NH, L Q1 is C1~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 8; FG' is a functional linking moiety; L 1 is the linker, D is an activator, W 2 is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 is selected from W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amide, O or N in the skeleton; R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently bonded, C1 to C 12 Alkylene and C1-C containing O in the skeleton12 alkylene; FG', L 1 and D is as defined above; r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; r and s are selected so that the total number of active agents per conjugate is 1 to 12.

[0085] If the integer v is 2, then two [ka] It is understood that the groups can be the same or different.

[0086] If the integer m is 2, then two W B1 and two R 24 It is also understood that the groups may be the same or different. B1 and two R 24 The groups are identical.

[0087] Preferably, m is 1.

[0088] Suitably, ring A is selected from phenyl, 1,3,5-triazine or 1,3,5 triazinane.

[0089] Preferably, W 1 is a group of formula IVc.

[0090] In one embodiment, W 1 is a group of formula IVc, W 1C is the formula W C1 It is based on.

[0091] In other embodiments, W 1 is a group of formula IVc, W 1C is the formula W C2 It is based on.

[0092] In one embodiment, LW is absent or selected from C1-C6 alkylene and C1-C6 alkylene containing one or more of O or N in the backbone.

[0093] W 1 is a group of formula IVc, and W 1C But, formula W C2 Preferably, t is an integer selected from 1 to 6, most preferably t is an integer selected from 1 to 4.

[0094] Therefore, the linker defined herein above, which relates to the first aspect of the present invention, can be represented by the formula L X1 or formula L X2 : [ka] [ka] (In the formula, Z 1 , Q 1 , Q 2 , Q 3 , W 1A , W 1B , W 1C , W 2A , W 2B , W 2C , W Q1 , W Q2 , R 23 , R 24 , R 25 , R 26 , R 27 , FG', L 1 , L W , L 2W , L Q1 , m, t, r, and s are as defined herein; [ka] indicates the position at which the linker is attached to the active agent).

[0095] Thus, in one embodiment, there is provided a conjugate comprising an antibody, a linker, and at least one active agent, i) a linker connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker; iv) the linker is of formula L X1 or formula L X2 The conjugate is provided in which the linker is

[0096] Preferably, the compound of formula L X1 and formula L X2 Each Z 1 are independently re-bridging moieties selected from the group consisting of formula IIIa, formula IIIb, formula IIIc, formula IIId, formula IIIe, formula IIIf, and formula IIIg, as defined hereinabove. More preferably, each Z 1 is a re-bridging moiety independently selected from the group consisting of Formula IIIa, Formula IIIb, and Formula IIIe, as defined hereinabove. Most preferably, each Z 1 is the re-bridging moiety of formula IIIa as defined hereinabove.

[0097] When the conjugate is attached to the antibody through 5 to 7 independent covalent bonds, Z of each re-bridging moiety of formula (III) A The functional linking group is [ka]

[0098] It is also understood that the attachment may be directly or indirectly to the antibody via one or both of the bonds set out herein above.

[0099] Preferably, r and s are selected so that the total number of active agents per conjugate is 1 to 10, more preferably 1 to 8, and most preferably 1 to 4.

[0100] Suitably, r and s are selected so that there are 1, 2, 3, 4, 5, 6, 7 or 8 active agents per conjugate.

[0101] In some embodiments, Q 1 is a bond.

[0102] In other embodiments, Q 1 is the formula Iva: [ka] (In the formula, Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=O)NR 7 , C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, and v is an integer selected from 0, 1 or 2.

[0103] Preferably, Q 1 is a group of formula IVa, Q 1A are C(=O), OC(=O), and NR 7 C(=O), C(=O)NR 7 and C(=NR 8 ) is selected from Q 1B N and CR 9 CR 10 is selected from R7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2;

[0104] More preferably, Q 1 is a group of formula IVa, Q 1A is C(=O), NR 7 C(=O) and C(=O)NR 7 is selected from Q 1B N and CR 9 CR 10 is selected from R 7 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ C6 alkylene; v is 1 or 2, preferably 1.

[0105] In some embodiments, Q 2 is N. In other embodiments, Q 2 is CR 11 and R 11 is selected from hydrogen and methyl.

[0106] In some embodiments, Q 3 is represented by formula IVb: [ka] (In the formula, Q 3A is NR 12 and O, Q3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent, or C(=O), NR 12 C(=O) and C(=O)NR 12 is selected from

[0107] In some embodiments, Q 3 is represented by formula IVb 1 : [ka] is the basis of Q 3A is NR 12 and O, Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently a C1 to C8 alkylene group and a C1 to C8 alkylene group containing O in the skeleton.

[0108] In some embodiments, Q 3 is represented by formula IVb 1 is the basis of Q 3A is O, Q 3B is a bond, Q 3C is NR 13 and R 13is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently C1 to C6 alkylene.

[0109] In some embodiments, W 1 is a group of formula IVc, W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or is selected from C1-C6 alkylene optionally containing O in the backbone, W 1B -OC(=O)-, -C(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 - and -NR 15 C(=O)NR 16 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 is independently selected from a bond and C1-C6 alkylene; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W C1 is the basis of FG' is a functional linking moiety; L 1 is the linker, D is an activator.

[0110] In other embodiments, W 1 is a group of formula IVc, W 1A is N, L W is absent or is C1-C6 alkylene, W 1B is -NR 15C(=O)- and -C(=O)NR 15 -, R 15 is selected from hydrogen and C1-C4 alkyl; R 23 and R 24 is independently selected from a bond and C1-C6 alkylene; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W C1 is the basis of FG' is a functional linking moiety; L 1 is the linker, D is an activator.

[0111] In other embodiments, W 1 is a group of formula IVc, W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1-C6 alkylene optionally containing O in the backbone, W 1B -OC(=O)-, -C(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 - and -NR 15 C(=O)NR 16 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, C1-C8 alkylene, and C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W C2 is the basis of WQ1 is the formula W C3 : [ka] (In the formula, X W1 is NH, X W2 is NR x1 R x2 and R x1 and R x2 are independently selected from hydrogen and C1-C4 alkyl; y is an integer selected from 0 or 1; Q W1 is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, Q W2 is selected from -NHC(=O)- and -C(=O)NH-; W Q2 is the formula W C4 : [ka] (In the formula, X W4 is NH, X W3 is NH,

[0112] [ka]

[0113] is X W4 But, Q W2A via the substituents, formula W Q2 indicates the position of attachment to another group of [ka] is X W4 But -C(=O)R X3 indicates the position of attachment to the R group. x3 is selected from hydrogen and C1-C4 alkyl; Q W1Ais absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, Q W2A is -C(=O)-, y is an integer selected from 0 or 1; L Q1 is a linker comprising one or more groups selected from an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 4; FG' is a functional linking moiety; L 1 is the linker, D is a group which is an activator.

[0114] In a further embodiment, W 1 is a group of formula IVc, W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1-C6 alkylene optionally containing O in the backbone, W 1B -OC(=O)-, -C(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 - and -NR 15 C(=O)NR 16 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W C2 is the basis of WQ1 is the formula W C3A : [ka] (In the formula, Q W1 is absent or is C1-C6 alkylene, Q W2 is selected from -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; W Q2 is the formula W C4A : [ka] is the basis of Q W1A is a C1-C6 alkylene optionally containing O in the skeleton, Q W2A is -C(=O)-, [ka] , NH, Q W2 via the substituents, formula W Q2 indicates the position of attachment to another group of [ka] indicates the position where NH is attached to -C(=O)CH3, L Q1 is the formula LQ1; [ka] is the basis of Y is O or NH; a is an integer selected from 1 to 6 (preferably, a is an integer selected from 1 to 4); b is an integer selected from 1 to 6 (preferably, b is an integer selected from 1 to 4, and most preferably, b is 4); t is an integer selected from 1 to 4; FG' is a functional linking moiety; L 1 is the linker, D is a group which is an activator.

[0115] W 1C But, formula W C2 When L is a group Q1 is the equation LQ2; [ka] It is based on.

[0116] In some embodiments, W 2 is a group of formula IVd, W 2A is selected from N, L 2W is absent or selected from C1-C6 alkylene optionally containing O or N in the backbone, W 2B N and CR 18 is a group selected from W 2C is -NR 17 C(=O)- or -C(=O)NR 17 -, R 17 is selected from hydrogen and C1-C4 alkyl; R 18 is selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 is independently selected from a bond and C1-C6 alkylene; FG', L 1 and D are as defined herein.

[0117] In other embodiments, W 2 is a group of formula IVd, W 2A is selected from N, L 2W is absent or is C1-C6 alkylene, W2B is N, W 2C is -C(=O)NH-, R 25 , R 26 and R 27 is independently selected from a bond and C1-C6 alkylene; FG', L 1 and D are as defined herein.

[0118] In certain embodiments, FG' is a bond or functional group formed from the reaction between i) a ketone or aldehyde and an alkoxyamine (e.g., hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) an amine and an acyl halide or carboxylic acid; iv) an electron-rich dienophile (e.g., a 1,3-nitrone alkene) and an electron-poor diene (e.g., a tetrazine); and iii) a strained alkene or alkyne (e.g., norbornene or cyclooctyne) and a tetrazine. Preferably, FG' is a bond or functional group formed from the reaction between an azide and an alkyne or an amine and an acyl halide or carboxylic acid. Most preferably, FG' is a functional group formed from the reaction between an azide and an alkyne.

[0119] Thus, in some embodiments, FG' is the bond [ka] is a functional group selected from -NH(=O)C- and -C(=O)NH-.

[0120] In another embodiment, FG' is a bond, [ka] is a functional group selected from -NH(=O)C- and -C(=O)NH-.

[0121] In some embodiments, FG' is [ka] is.

[0122] Preferably, FG' is [ka] is.

[0123] Most preferably, FG' is [ka] and N is L 1 is bonded to.

[0124] Linker L 1 Linker L 1 is a linker that attaches the functional linking moiety FG′ to the active agent D.

[0125] It will be appreciated that any suitable linker joining the functional linking moiety FG' to the active agent D may be used.

[0126] In one embodiment, the linker L 1 comprises one or more groups that are susceptible to enzymatic cleavage (e.g., proteolytic or peptidase cleavage, sulfatase cleavage, or galactosidase cleavage). Thus, in some embodiments, the linker L 1 comprises one or more amino-acid residues, dipeptide or tripeptide residues, one or more aryl sulfates, one or more aryl galactosides or combinations thereof. 1 comprises one or more amino-acid residues, dipeptide residues or tripeptide residues, where amino-acid residues, dipeptide residues or tripeptide residues are defined herein below.

[0127] In one embodiment, the linker L 1 contains one or more groups (e.g., disulfides or hydrazones) that are susceptible to chemical cleavage.

[0128] In some embodiments, the linker L 1 is C1~C 20 It comprises one or more groups selected from alkylene, alkylenediamine moieties, (poly)ethylene glycol moieties, amino acid residues, and combinations thereof.

[0129] In one embodiment, L 1 is a bond, C1~C 10 Alkylene, C1-C containing O in the skeleton 10 Alkylene, and Formula Va: [ka] (In the formula, L Q is a bond, C1~C 10 Alkylene and C1-C containing O or NH in the skeleton 10 alkylene; Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an active agent).

[0130] Preferably, L Q is a bond, C1~C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene.

[0131] In some embodiments, L Q In other embodiments, L is selected from a bond. Q is C1~C 10 Alkylene or C1-C containing O in the skeleton 10 Preferably, L is alkylene. Q is a C1-C group containing O in the skeleton. 10 Alkylene.

[0132] In one embodiment, L 1 is a bond, C1~C 10 Alkylene, C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] is selected from the group Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an active agent.

[0133] In one embodiment, L 1 is C1~C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene.

[0134] In other embodiments, L 1 is the formula Va or the formula Va 1 It is based on.

[0135] In some embodiments, Q 4 is a single bond.

[0136] In other embodiments, Q 4 teeth, [ka] is.

[0137] Q X In one embodiment, Q x is an amino acid residue. The amino acid may be a natural amino acid or an unnatural amino acid.

[0138] In one embodiment, Q x is selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, and Cit is citrulline.

[0139] In one embodiment, Q x comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then becomes the recognition site for the cathepsin.

[0140] In one embodiment, Q x teeth, NH -Phe-Lys- C=O , NH Val-Ala- C=O , NH Val-Lys- C=O , NH Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O and NH -Trp-Cit- C=O is selected from Cit is citrulline.

[0141] Preferably, Q x teeth, NH-Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O and NH -Val-Cit- C=O is selected from.

[0142] Most preferably, Q x teeth, NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0143] Other dipeptide combinations of interest are: NH -Gly-Gly- C=O , NH -Pro-Pro- C=O and NH -Val-Glu- C=O Includes.

[0144] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0145] In some embodiments, Q x is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and unnatural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then becomes the recognition site for the cathepsin.

[0146] In one embodiment, the amino acid side chains are chemically protected, if appropriate. The side chain protecting groups can be those discussed above. The protected amino acid sequence is enzymatically cleavable. For example, a dipeptide sequence containing a Boc side chain-protected Lys residue can be cleaved by cathepsin.

[0147] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalog and are discussed above.

[0148] In some embodiments, L D teeth, (a) a single bond, (b) -C(=O)-, (c) -NH-, and (d) [ka] is selected from.

[0149] In some of these embodiments, L D is a single bond. Q 4 If FG' is also a single bond, then FG' is directly attached to the activator.

[0150] In other of these embodiments, L D is —C(═O)— or —NH—.

[0151] In other of these embodiments, L D teeth, [ka] This group can act as a self-immolative group in conjunction with a cleavable linking group.

[0152] Certain Further Embodiments In some embodiments, the conjugate has formula (IIIA) shown below: [ka] (In the formula, each Z 1 is a re-bridging moiety of formula (IIIa), as defined herein above, wherein the re-bridging moiety is attached to the antibody at the position shown in formula (IIIa); Each Q 1 is represented by formula IVa: [ka] is the basis of Q 1A is C(=O), NR 7 C(=O) and C(=O)NR 7 is selected from Q 1B N and CR 9 CR 10 is selected from R 7 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is 1 or 2, preferably 1; Each Q 2 is N, Each Q 3 is represented by formula IVb: [ka] is the basis of Q 3A is O, Q 3B is a bond, Q 3C is NR 13 and R 13 is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently C1-C6 alkylene, W1 is represented by formula IVc1: [ka] is the basis of W 1A is N, L W is absent or is C1-C4 alkylene, W 1B is -NR 15 C(=O)- and -C(=O)NR 15 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 2 is represented by formula IVd [ka] is the basis of W 2A is selected from N, L 2W is absent or is C1-C4 alkylene W 2B is N, W 2c is C(=O)NH, R 25 , R 26 and R 27 are independently selected from a bond, C1-C8 alkylene, and C1-C8 alkylene containing O in the backbone; FG' is [ka] and N is L 1 is bound to L 1 is a bond, C1~C10 Alkylene, C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] is selected from the group Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an activator, D is an activator as defined herein; r and s are selected so that the total number of active agents per conjugate is 1 to 8).

[0153] In other embodiments, the conjugate has formula (IIIA) shown below: [ka] (In the formula, each Z 1 is a re-bridging moiety of formula (IIIa) as defined hereinabove, wherein the re-bridging moiety is attached to the antibody at the position shown in formula (IIIa); Each Q 1 is represented by formula IVa: [ka] is the basis of Q 1A is C(=O), NR 7 C(=O) and C(=O)NR 7 is selected from Q 1B N and CR 9 CR 10 is selected from R 7 , R9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is 1 or 2, preferably 1; Each Q 2 is N, Each Q 3 is represented by formula IVb: [ka] is the basis of Q 3A is O, Q 3B is a bond, Q 3C is NR 13 and R 13 is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently C1 to C6 alkylene and C1 to C8 alkylene containing O in the skeleton, W 1 is represented by formula IVc: [ka] is the basis of W 1A is N, L W is absent or is C1-C4 alkylene, W 1B is -NR 15 C(=O)- and -C(=O)NR 15 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W 2C is the basis of W Q1 is the formula W C3A : [ka] is the basis of Q W1 is absent or is C1-C6 alkylene, Q W2 is selected from -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; W Q2 is the formula W C4A : [ka] is the basis of Q W1A is a C1-C6 alkylene optionally containing O in the skeleton, Q W2A is -C(=O)-, [ka] is X W1 But, Q W2A Another W via a substituent Q2 indicates the position of attachment to the group, or [ka] is X W4 indicates the position of attachment to -C(=O)CH3, L Q1 is the equation LQ1 [ka] is the basis of Y is O or NH; a is an integer selected from 1 to 6; b is an integer selected from 1 to 6; t is an integer selected from 1 to 4; FG' is [ka] and N is L 1 is bound to L 1 is a bond, C1~C 10 Alkylene, C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] is selected from the group Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an activator, D is an activator as defined herein; r and s are selected so that the total number of active agents per conjugate is 1 to 8).

[0154] In certain embodiments, the conjugate comprises: [ka] [ka] [ka] [ka] [ka] [ka] is selected from one of [ka] indicates the location where the antibody is attached, The antibody is preferably trastuzumab or brentuximab, most preferably trastuzumab.

[0155] In some embodiments, the conjugate comprises: [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from one of [ka] [ka] indicates the location where the antibody is attached, The antibody is preferably trastuzumab or brentuximab, most preferably trastuzumab.

[0156] In some embodiments, the conjugate comprises: [ka] [ka] [ka] [ka] is selected from one of [ka] [ka] indicates the location where the antibody is attached, The antibody is preferably trastuzumab or brentuximab, most preferably trastuzumab.

[0157] In some embodiments, the conjugate is selected from ADC1 to ADC20, as described below.

[0158] Conjugation Reagents of the Invention According to another aspect, the present invention provides a compound of formula (II) shown below: (DL 1 -FG') n -L-(Z)8 (Formula II) (In the formula, FG' is a functional linking moiety; n is an integer selected from 1 to 20; L and L 1 is independently a linker, Z is a functional group capable of reacting with a sulfur atom of an antibody, D is an active agent), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0159] Preferably, Z is an electrophilic functional group capable of reacting with a sulfur atom of an antibody.

[0160] Preferably, n is an integer selected from 1 to 12, more preferably 1 to 10, even more preferably 1 to 8, and most preferably 1 to 4.

[0161] In certain embodiments, Z is a Michael acceptor (e.g., an alkene or alkyne, typically bearing an electron-withdrawing group) capable of reacting with a sulfur atom of an antibody via conjugate addition (Michael reaction). Conjugate addition reactions and Michael reactions are well known in the chemical arts. Thus, one of skill in the art can select a suitable Michael acceptor for use in the conjugation reagents of the present invention.

[0162] In certain embodiments, Z is a functional group reactive with a sulfur atom in an antibody selected from an alkene, an alkyne, a vinylarene (e.g., divinylpyrimidine), a maleimide, a halo-maleimide, a sulfone, an arylene-propiolonitrile, a pyridazinedione, a halo-(hetero)arene, a benzyl halide, a group containing a β-unsaturated ketone, a group capable of undergoing an SNAr reaction, or a group capable of alkylating sulfur.

[0163] In certain embodiments, Z is a functional group reactive with a sulfur atom in the antibody selected from a group comprising an alkene, alkyne, vinylarene (e.g., divinylpyrimidine), maleimide, halo-maleimide, sulfone, arylene-propiolonitrile, pyridazinedione, or β-unsaturated ketone.

[0164] In certain embodiments, the conjugation reagent has formula (IIa), shown below: (DL 1 -FG') n -L-(Z 2 )4 (Formula IIa) (In the formula, each Z 2is a re-bridging linking group containing two functional groups (Z) capable of reacting with sulfur atoms in an antibody, D, L, L 1 , FG′ and n are each as defined herein).

[0165] D, L, L 1 Preferred and suitable substituents for each of FG′ and n are the D, L, L of the conjugates of the invention described herein above. 1 , FG′ and n are similar to the preferred and suitable substituents for each of them.

[0166] In one embodiment, each Z 2 are independently one of the following groups: [ka] [ka] (In the formula, A 1 , A 2 , A 3 , X, R 1 , R 2 , R 3 , Q, Y 1 , Y 2 , p, and q are as defined herein above; Ts is tosylate, [ka] is a re-bridging linking group selected from:

[0167] In one embodiment, each Z 2 are independently represented by formula II ZA , formula II ZB , formula II ZE and Formula II ZH Preferably, each Z 2 are independently represented by formula II ZA , formula II ZB and Formula IIZE More preferably, each Z 2 is represented by formula II ZA is a re-bridging linking group.

[0168] In one embodiment, the following: [ka] [ka] (In the formula, D, L, L 1 , FG', A 1 , A 2 , A 3 , X, R 1 , R 2 , R 3 , Q, Y 1 , Y 2 , wherein each of p, q and n is as defined herein above.

[0169] In other embodiments, the conjugation reagent has formula (IIB) shown below: [ka] (In the formula, Z 2 , Q 1 , Q 2 , Q 3 , W 1 , W 2 , r and s are as defined herein above).

[0170] D, Q 1 , Q 2 , Q 3 , W 1 , W 2 Preferred and suitable substituents for each of r, r and s are the same as those described herein above for the D, Q groups of the conjugates of the invention. 1 , Q 2 , Q 3 , W 1 , W 2It is understood that the preferred and suitable substituents for each of r and s are similar. 2 It is understood that the groups are as described herein above.

[0171] Certain Further Embodiments In certain embodiments, the conjugation reagent has formula (IIB) shown below: [ka] (In the formula, each Z 2 is a compound of formula (II) ZA ) is the re-crosslinking portion, Each Q 1 is represented by formula IVa: [ka] is the basis of Q 1A is C(=O), NR 7 C(=O) and C(=O)NR 7 is selected from Q 1B N and CR 9 CR 10 is selected from R 7 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is 1 or 2, preferably 1; Each Q 2 is N, Each Q 3 is represented by formula IVb: [ka] is the basis of Q 3A is O, Q 3B is a bond, Q 3C is NR 13 and R 13 is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently C1-C8 alkylene or C1-C8 alkylene containing O in the skeleton, W 1 is represented by formula IVc1: [ka] is the basis of W 1A is N, L W is absent or is C1-C4 alkylene, W 1B is -NR 15 C(=O)- and -C(=O)NR 15 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 2 is represented by formula IVd: [ka] is the basis of W 2A is selected from N, L 2W is absent or is C1-C4 alkylene, W 2B is N, W 2C is -NHC(=O)- or -C(=O)NH-, R25 , R 26 and R 27 is independently selected from a bond and C1-C6 alkylene; FG' is [ka] and N is L 1 is bound to L 1 is a bond, C1~C 10 Alkylene, C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] is selected from the group Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an activator, D is an activator as defined herein; r and s are selected so that the total number of active agents per conjugate is 1 to 8).

[0172] In certain embodiments, the conjugation reagent has formula (IIB) shown below: [ka] (In the formula, each Z 2 is a compound of formula (II) ZA ) is the re-crosslinking portion, Each Q 1 is represented by formula IVa: [ka] is the basis of Q 1A is C(=O), NR 7 C(=O) and C(=O)NR 7 is selected from Q 1B N and CR 9 CR 10 is selected from R 7 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 independently, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is 1 or 2, preferably 1; Each Q 2 is N, Each Q 3 is represented by formula IVb: [ka] is the basis of Q 3A is O, Q 3B is a bond, Q 3C is NR 13 and R 13 is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently C1-C8 alkylene or C1-C8 alkylene containing O in the skeleton, W 1 is represented by formula IVc: [ka] is the basis of W 1A is N, L W is absent or is C1-C4 alkylene, W 1B is -NR15 C(=O)- and -C(=O)NR 15 -, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W Q1 is the formula W C3A : [ka] is the basis of Q W1 is absent or is C1-C6 alkylene, Q W2 is selected from -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; W Q2 is the formula W C4A : [ka] is the basis of Q W1A is a C1-C6 alkylene optionally containing O in the skeleton, Q W2A is -C(=O)-, [ka] , NH is Q W2 via the substituents, formula W Q2 indicates the position of attachment to another group of [ka] indicates the position where NH is attached to -C(=O)CH3, L Q1 is the equation LQ1 [ka] is the basis of Y is O or NH; a is an integer selected from 1 to 6; b is an integer selected from 1 to 6; t is an integer selected from 1 to 4; FG' is [ka] and N is L 1 is bound to L 1 is a bond, C1~C 10 Alkylene, C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] is selected from the group Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an activator, D is an activator as defined herein; r and s are selected so that the total number of active agents per conjugate is 1 to 8).

[0173] In other embodiments, the conjugation reagent is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from one of the following:

[0174] In some embodiments, the conjugation reagent is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] (In the formula, [ka] ) is selected from

[0175] Compounds of the Invention According to another aspect of the present invention, a compound of formula (I) shown below: (FG) n -L-(Z)8 (Formula I) (In the formula, FG is a functional group that is capable of reacting with another moiety to form a functional linking moiety; n is an integer selected from 0 to 20; L is a linker, Z is a functional group capable of reacting with a sulfur atom from a cysteine ​​moiety of an antibody), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0176] It is understood that the preferred and suitable substituents for each of FG, n, L and Z are similar to the preferred and suitable substituents for each of FG, n, L and Z of the conjugates and conjugating reagents of the invention described hereinabove.

[0177] Thus, in certain embodiments, FG is a functional group selected from alkenes, alkynes, azides, hydroxyls, amines, carboxylic acids, aldehydes, acyl halides, tetrazines, alkoxyamines (e.g., hydroxylamines), hydrazines, electron-rich dienophiles (e.g., 1,3-nitrone alkenes) and electron-poor dienes (e.g., tetrazines), nitrones, isocyanates, and isothiocyanates.

