Homogeneous antibody conjugates with high payload loading

Abstract

The present invention relates to homogeneous antibody conjugates with high payload loading (high DAR) obtained by site-specific conjugation to a single antibody N-glycan. The conjugates according to the present invention have a homogeneous, i.e., narrowly distributed, theoretical DAR or a DAR close to it, and do not require genetic modification of the antibody. The present invention also relates to a modular, non-genetic method for preparing such conjugates, which comprises three simple steps and starts from any antibody.

Description

Detailed Description of the Invention

[0001] [Field of the Invention]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to an antibody conjugate with a high payload, which is homogeneous and does not require genetic antibody modification. Such an antibody conjugate can be applied to more effectively treat diseases, especially cancer.

[0002] [background] Antibody-drug conjugates (ADCs), considered one of the major classes of targeted therapies, consist of antibodies to which pharmaceutical agents are attached. Antibodies (also known as binders or ligands) can be small protein formats (e.g., scFv, Fab fragments, DARPins, affibodies, etc.), but are generally IgG-type monoclonal antibodies (mAbs), selected for their high selectivity and affinity for a given antigen, their long circulating half-life, and little to no immunogenicity. Thus, mAbs, as ligands for carefully selected biological receptors, provide an ideal targeting platform for the selective delivery of pharmaceuticals. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic agents to tumors via binding, internalization, intracellular processing, and finally the release of active catabolites. The cytotoxic agents can be small molecule toxins, protein toxins, or other formats such as oligonucleotides. As a result, tumor cells can be selectively eradicated while sparing normal cells not targeted by the antibody. Similarly, chemical conjugation of antimicrobial agents (antibiotics) to antibodies can be applied to treat bacterial infections, while conjugates of anti-inflammatory drugs are under investigation for the treatment of autoimmune diseases, and the attachment of oligonucleotides to antibodies, for example, is a potentially promising approach for the treatment of neuromuscular diseases. Thus, the concept of targeted delivery of active pharmaceutical agents to optimal specific cellular locations is a powerful approach for the treatment of a wide range of diseases, with many beneficial aspects relative to the systemic delivery of the same drugs.

[0003] ADCs are prepared by conjugation of a linker-drug to a protein, a process known as bioconjugation. See GT Hermanson, "Bioconjugate Techniques," Elsevier, 3, incorporated by reference. rd Many techniques for bioconjugation are known, as summarized in Ed. 2013. Conceptually, the method for preparing an ADC by bioconjugation involves the reaction of x reactive moieties F present on an antibody with complementary reactive moieties Q present on a pharmaceutical (payload) (see Figure 1).

[0004] Typically, a chemical linker is present between Q and the payload. This linker must have several important attributes, including the need to be stable in plasma after prolonged drug administration. A stable linker allows localization of the ADC to the expected site or cell in the body and prevents premature release of the payload in the circulation, which could indiscriminately induce any kind of undesirable biological response, thereby reducing the therapeutic index of the ADC. Upon internalization, the ADC must be processed to ensure effective release of the payload so that it can exert its mode of action within the cell. The linker may also contain a spacer element. Two families of linkers exist: noncleavable and cleavable. Noncleavable linkers consist of a chain of atoms between the antibody and the payload that is completely stable under physiological conditions, regardless of which organ or biological compartment the antibody-drug conjugate resides in. As a result, release of the payload from an ADC with a noncleavable linker depends on complete (lysosomal) degradation of the antibody after internalization of the ADC into the cell. As a result of this degradation, the payload will be released, still bearing the linker and peptide fragments and / or amino acids from the antibody to which the linker was originally attached. Cleavable linkers exploit the intrinsic properties of cells or cell compartments for the selective release of payloads from ADCs, generally leaving no trace of the linker after processing. There are three commonly used mechanisms for cleavable linkers: (1) sensitivity to specific enzymes, (2) pH sensitivity, and (3) sensitivity to the redox state of the cell (or its microenvironment). Cleavable linkers can also contain, for example, self-immortalizing units based on para-aminobenzyl alcohol groups or para-hydroxybenzyl alcohol and their derivatives and / or analogs, or cyclization linkers based on, for example, 1,2-diaminoethane carbamate derivatives. Linkers can also contain additional elements, often referred to as spacer or stretcher units, to connect the linker to a reactive group for binding to an antibody via a reactive moiety F present on the antibody.

[0005]

[0005] The reactive moiety F may be naturally occurring in an antibody, for example, the reactive moiety may be a lysine or cysteine ​​side chain that can be used for acylation (lysine side chain) or alkylation (cysteine ​​side chain).

[0006] Acylation of the ε-amino group of a lysine side chain is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, such as SMCC, which is applied in the production of Kadcyla®. Given the fact that a given antibody may contain 60–90 lysines, a large proportion of which are reactive with the acylating agent, careful titration of the acylating agent is necessary; nevertheless, based on the stochastic distribution of drugs conjugated to the antibody, only average drug loading results in a highly heterogeneous mixture of conjugations. For example, Kadcyla® has an average drug-antibody ratio (DAR) of approximately 4, but in reality is composed of a mixture of components with DARs ranging from 0 to 12. Adding a larger amount of acylating agent results in a higher average DAR; for example, a DAR of 6 or 8 can be achieved based on the stochastic distribution containing higher DAR species (i.e., >DAR12).

[0007]

[0007] Various reagents are known for alkylating thiol groups in cysteine ​​side chains (see Figure 2). Among cysteine ​​alkylation methods, most are based on the use of maleimide reagents, as applied, for example, in the production of Adcetris®, Polivy®, and Padcev®. In addition to standard maleimide reagents, various maleimide variants are also applied for more stable cysteine ​​conjugation, as shown, for example, in James Christie et al., J. Contr. Rel. 2015, 220, 660-670 and Lyonetl., Nat. Biotechnol. 2014, 32, 1059-1062 (both incorporated by reference).Other approaches for cysteine ​​alkylation include, for example, nucleophilic substitution of haloacetamides (typically bromoacetamides or iodoacetamides) (see, e.g., Alleyetal., Bioconj. Chem. 2008, 19, 759-765, which are incorporated by reference), or reaction with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun. 2016, 7, 13128, and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both of which are incorporated by reference), reaction with phosphonamidates (see, e.g., Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, which are incorporated by reference), reaction with allenamides (see, e.g., Abbas et al., Bioconj. Chem. 2019, 58, 11625-11630, which are incorporated by reference), or reaction with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun. 2016, 7, 13128, and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both of which are incorporated by reference). al., Angew. Chem. Int. Ed, 2014, 53, 7491-7494), reaction with cyanoethynyl reagents (see, e.g., Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, which is incorporated by reference), reaction with vinyl sulfones (see, e.g., Gil de Montes et al., Chem. Sci. 2019, 10, 4515-4522, which is incorporated by reference), or reaction with vinyl pyridines (see, e.g., Seki et al., Chem. Sci., 2021, 12, 9060-9068 and https: / / iksuda.com / science / permalink / (accessed July 26, 2020)).

[0008]

[0008] As with lysine conjugation, the DAR is controlled by adjusting the amount of alkylating reagent reacted with the free cysteine ​​side chains (e.g., liberated by reduction of interchain disulfides with TCEP or DTT). The final DAR is typically an average number consisting of a stochastic mixture of different components, as with lysine conjugation. However, several notable differences can be noted: (a) different DAR species typically consist of multiples of 2 (i.e., 2, 4, 6, 8), and (b) the maximum achievable DAR is 8 (if all free interchain cysteine ​​side chains are reacted). This also means that a uniform DAR8 ADC can be achieved by comprehensive alkylation of all interchain cysteine ​​side chains. This is the most common method for generating DAR8 ADCs and is likely the only method used for all clinical ADCs with a DAR8. It should be noted that such approaches cannot produce uniform DAR6 ADCs or DAR>8 ADCs unless specific cysteines are removed from or added to the antibody sequence by recombinant DNA technology. It should also be noted that any method that includes a reduction step may result in antibody degradation (reduction of additional disulfide bonds) or fragment scrambling (e.g., due to light chain exchange).

[0009]

[0009] An alternative approach to antibody conjugation to interchain disulfide bridges involves the use of cysteine ​​cross-linking reagents, ie, reagents that react with two cysteine ​​side chains simultaneously. Examples of such crosslinkers include bis-sulfone reagents (see, e.g., Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both of which are incorporated by reference), mono- or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269, both of which are incorporated by reference), bis-maleimide reagents (see, e.g., WO 2014 / 114207), bis(phenylthio)maleimides (see, e.g., Schumacher et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269, both of which are incorporated by reference), and bis-maleimide reagents (see, e.g., WO 2014 / 114207). al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521), bis-bromopyridazinediones (see, e.g., Robinson et al. RSC Advances 2017, 7, 9073-9077, which is incorporated by reference), bis(halomethyl)benzenes (see, e.g., Ramos-Tomillero et al., Bioconj. Chem. 2018, 29, 1199-1208, which is incorporated by reference), or other bis(halomethyl)aromatic compounds (see, e.g., WO 2013 / 173391). Typically, ADCs prepared by cross-linking of cysteines have a drug-antibody loading of 4 (DAR4), which is achieved by complete alkylation of all cysteine ​​side chains liberated by reduction.

[0010] Another useful technique for conjugating to cysteine ​​side chains is to form new disulfide bonds by treating free cysteine ​​side chains with thiolation agents (i.e., asymmetric disulfide bonds in which one thiol is part of a good leaving group), resulting in bioactivatable linkages that have been utilized to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, for example, Pillow et al., Chem. Sci. 2017, 8, 366-370, which is incorporated by reference). Similar to cysteine ​​alkylation, when the native interchain disulfide bond is reduced, the average DAR of such ADCs can be adjusted to about 2 to 8.

[0011] In addition to conjugation to the side chains of the naturally occurring amino acids lysine or cysteine, various other conjugation techniques have been explored based on a two-step approach involving (a) the introduction of a new reactive group F, followed by (b) reaction with another complementary reactive group Q. For example, one method can be used to introduce a given number of reactive moieties F, which can be two, four, or eight, into an antibody (see Figure 3).