[0178] Preferably, FG is a functional group selected from alkene, alkyne, azide, hydroxyl, amine, carboxylic acid, aldehyde, acyl halide, tetrazine, alkoxyamine (e.g., hydroxylamine), and hydrazine. More preferably, FG is a functional group selected from alkyne, azide, hydroxyl, amine, carboxylic acid, aldehyde, and acyl halide. Most preferably, FG is a functional group selected from alkyne and azide.

[0179] In certain embodiments, the compound has formula (Ia), as shown below: (FG) n -L-(Z 2 )4 (Formula Ia) (In the formula, Z 2, L, FG and n are each as defined herein above).

[0180] In some embodiments, the compound is selected from the following groups: [ka] [ka] [ka] (In the formula, A 1 , A 2 , A 3 ,X,L,FG,n,Q,Y 1 , Y 2 , R 1 , R 2 , R 3 , q and p are each as defined herein above).

[0181] In certain embodiments, the compound is a compound of Formula Ib, Formula Ib, Formula If, or Formula Ij. Preferably, the compound is a compound of Formula Ib, Formula Ib, Formula If, or Formula Ij. More preferably, the compound is a compound of Formula Ib.

[0182] In some embodiments, the compound has formula (Ic) shown below: [ka] (In the formula, Z 2 , Q 1 , Q 2 , Q 3 , r and s are as defined herein; W 1' is of formula IVc or IVc2: [ka] is the basis of W 1A N and CR 14 Selected from R14 is selected from hydrogen and C1-C4 alkyl; L w is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amino, O or N in the skeleton, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); W 1C is the formula W C2 is the basis of W Q1 is the formula W C3A : [ka] is the basis of Q W1 is absent or is C1-C6 alkylene, Q W2 is selected from -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; W Q2 is the formula W C4A : [ka] is the basis of Q W1A is a C1-C6 alkylene optionally containing O in the skeleton, Q W2A is -C(=O)-, [ka] , NH is Q W2 via the substituents, formula W Q2 indicates the position of attachment to another group of [ka] indicates the position where NH is attached to -C(=O)CH3, L Q1 is the equation LQ1, [ka] is the basis of Y is O or NH; a is an integer selected from 1 to 6; b is an integer selected from 1 to 6; t is an integer selected from 1 to 4; FG is as defined herein; W 2' is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 Selected from R 17 is selected from hydrogen and C1-C4 alkyl; L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amino, O or N in the skeleton, W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; FG is as defined herein) of the compound.

[0183] In other embodiments, the compound has formula (Ic), shown below: [ka] (In the formula, Z 2 , Q 1 , Q 2 , Q 3 , r and s are as defined herein; W 1' is represented by formula IVc2: [ka] is the basis of W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L w is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15)- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); FG is as defined herein; W 2' is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 Selected from R 17 is selected from hydrogen and C1-C4 alkyl; L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; FG is as defined herein).

[0184] In other embodiments, the compound is: [ka] [ka] [ka] is selected from one of the following:

[0185] In some embodiments, the compound is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from one of the following:

[0186] In some embodiments, the compound is: [ka] [ka] [ka] is selected from one of the following:

[0187] Intermediate conjugates of the present invention According to another aspect of the invention, there is provided an intermediate conjugate comprising an antibody, a linker, and at least one functional group capable of reacting with another moiety to form a functional linking moiety, i) a linker connects at least one functional group to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) An intermediate conjugate is provided in which each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0188] In certain embodiments, the intermediate conjugate has formula (III) shown below: [ka] (In the formula, Z A is a functional linking group as defined herein; Pep indicates the position where the moiety is attached, directly or indirectly, to the antibody; [ka] indicates the position at which the moiety is attached to the linker) and includes 1 to 4 (preferably 2 to 4, more preferably 3 to 4, and most preferably 4) re-bridging moieties.

[0189] Preferred and preferred Z A The Z groups are preferred and suitable for the conjugates of the present invention as described herein above. A It is understood that the term is similar to the group

[0190] In other embodiments, the intermediate conjugate has formula (VI) shown below: [ka] (In the formula, Z 1 , Q 1 , Q 2 , Q 3 , W 1 ', W 2 ', r and s are as defined herein above).

[0191] In other embodiments, the intermediate conjugate is: [ka] [ka] [ka] is selected from one of [ka] indicates the location where the antibody is attached, The antibody is preferably trastuzumab or brentuximab, most preferably trastuzumab.

[0192] In other embodiments, the intermediate conjugate is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from one of [ka] indicates the location where the antibody is attached, The antibody is preferably trastuzumab or brentuximab, most preferably trastuzumab.

[0193] In other embodiments, the intermediate conjugate is selected from ALC1 to 12, described below.

[0194] How to treat The conjugates of the present invention, in which the active agent is a drug, can be used in methods of treatment. Also provided are methods of treatment, comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate according to the first aspect of the present invention, in which the active agent is a drug. The term "therapeutically effective amount" refers to an amount sufficient to demonstrate benefit to the patient. Such benefit may be at least the alleviation of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other medical doctors.

[0195] The conjugates may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (e.g., administration of active agents including drugs, surgery, and radiation therapy).

[0196] Pharmaceutical compositions according to and for use in accordance with the present invention may contain, in addition to the active ingredient, i.e., a conjugate according to the first aspect of the present invention, wherein the active agent is a drug, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other substance will depend on the route of administration, which may be oral, or by, for example, cutaneous, subcutaneous, or intravenous injection.

[0197] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol may be included. Capsules may contain a solid carrier such as gelatin.

[0198] For intravenous, cutaneous or subcutaneous injection, or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0199] The conjugates can be used to treat proliferative and autoimmune diseases. The term "proliferative disease" refers to unwanted or uncontrolled cell proliferation of unwanted, excessive, or abnormal cells, e.g., neoplastic or hyperplastic growth, whether in vitro or in vivo.

[0200] Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cell proliferations, including neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone disease, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. Other cancers of interest include, but are not limited to, blood cancers; malignant tumors, such as leukemia and lymphoma, including non-Hodgkin's lymphoma and subtypes, such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin's lymphoma, AML, and other cancers of B- or T-cell origin. Cells of any type that may be treated include, but are not limited to, lung, gastrointestinal (e.g., intestine, including colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

[0201] Examples of autoimmune diseases include: rheumatoid arthritis, autoimmune demyelinating diseases (e.g., multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Graves' disease, glomerulonephritis, autoimmune hepatopathy, and the like. disorder), inflammatory bowel disease (e.g., Crohn's disease), anaphylaxis, allergic reactions, Sjögren's syndrome, type 1 diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, polyendocrine deficiency, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus folliculorum, dermatitis herpetiformis, alopecia areata arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, digital sclerosis, and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-cardiotomy syndrome syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird fancier's lung, toxic epidermal necrolysis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behçet's disease, Kaplan's syndrome, Kawasaki disease, dengue fever, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, persistent erythema elevatum etdiutinum, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft-versus-host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's macroglobulinemia, Evans syndrome and autoimmune dysgonadism.

[0202] In some embodiments, the autoimmune disease is a disorder of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type 1 diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft-versus-host disease). Generally, disorders involving dendritic cells involve disorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

[0203] In certain embodiments, the conjugates can be used to treat microbial diseases. Suitably, the conjugates can be used to treat bacterial diseases such as tuberculosis (TB) and methicillin-resistant Staphylococcus aureus (MRSA).

[0204] It will also be understood that features, including optional, suitable and preferred features, relating to any one of the aspects of the invention (e.g., the conjugates of the invention) detailed above may also be features, including optional, suitable and preferred features, relating to any other aspect of the invention (e.g., the uses of the conjugates of the invention). [Brief explanation of the drawings]

[0205] [Figure 1] FIG. 1 shows analysis of conjugates by SDS-PAGE; (A) analysis of trastuzumab conjugates 36-39, (B) analysis of trastuzumab conjugates 40-46; (C) analysis of brentuximab conjugates 47-50, M = molecular weight marker. [Figure 2] LC-MS traces from the reaction between trastuzumab and compound 1 to produce conjugate 36: A) shows the undeconvoluted mass spectrometry trace; B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,536 Da, observed MW = 146,537 Da. [Figure 3] LC-MS traces from the reaction between trastuzumab and compound 8 to produce conjugate 37: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,674 Da, observed MW = 146,615 Da. [Figure 4] LC-MS traces from the reaction between trastuzumab and compound 11 to produce conjugate 38: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,812 Da, observed MW = 146,749 Da. [Figure 5]1 shows LCMS traces from the reaction between trastuzumab and compound 14 to produce conjugate 39: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 147,096 Da, observed MW 147,107 Da. [Figure 6] LC-MS traces from the reaction between trastuzumab and compound 19 to produce conjugate 40: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,369 Da, observed MW = 146,369 Da. [Figure 7] LC-MS traces from the reaction between trastuzumab and compound 21 to produce conjugate 41: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,507 Da, observed MW = 146,508 Da. [Figure 8] LC-MS traces from the reaction between trastuzumab and compound 22 to produce conjugate 42: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,605 Da, observed MW = 146,611 Da. [Figure 9] LC-MS traces from the reaction between trastuzumab and compound 24 to produce conjugate 43: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,838 Da, observed MW = 146,845 Da. [Figure 10]LC-MS traces from the reaction between trastuzumab and compound 28 to produce conjugate 44: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 147,077 Da, observed MW = 147,069 Da. [Figure 11] LC-MS traces from the reaction between trastuzumab and compound 31 to produce conjugate 45: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 147,524 Da, observed MW = 147,526 Da. [Figure 12] LC-MS traces from the reaction between trastuzumab and compound 33 to produce conjugate 46: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 147,295 Da, observed MW = 147,301 Da. [Figure 13] 1 shows LC-MS traces from the reaction between brentuximab and compound 1 to produce conjugate 47: A) shows the undeconvoluted mass spectrometry trace and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,549 Da, observed MW = 146,550 Da. [Figure 14] LC-MS traces from the reaction between brentuximab and compound 8 to produce conjugate 48: A) shows the undeconvoluted mass spectrometry trace; B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,687 Da, observed MW = 146,688 Da. [Figure 15]LC-MS traces from the reaction between brentuximab and compound 19 to produce conjugate 49: A) shows the undeconvoluted mass spectrometry trace, and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,393 Da, observed MW = 146,395 Da. [Figure 16] LC-MS traces from the reaction between brentuximab and compound 21 to produce conjugate 50: A) shows the undeconvoluted mass spectrometry trace and B) shows the deconvoluted mass spectrometry trace. Expected MW = 146,531 Da, observed MW = 146,533 Da. [Figure 17] (A) Size-exclusion chromatography (SEC) analysis of trastuzumab and trastuzumab conjugates 36-39; and (B) binding affinity comparison of trastuzumab and conjugates 36-39 via enzyme-linked immunosorbent assay (ELISA). Error bars represent standard deviation of biological quadruplicates. [Figure 18] Figure 1 shows UV / vis spectra of conjugates 51-54. Absorbance was normalized at 280 nm. Absorbance at 495 nm was used to calculate the fluorophore-to-antibody ratio (FAR). [Figure 19] Figure 1 shows stability analysis of conjugates 51-54 in human plasma by SDS-PAGE; M = molecular weight marker, P = human plasma, days of incubation on representative lane. No migration of AlexaFluor™ 488 to human serum albumin (66.5 kDa) or any other plasma protein is observed over the 14-day incubation period. [Figure 20] FIG. 1 shows hydrophobic interaction chromatography (HIC) analysis of ADC58-61. [Figure 21] FIG. 1 shows hydrophobic interaction chromatography (HIC) analysis of ADC62-65. [Figure 22] Figure 1 shows the cytotoxicity of ADC62-65 in HER2-positive SKBR3 cells, HER2-positive BT474 cells, HER2-negative MDA-MB-468 cells, and HER2-negative MCF7 cells. Viability data represent the average of three independent experiments, and error bars represent the standard error of the mean (SEM). [Figure 23] FIG. 1 shows live cell microscopy images of HER2-positive SKBR3 cells and HER2-negative MCF7 cells after treatment with conjugates 51 and 52, trastuzumab, or vehicle control. [Example]

[0206] General Experimental Details All solvents and reagents were used as received unless otherwise specified. Ethyl acetate, methanol, dichloromethane, acetonitrile, and toluene were distilled from calcium hydride. Diethyl ether was distilled from a mixture of lithium aluminum hydride and calcium hydride. Petroleum ether (PE) refers to the 40-60°C fraction during distillation. Tetrahydrofuran was dried using a Na wire and distilled from a mixture of lithium aluminum hydride and calcium hydride, along with triphenylmethane as an indicator.

[0207] Non-aqueous reactions were carried out using oven-dried glassware under a stream of dry nitrogen. An ice-water bath was used to maintain a temperature of 0° C. Room temperature (rt) refers to ambient temperature.

[0208] Yields refer to spectroscopically and chromatographically pure compounds unless otherwise specified. Reactions were monitored by thin layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). TLC was performed using Merck silica gel 60F. 254 UV fluorescence (λ) was measured using a glass plate pre-coated with maxThe retention factor (R = 254 nm) was visualized by quenching or by staining with potassium permanganate. f ) are quoted to the nearest 0.01. LC-MS was performed on a Waters ACQUITY H-Class UPLC with an ESI Multi-Mode Ionization Waters SQ Detector 2 spectrometer using MassLynx 4.1 software; ESI refers to electrospray ionization technology. LC system: Solvent A: 2 mM NHOAc in HO / MeCN (95:5); Solvent B: MeCN; Solvent C: 2% formic acid; Column: ACQUITY UPLC® CSH C18 (2.1 mm × 50 mm, 1.7 μm, 130 Å) at 40 °C; Gradient: 5 to 95% B over 1 min at a flow rate of 0.6 mL / min, constant at 5% C; Detector: PDA eλ Detector 220-800 nm, 1.2 nm interval.

[0209] Flash column chromatography was performed using a Combiflash Rf200 automated chromatography system with slurry-packed Merck 9385 Kieselgel 60 SiO2 (230-400 mesh) or Redisep® reversed-phase C18-silica flash columns (20-40 μm).

[0210] HPLC purification was performed by semi-preparative reversed-phase HPLC on an Agilent 1260 Infinity using a Supelcosil ABZ+PLUS column (250 mm × 21.2 mm, 5 μm) eluted with a linear gradient system (solvent A: 0.1% (v / v) TFA in water, solvent B: 0.05% (v / v) TFA in acetonitrile) over 20 min at a flow rate of 20 mL / min.

[0211] Analytical high performance liquid chromatography (HPLC) was performed at a flow rate of 1 mL / min for 20 min and with UV detection (λ maxThe separation was performed on an Agilent 1260 Infinity machine using a Supelcosil™ ABZ+PLUS column (150 mm × 4.6 mm, 3 μm) with a linear gradient system (solvent A: 0.05% (v / v) TFA in HO; solvent B: 0.05% (v / v) TFA in MeCN) at 220–254 nm (=220–254 nm).

[0212] Infrared (IR) spectra were recorded neat on a Perkin-Elmer Spectrum One spectrometer using an internal standard. Selected absorption maxima (ν max ) is the wave number (cm -1 ) has been reported.

[0213] 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX-400 (400 MHz, 101 MHz), a Bruker Avance 400 QNP (400 MHz, 101 MHz), and a Bruker Avance 500 Cryo Ultrashield (500 MHz, 126 MHz) using an internal deuterium lock. Tetramethylsilane was used as the internal standard. 1 In H NMR, the chemical shift (δ H ) are reported in parts per million (ppm) to 0.01 ppm and are referenced to the residual non-deuterated solvent peak (CDCl3: 7.26, DMSO-d6: 2.50, CD3OD: 3.31, DO: 4.79). Coupling constants (J) are reported in hertz (Hz) to 0.1 Hz. Data are reported as follows: chemical shift, multiplet (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet or combinations thereof, e.g., dd, dt), integral, and coupling constant. 13 In C NMR, the chemical shift (δ C ) are quoted in ppm to 0.1 ppm and are referenced to the residual non-deuterated solvent peak (CDCl3: 77.16, DMSO-d6: 39.52, CD3OD: 49.00).

[0214] Unless otherwise specified, high-resolution mass spectrometry (HRMS) measurements were recorded on a Micromass Q-TOF mass spectrometer or a Waters LCT Premier Time of Flight mass spectrometer. Mass values ​​are reported within error limits of ±5 ppm mass units. ESI refers to electrospray ionization technology.

[0215] Protein concentration and fluorophore-to-antibody ratio (FAR) were determined using UV-Vis spectroscopy using a nanodrop ND-1000 spectrometer. Sample buffer was used as a blank for baseline correction, and the extinction coefficient was ε for trastuzumab. 280 =215,380M -1 cm -1 and for AlexaFluor 488™ (AF488), ε 495 =71,000M -1 cm -1 The correction factor for AF488 absorption at 280 nm is 0.11. The FAR was calculated using the following formula:

number

[0216] Protein LC-MS was performed on a Xevo G2-S TOF mass spectrometer coupled to an Acquity UPLC system using an Acquity UPLC BEH300 C4 column (1.7 μm, 2.1 × 50 mm). HO with 0.1% formic acid (solvent A) and 95% MeCN with 5% HO with 0.1% formic acid (solvent B) were used as mobile phases at a flow rate of 0.2 mL / min. The gradient was programmed as follows: 0.93 min 95% A, then a gradient to 100% B over 4.28 min, then 100% B over 1.04 min, then a gradient to 95% A over 1.04 min. The electrospray source was operated at a capillary voltage of 2.0 kV and a cone voltage of 190 V. Nitrogen was used as the desolvation gas at a total flow rate of 850 L / h. All mass spectra were reconstructed from ion series using the MaxEnt algorithm preinstalled in MassLynx software (v4.2, Waters) according to the manufacturer's instructions. Trastuzumab samples were deglycosylated with PNGase F (New England Biolabs) before LC-MS analysis.

[0217] Example 1 Preparation of conjugation reagent 1 Step 1: Synthesis of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) [ka] 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) was synthesized as previously described (Bargh et al., Chem. Sci., 2020, 11, 2375-2380).

[0218] Step 2: Synthesis of di-tert-butyl(azanediylbis(ethane-2,1-diyl))dicarbamate (3) [ka] To a solution of diethylenetriamine (2.16 mL, 20.0 mmol) in THF (25 mL) at 0 °C was slowly added a solution of Boc-ON (9.92 g, 40.0 mmol) in THF (25 mL). The reaction was stirred at 0 °C under nitrogen for 1 h and then concentrated in vacuo. The crude yellow oil was dissolved in CHCl and washed sequentially with 10% aq. NaOH, water, and brine. The organic phase was dried over NaSO and concentrated in vacuo. Purification by column chromatography (10% MeOH / CHCl) afforded the product as a colorless oil (5.61 g, 18.5 mmol, 92% yield). f 0.08(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3336(m, NH), 2975(m, CH), 1687(s, C=O);δ H (400 MHz, CDCl3) 4.93 (br s, 2H), 3.24-3.19 (m, 4H), 2.73 (t, 4H, J = 5.7 Hz), 1.44 (s, 18H); δ C (101 MHz, CDCl3) 156.3, 79.4, 49.0, 40.4, 28.6; HRMS (ESI) m / z observed [M+H] + 304.2221, C 14 H 30 O4N3 + Calculated value 304.2231.

[0219] Step 3: Synthesis of methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) [ka] To a solution of di-tert-butyl (azanediylbis(ethane-2,1-diyl)) dicarbamate (3) (5.61 g, 18.5 mmol) in DMF (80 mL) was added DIPEA (3.86 mL, 22.2 mmol) and methyl bromoacetate (2.61 mL, 27.8 mmol). The reaction was stirred at room temperature for 6 h and then concentrated under a stream of nitrogen. The crude residue was purified by column chromatography (40-50% EtOAc / PE) to give the product as a colorless oil (6.56 g, 17.5 mmol, 94% yield). f 0.28(SiO2, 50% EtOAc / PE);ν max (neat / cm -1 )3345(m, NH), 2979(m, CH), 1740(s, C=O), 1688(s, C=O);δ H (400 MHz, CDCl3) 5.13 (br s, 2H, NH), 3.70 (s, 3H), 3.37 (s, 2H), 3.15 (q, 4H, J = 5.6 Hz), 2.72 (t, 4H, J = 5.9 Hz), 1.44 (s, 18H); δ C (101 MHz, CDCl3) 172.3, 156.3, 79.3, 55.1, 54.3, 51.8, 38.7, 28.6; LRMS (ESI) m / z observed [M+H] + 376.5.

[0220] Step 4: Synthesis of 2-bromo-N-(prop-2-yn-1-yl)acetamide (5) [ka] A solution of propargylamine (1.16 mL, 18.2 mmol) in CHCl (33 mL) and sat. NaHCO (33 mL) was cooled to −10° C. with maximal stirring, and 2-bromoacetyl bromide (2.42 mL, 27.2 mmol) was added dropwise over 15 min. The reaction mixture was allowed to slowly reach rt, and upon completion, the reaction mixture was concentrated. Following the addition of water (30 mL), the aqueous solution was extracted with EtOAc (2×80 mL), and the combined organic phases were washed with sat. NaHCO (30 mL), 5% HCl (30 mL), and brine (30 mL). The combined organic phases were dried over NaSO and concentrated in vacuo to afford the title compound as a pale yellow solid (2.97 g, 16.9 mmol, 93%). f 0.69(SiO2, 20%MeOH / CH2Cl2);ν max (neat / cm -1 )3289(m, NH), 3071(m, CH), 2120(w, C≡C), 1645(s, C=O);δ H (400 MHz, CDCl3) 6.68 (s, 1H), 4.09 (dd, J = 5.3, 2.6 Hz, 2H), 3.90 (s, 2H), 2.28 (t, J = 2.6 Hz, 1H); δ C (100 MHz, CDCl3) 165.1, 78.5, 72.3, 30.0, 28.7; LRMS (ESI) m / z observed [M+H] + 178.0.

[0221] Step 5: Synthesis of Compound 6 [ka] In a pre-dried microwave vial containing methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) (200 mg, 0.53 mmol) dissolved in dry MeOH (0.3 mL), diethylenetriamine (10.7 μL, 0.098 mmol) was added, the vial was capped, flushed with nitrogen, and stirred at 110 °C overnight. The reaction mixture was concentrated and purified by column chromatography (0-10% MeOH / CHCl) to give the desired compound as a colorless oil (60.6 mg, 0.077 mmol, 78%). f 0.14(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3316(m, NH), 2974(m, CH), 1687(s, C=O);δ H (400 MHz, CD3OD) 3.48 (t, 4H, J = 5.8 Hz), 3.21 (s, 4H), 3.14 (t, 8H, J = 6.3 Hz), 3.06 (t, 4H, J = 5.7 Hz), 2.61 (t, 8H, J = 6.3 Hz), 1.45 (s, 36H); δ C (101 MHz, CD3OD) 175.6, 158.6, 80.2, 59.9, 56.4, 49.5, 39.7, 38.4, 28.9; HRMS (ESI) m / z actual value [M+H] + 790.5425, C 36 H 72 O 10 N9 + Calculated value 790.5402.

[0222] Step 6: Synthesis of Compound 7 [ka] A suspension of compound 6 (59.3 mg, 0.075 mmol) and K2CO3 (20.7 mg, 0.15 mmol) in anhydrous MeCN (0.15 mL) in a pre-dried microwave vial was cooled to 0 °C using an ice bath. A solution of compound 5 (16.5 mg, 0.094 mmol) in MeCN (0.5 mL) was slowly added to the stirred suspension, after which the reaction was allowed to warm to rt and stirred under nitrogen overnight. The reaction mixture was concentrated and purified by flash chromatography (0-10% MeOH / CH2Cl2) to afford the title compound as a white solid (35.9 mg, 0.041 mmol, 54%). f 0.30(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3359(m, NH), 2985(m, CH), 2101(m, C≡C), 1724(s, C=O);δ H (600 MHz, CDCl3) 7.79 (s, 2H), 5.70 (s, 4H), 4.04 (dd, 2H, J = 2.4, 5.4 Hz), 3.37-3.32 (m, 4H), 3.24 (s, 2H), 3.20-3.14 (m, 8H), 3.14 (s, 4H), 2.75-2.70 (m, 4H), 2.60-2.55 (m, 8H), 2.25-2.22 (m, 1H), 2.10 (s, 1H), 1.42 (s, 36H); δ C (101 MHz, CDCl3) 172.2, 171.5, 156.7, 80.0, 79.4, 71.5, 59.5 (two peaks), 55.8 (two peaks), 38.8, 37.9, 29.0, 28.6; HRMS (ESI) m / z observed [M+H] + 885.5760, C 41 H 77 O 11 N 10 + Calculated value 885.5768.

[0223] Step 7: Synthesis of conjugating reagent 1 [ka] To a solution of compound 7 (69.0 mg, 0.078 mmol) in CHCl (0.5 mL) at 0 °C was added HCl (4 M in dioxane, 1.0 mL). The reaction was stirred under nitrogen for 6 h and then concentrated in vacuo to give the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was redissolved in CHCl (1 mL) and cooled to 0 °C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (72.8 mg, 0.312 mmol), triethylamine (217 μL, 1.56 mmol), and HBTU (118 mg, 0.312 mmol) in CHCl (2 mL) at 0 °C. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by column chromatography (2.5-10% MeOH / CH2Cl2) gave the product as a colorless oil (29.6 mg, 0.022 mmol, 28%, over two steps). f 0.15(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3290(m, NH), 2944(m, CH), 2187(m, C≡C), 1633(s, C=O);δ H (600 MHz, CD3OD) 6.67 (s, 4H), 6.59 (dd, 8H, J = 10.7, 17.4 Hz), 6.35 (d, 8H, J = 17.4 Hz), 5.55 (dd, 8H, J = 1.5, 10.7 Hz), 3.97 (d, 2H, J = 2.5), 3.45 (t, 8H, J = 6.8 Hz), 3.27 (t, 4H, J = 6.5 Hz), 3.24 (t, 8H, J = 6.3 Hz), 3.21 (s, 2H), 3.18 (s, 4H), 2.65 (t, 4H, J = 6.4 Hz), 2.62-2.59 (m, 1H), 2.60 (t, 8H, J = 6.2 Hz), 2.28 (t, 8H, J = 7.5 Hz), 1.90 (quintet, 8H, J = 7.2 Hz); δ C(101 MHz, CD3OD) 175.9, 174.1, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 80.8, 72.5, 59.7, 59.3, 55.9, 55.8, 44.0, 38.7, 38.5, 34.7, 29.4, 27.0; HRMS (ESI) m / z actual value [M+H] + 1345.7930, C 69 H 97 O7N 22 + Calculated value 1345.7905.

[0224] Example 2 Preparation of conjugation reagent 8 Step 1: Synthesis of Compound 9 [ka] In a pre-dried microwave vial containing methyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4) (203 mg, 0.54 mmol) dissolved in dry MeOH (0.3 mL), triethylenetetramine (14.9 μL, 0.100 mmol) was added, the vial was capped, flushed with nitrogen, and stirred at 110 °C overnight. The reaction mixture was concentrated and purified by column chromatography (0-30% MeOH / CHCl) to give the desired compound as a colorless oil (40.8 mg, 0.049 mmol, 49%). f 0.38(SiO2, 20%MeOH / CH2Cl2);ν max (neat / cm -1 )3335(m, NH), 2980(m, CH), 1690(s, C=O);δ H (600 MHz, CD3OD) 3.38 (t, 4H, J = 6.3 Hz), 3.17 (s, 4H), 3.13 (t, 8H, J = 6.1 Hz), 2.80-2.77 (m, 4H), 2.76 (s, 4H), 2.60 (t, 8H, J = 5.7 Hz), 1.45 (s, 36H); δ C(101 MHz, CD3OD) 174.5, 158.6, 80.2, 60.1, 56.4, 49.7, 49.4, 39.7, 39.7, 28.9; HRMS (ESI) m / z actual value [M+H] + 833.5843, C 38 H 77 O 10 N 10 + Calculated value 833.5824.

[0225] Step 2: Synthesis of Compound 10 [ka] A suspension of compound 9 (39.2 mg, 0.047 mmol) and K2CO3 (26.0 mg, 0.188 mmol) in anhydrous MeCN (0.1 mL) in a pre-dried microwave vial was cooled to 0 °C using an ice bath. A solution of compound 5 (20.8 mg, 0.118 mmol) in MeCN (0.4 mL) was slowly added to the stirred suspension, after which the reaction was brought to rt and stirred under nitrogen overnight. The reaction mixture was concentrated and purified by flash chromatography (0-10% MeOH / CH2Cl2) to afford the title compound as a white solid (30.0 mg, 0.029 mmol, 62%). f 0.18(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3296(m, NH), 2978(m, CH), 2157(m, C≡C), 1654(s, C=O);δ H (600 MHz, CD3OD) 4.20 (s, 4H), 4.08 (d, 4H, J = 2.5 Hz), 3.80 (s, 4H), 3.57 (t, 4H, J = 6.0 Hz), 3.51-3.44 (m, 16H), 3.27 (s, 4H), 3.21-3.17 (m, 4H), 2.72 (t, 2H, J = 2.5 Hz), 1.47 (s, 36H); δ C(101 MHZ, CD3OD) 169.8, 166.3, 159.1, 81.3, 80.3, 73.2, 57.0, 56.1, 56.0, 55.9, 53.3, 37.0, 36.7, 30.0, 28.8; Actual value [M+H] + 1023.6547, C 48 H 87 O 12 N 12 + Calculated value 1023.6566.

[0226] Step 3: Synthesis of conjugation reagent 8 [ka] To a solution of compound 10 (79.8 mg, 0.078 mmol) in CHCl (0.5 mL) at 0 °C was added HCl (4 M in dioxane, 1.0 mL). The reaction was stirred under nitrogen for 6 h and then concentrated in vacuo to give the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was redissolved in CHCl (1 mL) and cooled to 0 °C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (72.8 mg, 0.312 mmol), triethylamine (217 μL, 1.56 mmol), and HBTU (118 mg, 0.312 mmol) in CHCl (2 mL) at 0 °C. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by reverse-phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NH4OH(aq). Solvent B: MeCN) and lyophilization gave the product as a colorless oil (46.1 mg, 0.031 mmol, 40%, over two steps). max (neat / cm -1 )3289(m, NH), 2938(m, CH), 2186(m, C≡C), 1637(s, C=O);δ H(600 MHz, CD3OD) 6.67 (s, 4H), 6.59 (dd, 8H, J = 10.6, 17.4 Hz), 6.35 (d, 8H, J = 17.3 Hz), 5.56 (dd, 8H, J = 1.2, 10.7 Hz), 3.98 (d, 4H, J = 2.3), 3.45 (t, 8H, J = 6.7 Hz), 3.27 (t, 4H, J = 6.8 Hz), 3.24 (t, 8H, J = 6.4 Hz), 3.18 (s, 4H), 3.17 (s, 4H), 2.64-2.57 (m, 4H), 2.64-2.57 (m, 2H), 2.64-2.57 (m, 8H), 2.60 (s, 4H), 2.29 (t, 8H, J = 7.5 Hz), 1.91 (quintet, 8H, J = 7.1 Hz); δ C (101 MHz, CD3OD) 175.9, 174.1, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 80.9, 72.4, 59.7, 59.3, 55.8 (2つのピーク), 54.4, 41.7, 38.7, 38.4, 34.7, 29.4, 27.0; HRMS (ESI) m / z measurement value [M+H] + 1483.8687, C 76 H 107 O8N 24 + The calculated value is 1483.8698.

[0227] (Example 3) Preparation of コンジュゲートchemical test solution 11 Process 1: Synthesis of Compound 12

change

[0228] Step 2: Synthesis of Compound 13 [ka] In a pre-dried microwave vial, a suspension of compound 12 (184 mg, 0.21 mmol) and K2CO3 (174 mg, 1.26 mmol) in anhydrous MeCN (0.26 mL) was cooled to 0 °C using an ice bath. A solution of compound 5 (anh. MeCN, 0.5 M, 1.58 mL) was slowly added to the stirred suspension, after which the reaction was brought to rt and stirred under nitrogen overnight. The reaction mixture was concentrated, redissolved in CHCl2 (40 mL), and the organic phase was washed with water (2 × 10 mL), brine (1 × 20 mL), dried over Na2SO4, and purified by flash chromatography (5–10% MeOH / CHCl2) to give the title compound as a white foam (119 mg, 0.102 mmol, 49%). f 0.5(SiO2, 20%MeOH / CH2Cl2);ν max (neat / cm -1 )3309(m, NH), 2928(m, CH), 2158(m, C≡C), 1658(s, C=O);δ H (400 MHz, CD3OD) 4.05 (d, J = 2.6 Hz, 4H), 4.03 (d, J = 2.5 Hz, 2H), 3.39 - 3.32 (m, 7H), 3.27 (s, 4H), 3.25 (s, 2H), 3.20 (s, 4H), 3.17 - 3.13 (m, 8H), 2.77-2.67 (m, 8H), 2.66-2.58 (m, 8H), 1.47 (s, 36H); δ C (100 MHz, CD3OD) 174.3, 173.7, 173.6, 158.4, 80.9, 80.8, 80.1, 72.6, 72.3, 60.2, 59.3, 59.2, 56.5, 55.7, 54.4, 54.3, 39.7, 38.5, 29.5, 29.3, 28.9; HRMS (ESI) m / z actual value [M+H] + 1161.7365, C 55 H 97 O 13 N 14 + Calculated value 1161.7360.

[0229] Step 3: Synthesis of conjugation reagent 11 [ka] To a solution of compound 13 (25 mg, 22 μmol) in CHCl (150 μL) at 0 °C was added HCl (4 M in dioxane, 270 μL). The reaction was brought to rt and stirred for 1 h. The reaction mixture was concentrated to give the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt (23.0 mg, 22.0 μmol) was suspended in CHCl (150 μL) and cooled to 0 °C using an ice bath, and DIPEA (74.0 μL, 0.42 mmol) was added first, followed by 2 (25.0 mg, 0.11 mmol). The solution was stirred at rt for 10 min, after which a solution of BTFFH in CHCl (0.6 M, 205 μL) was added dropwise over 3 h with maximum stirring. After the addition of BTFFH was complete, the reaction was stirred at rt for an additional 12 h, concentrated, and purified by reverse-phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NHOH(aq). Solvent B: MeCN) to give, after lyophilization, the desired product (6.70 mg, 41.4 μmol, 20%, over two steps) as a pale yellow solid. max (neat / cm -1 )3305(m, NH), 2970(m, CH), 2156(m, C≡C), 1637(s, C=O);δ H (400 MHz, CD3OD) 6.68 (s, 4H), 6.60 (dd, J = 17.4, 10.7 Hz, 8H), 6.36 (d, J = 17.3 Hz, 8H), 5.56 (dd, J = 10.6, 1.6 Hz, 8H), 4.02 - 3.96 (m, 6H), 3.45 (t, J = 6.8 Hz, 8H), 3.35 (s, 4H), 3.29 - 3.15 (m, 21H), 2.67 - 2.56 (m, 20H), 2.29 (t, J = 7.5 Hz, 8H), 1.91 (quintet, J = 7.0 Hz, 8H); δ C(126 MHz, CD3OD) 175.9, 174.1, 173.8, 173.7, 165.3, 164.0, 137.1, 122.1, 105.8, 81.0 (two peaks), 72.5, 72.4, 59.8, 59.4, 59.3, 55.9, 55.8, 54.5 (two peaks), 54.3 (two peaks), 41.7, 38.7, 38.4, 34.7, 29.4, 29.3, 27.0; HRMS (ESI) m / z observed [M+H] + 1621.9491, C 83 H 117 N 26 O9 + Calculated value 1621.9497.

[0230] Example 4 Preparation of conjugation reagent 14 Step 1: Synthesis of tert-butyl bis(2-(prop-2-yn-1-yloxy)ethyl)carbamate (15) [ka] A solution of diethanolamine (2.44 g, 23.0 mmol) in CHCl (25 mL) was cooled to 0° C. using an ice bath, and a solution of di-tert-butyl dicarbonate (6.33 g, 29.0 mmol) in anh. CHCl (5 mL) was slowly added. The reaction was stirred at rt for 12 h, diluted with CHCl (40 mL), water (40 mL) was added, and the aqueous phase was extracted twice with CHCl (2×20 mL). The combined organic phases were washed with brine, dried over NaSO, and concentrated to give tert-butyl bis(2-hydroxyethyl)carbamate as a clear oil, which was used directly in the next step without further purification. 50% NaOH (aq)To the solution (11 mL) were added sequentially tert-butyl bis(2-hydroxyethyl)carbamate, tetrabutylammonium bisulfate (16.0 mg, 47.0 μmol), and propargyl bromide (80 wt % in toluene, 8.7 mL, 78.0 mmol) in toluene (11 mL), and the reaction was stirred at rt for 48 h under a nitrogen atmosphere. The organic layer was isolated and concentrated, and the desired compound was purified by flash chromatography (50% EtOAc / hexanes) to afford the title compound as a yellow viscous oil (2.30 g, 7.57 mmol, 35%, over two steps). f 0.60(SiO2, 50% EtOAc / hexane);ν max (neat / cm -1 )2975(m, CH), 2117(m, C≡C), 1686(s, C=O);δ H (400 MHz, CDCl3) 4.13 (d, J = 2.4 Hz, 4H), 3.66 - 3.57 (m, 4H), 3.52 - 3.38 (m, 4H), 2.41 (t, J = 2.4 Hz, 2H), 1.45 (s, 9H); δ C (100 MHz, CDCl3) 155.6, 79.9, 79.8, 74.5, 68.7, 58.3, 47.9, 28.6; LRMS (ESI) m / z observed [M+Na] + 304.2.

[0231] Step 2: Synthesis of tert-butyl (5-(bis(2-(prop-2-yn-1-yloxy)ethyl)amino)pentyl)carbamate (16) [ka] A solution of compound 15 (281 mg, 1.00 mmol) in CHCl (1.5 mL) was cooled to 0° C., followed by the addition of HCl (4 M in dioxane, 3 mL). The reaction was brought to rt and stirred for 2 h. The reaction mixture was concentrated to give the desired amine hydrochloride salt as a white solid. The solid was resuspended in MeCN (10 mL). Sodium carbonate (1.06 g, 10.0 mmol) was added. After stirring for 5 min, a solution of tert-butyl (5-bromopentyl)carbamate (399 mg, 1.50 mmol) in MeCN (2 mL) was added. The reaction mixture was then refluxed at 70° C. for 48 h. The reaction mixture was subsequently diluted with brine and extracted with EtOAc (3×). The combined organic layers were dried over Na2SO4, concentrated, and purified by flash chromatography (30-50% EtOAc / hexanes) to give the title compound as a pale yellow oil (242 mg, 66% over two steps). f 0.21(SiO2, 80% EtOAc / PE);ν max (neat / cm -1 )3342(m, NH), 2938(m, CH), 2111(m, C≡C), 1676(s, C=O);δ H (400 MHz, CDCl3) 4.54 (br s, 1H), 4.16 (d, J = 2.3 Hz, 4H), 3.60 (t, J = 6.0 Hz, 4H), 3.14 - 3.06 (m, 2H), 2.73 (t, J = 6.0 Hz, 4H), 2.55 - 2.49 (m, 2H), 2.42 (t, J = 2.2 Hz, 2H), 1.51 - 1.42 (m, 4H), 1.44 (s, 9H), 1.30 (quintet, J = 7.6 Hz, 2H); δ C (100 MHz, CDCl3) 156.0, 79.9, 79.0, 74.3, 68.3, 58.2, 55.0, 53.8, 40.5, 29.9, 28.4, 26.7, 24.5; HRMS (ESI) m / z observed [M+H] + 367.2587, C 20 H 34 O4N2 + Calculated value 367.2597.

[0232] Step 3: Synthesis of N-(5-(bis(2-(prop-2-yn-1-yloxy)ethyl)amino)pentyl)-2-bromoacetamide (17) [ka] To a solution of compound 16 (100 mg, 0.27 mmol) in CHCl (0.5 mL) at 0 °C was added HCl (4 M in dioxane, 1 mL). The reaction was stirred under nitrogen for 2 h and then concentrated in vacuo to give the desired amine hydrochloride salt as a white solid. The hydrochloride salt was redissolved in a mixture of CHCl (1 mL) and sat. NaHCO (1 mL) and cooled to -10 °C, followed by the dropwise addition of 2-bromoacetyl bromide (35.3 μL, 0.405 mmol). The reaction mixture was stirred for 1 h, then diluted with brine and extracted with CHCl (×3). The combined organic phases were dried over NaSO and concentrated in vacuo. Purification by flash chromatography (0–5% MeOH / CHCl) gave the title compound as a pale yellow oil (57.3 mg, 0.148 mmol, 55%, over two steps). R f 0.41(SiO2, 10%MeOH / CH2Cl2);ν max (neat / cm -1 )3219(m, NH), 2928(m, CH), 2111(m, C≡C), 1672(s, C=O);δ H (400 MHz, CDCl3) 6.55 (br s, 1H), 4.15 (d, J = 2.4 Hz, 4H), 3.87 (s, 2H), 3.62 (t, J = 5.5 Hz, 4H), 3.27 (q, 2H, J = 6.7 Hz), 2.78 - 2.75 (m, 4H), 2.58 - 2.54 (m, 2H), 2.43 (t, J = 2.4 Hz, 2H), 1.55 (quintet, J = 7.4 Hz, 2H), 1.50 (quintet, J = 7.4 Hz, 2H), 1.33 (quintet, J = 7.6 Hz, 2H); δ C(100 MHz, CDCl3) 165.5, 79.7, 74.8, 67.8, 58.4, 55.0, 53.9, 40.2, 29.8, 29.5, 29.1, 24.5; HRMS (ESI) m / z observed [M+H] + 387.1273, C 17 H 28 O3N2Br + Calculated value 387.1283.

[0233] Step 4: Synthesis of Compound 18 [ka] To a solution of compound 9 (49.3 mg, 59.2 μmol) in MeCN (1 mL) was added anh. KCO (32.7 mg, 237 μmol), followed by a solution of N-(5-(bis(2-(prop-2-yn-1-yloxy)ethyl)amino)pentyl)-2-bromoacetamide (17) (57.3 mg, 148 μmol) in MeCN (0.5 mL). The reaction was stirred at room temperature for 48 h. The reaction mixture was concentrated, and the desired compound was purified by flash chromatography (0% to 15% MeOH / CHCl) to afford the title compound as a clear oil (38.8 mg, 26.6 μmol, 45%). f 0.35(15%MeOH / CH2Cl2);ν max (neat / cm -1 )3283(m, NH), 2936(m, CH), 2155(m, C≡C), 1654(s, C=O);δ H (400 MHz, CD3OD) 4.17 (s, 4H), 3.66 (t, J = 5.5 Hz, 8H), 3.32 - 3.29 (m, 8H), 3.26 - 3.21 (m, 4H), 3.20 (s, 4H), 3.16 (s, 4H), 3.13 (t, J = 5.8 Hz, 8H), 2.89 - 2.84 (m, 8H), 2.70 - 2.65 (m, 12H), 2.59 (t, J = 5.7 Hz, 8H), 1.61 - 1.50 (m, 8H), 1.44 (s, 36H), 1.38 - 1.30 (m, 4H); δC (126 MHz, CD3OD) 174.3, 173.8, 158.5, 80.7, 80.2, 76.2, 68.3, 60.2, 59.6, 59.0, 56.5 (two peaks), 56.0 (two peaks), 54.6, 40.3, 39.7, 38.6, 30.5, 29.0, 26.8, 25.8; HRMS (ESI) m / z observed [M+H] + 1445.9692, C 72 H 129 N 14 O 16 + Calculated value 1445.9711.

[0234] Step 5: Synthesis of conjugation reagent 14 [ka] To a solution of compound 18 (13.0 mg, 8.99 μmol) in CHCl (0.25 mL) at 0 °C was added HCl (4 M in dioxane, 0.5 mL). The reaction was stirred under nitrogen for 2 h and then concentrated in vacuo to give the desired amine hydrochloride salt as a white solid. The amine hydrochloride salt was redissolved in CHCl (0.25 mL) and cooled to 0 °C. To this solution was added a solution of 4-((4,6-divinylpyrimidin-2-yl)amino)butanoic acid (2) (12.6 mg, 53.9 μmol), triethylamine (25 μL, 180 μmol), and HBTU (20.5 mg, 53.9 μmol) in CHCl (0.25 mL) at 0 °C. The reaction was stirred under nitrogen for 18 h and then concentrated in vacuo. Purification by reverse-phase flash column chromatography (35-70% solvent B in solvent A. Solvent A: 100 mM NH4OH(aq). Solvent B: MeCN) and lyophilization afforded the product as a white solid (1.80 mg, 0.94 μmol, 10%, over two steps). max (neat / cm -1 )3299(m, NH), 2928(m, CH), 2153(m, C≡C), 1645(s, C=O);δ H(400 MHz, CD3OD) 6.69 (s, 4H), 6.60 (dd, J = 17.4, 10.7 Hz, 8H), 6.36 (d, J = 17.3 Hz, 8H), 5.57 (dd, J = 10.7, 1.5 Hz, 8H), 4.23 (d, J = 2.4 Hz, 8H), 3.83 (t, J = 4.9 Hz, 8H), 3.45 (t, J = 6.8 Hz, 8H), 3.30 - 3.15 (m, 32H), 3.00 (t, J = 2.3 Hz, 4H), 2.65 - 2.60 (m, 24H), 2.29 (t, J = 7.5 Hz, 8H), 1.91 (quintet, J = 7.1 Hz, 8H), 1.56 (quintet, J = 7.5 Hz, 4H), 1.39 - 1.30 (m, 8H); δ C (126 MHz, CD3OD) 175.9, 174.2 (two peaks), 165.3, 164.0, 137.2, 122.2, 105.9, 89.9, 77.2, 68.8, 59.9 (two peaks), 59.2, 56.1, 56.0, 55.3, 54.0, 47.1, 41.8, 40.4, 38.8 (two peaks), 34.7, 30.2, 26.8, 27.1, 25.0; HRMS (ESI) m / z observed [M+H] + 1906.1851, C 100 H 149 N 26 O 12 + Calculated value 1906.1848.

[0235] Example 5 Preparation of conjugation reagent 19 Step 1: Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(4,5-dibromo-2-methyl-3,6-dioxo-3,6-dihydropyridazin-1(2H)-yl)propanoate (20) [ka] Dibromopyridazinedione (20) was synthesized as previously described (Bahou et al., Org. Biomol. Chem., 2018, 16, 1359-1366).

[0236] Step 2: Synthesis of conjugation reagent 19 [ka] Compound 7 (3.90 mg, 4.40 μmol) was dissolved in 4 M HCl in CHCl (0.25 mL) and 1,4-dioxane (0.25 mL), and the solution was stirred at rt for 1 h. The reaction mixture was then concentrated under a stream of N. To the crude residue was added dibromopyridazinedione 20 (10.0 mg, 22.0 μmol) in CHCl DMF (1:1, 1 mL), followed by KCO (3.00 mg, 22.0 μmol), and the reaction mixture was stirred at rt for 3 h. Upon completion, the reaction was concentrated under a stream of N and purified by preparative RP-HPLC (5-95% B) to afford the title compound (2.50 mg, 1.36 μmol, 31%) as a white solid. HPLC (5-95% MeCN / HO over 20 min) retention time: 7.433 min; LRMS (ESI) m / z observed [M+H] + 1837.0, C 53 H 69 Br8N 18 O 15 + Required value 1837.5, measured m / z value [M+HCOO] - 1881.1, C 54 H 69 Br8N 18 O 17 - Required value: 1881.5.

[0237] Example 6 Preparation of conjugation reagent 21 Step 1: Synthesis of conjugation reagent 21 [ka] Compound 10 (10.0 mg, 9.80 μmol) was dissolved in 4 M HCl in CHCl (0.25 mL) and 1,4-dioxane (0.25 mL), and the solution was stirred at rt for 1 h. The reaction mixture was then concentrated under a stream of N. To the crude residue was added dibromopyridazinedione 20 (22.0 mg, 48.9 μmol) in CHCl DMF (1:1, 1 mL), followed by KCO (6.80 mg, 48.9 μmol), and the reaction mixture was stirred at rt for 3 h. Upon completion, the reaction was concentrated under a stream of N and purified by preparative RP-HPLC (5-95% B) to afford the title compound (4.20 mg, 2.13 μmol, 22%) as a white solid. HPLC (5-95% MeCN / HO over 20 min) retention time: 7.507 min; LRMS (ESI) m / z observed [M+H] + July 1976, C 60 H 79 Br8N 20 O 16 + Required value 1975.7, measured m / z value [M+HCOO] - August 2018, C 61 H 79 Br8N 20 O 18 - Required value 2019.7.

[0238] Example 7 Preparation of conjugation reagent 22 Step 1: Synthesis of divinyltriazine 23 [ka] Divinyltriazine (23) was synthesized as previously described (Counsell et al., Org. Biomol. Chem., 2020, 18, 4739-4743).

[0239] Step 2: Synthesis of conjugating reagent 22 [ka] A solution of compound 10 (20.0 mg, 19.5 μmol) in HCl (4 M in 1,4-dioxane, 0.5 mL) and CHCl (0.5 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was concentrated in vacuo. To the deprotected residue was added HBTU (41.7 mg, 110 μmol), divinyltriazine 23 (24.2 mg, 110 μmol), and DMF (1 mL), followed by DIPEA (76.6 μL, 440 μmol), and the mixture was stirred at rt for 45 min. Upon completion, the reaction mixture was purified by automated reverse-phase FCC (10–60% solvent B in solvent A; solvent A: 0.1 M NHOH / HO, solvent B: MeCN) to afford the title compound (12.2 mg, 8.52 μmol, 44%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time: 8.257 min; LRMS (ESI) m / z observed [M+H] + 1432.9, C 68 H 95 N 28 O8 + Required value 1431.8, measured m / z value [M+HCOO] - 1477.1, C 69 H 95 N 28 O 10 - Required value: 1475.8.