[0012]

[0012] Examples of non-natural reactive functional groups F that can be used for linker-drug bioconjugation are oxime groups suitable for oxime ligation or azide groups suitable for click chemistry conjugation. Oximes can be introduced into antibodies by genetically encoding non-natural amino acids, such as p-acetophenylalanine, as shown, for example, in Axup et al. Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, which is incorporated by reference, or by enzymatic alkylation of cysteines present in the CAAX sequence with prenyl groups containing a remote keto group, as disclosed, for example, in WO 2012 / 153193. Azides can be introduced into antibodies by genetically encoding p-azidomethylphenylalanine or p-azidophenylalanine, as shown, for example, in Axup et al., Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, which is incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem. 2014, 25, 351-361, which is incorporated by reference, used cell-free protein synthesis to introduce p-azidomethylphenylalanine (AzPhe) into monoclonal antibodies for conversion to ADCs via metal-free click chemistry. Also, Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097, which is incorporated by reference, showed that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins using auxotrophic bacteria and further converted into protein conjugates via click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUAGenetic encoding of aliphatic azides in recombinant proteins using pairs has been reported in Nguyen et al., J. Am. Chem. Soc. 2009, 131, 8720-8721, which is incorporated by reference, and labeling was achieved by click chemistry via copper-catalyzed alkyne-azide cycloaddition (CuAAC) or strain-promoted alkyne-azide cycloaddition (SPAAC). In addition to CuAAC and SPAAC, bioconjugation of linker-drugs to antibodies (and other biomolecules such as glycans and nucleic acids) can be achieved by a variety of other metal-free click chemistries (see, e.g., Nguyen and Prescher, Nature Rev. Chem. 2020, 4, 476-489, which is incorporated by reference). For example, oxidation of certain tyrosines in proteins can yield ortho-quinones, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes (see, e.g., Bruins et al., Chem. Eur. J. 2017, 24, 4749-4756, incorporated by reference). In addition to cyclooctynes, certain cycloheptynes ​​are also suitable for metal-free click chemistry, as reported by Wetering et al., Chem. Sci. 2020, 11, 9011-9016, incorporated by reference. Tetrazine moieties can also be introduced into proteins or glycans by various means, such as genetic encoding or chemical acylation, and can also undergo cycloaddition with cyclic alkenes and cyclic alkynes. A list of functional group F and Q pairs for metal-free click chemistry is shown in Figure 4.

[0013] In SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne, and cycloaddition with an azide-modified antibody is facilitated by the relief of ring strain. Conversely, the linker-drug can be functionalized with an azide, and the antibody can be functionalized with a cyclic alkyne. Various strained alkynes suitable for metal-free click chemistry are shown in Figure 5.

[0014]

[0014] A method gaining popularity in the field of ADCs is based on the enzymatic introduction of a non-natural functional group F. For example, Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871, incorporated by reference, uses the bacterial enzyme transglutaminase (BTG or TGase) to introduce an azide moiety onto an antibody. A genetic method based on C-terminal TGase-mediated azide introduction, followed by conversion in ADCs by metal-free click chemistry, was reported in Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0015]

[0015] International Publication No. 2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12, and Wijdeven at al. MAbs 2022, 14, 2078466 (all incorporated by reference) demonstrate that enzymatic remodeling of the native antibody glycan at N297 allows for the introduction of azide-modified sugars suitable for the attachment of cytotoxic payloads using metal-free click chemistry (see Figure 6). Similarly, enzymatic glycan remodeling protocols can also be used to introduce free thiol groups into antibodies for conjugation based on any of the methods described above for cysteine ​​conjugation.

[0016] β-Glucuronidase is an enzyme involved in the degradation of endogenous glucuronic acid-containing glycosaminoglycans. It is normally localized in lysosomes in cells and is also present in lysosomes of first-pass tissues such as the liver and intestine. The concentration of β-glucuronidase in many solid tumors, including lung cancer, breast cancer, and gastrointestinal cancer, and in the tumor microenvironment has been reported to be higher than in normal tissues, and this enzyme is not found in the systemic circulation. Furthermore, lysosomal enzymes are released into the extracellular space from dying cells, resulting in elevated levels of these enzymes in necrotic areas of tumors. Lysosomal β-glucuronidase concentrations are also high in inflammatory immune cells, such as neutrophils and eosinophils, which can release the enzyme at inflammatory sites, such as the necrotic areas of human tumors. Exploiting the overexpression of β-glucuronidase in certain tumors is a well-known approach in oncology research, and β-glucuronidase-cleavable triggers have been applied to anthracyclines, auristatins, duocarmycins, and camptothecins, as well as multi-compound release prodrug approaches, PBD prodrugs, and ADCs. Similarly, β-galactosidase is an enzyme upregulated in the tumor microenvironment and can be used for the selective release of β-galactosides capped with phenolic positions, e.g., p-hydroxybenzyloxycarbonyl groups.

[0017]

[0017] Other enzymes upregulated in the tumor microenvironment, including cathepsins, matrix metalloproteinases (MMPs), legumain, and the serine protease elastase, have also been considered for the release of cytotoxic payloads, as summarized in, for example, M. Poreba, FEBS J., 2020, 287, 1936-1969. Investigations of these two TME-associated enzymes have led to the development of cleavable linkers based on specific peptide sequences, for example, Val-Cit or Val-Ala for cathepsins, Pro-Leu-Gly (PLG) for metalloproteinases (MMPs), Asn-Asn, Ala-Asn, or Asn-Ala for legumain, and Asn-Pro-Val (NPV) for the serine protease elastase.

[0018] Currently, cytotoxic payloads include, for example, microtubule-disrupting agents (e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and tubulysin), DNA-damaging agents (e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, duocarmycins, anthracyclines such as PNU-159,682), topoisomerase inhibitors (e.g., DXd, exatecan, and SN-38), or RNA polymerase II inhibitors (e.g., amanitin). ADCs that have achieved market approval include, for example, the payloads MMAE, MMAF, DM1, DM4, calicheamicin, SN-38, DXd, and PBD dimers, and various pivotal trials have been conducted on duocarmycin-based ADCs. A wider variety of payloads, such as eribulin, indolinobenzodiazepine dimers, PNU-159,682, amanitin, and hemiasterlin, are still undergoing clinical evaluation or have been clinically tested. Finally, various ADCs in early clinical or late preclinical stages are conjugated to novel payloads, such as KSP inhibitors, MMADs, and cryptophycins. Various other payloads, such as doxorubicin, vinca alkaloids, and duocarmycin SA, have been evaluated but abandoned due to lack of efficacy. Even less potent payloads have not undergone clinical evaluation for the same reason (lack of efficacy).

[0019] One way to expand the range of payloads available to ADCs by using drugs with lower potency than current arsenals is to increase the DAR of the ADC, thereby effectively delivering more drug to the tumor per internalization event. For example, Mersana Therapeutics developed the so-called Fleximer® technology, which enables ADCs with DARs of 12–15, and employed this in their lead program, XMT-1536 (UpRi). To achieve such a high DAR, a highly polar polymeric linker technology was first developed that can accommodate many hydrophobic payloads and avoid the aggregation / degradation of high-DAR ADCs, which is essential to avoid the significant impairment of their pharmacokinetic behavior. Indeed, Hamblett et al., Clin. CANC. Res. 2004, 10, 7063-7070 (incorporated by reference) confirmed that DAR8 ADCs exhibit better in vitro efficacy due to higher drug loading, but perform worse in vivo than comparable DAR4 ADCs due to the much faster clearance of DAR8 species. Such poor PK characteristics are revealed by measuring profiles with hydrophilic interaction chromatography (HIC), and in particular, a longer relative retention time compared to the naked antibody can be considered an indicator of poor PK performance, as disclosed in Burke et al., Mol. CANC. Ther. 2017, 16, 116-123 (incorporated by reference).Other solutions to alleviate poor PK properties and enable DAR8 ADCs typically involve inserting branched hydrophilic spacers to protect the hydrophobic payload, such as polyethylene glycol (PEG) as disclosed in Burke et al., Mol. CANC. Ther. 2017, 16, 116-123, which are incorporated by reference, and used in XGN-CD228A and XGN-CD48A, polysarcosine as disclosed in Viricel et al., Chem. Sci. 2019, 10, 4048-4053, or chitosan as disclosed in WO 2022 / 058548 and WO 2022 / 048883, and used in M1231.

[0020] To date, all high-DAR ADCs (approximately DAR 8 or greater) introduced into the clinic are based on conjugation techniques using cysteine ​​alkylation with a linear linker-drug and are therefore limited to a DAR of 8 unless additional cysteines are incorporated into the antibody by recombinant DNA technology. XMT-1536 is an exception to this rule, as it has a drug loading of 12-15, but it is also a highly heterogeneous ADC consisting of stochastically conjugated cysteines and highly heterogeneous polymers attached to each cysteine.

[0021] [Summary of the Invention]

[0021] The present inventors have succeeded for the first time in preparing homogeneous antibody conjugates with high payload loading (high DAR) by site-specific conjugation to a single antibody N-glycan. The conjugates according to the present invention have a uniform, i.e., narrowly distributed, theoretical DAR or a DAR close to it, and do not require genetic modification of the antibody. The conjugates according to the present invention have a payload-antibody ratio (DAR) of 6 or more, preferably 8 or more. Conjugates with a DAR of up to 64 can be easily prepared.

[0022] The present inventors have developed a modular, non-genetic method for preparing such conjugates, which involves three simple steps and starts from any antibody (see Figure 7). The present inventors have developed a modular, non-genetic method for preparing such conjugates, which involves three simple steps and starts from any antibody. These steps include (a) enzymatic remodeling of glycans to yield antibodies functionalized with two or four click probes (e.g., azides) per antibody; (b) click reaction (e.g., strain-promoted azide-alkyne cycloaddition) with a multivalent bifunctional reagent containing one click probe (e.g., cyclic alkyne) reactive with the remodeled antibody and at least two click probes unreactive with other click probes (e.g., tetrazines) (see Figures 8-11); and (c) separate click reactions (e.g., inverse electron-demand Diels-Alder cycloaddition) of the click probes with a branched linker-drug construct containing one click probe reactive with a previously unreactive click probe (e.g., cyclic alkyne or strained alkene), preferably linked to one or more payloads linked via cleavable linkers (see Figures 12-15). The resulting conjugates with DARs of 6 or greater are rapidly generated with high uniformity, close to theoretical DARs, and remarkable stability. Furthermore, the native glycosylation sites of the antibody can be used for site-specific conjugation, resulting in conjugates with high DAR without the need for genetic engineering of the antibody. In addition, the HIC profile of the resulting ADC, as well as in vitro and in vivo efficacy and tolerability studies, showed a short relative retention time, thus indicating high potential in the treatment of cancer.