[0240] Example 8 Preparation of conjugation reagent 24 Step 1: Synthesis of (9H-fluoren-9-yl)methyl(6-(2-bromoacetamido)hexyl)carbamate (25) [ka] To a solution of Fmoc-hexamethylenediamine hydrochloride (200 mg, 0.533 mmol) and bromoacetyl bromide (116 μL, 1.33 mmol) in CHCl (2 mL) was added saturated aqueous NaHCO (2 mL), and the mixture was stirred at rt for 24 h. The reaction mixture was then diluted with H0 and extracted with CHCl (3 × 50 mL). The combined organic fractions were washed with 1 M HCl (2 × 50 mL), dried (MgSO), and concentrated in vacuo. The crude residue was purified via FCC (50–100% EtOAc / PE) to afford the title compound (107 mg, 0.233 mmol, 44%) as a white solid. f 0.16(SiO2; 50% EtOAc / PE); δ H (400 MHz, DMSO-d6) 8.24 (t, 1H, J = 5.2 Hz), 7.89 (d, 2H, J = 7.2 Hz), 7.68 (d, 2H, J = 7.4 Hz), 7.41 (t, 2H, J = 7.4 Hz), 7.31 (td, 2H, J = 11.1, 0.7 Hz), 7.25 (t, 1H, J = 5.5 Hz), 4.29 (d, 2H, J = 7.5 Hz), 4.22-4.18 (m, 1H), 3.82 (s, 2H), 3.05 (q, 2H, J = 6.6 Hz), 2.96 (q, 2H, J = 6.6 Hz), 1.41-1.36 (m, 4H), 1.25-1.23 (m, 4H); δ C (101 MHz, DMSO-d6) 165.8, 156.1, 143.9, 140.7, 127.6, 127.0, 125.1, 120.1, 65.1, 46.8, 29.6, 29.3, 28.8, 26.0, 25.9. LRMS (ESI) m / z observed [M+H] + 459.3, C 23 H 28 Br1N2O3 + Calculated value 459.1.

[0241] Step 2: Synthesis of Compound 26 [ka] A solution of compound 6 (30.0 mg, 38.0 μmol), compound 25 (29.0 mg, 63.0 μmol), and DIPEA (8.60 μL, 49.4 μmol) in DMF (0.5 mL) was stirred at rt for 21 h. Upon completion, the reaction mixture was concentrated under a stream of N. The crude residue was purified by FCC (0-8% MeOH / CHCl) to afford the title compound (40.0 mg, 34.0 μmol, 90%) as a white solid. HPLC (5-95% Solvent B in Solvent A) retention time 10.824 min; LRMS (ESI) m / z observed [M+H]. + 1169.1, C 59 H 98 N 11 O 13 + Required value 1168.7, measured m / z value [M+HCOO] - 1213.1, C 60 H 98 N 11 O 15 - Required value 1212.7.

[0242] Step 3: Synthesis of Compound 27 [ka] A solution of compound 26 (44.0 mg, 38.0 μmol) and piperidine (7.50 μL, 76.0 μmol) in DMF (1 mL) was stirred at rt for 2 h. Upon completion, the reaction mixture was concentrated under a stream of N. To this crude residue was added HBTU (28.8 mg, 76.0 μmol) and DMF (1 mL), followed by N-PEG-COOH (114 μL, 57.0 μmol, 0.5 M in TBME) and DIPEA (13.2 μL, 76.0 μmol), and the mixture was stirred at rt for 20 h. Upon completion, the reaction mixture was concentrated under a stream of N, and the crude material was purified by FCC (0–10% MeOH / CHCl) to afford the title compound (38.0 mg, 31.5 μmol, 83%) as a clear oil. HPLC (5-95% solvent B in solvent A) retention time 10.471 min; LRMS (ESI) m / z observed [M+H] + 1205.8, C 54 H 105 N14 O 16 + Required value 1205.8, measured m / z value [M+HCOO] - 1250.7, C 55 H 105 N 14 O 18 - Required value: 1249.8.

[0243] Step 4: Synthesis of conjugation reagent 24 [ka] A solution of compound 27 (29.0 mg, 24.0 μmol) in HCl (4 M in 1,4-dioxane, 0.5 mL) and CHCl (0.5 mL) was stirred at rt for 1 h. Upon completion, the reaction mixture was concentrated in vacuo. To this deprotected residue was added HBTU (45.5 mg, 120 μmol), compound 2 (28.0 mg, 120 μmol), and DMF (1 mL), followed by DIPEA (83.8 μL, 480 μmol), and the mixture was stirred at rt for 1 h. Upon completion, the reaction mixture was purified by automated reverse-phase FCC (10–100% solvent B in solvent A; solvent A: 0.1 M NHOH / HO, solvent B: MeCN) to afford the title compound (12.0 mg, 7.20 μmol, 30%) as a pale yellow solid. HPLC (5-95% solvent B in solvent A) retention time: 8.401 min; LRMS (ESI) m / z observed [M+Na] + 1689.2, C 82 H 124 N 26 23 NaO 12 + Required value 1689.0, measured m / z value [M+HCOO] - 1711.1, C 83 H 125 N 26 O 14 - Required value 1711.0.

[0244] Example 9 Preparation of conjugation reagent 28 Step 1: Synthesis of Fmoc-Lys(PEG-N)-OH (29) [ka] Fmoc-Lys-OH·HCl (500 mg, 1.23 mmol) was suspended in DMF / CHCl (1:1, 3 mL), and a solution of N3-dPEG4-NHS ester (480 mg, 1.23 mmol) in DMF / CHCl (1:1, 3 mL) was added dropwise with stirring. KCO (324 mg, 2.35 mmol) was added, and the mixture was stirred for 23 h. The reaction mixture was diluted with CHCl and washed with aqueous LiCl (3 M, 2 × 15 mL). The combined aqueous layers were extracted with CHCl (15 mL), and the combined organic extracts were concentrated in vacuo. The crude organic fraction was purified by automated reverse-phase FCC (10-60% solvent B in solvent A; solvent A: 0.5% formic acid / HO, solvent B: MeCN) to give the product Fmoc-Lys(COdPEG-N)-OH (560 mg, 0.810 mmol, 66%) as a colorless oil. H (400 MHz, CDCl3) 7.72 (d, 2H, J = 7.5 Hz), 7.58 (t, 2H, J = 7.3 Hz), 7.36 (t, 2H, J = 7.4 Hz), 7.27 (t, 2H, J = 7.4 Hz), 6.90 (br s, 1H), 6.23 (br s, 1H), 5.90 (d, 1H, J = 7.1 Hz), 4.44-4.30 (m, 3H), 4.18 (t, 1H, J = 6.9 Hz), 3.72-3.63 (m, 2H), 3.60 (s, 10H), 3.56 (s, 4H), 3.34 (t, 2H, J = 4.9 Hz), 3.23 (qt, 2H, J = 6.1 Hz), 2.45 (t, 2H J = 5.5 Hz), 1.94-1.82 (m, 1H), 1.81-1.72 (m, 1H), 1.58-1.30 (m, 4H); δ c(101 MHz, CDCl3) 174.5, 172.6, 171.0, 156.3, 144.0, 143.8, 141.3, 127.7, 127.1, 125.2, 125.2, 120.0, 80.6, 70.6, 70.5, 70.5, 70.3, 70.2, 70.1, 70.0, 67.2, 66.9, 53.8, 50.6, 47.2, 38.9, 36.7, 36.3, 31.7, 28.8, 28.1, 22.1; LRMS (ESI) m / z observed [M+H] + 642.5, C 32 H 43 N5O9 + Calculated value 642.3.

[0245] Step 2: Synthesis of Ahx-Lyz-Gly-Lys(PEG4-N3)-Gly-Lys-Lys-CONH2 (30) [ka] The peptide was synthesized on a 0.1 mmol scale using a general Fmoc solid-phase peptide synthesis procedure to yield the desired peptide 30 (40.5 mg, 30.0 μmol, 27%) as a colorless viscous oil. HPLC (5-95% solvent B in solvent A) retention time: 4.313 min; LRMS (ESI) m / z observed [M+H]. + 1031.3, C 45 H 87 N 15 O 12 + Required value 1030.7.

[0246] Step 3: Synthesis of conjugation reagent 28 [ka] Compound 30 (35.0 mg, 24.0 μmol) was suspended in DMF / CHCl (1:1, 0.4 mL) and cooled to 0 °C. A suspension of compound 2 (27.5 mg, 120 μmol), HBTU (44.7 mg, 120 μmol), and NEt (66.0 μL, 470 μmol) in DMF / CHCl (1:1, 0.4 mL) was added, and the mixture was stirred for 3 h. The mixture was purified by automated reverse-phase FCC (10–60% solvent B in solvent A; solvent A: HO, solvent B: MeCN) to give the title compound (18.3 mg, 9.70 μmol, 40%) as a colorless viscous oil. HPLC (5–95% solvent B in solvent A) retention time: 8.084 min; LRMS (ESI) m / z observed [M+Na]. + 1914.2, C 93 H 139 23 Na1N 27 O 16 + Required value 1914.1, measured m / z value [M+HCOO - ] - 1936.3, C 94 H 140 N 27 O 18 - Required value 1936.1.

[0247] Example 10 Preparation of conjugation reagent 31 Step 1: Synthesis of Ahx-Lyz-Gly-Lys(PEG4-N3)-Gly-Lys(PEG4-N3)-Gly-Lys-Lys-CONH2 (32) [ka] The peptide was synthesized on a 0.1 mmol scale using general peptide synthesis procedures, yielding the product (30.6 mg, 16.0 μmol, 5%) as a colorless viscous oil. HPLC (5-95% solvent B in solvent A) retention time: 5.942 min; LRMS (ESI) m / z observed [M+H]. + 1489.8, C 64 H 122 N 21 O 19 +Required value 1488.9, measured m / z [M+HCOO - ] - 1532.9, C 65 H 122 N 21 O 21 - Required value: 1533.7.

[0248] Step 2: Synthesis of conjugation reagent 31 [ka] Compound 32 (23.3 mg, 12.0 μmol) was suspended in DMF (0.5 mL) and cooled to 0 °C. A suspension of compound 2 (14.0 mg, 60.0 μmol), HBTU (22.7 mg, 60.0 μmol), and NEt (33.4 μL, 240 μmol) in DMF (0.5 mL) was added, and the mixture was stirred for 5 h. The mixture was purified by automated reverse-phase FCC (40-55% MeCN in HO) to give the title compound (1.81 mg, 0.770 μmol, 5%) as a colorless viscous oil. HPLC (5-95% solvent B in solvent A) retention time 8.376 min; LRMS (ESI) m / z observed [M+2H]. 2+ 1176.3, C 112 H 175 N 33 O 23 2+ Required value: 1175.7.

[0249] Example 11 Preparation of conjugation reagent 33 Step 1: Synthesis of Ac-Lys(PEG4N3)-PEG2Glu-CONH2 (34) [ka] The peptide was synthesized on a 0.3 mmol scale using general peptide synthesis procedures to yield the desired product (44.1 mg, 60.0 μmol, 60%) as a colorless viscous oil. HPLC (5-95% solvent B in solvent A) retention time 5.985 min; LRMS (ESI) m / z observed [M+H]. + 735.5, C 30 H54 N8O 13 + Required value: 735.4.

[0250] Step 2: Synthesis of Compound 35 [ka] Compound 26 (20.0 mg, 17.0 μmol) was dissolved in DMF (0.5 mL) and piperidine (3.40 μL, 34.0 μmol) was added. The mixture was stirred for 90 min and then concentrated under N. The crude mixture was redissolved in DMF (0.5 mL) and compound 34 (18.9 mg, 26.0 μmol), HBTU (13.0 mg, 34.0 μmol), and DIPEA (6.00 μL, 34.0 μmol) were added. The reaction mixture was stirred for 3 h. The mixture was purified by automated reverse-phase FCC (10–100% solvent B in solvent A; solvent A: 0.1 M NHOH / HO, solvent B: MeCN) to give the title compound (11.4 mg, 6.90 μmol, 40%) as a pale yellow powder. HPLC (5-95% solvent B in solvent A) retention time: 8.538 min; LRMS (ESI) m / z observed [M+2H] 2+ 832.4, C 74 H 139 N 19 O 23 + Required value: 832.0.

[0251] Step 3: Synthesis of conjugation reagent 33 [ka] Compound 35 (7.00 mg, 4.20 μmol) was dissolved in CHCl (0.3 mL) and HCl (2 M in EtO, 0.3 mL) was added. The mixture was stirred for 1 h and then concentrated. The crude mixture was redissolved in DMF (0.5 mL) and compound 2 (7.00 mg, 30.0 μmol), HBTU (11.4 mg, 30.0 μmol), and DIPEA (10.5 μL, 60.0 μmol) were added. The mixture was stirred for 2 h and then purified twice via automated reverse-phase FCC (40-70% MeCN in HO) to give the desired product (0.120 mg, 0.600 μmol, 1.3%) as a yellow residue. HPLC (5-95% solvent B in solvent A) retention time 7.66 min; LRMS (ESI) m / z observed [M+Na]. + 2124.4, C 102 H 160 N 31 O 19 + Required value 2124.3, measured m / z value [M+HCOO - ] - 2169.0, C 103 H 160 N 31 O 21 - Required value 2168.2.

[0252] Example 12 Preparation of conjugates 36-39 To a solution of trastuzumab (198 μL, 17 μM, 2.5 mg / mL) in TBS (25 mM Tris-HCl pH 8, 25 mM NaCl, 0.5 mM EDTA), TCEP (10 equiv.) was added. The mixture was vortexed and incubated at 37°C for 1 h. A solution of conjugation reagent 1, 8, 11, or 14 (10 mM in DMSO) was added (34 μM final concentration, 2 equiv.), and the reaction mixture was incubated at 37°C for 4 h. Excess reagent was removed using a Zeba™ Spin desalting column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated hemodiafiltration into PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Samples were stored at 4°C until analysis. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding cross-linked conjugates 36, 37, 38 and 39.

[0253] Figure 1(A) shows the SDS-PAGE traces for conjugates 36, 37, 38 and 39. Figures 2 to 5 show the LC-MS traces for conjugates 36, 37, 38 and 39.

[0254] Example 13 Preparation of conjugates 40–46 To a solution of trastuzumab (50 μL, 17 μM, 2.5 mg / mL) in TBS (25 mM Tris-HCl pH 8, 25 mM NaCl, 0.5 mM EDTA), TCEP (10 equiv.) was added. The mixture was vortexed and incubated at 37°C for 1 h. A solution of conjugation reagent 19, 21, 22, 24, 28, 31, or 33 (5 mM in DMSO) was added (34 μM final concentration, 2 equiv.), and the reaction mixture was incubated at 37°C for 2 h. Excess reagent was removed using a Zeba™ Spin desalting column (40,000 MWCO, Thermo Fisher Scientific). Samples were stored at 4°C until analysis or flash-frozen and stored at -20°C. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding cross-linked conjugates 40, 41, 42, 43, 44, 45 and 46.

[0255] Figure 1(B) shows SDS-PAGE traces for conjugates 40, 41, 42, 43, 44, 45 and 46. Figures 6 to 12 show LC-MS traces for conjugates 40, 41, 42, 43, 44, 45 and 46.

[0256] Example 14 Preparation of conjugates 47-50 To a solution of brentuximab (50 μL, 17 μM, 2.5 mg / mL) in TBS (25 mM Tris-HCl pH 8, 25 mM NaCl, 0.5 mM EDTA), TCEP (10 equiv.) was added. The mixture was vortexed and incubated at 37°C for 1 h. A solution of conjugation reagent 1, 8, 19, or 21 (5 mM in DMSO) was added (34 μM final concentration, 2 equiv.), and the reaction mixture was incubated at 37°C for 2 h. Excess reagent was removed using Zeba™ Spin desalting columns (40,000 MWCO, Thermo Fisher Scientific). Samples were stored at 4°C until analysis or flash-frozen and stored at -20°C. LC-MS and SDS-PAGE analysis demonstrated >95% conversion to the corresponding cross-linked conjugates 47, 48, 49, and 50.

[0257] Figure 1(C) shows the SDS-PAGE traces for conjugates 47, 48, 49 and 50. Figures 13 to 16 show the LC-MS traces for conjugates 47, 48, 49 and 50.

[0258] Example 15 Size exclusion chromatography Size exclusion chromatography (SEC) was performed using a Superdex 200 10 / 300 GL column. Samples were injected at a concentration of 1 mg / mL and eluted with TBS buffer (25 mM Tris-HCl pH 8, 200 mM NaCl, 0.5 mM EDTA) at a flow rate of 0.5 mL / min.

[0259] Size exclusion chromatograms for conjugates 36, 37, 38 and 39 are shown in Figure 17(A).

[0260] Example 16 Enzyme-linked immunosorbent assay (ELISA) A 96-well plate was coated overnight at 4°C with 100 μL of a 0.25 μg / mL solution of HER2 (Sino Biological Co., Ltd., His-tagged). The coating solution was removed, and each well was washed with 200 μL of PBS. Each well was then blocked with 200 μL of 1% BSA in PBS for 1 h at room temperature. The blocking solution was then removed, and each well was washed with 300 μL of PBS. The wells were treated with serial dilutions of trastuzumab and trastuzumab conjugates 36, 37, 38, and 39 (100 μL of 90 nM, 30 nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, and 0 nM) in PBS and incubated at room temperature for 2 h. The conjugate solution was removed, and each well was washed with 0.1% Tween 20 in PBS (2 × 200 μL), followed by 3 × 200 μL of PBS. Next, 100 μL of detection antibody (mouse anti-human IgG-HRP, 1:500 dilution, ThermoFisher) in PBS was added to each well and incubated at room temperature for 1 h. Each well was washed with 0.1% Tween 20 in PBS (2 × 200 μL), followed by 3 × 200 μL of PBS. Finally, OPD solution (100 μL of solution prepared by dissolving one capsule in 9 mL HO and 1 mL stable peroxide substrate buffer (10×), ThermoFisher) was added to each well. After 15 min, 50 μL of 4 M HCl (aq.) was added to each well to quench the reaction. Absorbance was measured at 490 nm and 590 nm using a CLARIOstar microplate reader. Measurements were performed in quadruplicate with three independent replicates.

[0261] The ELISA results for conjugates 36, 37, 38 and 39 are shown in Figure 17(B).

[0262] Example 17 Preparation of antibody-fluorophore conjugates 51-54 To a solution of trastuzumab conjugate 36, 37, 38, or 39 in PBS, CuSO 5H O (20 equiv. per alkyne), THPTA (100 equiv. per alkyne), sodium ascorbate (150 equiv. per alkyne), and AlexaFluor™ 488 azide (Thermo Fisher Scientific) (5 mM in DMSO, 12 equiv. per alkyne) were added. The mixture was vortexed and incubated at 37°C for 4 h (36 and 37) or 6 h (38 and 39). Excess reagent was removed using a Zeba™ Spin desalting column (7,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration in PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). UV-vis analysis revealed the conversion of 36, 37, 38, and 39 to antibody-fluorophore conjugates 51, 52, 53, and 54, with average fluorophore-to-antibody ratios (FAR) of 1.0, 2.0, 2.9, and 4.0, respectively. Each reaction was repeated at least three times, with consistent results, with FAR values ​​within ±0.1 units of those reported above.

[0263] FIG. 18 shows the fluorophore-to-antibody ratio (FAR) measured for conjugates 51, 52, 53 and 54.

[0264] Example 18 Stability analysis To a solution of trastuzumab-AlexaFluor488 conjugates 51, 52, 53, and 54 (138 μL, 3.75 μM) in PBS, 12 μL of reconstituted human plasma (Sigma) was added. The mixture was incubated at 37°C for 14 days. Aliquots were removed every 2 days, flash-frozen, and stored at -80°C until analysis. SDS-PAGE was followed by in-gel fluorescence and Coomassie Brilliant Blue staining.

[0265] Figure 19 shows the stability analysis of conjugates 51, 52, 53, and 54 in human plasma by SDS-PAGE. No transfer of the fluorescent payload to plasma proteins was observed over the duration of the study, demonstrating that the conjugates of the present invention are stable under physiological conditions.

[0266] Example 19 Preparation of Payload 55 Step 1: Synthesis of Payload 55 [ka] Payload 55 was synthesized as previously described (Walsh et al., Chem. Sci., 2019, 10(3), pp. 694-700).

[0267] Example 20 Preparation of Payload 56 Step 1: Synthesis of Fmoc-Val-Cit-PABC-MMAE (57) [ka] A solution of Fmoc-Val-Cit-PABC-PNP (21.6 mg, 30.1 μmol), MMAE (30.0 mg, 39.1 μmol), HOBt HO (10.1 mg, 60.0 μmol), DIPEA (15.7 μL, 90.0 μmol), and pyridine (24.3 μL, 300 μmol) in DMF (1 mL) was stirred at rt for 24 h. Upon completion, the reaction mixture was purified by automated reverse-phase FCC (10–70% solvent B in solvent A. Solvent A: 0.1 M NHOH (aq). Solvent B: MeCN) and lyophilized to afford the desired compound (39.0 mg, 29.0 μmol, 96%) as a white solid. HPLC (5–95% solvent B in solvent A) retention time 13.189 min; LRMS (ESI) m / z observed [M + Na]. + 1368.6, C 73 H 104 N 10 23 NaO 14 + Required value: 1367.8.

[0268] Step 2: Synthesis of Payload 56 [ka] To a solution of Fmoc-Val-Cit-PABC-MMAE (57) (20.0 mg, 14.9 μmol) in DMF (2 mL) was added piperidine (7.30 μL, 74.3 μmol), and the mixture was stirred at rt for 2 h. Upon completion, the reaction was concentrated under a stream of N. To the free amine was added HBTU (11.3 mg, 29.8 μmol) and DMF (2 mL), followed by N-PEG-COH (50 μL of a 0.5 M solution in TBME, 22.4 μmol) and DIPEA (7.80 μL, 44.7 μmol), and the reaction mixture was stirred at rt for 18 h. Upon completion, the reaction was concentrated under a stream of N and purified by automated reverse-phase FCC (10-100% solvent B in solvent A. Solvent A: 0.1 M NHOH(aq). Solvent B: MeCN) and lyophilized to give the title compound (13.6 mg, 9.80 μmol, 66%) as a white solid. HPLC (5-95% solvent B in solvent A) retention time 11.319 min; LRMS (ESI) m / z observed [M+H]. + 1282.8, C 68 H 112 N 13 O 17 + Required value 1382.8, measured m / z value [M+HCOO] - 1428.0, C 69 H 112 N 13 O 19 - Required value: 1426.8.

[0269] Example 21 Preparation of non-cleavable antibody-drug conjugates 58-61 To a solution of trastuzumab conjugate 36, 37, 38, or 39 in PBS, CuSO₄·5H₂O (150 equiv.), THPTA (600 equiv.), sodium ascorbate (1000 equiv.), and compound 55 (20 mM in DMSO, 100 equiv.) were added. The mixture was vortexed and incubated at 37°C for 6 h. Excess reagent was removed using a Zeba™ Spin desalting column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration in PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Hydrophobic interaction chromatography (HIC) revealed the conversion of 36, 37, 38, and 39 to antibody-drug conjugates 58, 59, 60, and 61, with average drug-to-antibody ratios (DAR) of 0.8, 1.4, 2.1, and 2.8, respectively.

[0270] The hydrophobic interaction chromatograms for conjugates 58, 59, 60 and 61 are shown in FIG.

[0271] Example 22 Preparation of cleavable antibody-drug conjugates 62-65 To a solution of trastuzumab conjugate 36, 37, 38, or 39 in PBS, CuSO₄·5H₂O (150 equiv.), THPTA (600 equiv.), sodium ascorbate (1000 equiv.), and compound 56 (20 mM in DMSO, 100 equiv.) were added. The mixture was vortexed and incubated at 37°C for 6 h. Excess reagent was removed using a Zeba™ Spin desalting column (40,000 MWCO, Thermo Fisher Scientific), followed by repeated diafiltration in PBS using an Amicon-Ultra centrifugal filter (10,000 MWCO, Merck Millipore). Hydrophobic interaction chromatography (HIC) revealed the conversion of 36, 37, 38, and 39 to antibody-drug conjugates 62, 63, 64, and 65, with average drug-to-antibody ratios (DAR) of 0.5, 1.0, 1.6, and 2.4, respectively.

[0272] The hydrophobic interaction chromatograms for conjugates 62, 63, 64 and 65 are shown in FIG.

[0273] Example 23 In vitro cytotoxicity cell line HER2-positive SKBR3 and BT474 cells were obtained from the American Type Culture Collection (ATCC), and HER2-negative MCF7 and MDA-MB-468 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC) and ATCC, respectively. SKBR3 cells were maintained in high-glucose McCoy's 5A medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), GlutaMAX™, 50 U / mL penicillin, and 50 μg / mL streptomycin. MCF7 and MDA-MB-468 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin. BT474 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin. All cell lines were incubated at 37°C in 5% CO.

[0274] cell viability Cells were seeded in 96-well plates at 37°C and 5% CO2 for 24 h. SKBR3 cells were seeded at 15,000 cells / well, BT474 cells at 20,000 cells / well, MCF7 cells at 7,500 cells / well, and MDA-MB-468 cells at 10,000 cells / well. Serial dilutions of 62, 63, 64, and 65 and trastuzumab were added to the cells in complete growth medium and incubated at 37°C and 5% CO2 for 96 h. Cell viability was measured using the CellTiter-Glo Viability Assay (Promega) according to the manufacturer's instructions. Cell viability was plotted as a percentage of untreated cells. Each measurement was performed in triplicate and three independent replicates were performed.

[0275] FIG. 22 shows the cytotoxicity of conjugates 62, 63, 64, 65 and trastuzumab.

[0276] Each of the conjugates exhibited significantly increased cytotoxicity in HER2-positive cells compared to trastuzumab alone. In contrast, proliferation of HER2-negative cells was unaffected compared to vehicle controls, thus confirming selectivity for HER2-positive cells. Furthermore, the higher DAR conjugates (conjugates 64 and 65) exhibited increased cytotoxicity relative to the lower DAR conjugates (conjugates 62 and 63). This demonstrates the value of the DAR tunability provided by the present invention.

[0277] Example 24 Live cell microscopy SKBR3 or MCF7 cells were seeded at 40,000 cells / well onto 8-well chambered μ-slides (Ibidi, 80826) at 37°C for 48 h in 5% CO2. The slides were then placed on ice and washed with Ham's F12 nutrient mix medium containing 10% FBS, 2 mM L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin (3 × 200 μL). Antibody conjugates 51 and 52 (50 nM), trastuzumab (50 nM), or vehicle (PBS) were added to the cells in complete F12 growth medium and incubated at 4°C in the dark for 1 h. The cells were returned to ice and washed with complete F12 growth medium (3 × 200 μL). Complete F12 growth medium (200 μL) was added, and the cells were incubated at 37°C for 3.5 h in 5% CO2. After 3 h of incubation, Hoechst 33342 trihydrochloride trihydrate (1 μg / mL, Invitrogen, H3570) was added. Live-cell microscopy was performed using an Operetta CLS confocal microscope (Perkin Elmer) with a 40× water-immersion objective. Cells were maintained at 37°C and 5% CO2 in a humidified atmosphere throughout the analysis. Data analysis was performed using ImageJ (Fiji).

[0278] Microscopy images are shown in Figure 23. Both conjugates can be seen to selectively label and take up into HER2-positive SKBR3 cells. No labeling is observed in HER2-negative MCF7 cells.

[0279] Example 25 Synthesis of tetra-DVP scaffold A Step 1: Synthesis of Compound 202 [ka] Compound 3 (4.43 g, 14.6 mmol) was dissolved in DMF (49 mL) and benzyl bromoacetate (3.47 mL, 21.9 mmol), and DIPEA (3.05 mL, 17.5 mmol) was added dropwise. The reaction mixture was stirred for 16 h. The mixture was diluted with water (850 mL) and extracted with EtOAc (3 × 300 mL). The combined organic extracts were washed with brine (5 × 350 mL), aqueous LiCl (3 M, 2 × 350 mL), dried (MgSO), filtered, and concentrated in vacuo. The crude mixture was subjected to flash column chromatography (0–50% EtOAc / Pet. ether 40–60) to afford the product, benzyl bis(2-((tert-butoxycarbonyl)amino)ethyl)glycinate (4.99 g, 11.1 mmol, 76%), as a colorless oil. f :0.39 (50% EtOAc / Pet. Ether 40-60); 1 H NMR (500 MHz, CDCl3, 25℃): δ (ppm) = 7.39-7.31 (m, 5H), 5.16 (s, 2H), 3.49 (br s, 2H), 3.20 (br s, 4H), 2.81 (br s, 4H), 1.44 (s, 18H); HRMS (ESI) C 22 H 35 N3O6 m / z: [M+Na] + 474.2576 (calculated value 474.2574).

[0280] Step 2: Synthesis of Compound 203 [ka] Benzyl ester 202 (7.78 g, 17.2 mmol) was dissolved in MeOH (170 mL) and degassed with N for 15 min. Palladium on carbon (10 wt% Pd, 340 mg) was added, and the suspension was flushed with H for 10 min. The vent needle was removed, a new H balloon was applied, and the suspension was stirred for 20 h. The mixture was filtered through Super-Cel®, washed with MeOH (2 × 125 mL), and concentrated in vacuo to give the product, bis(2-((tert-butoxycarbonyl)amino)ethyl)glycine (5.81 g, 16.1 mmol, 93%), as a white solid. 1 H NMR (500 MHz, DMSO-d6, 25℃): δ (ppm) = 6.64 (t, J = 5.0 Hz, 2H), 3.27 (s, 2H), 2.95 (q, J = 6.0 Hz, 4H), 2.60 (t, J = 6.7 Hz, 4H), 1.37 (s, 18H); 13 C NMR (126 MHz, DMSO-d6): δ (ppm) = 172.6, 155.6, 77.5, 54.9, 53.4, 38.4, 28.2; HRMS (ESI) C 16 H 31 N3O6 m / z: [M+H] + 362.2271 (calculated value 362.2286).

[0281] Step 3: Synthesis of Compound 204 [ka] Acid 203 (100 mg, 0.28 mmol) was dissolved in DMF (1 mL). 11-Azido-3,6,9-trioxaundecanamine (55 μL, 0.28 mmol), HBTU (105 mg, 0.28 mmol), and DIPEA (96 μL, 0.55 mmol) were added, and the reaction mixture was stirred for 71 h. The mixture was diluted with aqueous HCl (1 M, 25 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (25 mL), dried (NaSO), filtered, and concentrated in vacuo. The crude residue was subjected to flash column chromatography (0–6% MeOH / CHCl) to afford product 204 (94 mg, 0.17 mmol, 60%) as a colorless residue. f :0.27(6%MeOH / CH2Cl2); 1 H NMR (400 MHz, CDCl3, 25℃): δ (ppm) = 7.47 (br s, 1H), 5.68 (br s, 2H), 3.68-3.57 (m, 12H), 3.46 (q, J = 3.2 Hz, 2H), 3.37 (t, J = 4.9 Hz, 2H), 3.26-2.99 (m, 5H), 2.58 (br s, 3H), 1.44 (s, 18H); 13 C NMR (101 MHz, CDCl3): δ (ppm) = 171.0, 156.7, 79.5, 70.8, 70.7, 70.6, 70.2, 70.1, 69.6, 55.3, 50.8, 39.2, 38.3, 28.6; 24 H 47 N7O8 m / z: [M+Na] + 584.3401 (calculated value 584.3378).

[0282] Step 4: Synthesis of Compound 205 [ka] N-Boc amine 204 (64 mg, 0.11 mmol) was suspended in HCl / dioxane (4 M, 1 mL) and stirred for 1.5 h. The solvent was removed under a stream of nitrogen, leaving a white solid, which was used without further purification. The intermediate was suspended in DMF (0.5 mL) and DIPEA (119 μL, 0.68 mmol), and acid 203 (103 mg, 0.28 mmol) and HBTU (108 mg, 0.28 mmol) were added. The reaction mixture was stirred for 22 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (2 × 20 mL). The combined organic extracts were washed with brine (4 × 50 mL), dried (NaSO), filtered, and concentrated. The crude product was subjected to flash column chromatography (0–8% MeOH / CHCl) to give product 205 (53.5 mg, 0.051 mmol, 46%) as a colorless residue. f :0.15(8%MeOH / CH2Cl2); 1 H NMR (500 MHz, CDCl3, 25℃): δ (ppm) = 6.77 (s, 3H), 3.61 (t, J = 5.0 Hz, 3H), 3.58-3.51 (m, 8H), 3.47 (t, J = 6.0 Hz, 2H), 3.39 (t, J = 5.0 Hz, 2H), 3.28 (q, J = 5.8 Hz, 2H), 3.24 (s, 1H), 3.12 (s, 1H), 1.39 (s, 36H).; 13 C NMR (125MHz, CDCl3): δ (ppm) = 155.7, 78.2, 69.8, 69.8, 69.7, 69.6, 69.3, 68.9, 54.1, 50.0, 28.2; LRMS (ESI) C 46 H 89 N 13 O 14 m / z: [M+H] + 1048.7 (calculated value 1048.7).

[0283] Step 5: Synthesis of Compound 206 (Scaffold A) [ka] Tetra-N-Boc amine 205 (20 mg, 0.019 mmol) was dissolved in CHCl (1.5 mL) and 4 M HCl in dioxane (0.5 mL) was added. The reaction mixture was stirred for 1.5 h, and then the solvent was removed in vacuo. The intermediate was redissolved in DMF (1 mL) and compound 2 (22.3 mg, 0.095 mmol), HBTU (36.2 mg, 0.095 mmol), and DIPEA (33 μL, 0.191 mmol) were added. The reaction mixture was stirred for 3.5 h. LCMS indicated incomplete conversion, so additional compound 2 (8.9 mg, 0.038 mmol), HBTU (14.5 mg, 0.038 mmol), and DIPEA (13 μL, 0.076 mmol) were added. The mixture was stirred for an additional 1.5 h and then subjected to reverse-phase Combiflash chromatography (10-70% MeCN in 0.1 M aqueous NH4OH) to give the product 206 (9.05 mg, 0.0060 mmol, 32%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3, 25℃): δ (ppm) = 7.73 (br s, 2H; H), 6.55 (dd, J = 16.9, 10.1 Hz, 14H), 6.39 (d, J = 17.3 Hz, 8H), 5.62 (d, J = 10.5 Hz, 8H), 2.38 (t, J = 7.1 Hz, 9H), 1.95 (qu, J = 6.6 Hz, 9H); 13 CNMR (125 MHz, CDCl3): δ (ppm) = 174.3, 163.4, 161.6, 136.4, 131.3, 121.3, 107.9, 105.2, 70.7, 70.7, 70.6, 70.6, 70.6, 70.2, LRMS (ESI) C 74 H 109 N 25 O 10 m / z: [M+ formate ion] - 1553.0 (calculated value 1552.7).

[0284] Example 26 Synthesis of tetra-DVP scaffold B Step 1: Synthesis of Compound 207 [ka] Tetra N-Boc amine 205 (35 mg, 0.033 mmol) was suspended in HCl / dioxane (4 M, 1 mL) and stirred for 1.5 h. The solvent was removed under a stream of nitrogen, leaving a white solid, which was used without further purification. The intermediate was suspended in DMF (0.3 mL), and Fmoc-8-amino-3,6-dioxaoctanoic acid (51 mg, 0.13 mmol), HBTU (50 mg, 0.13 mmol), and DIPEA (70 μL, 0.40 mmol) were added. The reaction mixture was stirred for 18 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (2 × 20 mL). The combined organic extracts were washed with aqueous LiCl (5 wt%, 30 mL), dried (NaSO), filtered, and concentrated. The crude product was subjected to flash column chromatography (4-10% MeOH / CH2Cl2) to give the product 207 (13 mg, 0.0059 mmol, 18%) as a white residue. f :0.10(10%MeOH / CH2Cl2); 1H NMR (400 MHz, MeOD, 25℃): δ (ppm) = 7.76 (d, J = 7.5 Hz, 8H), 7.61 (d, J = 7.4 Hz, 8H), 7.36 (t, J = 7.4 Hz, 8H), 7.27 (t, J = 7.4 Hz, 8H), 4.58 (s, 2H), 4.46 (s, 1H), 4.32 (d, J = 6.8 Hz, 7H), 4.16 (t, J = 6.7 Hz, 4H), 3.94 (s, 8H), 3.64-3.57 (m, 17H), 3.56-3.53 (m, 8H), 3.53-3.43 (m, 10H), 3.36 (t, J = 5.4 Hz, 2H), 3.27 (t, J = 5.9 Hz, 18H), 3.20 (s, 2H), 3.18 (s, 4H), 3.05 (br s, 1H), 2.66 (t, J = 6.5 Hz, 4H), 2.61 (t, J = 6.2 Hz, 8H); 13 C NMR (101 MHz, MeOD): δ (ppm) = 174.2, 172.7, 158.8, 145.3, 142.6, 128.8, 128.2, 126.2, 121.0, 72.0, 71.6, 71.5, 71.4, 71.2, 71.1, 71.0, 67.7, 59.7, 55.9, 55.6, 48.5, 41.7, 40.0, 38.3; LRMS (ESI) C 110 H 141 N 17 O 26 m / z: [M+ギ acid イオン] - 2161.4 (calculated value 2161.0).

[0285] Process 2: Synthesis of Compound 208 (Surface B)

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[0286] Example 27 Synthesis of tetra-DVP scaffold C Step 1: Synthesis of Compound 209 [ka] Compound 204 (385 mg, 0.69 mmol) was dissolved in HCl solution (4 M in dioxane, 4 mL) and CHCl (2 mL) and stirred for 3 h. The solvent was removed under a stream of nitrogen, and the resulting residue was redissolved in DMF (2.5 mL). Fmoc-8-amino-3,6-dioxaoctanoic acid (532 mg, 1.38 mmol), HBTU (523 mg, 1.38 mmol), and DIPEA (721 μL, 4.14 mmol) were added, and the reaction mixture was stirred for 17 h. Some acid starting material remained, so additional HBTU (261 mg, 0.69 mmol) and DIPEA (120 μL, 0.69 mmol) were added, and the mixture was stirred for an additional 1 h. The mixture was directly subjected to reverse-phase column chromatography (10-100% MeCN in HO) to give the product 209 (378 mg, 0.34 mmol, 50%) as a viscous orange residue. f : 0.18 (7% MeOH in CH2Cl2); IR (neat): ν max (cm -1 )=3313 (NH, medium), 2928 (CH, medium), 2870 (CH, medium), 2107 (N3, medium), 1714 (C=O, strong), 1658 (C=O, strong), 1529 (NH, strong), 1448 (CH, strong), 1247 (CN, strong), 1102 (CO, strong); 1H NMR (500 MHz, CDCl3, 25℃): δ (ppm) = 7.75 (d, J = 7.5 Hz, 4H), 7.60 (d, J = 7.2 Hz, 4H), 7.39 (t, J = 7.5 Hz, 4H), 7.30 (t, J = 7.4 Hz, 4H), 7.18 (br s, 2H), 5.71 (br s, 2H), 4.40 (d, J = 6.4 Hz, 4H), 4.20 (t, J = 6.7 Hz, 2H), 3.99 (s, 4H), 3.68-3.51 (m, 24H), 3.43 (q, J = 5.6 Hz, 2H), 3.40-3.30 (m, 10H), 3.15 (s, 2H), 2.64 (t, J = 6.0 Hz, 4H); 13 C NMR (125 MHz, CDCl3): δ (ppm) = 171.4, 170.5, 156.8, 144.1, 141.5, 127.8, 127.2, 125.2, 120.1, 77.4, 71.1, 70.8, 70.7, 70.6, 70.6, 70.4, 70.2, 70.1, 70.1, 69.7, 66.7, 59.2, 54.8, 50.8, 47.4, 41.0, 39.0, 37.2; HRMS (ESI) C 56 H 73 O 14 N9m / z: [M+H] + 1096.5346 (calculated value 1096.5350).

[0287] Process 2: Synthesis of Compound 210

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[0288] Step 3: Synthesis of Compound 211 (Scaffold C) [ka] Tetra-N-Boc amine 210 (25 mg, 0.019 mmol) was dissolved in HCl / dioxane (4 M, 1 mL) and stirred for 1 h. The solvent was removed under a stream of nitrogen. Compound 2 (26.1 mg, 0.112 mmol) and HBTU (42.5 mg, 0.112 mmol) were added, and the combined solid was suspended in DMF (0.75 mL). DIPEA (59 μL, 0.336 mmol) was added, and the reaction mixture was stirred for 1 h. The mixture was purified by reverse-phase column chromatography (0.1 M NH4OH). (aq) in 10-100% MeCN) to afford the product 211 (9.71 mg, 0.0054 mmol, 28%) as a brown solid. 1 H NMR (500 MHz, DMSO-d6, 25℃): δ (ppm) = 7.80-7.72 (m, 7H), 7.68 (t, J = 5.7 Hz, 2H), 7.04 (t, J = 5.6 Hz, 4H), 6.76 (s, 4H), 6.58 (dd, J = 10.6, 17.3 Hz, 8H), 6.35 (d, J = 16.6 Hz, 8H), 5.58 (dd, J = 1.4, 10.3 Hz, 8H), 3.85 (s, 4H), 3.61-3.57 (m, 2H), 3.56-3.45 (m, 16H), 3.44-3.36 (m, 9H), 3.29 (q, J = 6.6 Hz, 9H), 3.24 (q, J = 5.9 Hz, 6H), 3.17 (q, J = 6.3 Hz, 4H), 3.11 (q, J = 6.2 Hz, 8H), 3.08-3.03 (m, 6H), 2.56 (t, J = 6.8 Hz, 4H), 2.14 (t, J = 7.7 Hz, 8H), 1.76 (m, J = 7.2 Hz, 9H), 0.95 (d, J = 6.5 Hz, 4H); 13C NMR (125 MHz, DMSO-d6): δ (ppm) = 172.1, 170.6, 170.5, 169.2, 163.1, 162.4, 136.1, 121.4, 104.5, 70.1, 70.0, 69.8, 69.7, 69.7, 69.5, 69.3, 69.2, 69.0, 70.0, 58.3, 58.0, 54.2, 54.1, 50.0, 40.3, 40.1, 39.9, 39.8, 39.6, 38.1, 36.8, 36.4, 33.1; HRMS (ESI) C 86 H 132 O 16 N 27 m / z: [M+H] + 1799.0355 (calculated value 1799.0340).

[0289] Example 28 Synthesis of tetra-DVP scaffold D Step 1: Synthesis of Compound 212 [ka] Tetra-N-Boc amine 210 (102 mg, 0.076 mmol) was dissolved in HCl solution (4 M in dioxane, 2 mL) and stirred for 1 h. The solvent was removed under a stream of nitrogen, and the resulting white solid was redissolved in DMF (2 mL). Fmoc-8-amino-3,6-dioxaoctanoic acid (176 mg, 0.46 mmol), HBTU (173 mg, 0.46 mmol), and DIPEA (239 μL, 1.37 mmol) were added, and the reaction mixture was stirred for 19.5 h. The mixture was subjected to reverse-phase column chromatography (10–100% MeCN in HO) to give product 212 (50.6 mg, 0.021 mmol, 28%) as a white solid. 1H NMR (500 MHz, DMSO-d6, 25℃): δ (ppm) = 7.87 (d, J = 7.5 Hz, 8H), 7.77-7.71 (m, 3H), 7.68 (dをincludingむm, J = 7.5 Hz, 13H), 7.40 (t, J = 7.4 Hz, 8H), 7.31 (tをincludingむm, J = 7.4 Hz, 11H), 4.28 (d, J = 6.9 Hz, 8H), 4.20 (t, J = 6.8 Hz, 4H), 3.87-3.84 (m, 11H), 3.58-3.47 (m, 32H), 3.44-3.39 (tをincludingむm, J = 5.2 Hz, 13H), 3.36 (t, J = 4.9 Hz, 2H), 3.24 (q, J = 5.6 Hz, 6H), 3.15 (m, J = 6.4 Hz, 19H), 3.07 (s, 6H), 2.56 (t, J = 6.7 Hz, 11H); 13 C NMR (100 MHz, DMSO-d6): δ (ppm) = 170.6, 169.8, 169.3, 169.2, 142.6, 139.4, 137.4, 128.9, 127.3, 121.4, 120.0, 109.8, 70.2, 70.2, 70.0, 69.8, 69.7, 69.7, 69.5, 69.3, 69.0, 58.0, 54.1, 50.0, 40.3, 40.1, 39.8, 38.1, 36.4; HRMS (ESI) C 122 H 164 O 32 N 19 m / z: [M+H] + 2407.1743 (calculated value 2407.1784).

[0290] Project 2: Synthesis of Compound 213 (Surface D)

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[0291] Example 29 Synthesis of tetra-DVP scaffold E Step 1: Synthesis of Compound 216 [ka] To a mixture of compound 214 (10 g, 45.61 mmol) and compound 215 (4.9 g, 38.23 mmol) in DCM (100 mL) was added BuNBr (2.4 g, 7.44 mmol) and NaOH (9.85 g, 246.31 mmol). The reaction mixture was stirred at 15 °C for 3 h. The reaction mixture was poured into water (200 mL) and extracted with DCM (200 mL × 2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, and concentrated in vacuo to give a residue, which was purified by column (SiO, 30% to 40% EtOAc in petroleum) to give compound 216 (5.2 g, 32.8% yield) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ 3.73–3.69 (m, 2 H), 3.68–3.64 (m, 10 H), 3.64–3.59 (m, 4 H), 2.04 (s, 2 H), 1.44 (s, 9 H).

[0292] Step 2: Synthesis of Compound 217 [ka] A mixture of compound 216 (5.2 g, 14.97 mmol) in HCl / dioxane (4 M, 50 mL) was stirred at 20° C. for 2 hours. LCMS showed that the starting material was consumed and the desired mass was detected as the major product. The reaction mixture was concentrated in vacuo to give compound 217 (4.3 g, 98.6% yield) as a yellow oil. LCMS: rt=0.502 min, (292.2 [M+H] + ), 74.6% purity.

[0293] Step 3: Synthesis of Compound 219 [ka] To a mixture of compound 217 (4.3 g, 14.76 mmol) and compound 218 (2.8 g, 16.24 mmol) in DCM (60 mL) was added HATU (5.6 g, 14.76 mmol) and DPIEA (9.5 g, 73.81 mmol). The reaction mixture was stirred at 20 °C for 1 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B%: 30%-50%). The mixture was adjusted to pH = 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (200 mL × 2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, and concentrated in vacuo to give compound 219 (5 g, 75.7% yield) as a pale yellow oil. LCMS: rt = 0.740 min, (448.3 [M+H] + ), purity 100.00%. 1H NMR (400 MHz, methanol-d4) δ 3.72 (t, J= 6.0 Hz, 2 H), 3.69 - 3.61 (m, 14 H), 3.39 - 3.36 (m, 2 H), 3.21 (t, J= 6.8 Hz, 2 H), 3.07 (t, J= 6.8 Hz, 2 H), 2.43 (t, J= 6.0 Hz, 2 H), 1.66 - 1.58 (m, 2 H), 1.44 (s, 9 H).

[0294] Step 4: Synthesis of Compound 220 [ka] To a mixture of compound 219 (5 g, 11.17 mmol) in dioxane (30 mL) was added HCl / dioxane (4 M, 30 mL). The reaction mixture was stirred at 20° C. for 1 hour. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 220 (4.6 g, 97.9% yield) as a pale yellow oil. LCMS: rt=0.610 min, (348.2 [M+H] + ), 100.00% purity.

[0295] Step 5: Synthesis of Compound 221 [ka] To a mixture of compound 220 (2.3 g, 5.99 mmol) and compound 203 (2.17 g, 5.99 mmol) in DCM (50 mL) was added DIPEA (2.3 g, 17.97 mmol) and HATU (2.28 g, 5.99 mmol). The reaction mixture was stirred at 20 °C for 8 h. LCMS showed the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% HCl)-ACN; B% 10% to 54%). The mixture was adjusted to pH = 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (100 mL × 2). The combined organic layers were washed with brine, dried over NaSO, and concentrated in vacuo to give compound 221 (3.8 g, 88.5% yield) as a yellow oil. m / z 691.4 (M+H)+ (ES + ). LCMS: rt = 0.530 min, (691.4 [M+H] + ), purity 96.41%. 1 H NMR (400 MHz, methanol-d4) δ 3.76 (t, J= 6.0 Hz, 2 H), 3.69 - 3.61 (m, 14 H), 3.37 (t, J= 5.2 Hz, 2 H), 3.29 - 3.24 (m, 4 H), 3.19 - 3.11 (m, 6 H), 2.60 (t, J= 6.0 Hz, 2 H), 2.48 (t, J= 6.0 Hz, 2 H), 1.68 (q, J= 6.4 Hz, 2 H), 1.44 (s, 18 H).

[0296] Step 6: Synthesis of Compound 222 [ka] To a mixture of compound 221 (3.8 g, 5.50 mmol) in dioxane (20 mL) was added HCl / dioxane (4 M, 20 mL). The reaction mixture was stirred at 20° C. for 16 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 222 (3.2 g, 96.9% yield) as a pale yellow oil. LCMS: rt=0.404 min, (491.2 [M+H] + ), 82.75% purity.

[0297] Step 7: Synthesis of Compound 223 [ka] To a mixture of compound 222 (1.65 g, 2.75 mmol) and compound 203 (2.09 g, 5.78 mmol) in DCM (20 mL) was added DIPEA (2.13 g, 16.50 mmol) and HATU (2.20 g, 5.78 mmol). The reaction mixture was stirred at 20 °C for 1 h. LCMS showed the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B% 50% to 70%) and then concentrated in vacuo to give compound 223 (2.7 g, 79.8% yield) as a pale yellow solid. LCMS: rt = 0.739 min, (1178.0 [M+H] + ), purity 95.74%. 1H NMR (400 MHz, methanol-d4) δ 3.73 (t, J= 6.0 Hz, 2 H), 3.70 - 3.67 (m, 2 H), 3.66 - 3.65 (m, 4 H), 3.64 - 3.63 (m, 4 H), 3.62 - 3.58 (m, 4 H), 3.39 - 3.33 (m, 6 H), 3.29 - 3.22 (m, 6 H), 3.17 (s, 4 H), 3.13 (t, J= 6.0 Hz, 8 H), 2.74 (t, J= 6.4 Hz, 4 H), 2.60 (t, J= 6.0 Hz, 8 H), 2.46 (t, J= 6.0 Hz, 2 H), 1.75 - 1.67 (m, 2 H), 1.44 (s, 36 H).

[0298] Step 8: Synthesis of Compound 224 [ka] To a mixture of compound 223 (600 mg, 0.51 mmol) in dioxane (4 mL) was added HCl / dioxane (4 M, 2 mL). The reaction mixture was stirred at 20° C. for 8 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 224 (525 mg, 99.8% yield) as a pale yellow solid. LCMS: rt=0.390 min, (777.3 [M+H] + ), 93.39% purity.