[0023] [Detailed explanation] [Definition]

[0023] When used in this specification and claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded.

[0024] Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0025] A linker is defined herein as a moiety that connects (covalently bonds) two or more elements of a compound. A linker may include one or more spacer moieties. A spacer moiety is defined herein as a moiety that covalently bonds two (or more) parts of the linker with a certain spacing (i.e., providing a distance between them). A linker can be, for example, part of a linker-construct, linker-conjugate, linker-payload (e.g., linker-drug), or antibody-conjugate, as defined below.

[0026]

[0026] A "hydrophilic group" or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that impart improved polarity and therefore improved water solubility to the attached molecule. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonate groups, phosphate groups, acylsulfamide groups, or carbamoylsulfamide groups. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency, and less aggregation when incorporated into antibody-drug conjugates, improved pharmacokinetics, thereby resulting in higher efficacy and in vivo tolerability.

[0027] The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced with a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term "pharmaceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that has acceptable mammalian safety for a given administration regimen). Such salts may be derived from a pharmaceutically acceptable inorganic or organic base and a pharmaceutically acceptable inorganic or organic acid. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which can be derived from a variety of organic and inorganic counterions known in the art, such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0028]

[0028] The term "enediyne", or "enediyne antibiotic", or "enediyne-containing cytotoxin" refers to any cytotoxin characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule known in the art, including neocarzinostatin (NCS), C-1027, kedarcidin (KED), maduropeptin (MDP), N1999A2, sporolide (SPO), cyanosporacid (CYA and CYN), and phyziolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamemicin, shishijimicin, and uncialamicin (UCM).

[0029] As used herein, the term "alkylaminosugar" refers to a tetrahydropyranyl moiety linked via its 2-position to an alcohol functional group, thereby forming an acetal functional group, and further substituted with (at least) one N-alkylamino group at the 3-, 4-, or 5-position. In this context, an "N-alkylamino group" refers to an amino group bearing one methyl, ethyl, or 2-propyl group.

[0030] The term "click probe" refers to a functional moiety capable of undergoing a click reaction. That is, two compatible click probes undergo a click reaction with each other and become covalently linked in the product. Probes compatible with click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of the present invention, click probe Q in a compound according to the present invention can react with click probe F on a (modified) protein, and upon click reaction, a conjugate is formed in which the protein is conjugated to the compound of the present invention. Here, F and Q are compatible click probes. Click reactions are known in the art and typically refer to cycloaddition reactions such as [4 + 2] cycloaddition (e.g., Diels-Alder, inverse electron demand Diels-Alder) and [3 + 2] cycloaddition (e.g., 1,3-dipolar cycloaddition). In the context of the present invention, the term click reaction can also be referred to as cycloaddition.

[0031]

[0031] The term "(hetero)alkyl" refers to alkyl and heteroalkyl groups. Heteroalkyl groups are groups in which one or more carbon units (e.g., CH2, CH, or C) in the alkyl chain is replaced with O, S, S(O), S(O)2, or NR 4 In other words, alkyl chains containing O, S, S(O), S(O)2, and NR 4and n-butyl (n-butyl), n-butyl (n-butyl), or n-pentyl. Such interruptions are distinct from substituents, for example, pendant groups monovalently attached to a carbon atom of the alkyl chain, because they are present within the chain of the alkyl group. In preferred embodiments, the (hetero)alkyl group is an alkyl group, for example, ethyl (Et), isopropyl (i-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu), or n-pentyl.

[0032] Similarly, the term "(hetero)aryl" refers to aryl and heteroaryl groups. Heteroaryl groups are groups in which one or more carbon units in the ring (e.g., CH) are replaced with O, S, N, or NR 4 and aryl groups substituted with heteroatoms such as:

[0033] An "acylsulfamide moiety" is defined herein as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamoylated at one end of the molecule and N-alkylated (mono- or bis-) at the other end of the molecule. In the context of the present invention, particularly in the Examples, this group is also referred to as "HS."

[0034] A "domain" is generally defined based on sequence homology and can be any region of a protein that is often associated with a particular structural or functional entity. CEACAM family members are known to be composed of Ig-like domains. The term domain is used herein to refer to an individual Ig-like domain, e.g., an "N-domain," or a group of consecutive domains, e.g., an "A3-B3 domain."

[0035] A "coding sequence," or a sequence that "encodes" an expression product such as an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, produces that RNA, polypeptide, protein, or enzyme. That is, the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0036]

[0036] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term gene can refer to a genomic sequence that encodes a protein, i.e., a sequence that includes regulatory elements, promoter, intron, and exon sequences.

[0037] The term "glycoprotein" is used herein in its ordinary scientific sense to refer to a protein containing one or more monosaccharide or oligosaccharide chains ("glycans") covalently attached to the protein. The glycans can be attached to a hydroxyl group of the protein, such as that of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline (O-linked glycans), or to an amide functional group of a protein, such as asparagine or arginine (N-glycoproteins), or to a carbon of a protein, such as tryptophan (C-glycoproteins). A glycoprotein may contain two or more glycans, a combination of one or more monosaccharides and one or more oligosaccharide glycans, or a combination of N-linked, O-linked, and C-linked glycans. It is estimated that over 50% of all proteins have some form of glycosylation and are therefore considered glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0038] The term "glycan" is used herein in its ordinary scientific sense to refer to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term glycan refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of a single sugar, which may be unsubstituted (monosaccharide) or further substituted at one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when longer sugar chains are linked to a protein, the sugar chains are also considered glycans herein. The glycans of a glycoprotein can be monosaccharides. Typically, monosaccharide glycans of a glycoprotein consist of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man), or fucose (Fuc) covalently attached to the protein. A glycan can also be an oligosaccharide. The oligosaccharide chains of a glycoprotein can be linear or branched. In an oligosaccharide, the sugar directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to the protein and is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to the protein and is attached to a single other sugar, i.e., a sugar that does not have an additional sugar substituent on one or more of its other hydroxyl groups, is called a terminal sugar. For the avoidance of doubt, an oligosaccharide of a glycoprotein may have multiple terminal sugars, but only one core sugar. Glycans can be O-linked, N-linked, or C-linked glycans. In O-linked glycans, monosaccharides or oligosaccharide glycans are attached to the O-atom of an amino acid of the protein, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, monosaccharides or oligosaccharide glycans are attached to the protein via the N-atom of an amino acid of the protein, typically via the amide nitrogen of the side chain of asparagine (Asn) or arginine (Arg). In C-linked glycans, a monosaccharide or oligosaccharide glycan is attached to a C atom of an amino acid in a protein, typically the C atom of tryptophan (Trp).

[0039] The term "antibody" (AB) is used herein in its ordinary scientific sense. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is intended to include not only whole antibodies but also antibody fragments, such as antibody Fab fragments, F(ab')2, Fv or Fc fragments of truncated antibodies, scFv-Fc fragments, minibodies, diabodies, or scFv. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and recombinant antibodies can be obtained by methods known in the art.

[0040]

[0040] An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (K). Light chains contain two domains or regions, a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity for antigens. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties to the antibody chain, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g., human G1m1, G1m2, Gm3, non-G1m1 (that is, any allotype other than G1m1), G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of immunoglobulin molecule. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2, or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. Antibodies can be engineered in the Fc domain to enhance or impair binding to Fc-gamma receptors, as summarized in Saunders et al. Front. Immunol. 2019, 10, doi:10.3389 / fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131-141.For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. Elimination of binding to Fc-γ receptors can also be achieved by mutating the N297 amino acid to any other amino acid other than asparagine, the T299 amino acid to any other amino acid other than threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies, for example, with PNGase F or endoglycosidase. Immunoglobulins can be derived from any species, including human, mouse, or rabbit origin. Each chain contains distinct sequence domains.

[0041] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, and the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the full length of the reference sequence.

[0042] The term "CDR" refers to complementarity-determining region: the specificity of an antibody resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). Optionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and thus the binding site. Thus, complementarity-determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. Each immunoglobulin light and heavy chain has three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen-binding site contains six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0043]

[0043] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule of a single amino acid sequence, which is directed against a particular antigen, and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be produced recombinantly, i.e., by protein engineering.

[0044]

[0044] In its broadest sense, the term "chimeric antibody" refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal in association with the CH and CL domains of another antibody, in one embodiment a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also refer to multispecific antibodies that have specificity for at least two different antigens.

[0045] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified, for example by substituting certain amino acids in the framework regions of the VH and VL domains, to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most often human CH and CL domains. A "fragment" of a (traditional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0046] The term "multivalent" refers to a linker molecule or linker moiety of a larger molecule that has multiple linking groups. In the case of a linker molecule, these linking groups are formed by reactive groups that can be covalently bonded to other molecules (e.g., payloads or antibodies). In linker moieties of larger molecules such as conjugates, the linking group is a covalent bond to another part of the molecule (e.g., payloads or antibodies). A bivalent linker has two linking groups, a trivalent linker has three linking groups, etc. In the context of the present invention, bivalent is also referred to as 2-valent, trivalent is also referred to as 3-valent, etc.

[0047] The term "heterofunctional" refers to a linker molecule or linker portion of a larger molecule having linking groups that are not identical and have or result from different reactivities. Typically, such linking groups in a heterofunctional molecule are unreactive with each other.

[0048] [The present invention]

[0048] The present inventors have succeeded for the first time in preparing homogeneous antibody conjugates with high payload loading (high DAR) by site-specific conjugation to a single antibody N-glycan. The conjugates according to the present invention have a uniform, i.e., narrowly distributed, theoretical DAR or a DAR close to it, and do not require genetic modification of the antibody. The conjugates according to the present invention have a payload-antibody ratio (DAR) of 6 or more, preferably 8 or more. Conjugates with a DAR of up to 64 can be easily prepared.

[0049] The present inventors have developed a modular, non-genetic method for preparing such conjugates, starting from any antibody, that involves three simple steps: (a) enzymatic remodeling of glycans to obtain antibodies functionalized with two or four click probes (e.g., azides) per antibody, (b) click reaction (e.g., strain-promoted azide-alkyne cycloaddition) with a polyvalent bifunctional reagent containing one click probe (e.g., cyclic alkyne) reactive with the remodeled antibody and at least two click probes unreactive with other click probes (e.g., tetrazines), and (c) separate click reaction (e.g., inverse electron-demand Diels-Alder cycloaddition) of the click probe with a branched linker-drug construct containing one click probe reactive with a previously unreactive click probe (e.g., cyclic alkyne or strained alkene), preferably linked to one or more payloads linked via cleavable linkers. The resulting conjugates with DARs of 6 or greater are rapidly produced with high uniformity, close to the theoretical DAR, and surprising stability. Furthermore, the natural glycosylation sites of antibodies are used for site-specific conjugation, resulting in high DAR conjugates without the need for genetic engineering of the antibody. Additionally, the HIC profiles of the resulting ADCs, as well as in vitro and in vivo efficacy and tolerability studies, demonstrate short relative retention times, thus demonstrating their potential for cancer treatment.

[0050] In a first aspect, the present invention provides a process for preparing an antibody-payload conjugate having a payload-antibody ratio (DAR) of 6 or greater, comprising: (a) Structure Ab(F 1 ) z where Ab is an antibody, z is 2 or 4, and F 1 is a click probe); (b) Add z equivalents of Q 1 L A (F 2 ) y (In the formula, Q 1 is F 1 is a click probe that reacts with L A is a heterobifunctional (y+1)valent linker, where y is 2, 3, or 4; F 2 Q 1 (a click probe that does not react with Z) to form the structure Ab(Z 1 L A (F 2 ) y ) z (In the formula, Z 1 is F 1 and Q 1 wherein the linker group is obtained by reaction of (c) Binding the antibody-linker construct to z × y equivalents of Q 2 (L B )D x (In the formula, Q 2 is F 2 is a click probe that reacts with L B is an (x+1)-valent linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is a payload molecule) to form a compound of the structure Ab(Z 1 L A (Z 2 (L B )D x ) y ) z (In the formula, Z 2 is F 2 and Q 2 a step of obtaining a conjugate of The present invention relates to a process including:

[0051] In a second aspect, the present invention relates to a conjugate obtainable by the process according to the first aspect of the invention. Alternatively, the conjugate according to the second aspect may also be a conjugate of the structure Ab(Z 1 L A (Z 2 (L B )D x ) y ) z (In the formula, - Ab is an antibody; -Z 1 and Z 2 is a linking group obtained by reaction between two click probes; - x is 1, 2, 3, or 4. - y is 2, 3, or 4 and x+y is at least 4; - z is 2 or 4; L A is a heterobifunctional (y+1)valent linker; L B is a heterobifunctional (x+1)valent linker; - D is the payload) It can be defined as having

[0052]

[0052] The conjugate according to the second aspect of the present invention is ideally suited for targeting tumor cells, in particular for the treatment of cancer. Accordingly, the present invention also relates to a method for targeting tumor cells expressing a specific extracellular receptor, comprising contacting a conjugate according to the second aspect of the present invention with cells likely to express the extracellular receptor, wherein the antibody specifically targets the extracellular receptor. Similarly, the present invention also relates to a method for treating cancer, comprising administering to a subject in need thereof a conjugate according to the second aspect of the present invention, wherein the cancer cells specifically express the extracellular receptor.

[0053] The invention according to this aspect further relates to a pharmaceutical composition comprising a conjugate according to the second aspect of the invention and a pharmaceutically acceptable carrier.

[0054] In a third aspect, the present invention provides a compound of structure Q 1 L A (F 2 ) y (In the formula, - Q 1 is the click probe; -L A is a heterobifunctional (y+1)valent linker; - y is 2, 3, or 4; and -F 1 Q 1 (a click probe that does not react with The present invention relates to a heterobifunctional linker having the formula:

[0055] The heterobifunctional linkers according to the third aspect of the invention are ideally suited as intermediates in the synthesis of conjugates according to the second aspect.

[0056]

[0056] Salts, preferably pharmaceutically acceptable salts, of the conjugates and heterobifunctional linkers according to the invention are also considered to be within the scope of the present invention.

[0057]

[0057] Below, the conjugate according to the second aspect is first defined. The structural features of the conjugate according to the present invention also apply to the heterobifunctional linker according to the present invention and to the method for preparing the antibody-payload conjugate according to the present invention. As will be understood by those skilled in the art, the structural features of the conjugate according to the present invention also apply to the heterobifunctional linker according to the present invention. Those skilled in the art will understand that any structural feature that does not change in the conjugation reaction is equally defined for each of the molecules according to the present invention. In the conjugation reaction, only the reactive moieties F and Q are converted to the linking group Z. As will be understood by those skilled in the art, the definitions of the chemical moieties and their preferred embodiments apply to all aspects of the present invention.

[0058] [Conjugates of general structure (2)] The present invention relates to a compound having the general structure (2): [ka] (In the formula, - Ab is an antibody; -Z 1 and Z 2 is a linking group obtained by reaction between two click probes; - x is 1, 2, 3, or 4; - y is 2, 3, or 4 and x+y is at least 4; - z is 2 or 4; -L A is a heterobifunctional (y+1)valent linker; -L B is a heterobifunctional (x+1)valent linker; - D is the payload) Concerning the conjugate of

[0059] [Antibody Ab] Ab is an antibody. Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabodies, minibodies, affibodies, affilins, affimers, atrimers, finomers, cys-knots, DARPins, adnectins / centrins, knottins, anticalins, FN3, Kunitz domains, OBody, bicyclic peptides, and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of IgA, IgD, IgE, IgG, and IgM antibodies. Even more preferably, the Ab is an IgG antibody. The IgG antibody can be of any IgG isotype. The antibody can be of any IgG isotype, for example, IgG1, IgG2, IgI3, or IgG4. Preferably, the Ab is a full-length antibody, but the Ab can also be an Fc fragment.

[0060] The antibody Ab is typically specific for an extracellular receptor on a tumor cell, and preferably the extracellular receptor on a tumor cell is 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, or CD33. , CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tenascin.

[0061] The antibody may also be specific to an extracellular protein resulting from a viral infection, such as human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). The antibody may also be specific to Tn, STn, T-antigen, LDN, Lewis c (Le c ), Cialis-Lewis c (SLe c ), 6-Sialyl-Lewis c (6SLe c ), LN, alpha-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis a (Le a ), Cialis-Lewis a (SLe a ), 6-Sialyl-Lewis a (6SLe a ), Lewis b (Le b ), Cialis-Lewis b (SLe b ), 6-Sialyl-Lewis b (6SLe b ), Lewis x (Le x ), Cialis-Lewis x (SLe x ), 6-Sialyl-Lewis x (6SLe x ), Lewis y (Le y ), Cialis-Lewis y (SLe y ), 6-Sialyl-Lewis y (6SLe y ), and / or combinations thereof. The antibody may also be specific for both an extracellular protein and a TACA simultaneously.

[0062] The number of payloads (D) conjugated to a single antibody is known in the art as the drug-antibody ratio (DAR). In the context of the present invention, although DAR values ​​up to 64 are possible, it is preferred that the DAR be an even number ranging from 6 to 32, more preferably 6, 8, 10, or 12, even more preferably 8 or 12, and most preferably DAR=8. These are theoretical DAR values; it will be understood that in practice, the DAR may deviate slightly from this value due to incomplete conjugation. Thus, conjugates may be obtained as mixtures of conjugates with various DARs. In such mixtures, the DAR often refers to the average DAR of the mixture. This is well known in the art of bioconjugation. The conjugation technique used in the present invention site-specifically conjugates via the glycans of the antibody using a click reaction to provide conjugates with DAR values ​​close to the theoretical value and a narrow distribution with a narrow standard deviation. For example, if the theoretical DAR is 8, DAR values ​​greater than 7.4 or greater than 7.6 are easily obtained, indicating that most of the antibodies in the reaction mixture have reacted completely and have a DAR of 8. No antibody conjugates exist with a DAR greater than 8. Thus, the average DAR of the obtained conjugates is close to the theoretical value, preferably the average DAR is at most 15% lower than the theoretical DAR, more preferably the average DAR is at most 10% lower than the theoretical DAR, or even at most 5% lower than the theoretical DAR.

[0063] In one embodiment of a conjugate according to the invention, only conserved glycosylation sites are used for conjugation as described below (i.e., z=2), and the antibody is functionalized with 2×x×y occurrences of payload D. Herein, x is 1, 2, 3, or 4, y is 2, 3, or 4, and x+y is at least 4. Thus, x+y is an integer ranging from 4 to 8. The exact numbers of x and y determine the DAR. In a preferred embodiment, y=2 and x=1, 2, 3, or 4, preferably x=2, 3, or 4, more preferably x=2. In another preferred embodiment, x=2 and y=2, 3, or 4, preferably y=2 or 4, more preferably y=2. In another preferred embodiment, x=y, preferably x=y=2 or 4, more preferably x=y=2.

[0064] In an alternative embodiment of a conjugate according to the invention, both the conserved glycosylation site and the second glycosylation site are used for conjugation as described below (i.e., z=4), and the antibody is functionalized with a 4×x×y occurrence of payload D. Herein, x is 1, 2, 3, or 4, y is 2, 3, or 4, and x + y is at least 4. Thus, x + y is an integer ranging from 4 to 8. The exact numbers of x and y determine the DAR. In a preferred embodiment, y=2 and x=1, 2, 3, or 4, preferably x=1, 2, or 3, more preferably x=2. In another preferred embodiment, x=1 and y=2, 3, or 4, preferably y=2 or 4, more preferably y=2. In another preferred embodiment, x=y, preferably x=y=2 or 4, more preferably x=y=2.