[0299] Step 9: Synthesis of Compound 225 (Scaffold E) [ka] To a mixture of compound 2 (593.2 mg, 2.54 mmol) and compound 224 (525 mg, 0.51 mmol) in DCM (8 mL) was added DIPEA (1.31 g, 10.17 mmol). HATU (773.58 mg, 2.03 mmol) was then slowly added to the above solution. The reaction mixture was stirred at 20 °C for 1 h. LCMS showed the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Waters Xbridge 150 mm × 25 mm × 5 μm; mobile phase: [water (ammonia hydroxide v / v)-ACN]; B%: 37% to 67%, 9 min) and lyophilized to give 225 scaffold E (236 mg, 25.7% yield) as a yellow oil. The product was then immediately dissolved in DMSO (13 mL). LCMS:rt=1.022min, (1638.9[M+H] + ), 93.22% purity.

[0300] Example 30 Synthesis of tetra-DVP scaffold F Step 1: Synthesis of Compound 226 [ka] To a mixture of compound 202 (10 g, 22.15 mmol) in dioxane (50 mL) was added HCl / dioxane (4 M, 50 mL). The reaction mixture was stirred at 20° C. for 8 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 226 (7.9 g, 98.9% yield) as a white solid. LCMS: rt=0.435 min, (252.3 [M+H] + ), 100.00% purity.

[0301] Step 2: Synthesis of Compound 228 [ka] To a mixture of compound 226 (5 g, 12.59 mmol) and compound 227 (6.96 g, 26.44 mmol) in DCM (100 mL) was added DIPEA (11.39 g, 88.12 mmol) and HATU (10.05 g, 26.44 mmol). The reaction mixture was stirred at 20 °C for 2 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase combiflash (water (0.1% FA)-ACN; B% 70%-90%). The mixture was adjusted to pH 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (100 mL × 2). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, and concentrated in vacuo to give compound 228 (6.1 g, 57.4% yield) as a yellow oil. LCMS: rt = 0.555 min, (742.6 [M+H] + ), purity 87.94%. 1 H NMR (400 MHz, methanol-d4) δ 7.39 - 7.31 (m, 5 H), 5.16 (s, 2 H), 3.99 (s, 4 H), 3.63 - 3.69 (m, 8 H), 3.55 (s, 2 H) 3.51 (t, J= 5.6 Hz, 4 H), 3.34 - 3.31 (m, 4 H), 3.22 (t, J= 5.6 Hz, 4 H), 2.79 (t, J= 6.0 Hz, 4 H), 1.43 (s, 18 H).

[0302] Step 3: Synthesis of Compound 229 [ka] To a mixture of compound 228 (6.1 g, 8.22 mmol) in MeOH (200 mL) under N was added Pd / C (800 mg, 10% purity). The reaction mixture was stirred at 20 °C under H (15 psi) for 3 h. LCMS showed the reaction was complete. The catalyst was filtered off. The filtrate was concentrated in vacuo to give compound 229 (5.3 g, 98.9% yield) as a colorless oil. LCMS: rt = 0.823 min, (652.5 [M+H] + ), purity 100.00%. 1 H NMR (400 MHz, methanol-d4) δ 4.04 (s, 4 H), 3.74–3.68 (m, 4 H), 3.68–3.62 (m, 6 H), 3.55 (t, J = 6.0 Hz, 8 H), 3.26–3.17 (m, 8 H), 1.44 (s, 18 H).

[0303] Step 4: Synthesis of Compound 230 [ka] To a mixture of compound 229 (700 mg, 1.17 mmol) and compound 221 (1.52 g, 2.33 mmol) in DCM (20 mL) was added DIPEA (904.73 mg, 7.00 mmol) and HATU (887.23 mg, 2.33 mmol). The reaction mixture was stirred at 20 °C for 2 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B% 50%-70%) and lyophilized to give compound 230 (800 mg, 39.0% yield) as a yellow oil. 1H NMR (400 MHz, methanol-d4) δ 4.02 (s, 8 H), 3.73 (t, J= 6.0 Hz, 2 H), 3.71 - 3.68 (m, 10 H), 3.67 - 3.66 (m, 10 H), 3.65 - 3.61 (m, 10 H), 3.53 (t, J= 6.0 Hz, 8 H), 3.43-3.40 (m, 2 H), 3.35 (t, J= 6.0 Hz, 12 H), 3.27 - 3.22 (m, 18 H), 2.75 - 2.67 (m, 12 H), 2.47 (t, J= 6.0 Hz, 2 H), 1.74 - 1.67 (m, 2 H), 1.44 (s, 36 H).

[0304] Step 5: Synthesis of Compound 231 [ka] To a mixture of compound 230 (800 mg, 0.46 mmol) in dioxane (5 mL) was added HCl / dioxane (4 M, 5 mL). The reaction mixture was stirred at 20° C. for 4 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 231 (700 mg, 95.4% yield) as a yellow oil. LCMS: rt=0.471 min, (1357.7 [M+H] + ), 59.49% purity.

[0305] Step 6: Synthesis of Compound 232 (Scaffold F) [ka] To a mixture of compound 2 (343.6 mg, 1.47 mmol) and compound 231 (400 mg, 294.64 μmol) in DCM (20 mL) was added DIPEA (571.2 mg, 4.42 mmol). HATU (448.1 mg, 1.18 mmol) was then slowly added to the above solution. The reaction mixture was stirred at 20 °C for 1 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Waters Xbridge 150 mm × 25 mm × 5 μm; mobile phase: [water (ammonia hydroxide v / v)-ACN]; B%: 30% to 60%, 9 min) and lyophilized to give 232 scaffold F (155 mg, 22.7% yield) as a yellow oil. The product was then immediately dissolved in DMSO (7 mL). LCMS:rt=0.961min, (1110.3[[M+H] / 2] + ), 97.65% purity.

[0306] Example 31 Synthesis of tetra-DVP scaffold G Step 1: Synthesis of Compound 233 [ka] To a mixture of compound 229 (3 g, 4.60 mmol) and compound 219 (1.93 g, 4.60 mmol) in DCM (30 mL) was added DIPEA (2.97 g, 23.02 mmol) and HATU (1.75 g, 4.60 mmol). The reaction mixture was stirred at 20 °C for 1 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B% 40%-60%). The mixture was adjusted to pH = 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (200 mL × 2). The combined organic layers were washed with brine (200 mL), dried over sodium sulfate, and concentrated in vacuo to give compound 233 (2.0 g, 44.3% yield) as a yellow oil. LCMS: rt = 0.533 min, (981.4 [M+H] + ), purity 94.19%. 1 H NMR (400 MHz, methanol-d4) δ 4.00 (s, 4 H), 3.74 (t, J= 6.0 Hz, 2 H), 3.69 - 3.61 (m, 22 H), 3.53 (t, J= 6.0 Hz, 4 H), 3.40 - 3.34 (m, 6 H), 3.28 - 3.22 (m, 10 H), 2.71 (t, J= 6.8 Hz, 4 H), 2.46 (t, J= 6.0 Hz, 2 H), 1.70 (q, J= 6.4 Hz, 2 H) 1.44 (s, 18 H).

[0307] Step 2: Synthesis of Compound 234 [ka] To a mixture of compound 233 (2 g, 2.04 mmol) in dioxane (10 mL) was added HCl / dioxane (4 M, 9.1 mL). The reaction mixture was stirred at 20° C. for 4 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 234 (1.81 g, 100% yield) as a yellow oil. LCMS: rt=0.354 min, (781.5 [M+H] + ), 55.4% purity.

[0308] Step 3: Synthesis of Compound 235 [ka] To a mixture of compound 203 (487.1 mg, 1.35 mmol) in DCM (5 mL) was added HATU (512.5 mg, 1.35 mmol) and DIPEA (348.4 mg, 2.70 mmol). The reaction mixture was stirred at 20 °C for 0.5 h. Then, a solution of compound 234 (600 mg, 0.67 mmol) and DIPEA (522.6 mg, 4.04 mmol) in DCM (5 mL) was added dropwise to the above solution. The reaction mixture was stirred at 20 °C for another 0.5 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B% 40%-60%). The mixture was adjusted to pH = 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (100 mL x 2). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, and concentrated in vacuo to give compound 235 (360 mg, 34.5% yield) as a yellow oil. LCMS: rt = 0.739 min, (1468.1 [M+H] + ), purity 100.00%. 1H NMR (400 MHz, methanol-d4) δ 4.01 (s, 4 H), 3.76 - 3.70 (m, 4 H), 3.70 - 3.59 (m, 28 H), 3.45 (t, J= 5.6 Hz, 4 H), 3.40 - 3.34 (m, 6 H), 3.29 - 3.22 (m, 6 H), 3.18 - 3.10 (m, 14 H), 2.71 (t, J= 6.6 Hz, 4 H), 2.60 (br t, J= 6.0 Hz, 8 H), 2.46 (t, J= 6.0 Hz, 2 H), 1.74 - 1.66 (m,2 H), 1.45 (s, 36 H).

[0309] Step 4: Synthesis of Compound 236 [ka] To a mixture of compound 235 (250 mg, 0.17 mmol) in dioxane (4 mL) was added HCl / dioxane (4 M, 1 mL). The reaction mixture was stirred at 20° C. for 2 hours. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give compound 236 (225 mg, 99% yield) as a yellow oil. LCMS: rt=0.354 min, (1167.8 [M+H] + ), 75.55% purity.

[0310] Step 5: Synthesis of Compound 237 (Scaffold G) [ka] To a mixture of compound 236 (225 mg, 0.17 mmol) in DCM (10 mL), DIPEA (439.7 mg, 3.40 mmol) and compound 2 (198.4 mg, 0.85 mmol) were added. HATU (323.5 mg, 0.85 mmol) was then slowly added portionwise to the above solution. The reaction mixture was stirred at 20 °C for 1 h. LCMS showed that the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Waters Xbridge 150 mm × 25 mm × 5 μm; mobile phase: [water (ammonia hydroxide v / v)-ACN]; B%: 36% to 66%, 9 min) and lyophilized to give 237 scaffold G (278 mg, 78.2% yield) as a yellow oil. The product was then immediately dissolved in DMSO (13 mL). LCMS:rt=0.958min, (1929.4[M+H] + ), 95.17% purity.

[0311] Example 32 Synthesis of tetra-DVP scaffold H Step 1: Synthesis of Compound 238 [ka] To a mixture of compound 229 (878.5 mg, 1.35 mmol) in DCM (10 mL) was added DIEA (174.2 mg, 1.35 mmol) and HATU (512.5 mg, 1.35 mmol). The reaction mixture was stirred at 20 °C for 0.5 h. Then, a solution of compound 234 (600 mg, 0.67 mmol) and DIEA (522.61 mg, 4.04 mmol) in DCM (5 mL) was added to the above solution. The reaction mixture was stirred at 20 °C for another 0.5 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by reverse-phase Combiflash (water (0.1% FA)-ACN; B% 60%-80%). The mixture was adjusted to pH = 8 with aq. NaHCO solution and concentrated in vacuo to remove CHCN. The mixture was then extracted with EtOAc (100 mL x 2). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate, and concentrated in vacuo to give compound 238 (360 mg, 26.1% yield) as a yellow oil. LCMS: rt = 0.740 min, (1025.2 [[M+H] / 2] + ), purity 100.00%. 1 H NMR (400 MHz, methanol-d4) δ 4.02 (s, 12 H), 3.76 - 3.71 (m, 4 H), 3.70 - 3.64 (m, 32 H), 3.63 - 3.58 (m, 8 H), 3.53 (t, J= 5.6 Hz, 8 H), 3.47 - 3.42 (m, 4 H), 3.41 - 3.34 (m, 14 H), 3.28 - 3.22 (m, 18 H), 2.71 (t, J= 6.4 Hz, 12 H), 2.47 (t, J= 6.0 Hz, 2 H), 1.74-1.67 (m, J=6.4 Hz, 2 H), 1.44 (s, 36 H).

[0312] Step 2: Synthesis of Compound 239 [ka] To a mixture of compound 238 (300 mg, 0.15 mmol) in dioxane (5 mL) was added HCl / dioxane (4 M, 5 mL). The reaction mixture was stirred at 20° C. for 4 hours. The reaction mixture was concentrated in vacuo to give compound 239 (270 mg, 96.9% yield) as a yellow oil. LCMS: rt=0.414 min, (1647.9 [M+H] + ), 44.49% purity.

[0313] Step 2: Synthesis of Compound 240 (Scaffold H) [ka] To a mixture of compound 239 (270 mg, 0.14 mmol) and compound 2 (165.5 mg, 0.71 mmol) in DCM (5 mL) was added DIEA (366.7 mg, 2.84 mmol). HATU (215.8 mg, 0.57 mmol) was then added to the above solution. The reaction mixture was stirred at 20 °C for 1 h. LCMS showed the reaction was complete. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Waters Xbridge 150 mm × 25 mm × 5 μm; mobile phase: [water (ammonia hydroxide v / v)-ACN]; B%: 34% to 64%, 9 min) and lyophilized to give 240, scaffold H (131 mg, 34.4% yield) as a yellow oil. The product was then immediately dissolved in DMSO (4.7 mL). LCMS:rt=0.946min, (1255.3[[M+H] / 2] + ), 100.00% purity.

[0314] Example 32 Synthesis of DBCO Reagent 247 Step 1: Synthesis of Fmoc-Val-Cit-OH(241) [ka] L-Citrulline (85 mg, 0.486 mmol) in DME (1.5 mL) was added to a solution of Fmoc-Val-OSu (202 mg, 0.463 mmol) and NaHCO (42.8 mg, 0.509 mmol) in HO (3 mL) and THF (4 mL) at 0 °C. The reaction was warmed to rt and stirred for 48 h. Upon completion, the reaction was adjusted to pH 10 with sat. aq. KCO and washed with EtOAc (2 × 20 mL). The aqueous layer was acidified to pH 4 with 10% aq. citric acid, and the formed gelatinous mixture was filtered and dried in vacuo to give Fmoc-Val-Cit-OH 241 (141 mg, 0.284 mmol, 61%) as an off-white solid. 1 H NMR (700 MHz, DMSO-d6) δ ppm: 7.89 (d, 2H, J = 7.6 Hz), 7.75 (dd, 2H, J = 11.7, 7.8 Hz), 7.41 (t, 2H, J = 7.4 Hz), 7.34-7.30 (m, 2H), 6.00 (s, 1H), 4.31-4.26 (m, 1H), 4.27-4.19 (m, 2H), 4.17-4.13 (m, 1H), 3.94-3.87 (m, 1H), 2.99-2.92 (m, 2H), 1.98 (apparently sx, 1H, J = 6.8 Hz), 1.74-1.66 (m, 1H), 1.61-1.53 ​​(m, 1H), 1.46-1.35 (m, 2H), 0.89 (d, 3H, J = 6.8 Hz), 0.86 (d, 3H, J = 6.8 Hz). 13 C NMR (176 MHz, DMSO-d6) δ ppm: 173.4, 171.3, 158.8, 156.1, 143.9, 143.9, 140.7, 127.7, 127.1, 125.4, 120.1, 65.7, 59.8, 51.9, 46.7, 38.8, 30.6, 28.4, 26.6, 19.2, 18.2. HRMS (ESI) m / z actual value [M+H] + 497.2394, C 26 H 33 N4O6 + Calculated value: 497.2395.

[0315] Step 2: Synthesis of Fmoc-Val-Cit-PABA(242) [ka] A solution of Fmoc-Val-Cit-OH 241 (104 mg, 0.210 mmol), 4-aminobenzyl alcohol (PABA, 52.0 mg, 0.419 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 104 mg, 0.419 mmol) in DCM (2.5 mL) and MeOH (1.5 mL) was stirred at rt for 22 h. Upon completion, the mixture was diluted with EtO (10 mL), filtered, and washed with EtO (2 × 4 mL) to give Fmoc-Val-Cit-PABA 242 (66.4 mg, 0.110 mmol, 53%) as a white solid. 1 H NMR (700 MHz, DMSO-d6) δ ppm: 9.98 (s, 1H), 8.12 (d, 1H, J = 7.5 Hz), 7.89 (d, 2H, J = 7.6 Hz), 7.74 (t, 2H, J = 8.1 Hz), 7.54 (d, 2H, J = 8.4 Hz), 7.46-7.39 (m, 3H), 7.34-7.30 (m, 2H), 7.23 (d, 2H, J = 8.4 Hz), 6.00 (t, 1H, J = 5.8 Hz), 5.41 (s, 2H), 5.09 (t, 1H, J = 5.5 Hz), 4.43-4.39 (m, 3H), 4.33-4.20 (m, 3H), 3.95-3.91 (m, 1H), 3.06-2.89 (m, 2H), 2.04-1.95 (m, 1H), 1.74-1.55 (m, 2H), 1.50-1.32 (m, 2H), 0.89-0.84 (m, 6H); 13C NMR (176 MHz, DMSO-d6) δ ppm: 171.3, 170.4, 158.9, 156.1, 143.9, 140.7, 137.5, 137.4, 127.7, 127.1, 126.9, 125.4, 120.1, 118.9 , 65.7, 62.6, 60.1, 53.1, 46.7, 38.6, 30.5, 29.5, 26.8, 19.2, 18.3; HRMS (ESI) m / z actual measurement value [M+H] + 602.2968, C 33 H 40 N5O6 + Calculated value 602.2973.

[0316] Step 3: Synthesis of Boc-PEG-Val-Cit-PABA (243) [ka] A solution of Fmoc-Val-Cit-PABA 242 (100 mg, 0.166 mmol) and diethylamine (343 μL, 3.33 mmol) in DMF (1.5 mL) was stirred at rt for 19 h. Upon completion, the solvent was evaporated under reduced pressure, and the product was crashed out with DCM, filtered, and washed with EtOAc and EtO to give H-Val-Cit-PABA (52.3 mg, 0.138 mmol, 83%) as a white solid, which was carried forward without further purification. A solution of H-Val-Cit-PABA (52.3 mg, 0.138 mmol), Boc-PEG-COOH (50.0 mg, 0.138 mmol), HBTU (52.5 mg, 0.138 μmol), and DIPEA (48.0 μL, 0.276 μmol) in DMF (3 mL) was stirred at rt for 2 h. Upon completion, the reaction was concentrated under a stream of N, and the crude residue was purified by reverse-phase flash column chromatography (5–100% solvent B in solvent A; solvent A: 0.1 M NHOH(aq), solvent B: MeCN) and lyophilized to give Boc-PEG-Val-Cit-PABA 243 (50.5 mg, 69.4 μmol, 51%) as a white solid. 1H NMR (700 MHz, DMSO-d6) δ ppm: 9.87 (s, 1H), 8.07 (d, 1H, J = 7.6 Hz), 7.86 (d, 1H, J = 8.7 Hz), 7.53 (d, 2H, J = 8.5 Hz), 7.21 (d, 2H, J = 8.6 Hz), 6.73 (t, 1H, J = 5.0 Hz), 6.00 (t, 1H, J = 5.8 Hz), 5.39 (s, 2H), 5.09 (t, 1H, J = 5.7 Hz), 4.41 (d, 2H, J = 5.5 Hz), 4.39-4.31 (m, 1H), 4.23-4.17 (m, 1H), 3.63-3.53 (m, 2H), 3.50-3.43 (m, 12H), 3.07-2.96 (m, 3H), 2.96-2.87 (m, 1H), 2.47-2.42 (m, 1H), 2.40-2.31 (m, 1H), 1.95 (h, 1H, J = 6.8 Hz), 1.76-1.63 (m, 1H), 1.62-1.52 (m, 1H), 1.45-1.38 (m, 2H), 1.37-1.35 (s, 9H), 0.82 (dd, 6H, J = 6.8 Hz); 13 C NMR (176 MHz, DMSO-d6) δ ppm: 171.6, 170.8, 159.3, 156.0, 137.9, 127.4, 119.3, 78.1, 70.2, 70.2, 70.1, 69.9, 69.9 69.6, 67.4, 63.0, 58.0, 53.5, 39.0, 36.4, 31.0, 29.8, 28.7, 27.3, 19.6, 18.5; LRMS (ESI) m / z found [M+H] + 726.8, C 34 H 59 N6O 11 + The calculated value is 727.4273.

[0317] Process 4: Synthesis of Boc-PEG4-Val-Cit-PABC-MMAE(244)

change

[0318] Step 5: Synthesis of Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2(245) [ka] A solution of Boc-PEG4-Val-Cit-PABC-MMAE 244 (40.0 mg, 27.2 μmol), TIPS (100 μL), TFA (500 μL), and DCM (950 μL) was stirred at rt for 20 min. Upon completion, the reaction mixture was concentrated under a stream of N2 and carried forward without further purification. A solution of H-PEG4-Val-Cit-PABC-MMAE (37.3 mg, 27.2 μmol), Fmoc-PEG2-Glu-PEG2-Glu-NH2 (8.57 mg, 10.9 μmol), HATU (18.7 mg, 49.1 μmol), and DIPEA (12.3 μL, 70.9 μmol) in DMF (2 mL) was stirred at rt for 48 h. Upon completion, the reaction was concentrated under a stream of N2. 、 The crude product was purified by preparative HPLC and lyophilized to give Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE) 245 (1.10 mg, 0.315 μmol, 3%) as a white solid. HPLC (5-95% MeCN / HO over 20 min) retention time: 12.721 min; LRMS (ESI) m / z observed [M+2H]. + 1165.3, [M+3H] + 1747.7, [M+4H] + 873.9, C 175 H 276 N 27 O 46 + Required value: 3492.0073.

[0319] Step 6: Synthesis of Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2(246) [ka] A solution of Boc-PEG-Val-Cit-PABC-MMAE 244 (38.0 mg, 25.8 μmol), TIPS (100 μL), TFA (500 μL), and DCM (950 μL) was stirred at rt for 20 min. Upon completion, the reaction mixture was concentrated under a stream of N, dissolved in HO (5 mL), lyophilized, and carried forward without further purification. A solution of Fmoc-PEG-Glu-PEG-Glu-NH (10.9 mg, 11.7 μmol), BTTFH (7.77 mg, 24.6 μmol), and DIPEA (8.55 μL, 49.2 μmol) in DMF (0.5 mL) was stirred at 0 °C for 1.5 h. To the solution was added TFA.HN-PEG4-Val-Cit-PABC-MMAE (33.7 mg, 23.0 μmol) and DIPEA (6.09 μL, 35.0 μmol) in DMF (0.5 mL), and the reaction mixture was stirred at rt for 24 h. Upon completion, the reaction was concentrated under a stream of N. 、 The crude product was purified by preparative HPLC and lyophilized to give Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2246 (6.39 mg, 1.76 μmol, 15%) as a white solid. HPLC (5-95% MeCN / HO over 20 min) retention time 12.720 min; HRMS (ESI) m / z observed [M+H]. + 3637.140, C 181 H 387 N 28 O 49 + Required value: 3637.0773.

[0320] Step 7: Synthesis of DBCO-PEG-PEG-Glu(-PEG-Val-Cit-PABC-MMAE)-PEG-PEG-Glu(-PEG-Val-Cit-PABC-MMAE)-NH (247) [ka] Polymer-bound piperazine (30 mg, mmol, 200-400 mesh, 1.0-2.0 mmol / g loading, 2% cross-linked with divinylbenzene) was added to a solution of Fmoc-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2 (3.60 mg, 0.989 μmol) in DMF (200 μL), followed by the addition of 0.1% DBU. The resulting mixture was stirred for 2 h at room temperature. Upon completion, the solvent was evaporated under a stream of N2, and the residue was used without further purification. A solution of H-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE) (3.38 mg, 0.989 μmol), DBCO-PEG5-COOH (1.60 mg, 2.68 μmol), HATU (1.02 mg, 2.68 μmol), and DIPEA (0.932 μL, 5.36 μmol) in DMF (100 μL) was stirred at rt for 24 h. Upon completion, the reaction was concentrated under a stream of N2. 、 The crude product was purified by preparative HPLC to give DBCO-PEG5-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-PEG2-PEG2-Glu(-PEG4-Val-Cit-PABC-MMAE)-NH2247 (0.60 mg, 0.150 μmol, 16%) as a white solid. HPLC (5-95% MeCN / HO over 20 min) retention time: 13.208 min; HRMS (ESI) m / z observed [M+H]. + 3995.4700, [M+2H] + 1997.1381, [M+3H] + 1331.7620 and [M+4H] + 999.0757, C 198 H 315 N 30 O 55 + Required value: 3993.2773.

[0321] Example 33 Peptide synthesis Manual peptide synthesis: Manual peptide synthesis was performed on Merck LL MHBA low-loading Rink amide resin (0.308 mmol / g, 100-200 mesh, 1 eq) to obtain the C-terminal amide. Amino acid coupling was performed for 1-3 h using Fmoc-protected amino acids (3 eq), HATU (3 eq), and DIPEA (6 eq) in DMF. Fmoc deprotection was performed for 10 min using 20% ​​piperidine in DMF.

[0322] Resin cleavage: Side chain deprotection and cleavage from the resin were achieved using a TFA cleavage cocktail containing TFA / TIPS / HO (95:2.5:2.5) for 3 h at rt. After filtration and evaporation under a stream of N, the peptide was precipitated in ice-cold EtO. The crude peptide was lyophilized and analyzed by mass spectrometry using LCMS, and purity was determined by analytical HPLC. The crude peptide was carried forward without further purification.

[0323] LCMS and peptide purity The peptide sequences, mass observed LCMS, peptide purity and yield, along with retention times as determined by analytical HPLC, are shown in Tables 1 and 2 below.