[0065] These values ​​of z, x, and y readily allow the formation of conjugates having DARs ranging from 6 to 64. Conjugates according to the present invention have even DARs. In a preferred embodiment, DAR=6 (z=2, x=1, and y=3). In another preferred embodiment, DAR=8 (z=2, x=2, and y=2). In another preferred embodiment, DAR=8 (z=2, x=1, and y=4). In another preferred embodiment, DAR=12 (z=2, x=2, and y=3). In another preferred embodiment, DAR=16 (z=2, x=2, and y=4). In another preferred embodiment, DAR=32 (z=2, x=4, and y=4). In another preferred embodiment, DAR=24 (z=2, x=3, and y=4, or z=2, x=4, and y=3). In another preferred embodiment, DAR=12 (z=4, x=1, and y=3). In another preferred embodiment, DAR=16 (z=4, x=2, and y=2). In another preferred embodiment, DAR=16 (z=4, x=1, and y=4). Preferred conjugates have a DAR in the range of 6 to 32, more preferably in the range of 6 to 16, and even more preferably in the range of 6 to 12. In a further preferred embodiment, the DAR is at least 8, preferably in the range of 8 to 32, more preferably in the range of 8 to 16, and even more preferably in the range of 8 to 12. Most preferably, DAR=8.

[0066] The conjugates according to the invention comprise two linking groups Z 1 and is formed during the click reaction, wherein the structure Ab(F 1 ) z Antibodies and Structure Q 1 L A (F 2 ) y In the first click reaction, F 1 and Q 1 reacts to form the linking group Z 1 by forming a z × y moiety F 2 It forms a covalent bond with

[0067]

[0067] As used herein, "(y+1) valence" refers to the number of attachment points of the reactive groups F or Q (before reaction) or the linking group Z (after reaction). Thus, when y=2, the linker construct has one click probe Q 1 and two click probes F 2 and the linker has a valence of 2+1=3. Thus, when y=2, L A is trivalent. Thus, when y=3, the linker construct contains one click probe Q 1 and three click probes F 2 and the linker has a valency of 3+1=4. Thus, when y=3, L A is tetravalent. Thus, when y=4, the linker construct contains one click probe Q 1 and four click probes F 2 and the linker has a valence of 4+1=5. Thus, when y=4, L A is pentavalent.

[0068]

[0068] Some antibodies are click probes 1 or a linking group Z 1 a linker L connecting the peptide portion of the cell-binding agent to 6 Preferably, the linking group Z 1 is linked to the cell-binding agent CB via the glycan of the Ab. Thus, the conjugate according to the invention preferably comprises [ka] (In the formula, -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0 or 1, and L 7is -N(H)C(O)CH2-, -N(H)C(O)CF2-, or -CH2-) It is expressed as:

[0069] [Linker L 6 ] Linker L 6 is preferably present, where the reactive group F 1 can be introduced into specific positions of the antibody, for example, by using transglutaminase, by using sortase, or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase), or by introducing an artificially introduced reactive group F 1 For example, conjugation via a modified sugar residue S(F 1 )2 can be introduced into the glycan, allowing the glycan to be elongated with one monosaccharide residue S, thereby providing two reactive groups F on the glycan of the antibody. 1 In the most preferred embodiment, conjugation occurs via the glycans of the antibody. The site of conjugation is preferably in the heavy chain of the antibody.

[0070] All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at an asparagine residue at or near position 297 (Kabat numbering) of the heavy chain that is modified with complex-type glycans. This naturally occurring glycosylation site of the antibody is preferably used, although other glycosylation sites, including artificially introduced ones, may also be used in the linker L. 6 Thus, in a preferred embodiment, L 6 is linked to an amino acid of the antibody located at positions ranging from 250 to 350 of the heavy chain, preferably at positions ranging from 280 to 310 of the heavy chain, more preferably at positions ranging from 295 to 300 of the heavy chain, and most preferably at position 297 of the heavy chain. Using this conserved glycosylation position of the antibody, the resulting conjugate is formed as a symmetric dimer, in which each half-antibody contains one F 2Both click probes contain F 2 form arms bearing three or more payloads. Some antibodies may have a second glycosylation site per half antibody, which is not used as a conjugation site in embodiments where z=2. One skilled in the art can perform the enzymatic conversion in a way that only the primary glycosylation site is available for conjugation. Alternatively, one skilled in the art can perform the enzymatic conversion in a way that the secondary glycosylation site is also available for conjugation, thereby doubling the DAR of the antibody-drug conjugate. In this embodiment, z=4, and each half antibody contains two F 2 , and all four click probes F 2 forms an arm with three or more payloads.

[0071]

[0071] L 6 Ab F 1 or Z 1 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - (wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0 or 1, and L 7 is —N(H)C(O)CH—, —N(H)C(O)CF—, or —CH—. Typically, L 6 is formed at least in part by the glycans of the antibody.

[0072]

[0072] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan or a part thereof. Thus, -GlcNAc(Fuc) of the glycan w -(G) j- is typically derived from the original antibody, GlcNAc is an N-acetylglucosamine moiety, and Fuc is a fucose moiety. Fuc is typically attached to GlcNAc via an α-1,6-glycosidic bond. Usually, antibodies can be fucosylated (w=1) or non-fucosylated (w=0). In the context of the present invention, the presence of the fucosyl moiety is irrelevant, and fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates achieve similar results. The GlcNAc residue, sometimes referred to as the core-GlcNAc residue, is the monosaccharide that is directly attached to the peptide portion of the antibody.

[0073] S is core-GlcNAc(Fuc) w The moiety can be directly linked to the core-GlcNAc(Fuc) moiety, i.e., j=0, which means that the remainder of the glycan is core-GlcNAc(Fuc) before S is attached. w Such glycan trimming is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, the core-GlcNAc(Fuc) w There are one or more monosaccharide residues between the moiety and S, i.e., j is an integer ranging from 1 to 10, preferably j=1 to 5. In one preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer ranging from 2 to 5. In another preferred embodiment, (G) j is the oligosaccharide fraction containing j monosaccharide residues G, where j is 1. (G) j is typically linked to GlcNAc(Fuc) via a β-1,4-linkage w In a preferred embodiment, j is 0, 1, 3, 4, or 5, more preferably j is 0 or 1, and most preferably j is 0.

[0074]

[0074] Any monosaccharide that may be present in a glycan can be used as G, but each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and N-acetylneuraminic acid. More preferred choices for G are galactose, N-acetylglucosamine, and mannose. When j=1, it is preferred that G=galactose and S=N-acetylneuraminic acid.

[0075]

[0075] When j is 3 to 10, (G) j can be linear or branched. Branched oligosaccharides (G) j Preferred examples are (a), (b), (c), (d), (e), (f), (g), and (h) shown below. [ka]

[0076] (G) where j>2 j When present, it is preferred that it terminates with GlcNAc or Gal, preferably GlcNAc. In other words, the monosaccharide residue directly linked to S is preferably GlcNAc or Gal. The presence of a GlcNAc moiety facilitates the synthesis of functionalized antibodies, since the monosaccharide derivative S can be easily introduced to a terminal GlcNAc residue by glycosyl transfer. The presence of a Gal moiety facilitates the synthesis of functionalized antibodies, since the monosaccharide derivative S sialic acid can be easily introduced to a terminal Gal residue by sialyltransferase. (G) having structures (a) to (h) j In the preferred embodiment described above for, the moiety S can be linked to any of the terminal GlcNAc residues, ie, not those with a wavy bond linked to a core GlcNAc residue on the antibody.

[0077] Antibodies and antibody conjugates with j=0 or 1 do not bind or have significantly reduced binding to Fc-gamma receptors, whereas antibodies and antibody conjugates with j in the range of 4 to 10 bind to Fc-gamma receptors. Thus, by selecting a particular value for j, a desired degree of binding to Fc-gamma receptors can be obtained. Thus, j=0, 1, 4, 5, 6, 7, 8, 9, or 10 is preferred, j=0, 1, 4, or 5 is more preferred, and the antibody is most preferably trimmed so that j=0.

[0078]

[0078] S is a sugar or sugar derivative. The term "sugar derivative" is used herein to refer to a derivative of a monosaccharide sugar, i.e., a monosaccharide sugar containing a substituent and / or functional group. Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH), galactosamine (GalNH), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), also known as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Preferably, S is selected from Gal, GlcNAc, GalNAc, and NeuNAc. In a particularly preferred embodiment, S is GalNAc.

[0079]

[0079] Linking group Z 1 or reactive group F 1 may be directly bonded to S, or S and Z 1 Or F 1 Between them is the linker L 7 may be present. Therefore, L 7 may be present (w'=1 or 2) or absent (w'=0). Typically, each moiety Z is 7 and thus in one embodiment, x with w'=0. Preferably, L 7is absent and each linking moiety Z is directly bonded to S. If present, L 7 may be selected from -N(H)C(O)CH-, -N(H)C(O)CF-, or -CH-. In a preferred embodiment, x=1 and w'=0 or 1, and most preferably, x=1 and w'=0.