[0324] [Table 1]

[0325] [Table 2]

[0326] Bioconjugation General method for trastuzumab bioconjugation TBS buffer (1×, pH8, [Tras]=2.5mgml -1To a solution of trastuzumab in 200 μL of TCEP (5 mM stock in TBS, 6.8 μL, 34 nmol, 10 equiv.) was added, and the mixture was incubated for 1 h at 37°C on a thermoshaker at 400 rpm. DMSO was added to ensure a final organic solvent concentration of 5-10%. Linker / scaffold (1-10 mM stock in DMSO, 2-10 equiv.) was added, and the mixture was incubated for 4 h at 37°C on a thermoshaker at 400 rpm. The conjugate was purified using a Zeba™ Spin desalting column (MW cutoff 40,000 Da, Thermo Fisher Scientific), which was equilibrated in PBS (3 × 300 μL). Organic solvents were reduced to <0.01% by PBS diafiltration using Amicon-Ultra centrifugal filters (MW cutoff 10,000 Da, Merck Millipore).

[0327] General methods of click chemistry PBS ([Tras] = 1.0 mg ml -1 To a solution of trastuzumab-linker conjugate in DMSO (10 mM stock concentration, 60 μL, 0.4 nmol), a solution of DBCO click reagent in DMSO (10 mM stock concentration, 0.4 μL, 4 nmol, 10 equiv.) was added, and the mixture was incubated at 37°C on a thermal shaker at 400 rpm for 4 to 24 hours. The conjugate was purified using a Zeba™ Spin desalting column (MW cutoff 40,000 Da, Thermo Fisher Scientific), which was equilibrated in PBS (3 × 300 μL). The organic solvent was reduced to <0.01% by PBS diafiltration using an Amicon-Ultra centrifugal filter (MW cutoff 10,000 Da, Merck Millipore).

[0328] Example 34 Synthesis of ALC1 Bioconjugation was performed using scaffold A (206). SDS-PAGE (12% polyacrylamide gel, run at 200V for 50 min and visualized with Coomassie brilliant blue staining) indicated that the fully re-crosslinked antibody was the major product. HRMS (ESI) [M+H] + 146,687 Da (calculated value 146,687 Da).

[0329] Example 35 Synthesis of ALC2 Bioconjugation was performed using scaffold B (208). SDS-PAGE (12% polyacrylamide gel, run at 200V for 50 min and visualized with Coomassie brilliant blue staining) indicated that the fully re-crosslinked antibody was the major product. HRMS (ESI) [M+H] + 147,269 Da (calculated value 147,267 Da).

[0330] Example 36 Synthesis of ALC3 Bioconjugation was performed using scaffold C(211). SDS-PAGE (12% polyacrylamide gel, run at 200V for 50 min and visualized with Coomassie brilliant blue staining) indicated that the fully re-crosslinked antibody was the major product. HRMS (ESI) [M+H] + 146,979 Da (calculated value 146,978 Da).

[0331] Example 37 Synthesis of ALC4 Bioconjugation was performed using linker scaffold D (213). SDS-PAGE (12% polyacrylamide gel, run at 200V for 50 min, visualized with Coomassie brilliant blue staining) indicated that fully re-crosslinked antibody was the major product. HRMS (ESI) [M+H] + 147,559 Da (calculated value 147,557 Da).

[0332] (Examples 38 to 41) Synthesis of ADC1-4 Click reactions were carried out using ALC1, ALC2, ALC3, and ALC4, respectively, with DBCO-PEG5-Val-Cit-PAB-MMAE(248) as the click partner in each reaction, and the mixtures were incubated for 24 h to generate the desired ADCs. ADC1:HRMS(ESI)[M+H]+148,390Da (calculated value 148,388Da). ADC2:HRMS(ESI)[M+H]+148,971Da (calculated value 148,968Da). ADC3: HRMS (ESI) [M+H]+ 148,681 Da (calculated 148,679 Da). ADC4:HRMS(ESI)[M+H]+149,262Da (calculated value 149,258Da).

[0333] Compound 248: [ka]

[0334] Examples 42 to 45 Synthesis of ALC5-8 A solution of brentuximab (5.7 mg / mL) in PBS was pH adjusted with 5% v / v addition of 500 mM Tris and 25 mM EDTA (pH 8.5). The mixture was reduced by adding 10 eq of TCEP at room temperature for 90 minutes. The solution was then further diluted to 2.5 mg / mL with 50 mM Tris (pH 8.0). Five eq of a 10 mM solution of conjugation reagents (Scaffolds E, F, G, and H) in DMSO, along with additional solvent, was added to a final concentration of 5% v / v. The reaction was allowed to proceed for 24 hours at room temperature. The mixture was then desalted into PBS (pH 7.4) using a NAP25 desalting column to remove excess reagent, and re-crosslinking was confirmed by RP-HPLC (PLRP) and HRMS to yield ALC5, ALC6, ALC7, and ALC8, respectively. ALC5: HPLC retention time 13.59 min; HRMS observed Mw 149,727 Da (calculated 149,728 Da). ALC6: HPLC retention time 13.60 min; HRMS observed Mw 150,308 Da (calculated 150,308 Da). ALC7: HPLC retention time 13.59 min; HRMS observed Mw 150,017 Da (calculated 150,018 Da). ALC8: HPLC retention time 13.58 min; HRMS observed Mw 150,599 Da (calculated 150,599 Da).

[0335] (Examples 46 to 49) Synthesis of ADC5-8 Each recrosslinked mAb sample (ALC5, ALC6, ALC7, and ALC8) was conjugated with commercially available DBCO-VC-MMAE using 10 eq of DBCO-linker-warhead and incubated at room temperature for 24 h. Conjugation was confirmed by RP-HPLC (PLRP) and HRMS to give ADCs 5–8, respectively. ADC5: HPLC retention time 13.96 min; HRMS observed Mw 151,170 Da (calculated 151,167 Da). ADC6: HPLC retention time 13.96 min; HRMS observed Mw 151,751 Da (calculated 151,747 Da). ADC7: HPLC retention time 13.97 min; HRMS observed Mw 151,459 Da (calculated 151,650 Da). ADC8: HPLC retention time 13.98 min; HRMS observed Mw 152,041 Da (calculated 152,038 Da).

[0336] (Examples 50 to 53) Synthesis of ADC9-12 Each re-crosslinked mAb sample (ALC5, ALC6, ALC7, and ALC8) was conjugated with the commercially available DBCO-PBD using 10 eq of DBCO-linker-warhead and incubated at room temperature for 24 h. Conjugation was confirmed by RP-HPLC (PLRP) and HRMS to give ADCs 9–12, respectively. ADC9: HPLC retention time 13.92 min; HRMS observed Mw 151,368 Da (calculated 151,360 Da). ADC10: HPLC retention time 13.92 min; HRMS observed Mw 151,948 Da (calculated 151,942 Da). ADC11: HPLC retention time 13.94 min; HRMS observed Mw 151,658 Da (calculated 151,650 Da). ADC12: HPLC retention time 13.93 min; HRMS observed Mw 152,239 Da (calculated 152,232 Da).

[0337] (Examples 54 to 57) Synthesis of ALC9-12 A solution of trastuzumab (25.6 mg / mL) in PBS was pH adjusted with 5% v / v addition of 500 mM Tris and 25 mM EDTA (pH 8.5). The mixture was reduced by adding 6 eq of TCEP at room temperature for 90 min. The solution was then further diluted to 2.5 mg / mL with 50 mM Tris (pH 8.0). Five eq of a 10 mM solution of conjugation reagents (scaffolds E, F, G, and H) in DMSO, along with additional solvent, was added to a final concentration of 5% v / v. The reaction was allowed to proceed for 24 h at room temperature. The mixture was then desalted into PBS (pH 7.4) using a NAP25 desalting column to remove excess reagent, and re-crosslinking was confirmed by RP-HPLC (PLRP) and HRMS to yield ALC9, ALC10, ALC11, and ALC12, respectively. ALC9: HPLC retention time 12.64 min; HRMS observed Mw 149,866 Da (calculated 149,867 Da). ALC10: HPLC retention time 12.63 min; observed Mw 150,447 Da by HRMS (calculated 150,447 Da). ALC11: HPLC retention time 12.60 min; HRMS observed Mw 150,157 Da (calculated 150,157 Da). ALC12: HPLC retention time 12.62 min; HRMS observed Mw 150,738 Da (calculated 150,738 Da).

[0338] Examples 58-61 Synthesis of ADC13-16 Each re-crosslinked mAb sample (ALC9, ALC10, ALC11, and ALC12) was conjugated with commercially available DBCO-VC-MMAE using 10 eq of DBCO-linker-warhead and incubated at room temperature for 24 h. Conjugation was confirmed by SEC, HIC, PLRP, and MS to give ADC13–16, respectively. ADC13: HPLC retention time 13.15 min; HRMS observed Mw 151,308 Da (calculated 151,306 Da). ADC14: HPLC retention time 13.20 min; HRMS observed Mw 151,888 Da (calculated 151,886 Da). ADC15: HPLC retention time 13.20 min; HRMS observed Mw 151,598 Da (calculated 151,596 Da). ADC16: HPLC retention time 13.19 min; observed Mw 152,179 Da by HRMS (calculated 152,177 Da).

[0339] (Examples 50 to 53) Synthesis of ADC17-20 Each re-crosslinked mAb sample (ALC9, ALC10, ALC11, and ALC2) was conjugated with the commercially available DBCO-PBD using 10 eq of DBCO-linker-warhead and incubated at room temperature for 24 h. Conjugation was confirmed by SEC, HIC, PLRP, and MS to give ADC17–20, respectively. ADC17: HPLC retention time 13.07 min; HRMS observed Mw 151,506 Da (calculated 151,500 Da). ADC18: HPLC retention time 13.08 min; HRMS observed Mw 152,086 Da (calculated 152,080 Da). ADC19: HPLC retention time 13.10 min; HRMS observed Mw 151,796 Da (calculated 151,790 Da). ADC20: HPLC retention time 13.10 min; HRMS observed Mw 152,377 Da (calculated 152,371 Da).

[0340] DBCO-VC-MMAE: [ka] DBCO-VC-MMAE was purchased from Syntabio LLC.

[0341] DBCO-PBD: [ka] DBCO-PBD was purchased from Levena biopharma, catalog code SET0317.

[0342] For each of Examples 42 to 61, the following reversed-phase-HPLC (PLRP) conditions were used: Column: Polymer Labs PLRP-S 2.1 mm x 50 mm, 5 μm, 1000 Å Mobile phase A: 0.1% v / v TFA / water Mobile phase B: 0.1% v / v TFA / acetonitrile Column temperature: 80℃ Detection wavelength: 214 nm Reference values: 440nm, 80nm Peak width: >0.4 min, slit 8 nm Reported value: 214 nm Collects spectra from 200 to 600 nm in 1.2 nm steps For each of Examples 42 to 61, the following HRMS conditions were used: Mass spectrometer: SCIEX X500B Q-ToF Software: SCIEX OS UHPLC:ExionLC AD LC column: bioZen™ 3.6 μm Intact XB-C8, 50 × 2.1 mm

[0343] The measured masses of the major glycoforms of each antibody were also used to calculate the theoretical (calculated) molecular mass.

[0344] Example 54 Re-crosslinking and conjugation trial with brentuximab Brentuximab at 5.7 mg / ml was pH adjusted with the addition of 5% v / v of 500 mM Tris, 25 mM EDTA pH 8.5, then reduced with 10 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 90 minutes at room temperature. RP-HPLC analysis indicated complete reduction was achieved. The reduced brentuximab was diluted to 2.5 mg / ml with 50 mM Tris pH 8.0 and then re-crosslinked by adding 5 equiv of 10 mM DMSO and additional scaffolds (Scaffolds E, F, G, and H, respectively) in solvent to a total of 5% v / v. The re-crosslinking reaction was allowed to proceed for 24 hours at room temperature. The re-crosslinked antibody sample was then desalted into PBS pH 7.4 using a NAP 25 desalting column to remove solvent and residual scaffold. UV analysis showed that all samples had approximately [P] = 2 mg / ml after desalting. Analysis was performed using SEC, HIC, PLRP (intact), and MS (intact). Each re-crosslinked antibody sample was divided and conjugated with the following click compounds: DBCO-sulfo-Gly3, DBCO-VC-MMAE, and DBCO-PBD, using 10 equivalents of DBCO-compound in 10% v / v DMA. The conjugation reactions were incubated overnight at room temperature, and SEC, HIC, PLRP (intact), and MS (intact) analyses were repeated.

[0345] The results are summarized in Table 1 below.

[0346] In the table below: - "L" refers to the equivalent amount of antibody light chain; - "HHL" refers to a substantial amount of antibody heavy-heavy-light chain; - "intact" refers to a substantial amount of fully recrosslinked (intact) antibody; - "[P]" refers to the protein concentration measured after desalting by UV analysis. [Table 3]

[0347] Example 55 Recrosslinking and conjugation trial with trastuzumab Trastuzumab (25.6 mg / ml) was pH adjusted with the addition of 5% v / v of 500 mM Tris, 25 mM EDTA pH 8.5, and then reduced with 6 equivalents of TCEP for 90 minutes at room temperature. RP-HPLC analysis indicated complete reduction was achieved. The reduced trastuzumab was then diluted to 2.5 mg / ml in 50 mM Tris, pH 8.0, and then re-crosslinked by adding 5 equivalents of 10 mM scaffold (scaffolds E, F, G, and H, respectively) in DMSO and additional solvent to a total of 5% v / v. The re-crosslinking reaction proceeded for 23 hours at room temperature. The re-crosslinked antibody samples were then desalted in PBS, pH 7.4, using a NAP 25 desalting column to remove solvent and residual scaffold. UV analysis indicated that all samples had a [P] of 1.8 mg / ml after desalting. Analysis was performed using SEC, HIC, PLRP (intact), and MS (intact). Each re-crosslinked antibody sample was divided and conjugated with the following click compounds: DBCO-Sulfo-Gly3, DBCO-vcE, and DBCO-PBD, using 10 equivalents of DBCO-compound in 10% v / v DMA. The conjugation reactions were incubated overnight at room temperature, and SEC, HIC, PLRP (intact), and MS (intact) analyses were repeated.

[0348] The results are summarized in Table 2 below: [Table 4]

[0349] While specific embodiments of the present invention have been described for purposes of reference and illustration, various modifications will become apparent to those skilled in the art without departing from the scope of the invention as defined by the appended claims.

[0350] Numbered paragraphs The following numbered paragraphs describe certain aspects and embodiments of the present invention: (1) A conjugate comprising an antibody, a linker, and at least one active agent, i) a linker connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) Conjugates, wherein each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0351] (2) A conjugate according to numbered paragraph (1), wherein the sulfur atom in the antibody is a sulfur atom present in a cysteine ​​residue of the antibody.

[0352] (3) A conjugate according to numbered paragraph (1) or (2), wherein the functional group that reacts with a sulfur atom on the antibody is a Michael acceptor.

[0353] (4) A conjugate according to any one of numbered paragraphs (1) to (3), wherein the linker is attached to the antibody via six to eight (preferably seven to eight) independent covalent bonds, such that the linker re-bridges two to four (preferably three to four) reduced interchain disulfide bonds in the antibody.

[0354] (5) The conjugate according to any one of numbered paragraphs (1) to (4), wherein the functional group reactive with a sulfur atom in the antibody is selected from a group containing an alkene, alkyne, maleimide, halo-maleimide, sulfone, arylene-propiolonitrile, pyridazinedione, or β-unsaturated ketone.

[0355] (6) The conjugate has formula (III) shown below: [ka] (In the formula, Z A is a functional linking group, Pep indicates the position where the moiety is attached, directly or indirectly, to the antibody; [ka] A conjugate according to any one of numbered paragraphs (1) to (5), comprising 1 to 4 (preferably 2 to 4 or 3 to 4) re-bridging moieties (where " indicates the position at which the moiety is attached to the linker).

[0356] (7) The re-bridging moieties of formula (III) are independently selected from the group consisting of: [ka] (In the formula, A 1 , A 2 and A 3 One or two of these are N, and the other A 1 , A 2 and A 3 One or two of are CH, or A 1 , A 2 and A 3 All three of these are N or CH, a and b are integers selected from 0 or 1; X is N, NR N , O and S; R N is H or C 1~2 is alkyl, R1 and R 2 independently, C 1~ selected from C6 alkylene and C1 to C6 alkylene containing O in the skeleton, R 3 is selected from hydrogen or C1-C4 alkyl, Y 1 and Y 2 is independently absent, or O, NR 4 , C(=O), C(=O)NR 4 Or NR 5 C(=O), p and q are independently integers selected from 0 or 1; Q is CR 6 , N or aryl; R 4 , R 5 and R 6 are independently selected from hydrogen and C1-C4 alkyl; Pep indicates the position at which the moiety is linked, directly or indirectly, to the antibody; [ka] indicates the position where the moiety is attached to the linker). (8) The re-bridging moieties each have the general formula (IIIa) shown below: [ka]

[0357] (In the formula, A 1 , A 2 , A 3 , X, Pep and [ka] A conjugate according to any one of numbered paragraphs (1) to (7), wherein each of

[0358] (9) A conjugate according to any one of numbered paragraphs (1) to (8), wherein the antibody is a monoclonal antibody.

[0359] (10) A conjugate according to any one of numbered paragraphs (1) to (9), wherein the antibody is directed against a tumor-associated antigen.

[0360] (11) A conjugate according to any one of numbered paragraphs (1) to (10), wherein the active agent is a labeling moiety.

[0361] (12) The conjugate according to numbered paragraph (11), wherein the labeling moiety is a fluorophore, a biotin tag, or a PET tracer.

[0362] (13) A conjugate according to any one of numbered paragraphs (1) to (10), wherein the active agent is a drug.

[0363] (14) A conjugate according to numbered paragraph (13), wherein the drug is a cytotoxin.

[0364] (15) The conjugate according to numbered paragraph (14), wherein the cytotoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, calicheamicins, duocarmycins, pyrrolobenzodiazepines, camptothecin analogs, and doxorubicin.

[0365] (16) A conjugate according to any one of numbered paragraphs (1) to (15), wherein the conjugate comprises 1 to 8 active agents.

[0366] (17) A conjugate according to any one of numbered paragraphs (6) to (16), wherein at least two of the re-bridging moieties are separated by 10 to 60 atoms.

[0367] (18) A conjugate according to any one of numbered paragraphs (6) to (17), wherein at least two of the re-bridging moieties are separated by 20 to 40 atoms.

[0368] (19) The conjugate according to any one of numbered paragraphs (1) to (18), wherein the linker comprises one or more groups selected from one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue, an arylene-containing moiety, a heteroarylene-containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety, and combinations thereof.

[0369] (20) The conjugate according to any one of numbered paragraphs (1) to (19), wherein the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, and an amino acid residue.

[0370] (21) The conjugate has formula (IIIA) shown below: [ka] (In the formula, Z 1 is a re-bridging moiety of formula (III) as defined in numbered paragraph (7) or (8) above, wherein the re-bridging moiety is attached to the antibody at the position indicated in formula (III); Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=O)NR 7 , C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene; v is an integer selected from 0, 1, or 2; Each Q 2 are, independently, N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently bonded, C1 to C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is of formula IVc or formula IVe: [ka] is the basis of W 1A N and CR 14 Selected from R 14is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, Ring A is a 6-membered aryl, 6-membered heteroaryl, 6-membered heterocyclyl, or 6-membered cycloalkyl; each X is independently selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently bonded, C1 to C 12 Alkylene and C1-C containing O in the skeleton 12 alkylene; m is an integer selected from 1 or 2; W 1C is the formula W C1 or formula W C2 : [ka] is selected from W Q1 is the formula W C3 : [ka] is the basis of Q W1 does not exist, or C1~C 12 Alkylene and C1-C containing O or N in the backbone 12 alkylene; Q W2is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; X W1 is selected from O or NH, X W2 is hydrogen, C1-C4 alkyl, OR x1 and NR x1 R x2 Selected from R x1 and R x2 are independently selected from hydrogen and C1-C4 alkyl; [ka] is X W1 Formula W Q2 indicates the position of the bond to the base of [ka] is W Q1 is the formula R 24 indicates the position of the bond to the base of W Q2 is the formula W C4 : [ka] is the basis of Q W1A does not exist, or C1 to C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2A is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; X W3 is selected from O or NH, [ka] is X W4 Q W2A via the substituents, formula W Q2indicates the position of attachment to another group of [ka] is X W4 is the following group, hydrogen or -C(=O)R X3 Point to the location where one of the x3 is selected from hydrogen and C1-C4 alkyl; [ka] Q W2A But X W1 via the group W Q1 indicates the position of the bond to the base of X W4 is selected from O or NH, L Q1 is C1~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 8; FG' is a functional linking moiety; L 1 is the linker, D is an activator as defined in any one of numbered paragraphs (13) to (15); W 2 is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 is selected from W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently bonded, C1 to C 10 Alkylene and C1-C containing O in the skeleton 10 alkylene; FG', L 1 and D is as defined above; r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; A conjugate according to any one of numbered paragraphs (1) to (20), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 12.

[0371] (22) The conjugate has formula (IIIA) shown below: [ka] (In the formula, Z 1 is a re-bridging moiety of formula (III) as defined in numbered paragraph (7) or (8) above, wherein the re-bridging moiety is attached to the antibody at the position indicated in formula (III); Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1Ais absent or C(=O), OC(=O), NR 7 C(=O), C(=O)NR 7 , C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton; v is an integer selected from 0, 1, or 2; Each Q 2 are independently N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR12 is selected from W 1 is of formula IVc1 or formula IVe1: [ka] is the basis of W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amide, O or N in the skeleton; Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X is selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2; FG' is a functional linking moiety; L 1 is the linker, D is an activator as defined in any one of numbered paragraphs (13) to (15); W 2 is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 is selected from W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; FG', L 1 and D is as defined above; r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; A conjugate according to any one of numbered paragraphs (1) to (21), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 12.

[0372] (23) The conjugate has formula (IIIA) shown below: [ka] (In the formula, Z 1is a re-bridging moiety of formula (III) as defined in numbered paragraph (7) or (8) above, wherein the re-bridging moiety is attached to the antibody at the position indicated in formula (III); Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2; Each Q 2 are independently N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is represented by formula IVc1: [ka] is the basis of W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); FG' is a functional linking moiety; L 1 is the linker, D is an activator as defined in any one of numbered paragraphs (13) to (15); W 2 is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 is selected from W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); FG', L 1 and D is as defined above; r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; A conjugate according to any one of numbered paragraphs (1) to (22), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 12.

[0373] (24) FG' is a bond, [ka] and —C(═O)NH—.

[0374] (25)L 1 But, bond, C1~C 10 Alkylene and C1-C containing O in the skeleton 10 Alkylene and Formula Va 1 : [ka] (In the formula, Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an active agent).

[0375] (26)Q 1 is a group of formula IVa, Q 1A C(=O) and NR 7 C(=O), Q 1B However, N and CR 9 CR 10 is selected from R 7 , R 9 and R 10are independently selected from hydrogen or C1-C4 alkyl; R 20 and R 21 But independently, C 1~ selected from C6 alkylene and C1 to C6 alkylene containing O in the skeleton, v is 1, Q 2 But N or CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Q 3 is a group of formula IVb, Q 3A is O, Q 3B is the bond, Q 3C NR 13 and R 13 is selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from C1 to C8 alkylene and C1 to C8 alkylene containing O in the skeleton; m is an integer selected from 1 or 2 (preferably m is 1); W 1A is N, L W is C1-C4 alkylene, W 1B But, -NR 15 C(=O)- and -C(=O)NR 15 is a group selected from R 15 is selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, C1-C8 alkylene, and C1-C8 alkylene containing O in the backbone; W 2A is N, W 2B is N, W 2C is -C(=O)NH-, L2W is C1-C4 alkylene, R 25 and R 26 are independently selected from a bond, C1-C8 alkylene, and C1-C8 alkylene containing O in the backbone; r is an integer selected from 0 to 10; s is an integer selected from 0 to 5; A conjugate according to any one of numbered paragraphs (21) to (25), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 10.

[0376] (27)Z 1 is represented by the formula (IIIa) shown below: [ka] (In the formula, A 1 , A 2 , A 3 , X, Pep and [ka] A conjugate according to any one of numbered paragraphs (21) to (26), wherein each of

[0377] (28) A conjugate according to any one of numbered paragraphs (1) to (27), wherein the conjugate is selected from any one of the conjugate compounds listed on pages 49 to 60 (preferably pages 49 to 54) above.

[0378] (29) Formula (I) shown below: (FG) n -L-(Z)8 (Formula I) (In the formula, FG is a functional group that is capable of reacting with another moiety to form a functional linking moiety; n is an integer selected from 0 to 20; L is a linker, Z is a functional group capable of reacting with a sulfur atom from a cysteine ​​moiety of an antibody, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0379] (30) A compound according to numbered paragraph (29), wherein at least two of the Z groups are separated by 10 to 60 atoms.

[0380] (31) A compound according to numbered paragraph (29) or (30), wherein at least two of the Z groups are separated by 20 to 40 atoms.

[0381] (32) The compound according to any one of numbered paragraphs (29) to (31), wherein the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, and an amino acid moiety.

[0382] (33) The compound according to any one of numbered paragraphs (29) to (32), wherein FG is a functional group selected from alkenes, alkynes, azides, hydroxyls, amines, carboxylic acids, aldehydes, acyl halides, tetrazines, alkoxyamines (e.g., hydroxylamines), hydrazines, electron-rich dienophiles (e.g., 1,3-nitrone alkenes) and electron-poor dienes (e.g., tetrazines), nitrones, isocyanates, and isothiocyanates.

[0383] (34) The compound according to any one of numbered paragraphs (29) to (33), wherein FG is a functional group selected from alkyne, azide, hydroxyl, amine, carboxylic acid, aldehyde, and acyl halide.

[0384] (35) The compound according to any one of numbered paragraphs (29) to (34), wherein FG is a functional group selected from amine, carboxylic acid, azide, and alkyne.

[0385] (36) The compound according to any one of numbered paragraphs (29) to (35), wherein Z is a Michael acceptor.

[0386] (37) The compound according to any one of numbered paragraphs (29) to (36), wherein Z is selected from the group comprising an alkene, an alkyne, a maleimide, a halo-maleimide, a sulfone, an arylene-propiolonitrile, a pyridazinedione, a dibromomethylene-pyridine, or a β-unsaturated ketone.

[0387] (38) The compound has formula (Ia) shown below: (FG) n -L-(Z 2 )4 (Formula Ia) (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, A compound according to any one of numbered paragraphs (29) to (37), wherein L, FG, and n are as defined in numbered paragraph (29) above.

[0388] (39) The compound is selected from the group: [ka] [ka] (In the formula, A 1 , A 2 and A 3 One or two of these are N, and the other A 1 , A 2 and A 3 One or two of are CH, or A 1 , A 2 and A 3 All three of these are N, X is N, NR N , O and S; R N is H or C 1~2is alkyl, R 1 and R 2 independently, C 1~ selected from C6 alkylene and C1 to C6 alkylene containing O in the skeleton, R 3 is selected from hydrogen or C1-C4 alkyl, Y 1 and Y 2 is independently absent, or O, NR 10 , C(=O), C(=O)NR 10 Or NR 11 C(=O), Q is CR 12 , N or aryl; p and q are independently integers selected from 0 or 1; R 10 , R 11 and R 12 are independently selected from hydrogen and C1-C4 alkyl; Ts is tosylate, A compound according to any one of numbered paragraphs (29) to (38), wherein L and FG are as defined in numbered paragraph (29) above.

[0389] (40) The compound has formula (Ib) shown below: [ka] (In the formula, A 1 , A 2 and A 3 Two of them are N and A 1 , A 2 and A 3 The other one is CH, X is selected from N, O and S; A compound according to any one of numbered paragraphs (29) to (39), wherein FG, L and n are as defined in numbered paragraph (29).

[0390] (41) The compound has the formula (Ic) shown below: [ka] (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2; Each Q 2 are independently N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3Band Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is of formula IVc2, formula IVe2 or formula IVc: [ka] is the basis of W 1A and W 1c are independently N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amino, O or N in the skeleton, Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X is selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2; W 1C is the formula W C2 : [ka] is the basis of W Q1 is the formula W C3 : [ka] is the basis of Q W1 does not exist, or C1 to C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2 is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; X W1 is selected from O or NH, X W2 is hydrogen, C1-C4 alkyl, OR x1 and NR x1 R x2 Selected from R x1 and R x2 are independently selected from hydrogen and C1-C4 alkyl; [ka] is X W1 Formula W Q2 indicates the position of the bond to the base of [ka] is W Q1 is the formula R 24 indicates the position of the bond to the base of W Q2is the formula W C4B : [ka] is the basis of Q W1A does not exist, or C1 to C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2A is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; X W3 is selected from O or NH, [ka] is X W4 But, Q W2A via the substituents, formula W Q2 indicates the position of attachment to another group of [ka] is X W4 is the following group, hydrogen or -C(=O)R X3 Point to the location where one of the x3 is selected from hydrogen and C1-C4 alkyl; [ka] Q W2A But X W1 via the group W Q1 indicates the position of the bond to the base of X W4 is selected from O or NH, L Q1 is C1~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 8; FG is as in any one of numbered paragraphs (29) or (33) to (35); r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; The compound according to any one of numbered paragraphs (29) to (40), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 12.

[0391] (42) The compound has the formula (Ic) shown below: [ka] (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2; Each Q 2 are independently N and CR11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1' is represented by formula IVc1: [ka] is the basis of W 1A N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C6 alkylene, and a C1-C6 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably m is 1); FG is as defined in any one of numbered paragraphs (29) or (33) through (35); W 2' is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 Selected from R 17 is selected from hydrogen and C1-C4 alkyl; W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 , R 26 and R 27are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; FG is as defined in numbered paragraph (30); r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; The compound according to any one of numbered paragraphs (29) to (41), wherein r and s are selected such that the total number of active agents per conjugate is 1 to 12.

[0392] (43)Z 2 is represented by formula III shown below: [ka] (In the formula, A 1 , A 2 and A 3 Two of them are N and A 1 , A 2 and A 3 The other one is CH, X is N, NR N , O and S; R N is H or C 1~2 is alkyl, [ka] Q 1 A compound according to numbered paragraph (41) or (42), wherein the group represents the point of attachment to

[0393] (44) A compound according to any one of numbered paragraphs (29) to (43), wherein the compound is selected from any one of the compounds listed on pages 95 to 103 above (preferably pages 95 to 97).

[0394] (45) Formula (II) shown below: (DL 1 -FG') n -L-(Z)8 (Formula II) (In the formula, FG' is a functional linking moiety; n is an integer selected from 0 to 20; L and L' are independently a linker; Z is a functional group capable of reacting with a sulfur atom from a cysteine ​​moiety of an antibody; D is an active agent), or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0395] (46) A conjugation reagent according to numbered paragraph (45), wherein at least two of the Z groups are separated by 10 to 60 atoms.

[0396] (47) The conjugation reagent according to any one of numbered paragraphs (45) to (46), wherein the linker comprises one or more of the group selected from an alkylenediamine moiety, a polyethylene glycol moiety, and an amino acid moiety.

[0397] (48)FG' is [ka] A conjugation reagent according to any one of numbered paragraphs (45) to (47), selected from —C(═O)NH— and —C(═O)NH—.

[0398] (49)L 1 But, bond, C1~C 10 Alkylene and C1-C containing O in the skeleton 10 Alkylene, and Formula Va: [ka] (In the formula, L Q But, bond, C1~C 10 Alkylene and C1-C containing O or NH in the skeleton 10 alkylene; Q 4 is a single bond, or [ka] and Q X Q 4 becomes an amino acid residue, a dipeptide residue, or a tripeptide residue, L D is a group for attachment to an active agent).

[0399] (50) A conjugation reagent according to any one of numbered paragraphs (45) to (49), wherein Z is a Michael acceptor.

[0400] (51) The conjugation reagent is represented by formula (IIa) shown below: (DL 1 -FG') n -L-(Z 2 )4 (Formula IIa) (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, L, L 1 , FG' and n are as defined in any one of numbered paragraphs (45) to (49).

[0401] (52) The conjugation reagent is selected from the following group: [ka] [ka] (In the formula, A 1 , A 2 and A 3 One or two of these are N, and the other A 1 , A 2 and A 3One or two of are CH, or A 1 , A 2 and A 3 All three of these are N, X is N, NR N , O and S; R N is H or C 1~2 is alkyl, R 1 and R 2 independently, C 1~ selected from C6 alkylene and C1 to C6 alkylene containing O in the skeleton, R 3 is selected from hydrogen or C1-C4 alkyl, Y 1 and Y 2 is independently absent, or O, NR 10 , C(=O), C(=O)NR 10 Or NR 11 C(=O), Q is CR 12 , N or aryl; p and q are independently integers selected from 0 or 1; R 10 , R 11 and R 12 are independently selected from hydrogen and C1-C4 alkyl; Ts is tosylate, FG', L, L 1 , n and D are as defined in any one of numbered paragraphs (45) to (51).

[0402] (53) The conjugation reagent is represented by formula (IIb) shown below: [ka] (In the formula, A 1 , A 2 and A 3 Two of them are N and A 1 , A2 and A 3 The other one is CH, X is NR N , O and S; R N is H or C 1~2 is alkyl, FG', L, L 1 , n and D are as defined in any one of numbered paragraphs (45) to (51).

[0403] (54) The conjugation reagent is represented by formula (IIc) shown below: [ka] (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, Each Q 1 is independently a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2; Each Q 2 are independently N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 independently represents a group of formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is of formula IVc or formula IVe: [ka] is the basis of W 1A and W 1c are independently N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; L W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amino, O or N in the skeleton, Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X is selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2; W 1C is the formula W C1 or formula W C2 : [ka] is selected from W Q1 is the formula W C3 : [ka] is the basis of Q W1 does not exist, or C1~C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2 is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; X W1 is selected from O or NH, X W2is hydrogen, C1-C4 alkyl, OR x1 and NR x1 R x2 Selected from R x1 and R x2 are independently selected from hydrogen and C1-C4 alkyl; [ka] is X W1 Formula W Q2 indicates the position of the bond to the base of [ka] is W Q1 is the formula R 24 indicates the position of the bond to the base of W Q2 is the formula W C4 : [ka] is the basis of Q W1A does not exist, or C1~C 12 Alkylene and C1-C containing O or N in the skeleton 12 alkylene; Q W2A is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; X W3 is selected from O or NH, [ka] is X W4 But, Q W2A via the substituents, the formula W Q2 indicates the position of attachment to another group of [ka] is X W4 is the following group, hydrogen or -C(=O)R X3 Point to the location where one of the x3is selected from hydrogen and C1-C4 alkyl; [ka] Q W2A But X W1 via the group W Q1 indicates the position of the bond to the base of X W4 is selected from O or NH, L Q1 is C1~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 4; FG', L 1 and D is as defined in any one of numbered paragraphs (45) to (50); W 2' is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 Selected from R 17 is selected from hydrogen and C1-C4 alkyl; W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing amino, O or N in the skeleton, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 and R 26 are independently selected from a bond, a C1-C6 alkylene, and a C1-C6 alkylene containing O in the backbone; FG', L 1 and D is as defined in any one of numbered paragraphs (45) to (50); r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; A conjugation reagent according to any one of numbered paragraphs (45) to (53), wherein r and s are selected such that the total number of active agents per conjugation reagent is 1 to 12.

[0404] (55) The conjugation reagent is represented by formula (IIc) shown below: [ka] (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine ​​moieties of an antibody, Each Q 1 is a bond or a group of formula IVa: [ka] is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10are independently selected from hydrogen and C1-C4 alkyl; R 20 and R 21 are independently bonded, C 1~ selected from C8 alkylene and C1 to C8 alkylene containing O in the skeleton, v is an integer selected from 0, 1, or 2; Each Q 2 N and CR 11 Selected from R 11 is selected from hydrogen and C1-C4 alkyl; Each Q 3 is represented by formula IVb: [ka] is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently selected from hydrogen and C1-C4 alkyl; R 22 and R 23 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1' is of formula IVc1 or formula IVe1: [ka] is the basis of W 1A and W 1c are, independently, N and CR 14 Selected from R 14 is selected from hydrogen and C1-C4 alkyl; LW is absent or selected from C1 to C6 alkylene and C1 to C6 alkylene containing O or N in the skeleton, Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X is selected from CH or N; W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently selected from hydrogen and C1-C4 alkyl; R 23 and R 24 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; m is an integer selected from 1 or 2 (preferably, m is 1); FG', L 1 and D is as defined in any one of numbered paragraphs (45) to (50); W 2' is represented by formula IVd: [ka] is the basis of W 2A N and CR 17 Selected from R 17 is selected from hydrogen and C1-C4 alkyl; W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W is absent or selected from C1-C6 alkylene and C1-C6 alkylene containing one or more O or N in the backbone, R 17 and R 18 are independently selected from hydrogen and C1-C4 alkyl; R 25 and R 26 are independently selected from a bond, a C1-C8 alkylene, and a C1-C8 alkylene containing O in the backbone; FG', L 1 and D is as defined in any one of numbered paragraphs (45) to (50); r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; A conjugation reagent according to any one of numbered paragraphs (45) to (53), wherein r and s are selected such that the total number of active agents per conjugation reagent is 1 to 12.

[0405] (56)Z 2 is represented by formula III shown below: [ka] (In the formula, A 1 , A 2 and A 3 Two of them are N and A 1 , A 2 and A 3 The other one is CH, X is NR N , O and S; R N is H or C 1~2 is alkyl, [ka] Q 1 A conjugation reagent according to numbered paragraph (54) or (55), wherein the group represents the point of attachment to

[0406] (57) A conjugation reagent according to any one of numbered paragraphs (45) to (56), wherein the conjugation reagent is selected from any one of the conjugation reagents listed on pages 74 to 86 (preferably pages 74 to 79) above.

[0407] (58) An intermediate conjugate comprising an antibody, a linker, and at least one functional group capable of reacting with another moiety to form a functional linking moiety, i) the linker connects at least one functional group to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) An intermediate conjugate in which each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

[0408] (59) The intermediate conjugate is represented by formula (III) shown below: [ka] (In the formula, Z A is a functional linking group as defined in any one of numbered paragraphs (6) through (8) above; Pep indicates the position where the moiety is attached, directly or indirectly, to the antibody; [ka] indicates the position at which the moiety is attached to the linker).

[0409] (60) The intermediate conjugate is represented by formula (VI) shown below: [ka] (In the formula, Z 1 , Q 1 , Q 2 , Q 3 is defined in any one of numbered paragraphs (21) to (23) above; W 1 ', W 2 An intermediate conjugate according to numbered paragraph (58) or (59), wherein ', r and s are defined in any one of numbered paragraphs (41) to (42) above).

[0410] (61) An intermediate conjugate according to any one of numbered paragraphs (58) to (60), wherein the intermediate conjugate is selected from any one of the intermediate conjugates listed on pages 105 to 1134 (preferably pages 105 to 107) above.

[0411] (62) A pharmaceutical composition comprising a conjugate according to any one of numbered paragraphs (1) to (28) and a pharmaceutically acceptable carrier, excipient, or diluent.

[0412] (63) A conjugate according to any one of numbered paragraphs (1) to (28) for use in treating a proliferative disease.

[0413] (64) The conjugate according to numbered paragraph (63), wherein the proliferative disease is cancer. (References) TIFF2024528719000352.tif237170TIFF2024528719000353.tif254169TIFF2024528719000354.tif25316 9TIFF2024528719000355.tif254169TIFF2024528719000356.tif253169TIFF2024528719000357.tif95169

Claims

1. A conjugate comprising an antibody, a linker, and at least one active agent, i) a linker connects at least one active agent to the antibody; ii) the linker is attached to the antibody through 5 to 8 independent covalent bonds; iii) Conjugates, wherein each covalent bond between the linker and the antibody is formed from the reaction between a sulfur atom in the antibody and a functional group in the linker.

2. 2. The conjugate of claim 1, wherein the linker is attached to the antibody via 6 to 8 independent covalent bonds such that the linker re-bridges 2 to 4 reduced interchain disulfide bonds in the antibody.

3. 2. The conjugate of claim 1, wherein the linker is attached to the antibody via eight independent covalent bonds such that the linker re-bridges four reduced interchain disulfide bonds in the antibody.

4. The conjugate may have formula (III) shown below: 【Chemical 1】 (In the formula, Z A is a functional linking group, Pep indicates the position where the moiety is attached, directly or indirectly, to the antibody; 【Chemistry 2】 10. The conjugate of claim 1, comprising 1 to 4 re-bridging moieties of the formula: (wherein indicates the position where the moiety is attached to the linker).

5. 5. The conjugate of claim 4, wherein the conjugate comprises 3 to 4 re-bridging moieties of formula (III).

6. The re-bridging moieties of formula (III) are independently selected from the following group: 【Chemistry 3-1】 【Chemistry 3-2】 (In the formula, A 1 , A 2 and A 3 One or two of these are N, and the other A 1 , A 2 and A 3 One or two of are CH, or A 1 , A 2 and A 3 All three of these are N or CH, a and b are integers selected from 0 or 1; X is N, NR N , O and S; R N But H or C 1～2 is alkyl, R 1 and R 2 independently, C 1～ C 6 Alkylene and C containing O in the skeleton 1 ~C 6 alkylene; R 3 is hydrogen or C 1 ~C 4 alkyl, Y 1 and Y 2 is independently absent, or O, NR 4 , C(=O), C(=O)NR 4 Or NR 5 C(=O), p and q are independently integers selected from 0 or 1; Q is CR 6 , N or aryl; R 4 , R 5 and R 6 are independently hydrogen and C 1 ~C 4 alkyl, Pep indicates the position at which the moiety is linked, directly or indirectly, to the antibody; 【Chemistry 4】 indicates the position where the moiety is attached to the linker).

7. The re-bridging moieties each have the general formula (IIIa) shown below: 【Chemistry 5】 (In the formula, A 1 , A 2 , A 3 , X, Pep and 【Chemistry 6】 each of which is defined in claim 6) 7. The conjugate of claim 6, having the formula:

8. The conjugate may have formula (IIIA) shown below: 【Chemistry 7】 (In the formula, Z 1 is a re-bridging moiety of formula (III) as defined in claim 6 above, wherein the re-bridging moiety is attached to the antibody at the position shown in formula (III); Each Q 1 is independently a bond or a group of formula IVa: 【Chemistry 8】 is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently hydrogen and C 1 ~C 4 alkyl, R 20 and R 21 are independently bonded, C 1～ C 10 Alkylene and C containing O in the skeleton 1 ~C 10 alkylene; v is an integer selected from 0, 1, or 2; Each Q 2 are, independently, N and CR 11 Selected from R 11 But hydrogen and C 1 ~C 4 alkyl, Each Q 3 independently represents a group of formula IVb: 【Chemistry 9】 is the basis of Q 3A is NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently hydrogen and C 1 ~C 4 alkyl, R 22 and R 23 are independently bonded, C 1 ~C 10 Alkylene and C containing O in the skeleton 1 ~C 10 alkylene; Q 3D is absent or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 is of formula IVc or formula IVe: 【Chemistry 10】 is the basis of W 1A N and CR 14 Selected from R 14 is hydrogen and C 1 ~C 4 alkyl, L W does not exist or C 1 ~C 6 Alkylene and C containing amino, O or N in the skeleton 1 ~C 6 alkylene; Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X 1 is selected from CH or N, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently hydrogen and C 1 ~C 4 alkyl, R 23 and R 24 are independently bonded, C 1 ~C 12 Alkylene and C containing O in the skeleton 1 ~C 12 alkylene; m is an integer selected from 1 or 2; W 1C is the formula W C1 or formula W C2 : 【Chemistry 11】 is selected from W Q1 is the formula W C3 : 【Chemistry 12】 is the basis of Q W1 does not exist or C 1 ~C 12 Alkylene and C containing O or N in the skeleton 1 ~C 12 alkylene; Q W2 is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; y is an integer selected from 0 or 1; X W1 is selected from O or NH, X W2 is hydrogen, C 1 ~C 4 Alkyl, OR x1 and NR x1 R x2 Selected from R x1 and R x2 are independently hydrogen and C 1 ~C 4 alkyl, 【Chemistry 13】 is X W1 Formula W Q2 indicates the position of the bond to the base of 【Chemistry 14】 is W Q1 is the formula R 24 indicates the position of the bond to the base of W Q2 is the formula W C4 : 【Chemistry 15】 is the basis of Q W1A does not exist or C 1 ~C 12 Alkylene and C containing O or N in the skeleton 1 ~C 12 alkylene; Q W2A is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH-; L Q1 is C 1 ~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; X W3 is selected from O or NH, 【Chemistry 16】 is X W4 But, Q W2A via the substituents, formula W Q2 indicates the position of attachment to another group of 【Chemistry 17】 is XW 4 is selected from the following groups: hydrogen or -C(=O)R X3 Point to the location attached to one of the R x3 is hydrogen and C 1 ~C 4 alkyl, 【Chemistry 18】 Q W2A But X W1 via the group W Q1 indicates the position of the bond to the base of X W4 is selected from O or NH; L Q1 But C 1 ~C 20 a linker comprising one or more groups selected from an alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue, and combinations thereof; t is an integer selected from 1 to 8; FG' is a functional linking moiety; L 1 is the linker, D is an activator, W 2 is represented by formula IVd: 【Chemistry 19】 is the basis of W 2A N and CR 17 is selected from W 2B N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W does not exist or C 1 ~C 6 Alkylene and C containing amino, O or N in the skeleton 1 ~C 6 alkylene; R 17 and R 18 are independently hydrogen and C 1 ~C 4 alkyl, R 25 and R 26 are independently bonded, C 1 ~C 6 Alkylene and C containing O in the skeleton 1 ~C 6 alkylene; FG', L 1 and D is as defined above, r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; 7. The conjugate of claim 6, wherein r and s are selected so that the total number of active agents per conjugate is 1 to 12.

9. The conjugate may have formula (IIIA) shown below: 【Chemistry 20】 (In the formula, Z 1 is a re-bridging moiety of formula (III) as defined in claim 6 above, wherein the re-bridging moiety is attached to the antibody at the position shown in formula (III); Each Q 1 is independently a bond or a group of formula IVa: 【Chemical 21】 is the basis of Q 1A is absent or C(=O), OC(=O), NR 7 C(=O), C(=NR 8 ) and C(=S), Q 1B are O, N, S and CR 9 CR 10 is selected from R 7 , R 8 , R 9 and R 10 are independently hydrogen and C 1 ~C 4 alkyl, R 20 and R 21 are independently bonded, C 1～ C 8 Alkylene and C containing O in the skeleton 1 ~C 8 alkylene; v is an integer selected from 0, 1, or 2; Each Q 2 are, independently, N and CR 11 Selected from R 11 is hydrogen and C 1 ~C 4 alkyl, Each Q 3 independently represents a group of formula IVb: 【Chemical 22】 is the basis of Q 3A But NR 12 , O and S; Q 3B and Q 3C are independently a bond, O, and NR 13 is selected from R 12 and R 13 are independently hydrogen and C 1 ~C 4 alkyl, R 22 and R 23 But independently, bond, C 1 ~C 8 Alkylene and C containing O in the skeleton 1 ~C 8 alkylene; Q 3D is not present, or C(=O), OC(=O), NR 12 C(=O) and C(=O)NR 12 is selected from W 1 Formula IVc 1 or Formula IVe 1 : 【Chemical 23】 is the basis of W 1A N and CR 14 Selected from R 14 is hydrogen and C 1 ~C 4 alkyl, L W does not exist or C 1 ~C 6 Alkylene and C containing O or N in the skeleton 1 ~C 6 alkylene; Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl, or a 6-membered cycloalkyl; X 1 is selected from CH or N, W 1B -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 15 C(=O)-, -C(=O)NR 15 -, -NR 15 C(=O)NR 16 -, -C(=NR 15 )- and -C(=S)-, R 15 and R 16 are independently hydrogen and C 1 ~C 4 alkyl, R 23 and R 24 are independently bonded, C 1 ~C 8 Alkylene and C containing O in the skeleton 1 ~C 8 alkylene; m is an integer selected from 1 or 2; FG' is a functional linking moiety; L 1 is the linker, D is an activator, W 2 is represented by formula IVd: 【Chemistry 24】 is the basis of W 2A However, N and CR 17 is selected from W 2B But, N, CR 18 , -C(=O)N- and -C(=O)CR 18 -, W 2C is -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR 17 C(=O)-, -C(=O)NR 17 -, -NR 17 C(=O)NR 18 -, -C(=NR 17 )- and -C(=S)-, L 2W does not exist, or C 1 ~C 6 Alkylene and C containing O or N in the skeleton 1 ~C 6 alkylene; R 17 and R 18 are independently hydrogen and C 1 ~C 4 alkyl, R 25 and R 26 But independently, bond, C 1 ~C 8 Alkylene and C containing O in the skeleton 1 ~C 8 alkylene; FG', L 1 and D is as defined above, r is an integer selected from 0 to 12; s is an integer selected from 0 to 6; 7. The conjugate of claim 6, wherein r and s are selected so that the total number of active agents per conjugate is 1 to 12.

10. Z 1 is represented by the formula (IIIa) shown below: 【Chemistry 25】 (In the formula, A 1 , A 2 , A 3 , X, Pep and 【Chemical 26】 9. The conjugate of claim 8, wherein each of is a re-bridging moiety as defined in claim 6.

11. The conjugate of claim 1, wherein the antibody is a monoclonal antibody.

12. 5. The conjugate of claim 4, wherein at least two of the re-bridging moieties are separated by 10 to 60 atoms.

13. The conjugate is a conjugate of: 【Chemistry 27-1】 【Chemistry 27-2】 【Chemistry 27-3】 【Chemistry 27-4】 【Chemistry 27-5】 【Chemistry 27-6】 is selected from one of 【Chemical 28】 indicates the location where the antibody is attached, 2. The conjugate of claim 1, wherein the antibody is trastuzumab or brentuximab, preferably trastuzumab.

14. Formula (II) shown below: (D-L 1 -FG') n -L-(Z) 8 (Formula II) (In the formula, FG' is a functional linking moiety; n is an integer selected from 0 to 20; L and L' are independently a linker; Z is a functional group capable of reacting with a sulfur atom from a cysteine of an antibody, D is an activator; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

15. The conjugation reagent may have the formula (IIa) shown below: (D-L 1 -FG') n -L-(Z 2 ) 4 (Formula IIa) (In the formula, Z 2 is a re-bridging linking group containing two functional groups capable of reacting with sulfur atoms from cysteine moieties of an antibody, 15. The conjugation reagent of claim 14, wherein L, L', FG' and n are as defined in claim 14.

16. The conjugation reagent is selected from the group consisting of: 【Chemistry 29-1】 【Chemistry 29-2】 is selected from one of A 1 , A 2 , A 3 , X, R 1 , R 2 , R 3 , Y 1 , Y 2 , Q, p and 1 are each defined in claim 6; Ts is tosylate; FG', L, L 1 15. The conjugation reagent of claim 14, wherein n and D are as defined in claim 14.

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