[0080] [Linking group Z 1 and Z 2 ]

[0080] Z 1 and Z 2 is a linking group that covalently links the antibody to the payload of the conjugate according to the invention. The term "linking group" herein refers to a structural element resulting from the reaction between Q and F that links one part of a conjugate to another part of the same conjugate, as used herein. Z 1 Q 1 and F 1 Similarly, Z 2 Q 2 and F 2 As used herein, Z is formed by a click reaction between Z 1 and Z 2 Q refers to Q 1 and Q 2 F refers to F 1 and F 2 refers to

[0081] As will be appreciated by those skilled in the art, the exact nature of the linking group will depend on the exact structures of click probes Q and F. Those skilled in the art will be able to identify suitable reaction partners Q that will react with each other. 1 / F 1 and Q 2 / F 2For example, when F includes or is an alkynyl group, the complementary group Q includes an azido group. For example, when F includes or is an azido group, the complementary group Q includes an alkynyl group. For example, when F includes or is a cyclopropenyl group, trans-cyclooctene group, cycloheptyne, or cyclooctyne group, the complementary group Q includes a tetrazinyl group. In these particular cases, as shown in Figure 4, Z is merely an intermediate structure, releasing N2, thereby generating a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0082] The linking group Z is obtained by a cycloaddition reaction, preferably a [4 + 2] cycloaddition or a 1,3 dipolar cycloaddition. Conjugation reactions via cycloaddition are known to those skilled in the art, and those skilled in the art will be able to select appropriate reaction partners F and Q and understand the nature of the resulting linking group Z. A preferred cycloaddition is a [4 + 2]-cycloaddition (e.g., Diels-Alder reaction) or a [3 + 2]-cycloaddition (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition, more preferably an alkyne-azide cycloaddition, is used. Cycloadditions such as the Diels-Alder reaction and the 1,3-dipolar cycloaddition are known in the art and one of ordinary skill in the art would know how to perform them.

[0083] Preferably, Z contains a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, or pyridazine, more preferably triazole, isoxazoline, or pyridazine. A triazole moiety is preferably present in Z. In one embodiment, Z comprises a (hetero)cycloalkene moiety, i.e., is formed from Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z comprises a (hetero)cycloalkane moiety, i.e., is formed from Q comprising a (hetero)cycloalkene moiety. As used herein, aromatic rings such as triazole rings are considered heterocycloalkane rings because they are formed by the reaction of an alkyne moiety with an azide moiety.

[0084] In a preferred embodiment, Z has the structure (Z1): [ka] where: [ka] A bond shown as is a single or double bond. Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za) to (Zm) as defined below, where the carbon atom marked with ** is the carbon atom of (Z1) to which ring Z is fused. [ka] corresponding to the two carbon atoms of the bond shown as: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, - CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 , or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked via L; - u is 0, 1, 2, 3, 4, or 5; u' is 0, 1, 2, 3, 4, or 5, where u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; - v=an integer in the range 8 to 16; Ring Z is formed by cycloaddition and is preferably selected from (Za) to (Zm).

[0085] In a preferred embodiment, u+u′=0, 4, 5, 6, 7, or 8, more preferably 0, 4, or 5. [ka] When the bond shown as: is a double bond, it is preferred that u+u'=4, 5, 6, 7, or 8, and more preferably u+u'=4 or 5. [ka] When the bond shown as is a single bond, it is preferred that u+u'=0 or 5. Preferably, the wavy bond marked with * is optionally L 6 The bond is connected to CB via , and the wavy bond labeled with ** is connected to L.

[0086] Z comprises a (hetero)cycloalkene moiety, i.e. [ka] It is particularly preferred that the bond shown as is a double bond. In a preferred embodiment, Z is selected from structures (Z2) to (Z20) shown below: [ka]

[0087]

[0087] where the connection to L is indicated by a wavy bond. (-) is an anion, preferably a pharmaceutically acceptable anion. (+)is a cation, preferably a pharmaceutically acceptable cation. Ring Z is formed by a cycloaddition reaction and is preferably triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za) to (Zm) shown below, where the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z2) to (Z20) to which ring Z is fused. Since the linking group Z is formed by reaction with a (hetero)cycloalkyne in the context of this embodiment, [ka] A bond shown as is a double bond. [ka]

[0088] where R 29 is hydrogen, C 1~6 Alkyl, aryl, C(O)C 1~6 Alkyl, C(O)-aryl, C(O)C 1~6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1~6 Alkyl, and C(O)-NR 33 -aryl, and R 33 is H or C 1~4 Preferably, R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It has been found that a hydrogen atom provides optimal reactivity for the cycloaddition reaction, particularly when the ring (Zl) is formed. Thus, in a preferred embodiment, the ring Z is (Zl), where R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl, more preferably R 29is hydrogen.

[0089]

[0089] When Z contains a (hetero)cycloalkene moiety, ring Z is preferably selected from (Za), (Zj), (Zk), or (Zl), and more preferably ring Z is according to structure (Za) or (Zl).

[0090] In further preferred embodiments, Z is selected from structures (Z21) to (Z38a) shown below: [ka]

[0091]

[0091] Here, the connection to L is shown as a wavy bond. Structure (Z29) can be in the endo or exo configuration, preferably in the endo configuration. In structure (Z38), B (-) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) to (Zm) defined above.

[0092] In preferred embodiments, Z comprises an optionally substituted (hetero)cyclooctene or (hetero)cycloheptene moiety, preferably represented by the structures (Z), (Z), (Z), (Z), (Z), (Z), (Z), or (Z). Each of these preferred options for Z is further defined herein below.

[0093]

[0093] Thus, in a preferred embodiment, Z comprises an optionally substituted heterocycloheptene moiety represented by structure (Z37): Preferably, the heterocycloheptene moiety represented by structure (Z37) is unsubstituted.

[0094] In a preferred embodiment, Z comprises a (hetero)cyclooctene moiety represented by structure (Z8), more preferably represented by (Z29), which is optionally substituted. Preferably, the cyclooctene moiety represented by structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z is preferably a (hetero)cyclooctene moiety represented by structure (Z39) shown below, where V is (CH2) l where l is an integer ranging from 0 to 10, preferably from 0 to 6. More preferably, l is 0, 1, 2, 3, or 4, more preferably, l is 0, 1, or 2, and most preferably, l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z has structure (Z42), further defined below.

[0095] In an alternative preferred embodiment, Z comprises a (hetero)cyclooctene moiety represented by structure (Z26), (Z27) or (Z28), which are optionally substituted. In the context of this embodiment, Z preferably comprises structure (Z40) or (Z41) shown below, where Y 1 is O or NR 11 and R 11 are independently hydrogen, straight-chain or branched C1-C 12 Alkyl group or C4-C 12 (Hetero)cyclooctene moieties represented by structure (Z40) are optionally O-sulfated at one or more positions, while the rings of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moieties represented by structure (Z40) or (Z41) are not further substituted. Also preferably, Z is represented by structure (Z43), further defined below.

[0096] In an alternative preferred embodiment, Z comprises a heterocycloheptenyl group and is represented by structure (Z37). [ka]

[0097] In a particularly preferred embodiment, Z comprises a cyclooctenyl group and has the structure (Z42): [ka] It is expressed as: where: - the bond labeled * is connected to CB, and the wavy bond labeled ** is connected to L; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, - CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 19are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)alkyl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is a second occurrence of Z (or Q) or D linked via a spacer moiety; and - l is an integer ranging from 0 to 10.

[0098] In a preferred embodiment of the group represented by structure (Z42), R 15 is hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the group represented by structure (Z42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the group represented by structure (Z42), R 19 is H. In preferred embodiments of the group represented by structure (Z42), l is 0 or 1, and more preferably l is 1.

[0099] In a particularly preferred embodiment, Z comprises a (hetero)cyclooctenyl group and has the structure (Z43): [ka] It is expressed as: where: - the bond labeled * is connected to CB, and the wavy bond labeled ** is connected to L; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, - CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y is N or CR 15 and; A carbon atom in the fused aromatic ring may be replaced by a nitrogen atom as in (Z6a) to (Z6d), preferably Y is CR 15 is.

[0100] In a preferred embodiment of the group represented by structure (Z43), R 15 is hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 is hydrogen and -S(O)3 (-)In preferred embodiments of the group represented by structure (Z43), Y is N or CH, more preferably Y=N.

[0101] In particularly preferred embodiments, Z comprises a heterocycloheptenyl group and is represented by structure (Z37) or (Z38a), where ring Z is a triazole. [ka]

[0102] In an alternative preferred embodiment, the linking group Z comprises a (hetero)cycloalkane moiety, i.e. [ka] The bond shown as is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkyl group or a cycloalkyl group, preferably a cycloalkyl group, where the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornenyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, all of which may be optionally substituted. Particularly preferred are a (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, where the (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group is optionally substituted. Preferably, Z comprises a cyclopropyl moiety represented by structure (Z44), a heterocyclobutane moiety represented by structure (Z45), a norbornane or norbornene group represented by structure (Z46), a (hetero)cycloheptyl moiety represented by structure (Z47), or a (hetero)cyclooctyl moiety represented by structure (Z48), where Y 3 is C(R 23 )2, NR 23 , or O, and each R 23are individually hydrogen, C1-C6 alkyl, or optionally connected to L via a spacer, and [ka] is a single bond or a double bond. In a further preferred embodiment, a cyclopropyl group is represented by structure (Z49). In another preferred embodiment, a (hetero)cycloheptane group is represented by structure (Z50) or (Z51). In another preferred embodiment, a (hetero)cyclooctane group is represented by structure (Z52), (Z53), (Z54), (Z55), or (Z56). [ka]

[0103]

[0103] Here, the R group on Si in (Z50) and (Z51) is typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is formed in a cycloaddition reaction and is typically selected from structures (Zn) to (Zu), the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z44) to (Z56) to which ring Z is fused, and the carbon marked with * is linked to CB. Since the linking group Z is formed by reaction with a (hetero)cycloalkene in the context of this embodiment, [ka] The bond shown above as is a single bond. [ka]

[0104] where R 29 is hydrogen, C 1~6 Alkyl, aryl, C(O)C 1~6 Alkyl, C(O)-aryl, C(O)C 1~6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C1~6 Alkyl, and C(O)-NR 33 -aryl, and R 33 is H or C 1~4 Preferably, R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It has been found that a hydrogen atom provides optimal reactivity for the cycloaddition reaction, especially when the ring (Zu) is formed. Thus, in a preferred embodiment, the ring Z is (Zu), where R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl, more preferably R 29 is hydrogen.

[0105]

[0105] When Z contains a (hetero)cycloalkane moiety, ring Z is preferably selected from (Zn), (Zs), (Zt) or (Zu), and most preferably ring Z is represented by the structure (Zu).

[0106]

[0106] In a preferred embodiment, the linking group Z comprises a moiety selected from (Z1) to (Z56), and the ring Z is selected from (Za) to (Zu).

[0107] In the present invention, Z 1 and Z 2 The exact structure of Z 1 Q 1 and F 1 while Z is formed by reaction with 2 Q 2 and F 2 In this specification, F 2 Q 2 It reacts with Q 1 Since it does not react with Q 1 and Q 2 should be different.

[0108] In a preferred embodiment, F 1 is azide, and Q 1is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F 2 is a tetrazine or nitrone, and Q 2 is a bicyclononyne or a cycloalkene, such as trans-cyclooctene or cyclopropene. More preferably, F 1 Azide, Q 1 is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F 2 is a tetrazine and Q 2 is bicyclononyne.

[0109]

[0109] where F 1 and Q 1 The reaction with is preferably carried out with a linking group Z represented by structure (Z5), (Z6), (Z7), (Z11), (Z17), (Z18), (Z19), or (Z19a) (wherein ring Z is represented by structure (Za)), preferably represented by structure (Z26), (Z27), (Z28), (Z32), (Z37), (Z38), or (Z38a), more preferably represented by structure (Z40), (Z41), or (Z43), or a linking group Z represented by structure (Z37) or (Z43). 1 where Q 2 is preferred, where F 2 and Q 2 is preferably a linking group Z represented by structure (Z8), (Z44), (Z47), (Z48), (Z49), (Z54), (Z55), or (Z56), more preferably represented by structure (Z29), (Z48), or (Z49), and most preferably represented by structure (Z42). 2 Here, ring Z is represented by structure (Zd), (zl), (zq), or (Zu), preferably represented by structure (Zl) or (Zu), and most preferably represented by structure (Zl). Alternatively, the linking group Z 2 is represented by the structure (Z8) (wherein ring Z is represented by the structure (Z1)), preferably represented by the structure (Z29), and more preferably represented by the structure (Z42).

[0110] [Linker L A ] Linker L A The payload D is B and linking group Z 2 via the linking group Z 1 (in a conjugate according to the invention) or to the Ab via a reactive group F 2 a reactive group Q 1 (in a linker according to the present invention). Linkers are known in the art and may be cleavable or non-cleavable. A is preferably not a cleavable linker, while the linker L B is preferably cleavable.

[0111] Linker L A is Z 1 (or Q 1 ) and one occurrence of Z 2 (or F 2 ) is linked to the occurrence of y in the linker L A The valency of is y+1, i.e., "(y+1)-valent." Here, "(y+1)-valent" refers to the number of connection points of the reactive group F or Q (before reaction) or the linking group Z (after reaction). Thus, when y=2, the linker has one Z 1 / Q 1 and two Zs 2 / F 2 and the linker has a valency of 2+1=3. Thus, when y=2, L A is trivalent. Therefore, when y=3, the linker has one Z 1 / Q 1 and three Zs 2 / F 2 and the linker has a valency of 3+1=4. Therefore, when y=3, L A is tetravalent. Therefore, when y=4, the linker has one Z 1 / Q 1 and four Zs 2 / F 2 and the linker has a valency of 4+1=5. Thus, when y=4, L A is pentavalent.

[0112] Linker L A can be referred to as "heterobifunctional", which means that Z 1 / Q 1 and Z 2 / F 2 This means that the compound contains two different functionalities, which refer to chemically different bonding properties to the compound.

[0113] In a preferred embodiment, L A is the structure -(L 11 )-BM(L 12 -) y (In the formula, -L 11 Q 1 or Z 1 Each L 12 is F 2 or Z 2 is connected to L 11 and L 12 are individually, C(R 13 )2, C(R 13 )=C(R 13 ), C≡C, C(O), NR 13 ,O,S,S(O),S(O)2,PR 13 , and P(O)R 13 Preferably, the building blocks are selected from CH, CH=CH, C(O), NR 13 , O, S, S(O), and S(O)2; - Each R 13 is as defined below in structure (23), preferably R 13 is H; and - BM is the branching part) The branching moiety is further defined below.

[0114] In a more preferred embodiment, L A is the structure -(L 11 )-BM(L12 -)(L 13 -) (In the formula, -L 11 Q 1 or Z 1 is connected to L 12 and L 13 is F 2 or Z 2 is connected to L 11 , L 12 , and L 13 are individually, C(R 13 )2, C(R 13 )=C(R 13 ), C≡C, C(O), NR 13 ,O,S,S(O),S(O)2,PR 13 , and P(O)R 13 Preferably, the building blocks are selected from CH, CH=CH, C(O), NR 13 , O, S, S(O), and S(O)2; and - Each R 13 is as defined below in structure (23), preferably R 13 is H; and - BM is the branching part) The branching moiety is further defined below.

[0115]

[0114] L 11 , L 12 , and L 13 is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ , and NR 21 wherein y′ is 0, 1, or 2, preferably y′=2; and R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 In one embodiment, the optional substituent may be selected from the group consisting of a polar group, for example, an oxo group, a (poly)ethylene glycol diamine, a (poly)ethylene glycol or (poly)ethylene oxide chain, a (poly)propylene glycol or (poly)propylene oxide chain, a carboxylic acid group, a carbonate group, a carbamate group, a cyclodextrin, a crown ether, a sugar (e.g., a monosaccharide, an oligosaccharide), a phosphate or an ester thereof, a phosphonic acid or an ester thereof, a phosphinic acid or an ester thereof, a sulfoxide, a sulfone, a sulfonic acid or an ester thereof, a sulfinic acid, or a sulfenic acid.

[0116] In the context of the present invention, the structure Q 1 L A (F 2 ) y The linker construct of the structure Ab(F 1 ) z reacting with F per antibody Aba 1 and Q 1 Thus, z linking groups Z are used per antibody Ab. 1 When the reaction is complete, z × y click probes F are formed per antibody.2 The antibody linker construct thus formed contains the structure Ab(Z 1 L A (F 2 ) y ) Z It has.

[0117] [Branch part BM]

[0116] A "branched moiety" in the context of this invention refers to a moiety embedded in a linker that connects three moieties. In other words, the branched moiety contains at least three bonds to other moieties.

[0118] Any moiety containing at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. Suitable branched moieties include carbon atoms (BM-1), nitrogen atoms (BM-3), phosphorus atoms (phosphines (BM-5) and phosphine oxides (BM-6)), aromatic rings such as phenyl rings (BM-7) or pyridyl rings (BM-9), (hetero)rings (BM-11 and BM-12), and polycyclic moieties (BM-13, BM-14, and BM-15). Preferably, BM is selected from carbon atoms, nitrogen atoms, phosphorus atoms, (hetero)aromatic rings, (hetero)rings, or polycyclic moieties, more preferably, BM is a carbon atom or a nitrogen atom. When BM is a nitrogen atom, the branched nitrogen can be connected to Z, for example, via a C=O group. 1 or Q 1 and via two substituents on the nitrogen 2 or F 2 When BM is a carbon atom, the carbon atom is preferably part of a trivalent amino acid such as lysine, aspartic acid, or glutamic acid.

[0119]

[0118] Suitable branching moieties BM are selected from the structures (BM-1) to (BM-15) shown below, where the three branches, i.e., bonds to other moieties defined above, are indicated by * (bonds marked with *). [ka]

[0120] In (BM-1), one of the branches marked with an * is [ka] The bond may be a single bond or a double bond, as shown in In (BM-11) to (BM-15), the following applies: - each of n, p, q, and q is individually an integer ranging from 0 to 5, preferably 0 or 1, most preferably 1; -W 1 , W 2 , and W 3 Each of C(R 21 ) w and N are independently selected; -W 4 , W 5 , and W 6 Each of C(R 21 ) w+1 , N(R 22 ) w , O, and S; - each [ka] represents a single or double bond; - w is 0 or 1 or 2, preferably 0 or 1; - Each R 21 are independently hydrogen, OH, C1-C 24 Alkyl groups, C1-C 24 Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl groups, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, wherein C1-C 24 Alkyl groups, C1-C 24Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 3 and optionally interrupted by one or more heteroatoms selected from, where R 3 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; - Each R 22 are independently hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, C1-C 24 Alkyl groups, C1-C 24 Alkoxy group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 3 and optionally interrupted by one or more heteroatoms selected from, where R 3 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

[0121] Those skilled in the art will appreciate that the value of w, and [ka] The join order of the bond represented by It is understood that they are interdependent. Thus, whenever an occurrence of W is attached to an endocyclic double bond, w=1 for that occurrence of W, and whenever an occurrence of W is attached to two endocyclic single bonds, w=0 for that occurrence of W. For BM-12, at least one of o and p is not 0.

[0122] Representative examples of branched moieties according to structures (BM-11) and (BM-12) include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, aziridine, azetidine, diazetidine, oxetane, thietane, pyrrolidine, dihydropyrrolyl, tetrahydrofuranyl, dihydrofuranyl, thiolanyl, imidazolinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, dioxolanyl, dithiolanyl, piperidinyl, oxanyl, thianyl, piperazinyl, morpholino, thiomorpholino, dioxanyl, trioxanyl, dithianyl, trithianyl, azepanyl, oxepanyl, and thiepanyl. Preferred cyclic moieties for use as branching moieties include cyclopropenyl, cyclohexyl, oxanyl (tetrahydropyran), and dioxanyl. The substitution pattern of the three branches determines whether the branching moiety is of structure (BM-11) or structure (BM-12).

[0123]

[0122] Representative examples of branched moieties having structures (BM-13) to (BM-15) include decalin, tetralin, dialin, naphthalene, indene, indane, isoindene, indole, isoindole, indoline, and isoindoline.

[0124] In a preferred embodiment, BM is a carbon atom. If the carbon atom has structure (BM-1) and all four bonds to separate moieties, the carbon atom is chiral. The stereochemistry of the carbon atom is not critical to the present invention and can be either S or R. The same applies to phosphine (BM-6). Most preferably, the carbon atom has structure (BM-1). In the carbon atom of structure (BM-1), one of the branches indicated by * can be a double bond, in which case the carbon atom can be part of an alkene or imine. When BM is a carbon atom, it can be part of a larger functional group such as an acetal, ketal, hemiketal, orthoester, orthocarbonate ester, amino acid, etc. Preferred amino acids in this regard are Asp, Gly, Lys, and iGlu. This also applies when BM is a nitrogen or phosphorus atom, in which case it can be part of an amide, imide, imine, phosphine oxide (as in BM-6), or phosphotriester.

[0125]

[0124] In a preferred embodiment, BM is a phenyl ring. Most preferably, the phenyl ring is according to structure (BM-7). The substitution pattern of the phenyl ring can be of any regiochemistry, such as a 1,2,3-substituted phenyl ring, a 1,2,4-substituted phenyl ring, or a 1,3,5-substituted phenyl ring. To allow optimal flexibility and conformational freedom, the phenyl ring is preferably according to structure (BM-7), and most preferably, the phenyl ring is 1,3,5-substituted. The same applies to the pyridine ring of (BM-9).

[0126] In the preferred structure for the BM defined above, the BM typically contains three linking points. Those skilled in the art can determine the corresponding BMS having four or five linking points. For example, (BM-1) and (BM-7) to (BM-15) are also suitable as BMs having more linking points. Alternatively, the linker may contain two or more BMs to form four or five linking points.

[0127]

[0126] In preferred embodiments, the branching moiety BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring, or a polycyclic moiety, more preferably selected from a carbon atom or a nitrogen atom.

[0128] [Linker L B ] Linker L B The payload D is linked to Ab and the linking group Z 2 and linker L A either via a linking group (in a conjugate according to the invention) or by attaching a payload D to a reactive group Q 2 (in a payload linker construct). Linkers are known in the art and may be cleavable or non-cleavable. B is preferably a cleavable linker, while the linker L A is preferably not cleavable.

[0129] Linker L B is Z 2 (or Q 2 ) and an occurrence of x in D. Thus, the linker L B The valence of is x+1, that is, "(x+1) valence". Here, "(x+1) valence" refers to the reactive group Q 2 (before reaction) or linking group Z 2 This refers to the number of linkage points (after reaction). Therefore, when x=1, the linker is a single Z 2 / Q 2 and one payload D, and the linker has a valency of 1+1=2. Therefore, when x=1, L B is divalent. Therefore, when x=2, the linker has one Z 2 / Q 2 and two payloads D, and the linker has a valency of 2+1=3. Therefore, when x=2, L B is trivalent. Therefore, when x=3, the linker has one Z 2 / Q 2and three payloads D, the linker has a valency of 3+1=4. Therefore, when x=3, L B is tetravalent. Therefore, when x=4, the linker has one Z 2 / Q 2 and linked to four payloads D, the linker has a valency of 4+1=5. Therefore, when x=4, L B is pentavalent.

[0130] Linker L B can be referred to as "heterobifunctional", which means that Z 2 / Q 2 and D.

[0131] In a preferred embodiment, L B is the structure -(L 1 )-(BM) r’ -[(L 2 ) o -(L 3 ) p -(L 4 ) q -] x (In the formula, -L 1 Q 2 or Z 2 is connected to L 4 All occurrences of are concatenated to D; -L 1 , L 2 , L 3 , and L 4 are individually, Q 2 or Z 2 is a linker that connects together D; - o, p and q are each independently 0 or 1, preferably o=p=1; - r' is 0 or 1, where r' is 0 when x is 1, or r' is 1 when x is 2, 3, or 4; - BM is the branching part) The branching moiety is further defined above.

[0132] In an alternative preferred embodiment, x=1 and L B is the structure -(L 1 )-(L 2 ) o -(L 3 ) p -(L 4 ) q - (In the formula, -L 1 Q 2 or Z 2 and L 4 is concatenated to D; -L 1 , L 2 , L 3 , and L 4 are individually, Q 2 or Z 2 is a linker that connects together D; - o, p and q are each independently 0 or 1, preferably o=p=1; - BM is the branching part) The branching moiety is further defined above.

[0133] In a more preferred embodiment, x=2 and L B is the structure -(L 1 )-(BM) ’ -[(L 2 ) o -(L 3 ) p -(L 4 ) q -]2 (In the formula, -L 1 Q 2 or Z 2 and L 4 is concatenated to D; -L 1、 L 2 , L3 , and L 4 are individually, Q 2 or Z 2 is a linker that connects together D; - o, p and q are each independently 0 or 1, preferably o=p=1; - BM is the branching part) The branching moiety is further defined above.

[0134]

[0133] Alternatively, L B is the structure -(L 1 )-[(L 2 ) o -(L 3 ) p -(L 4 ) q -] x (wherein BM is a linker L when x=2, 3, or 4) 1 embedded in is a heterobifunctional linker of the formula:

[0135] In the context of the present invention, the structure Q 2 (L B )D x The payload linker construct has the structure Ab(Z 1 L A (F 2 ) y ) z 1 antibody-linker construct, thereby reacting with F per antibody Ab. 2 and Q 2 A click reaction of z×y occurs between the antibody Ab and the linking group Z. 2 Upon completion of the reaction, an antibody conjugate containing z x y x x payload D per antibody is formed. The antibody conjugate thus formed has the structure Ab(Z 1 L A (Z 2 (L B )D x ) y ) zIt has.

[0136]

[0135] L 1 , L 2 , L 3 and L 4 is a linker or linking unit, and each of o, p and q is individually 0 or 1, preferably o=p=1. In a preferred embodiment, at least the linker L 1 and L 2 is present (i.e., o=1; p=0 or 1; q=0 or 1), and more preferably, a linker L 1 , L 2 , and L 3 (i.e., o=1; p=1; q=0 or 1). In one embodiment, L 1 , L 2 , L 3 , and L 4 are each independently (hetero)aryl, CH, CH=CH, C≡C, C(O), NR 13 ,O,S,S(O),S(O)2,PR 13 , and P(O)R 13 and preferably the building blocks are selected from aryl, CH, CH=CH, C(O), NR 13 , O, S, S(O), and S(O)2. 1 , L 2 , L 3 , and L 4 Preferred embodiments for each are shown below.

[0137] Linkers, especially linkers L 1 may contain one or more branching points for the attachment of multiple payloads to a single linking group. In a preferred embodiment, the linker of the conjugate according to the present invention contains a branching moiety. A "branching moiety" in the context of the present invention refers to a moiety embedded in a linker that connects three moieties. In other words, a branching moiety has at least three bonds to other moieties, typically Z 1or Q, one bond to a payload D, and one bond to a second payload D. The branching moiety, if present, is connected to the linker L 1 and preferably Sp 3 Part of or NR 13 Preferably, the branched moiety is embedded as a nitrogen atom of the formula (I). Any moiety containing at least three bonds to other moieties is suitable as a branched moiety in the context of the present invention. In preferred embodiments, the branched moiety is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring, or a polycyclic moiety. Most preferably, the branched moiety is a nitrogen atom.

[0138] [Linker L 1 ]

[0137] L 1 is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group, and arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ , and NR 21 wherein y′ is 0, 1, or 2, preferably y′=2; and R 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24(Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 In one embodiment, the optional substituent may be selected from the group consisting of a polar group, for example, an oxo group, a (poly)ethylene glycol diamine, a (poly)ethylene glycol or (poly)ethylene oxide chain, a (poly)propylene glycol or (poly)propylene oxide chain, a carboxylic acid group, a carbonate group, a carbamate group, a cyclodextrin, a crown ether, a sugar (e.g., a monosaccharide, an oligosaccharide), a phosphate or an ester thereof, a phosphonic acid or an ester thereof, a phosphinic acid or an ester thereof, a sulfoxide, a sulfone, a sulfonic acid or an ester thereof, a sulfinic acid, or a sulfenic acid.

[0139] In a preferred embodiment, the linker L 1 contains a polar group, which also 1 Such polar groups can be present in the chain of (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,x'-diaminoalkanes (where x' is the number of carbon atoms in the alkane, preferably x'=1 to 10), -(O) a -C(O)-NH-S(O)2-NR 13 - (see structure (23) as further defined below), -C(S(O)3 (-) )-, - C(C(O)2 (-) )-, -S(O)2-, -P(O)2 (-) -, -O(CH2CH2O) t -, -NR 30 (CH2CH2NR 30 ) t - and may be selected from the following two structures: [ka]

[0140]

[0139] With respect to the polar groups defined herein above, which terminus is Z 1 Which end is linked to (L 2 ) o It is irrelevant whether the

[0141] The polar group may also contain an amino acid, preferably selected from Arg, Glu, Asp, Ser, and Thr, where R 13 is further defined below in structure (23), where t is an integer ranging from 0 to 15, preferably from 1 to 10, more preferably from 2 to 5, and most preferably t=2 or 4. Each R 30 are individually, H, C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkaryl, or C 1~12 Aralkyl. Linker L 1 may contain two or more such polar groups, such as at least two polar groups. The polar groups may also be linked to a linker, L, branched from a branching moiety as defined elsewhere. 1 It can be present in the branch of L 1 Preferably, nitrogen or carbon atoms are used as branching moieties in connection with -O(CH2CH2O) t The presence of polar groups is particularly preferred.

[0142] In a preferred embodiment, the linker L 1 is or includes a sulfamide group, preferably a sulfamide group represented by structure (23): [ka]

[0143] The wavy line indicates the remainder of the compound, typically Q 2 or Z 2 to, and L 2 , L 3 , L 4or a linkage to D. Preferably, (O) a The C(O) part is Q 2 or Z 2 linked to NR 13 Part is L 2 , L 3 , L 4 , or D, preferably L 2 is linked to.

[0144] In structure (23), a=0 or 1, preferably a=1, and R 13 is hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR 14 and optionally interrupted by one or more heteroatoms selected from, where R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; 13 is preferably Sp as defined below, optionally via a spacer moiety. 2 and in one embodiment, D is linked to N via -(B) e -(A) f -(B) g is linked to N via —C(O)—; or R 13 is linked to another position in the linker, optionally via a spacer moiety, to form a cyclic structure. For example, R 13 may be linked to a linker via a CH2CH2 spacer moiety to form a piperazinyl ring, and the linkage to D is via the second nitrogen of the piperazinyl ring.

[0145] In a preferred embodiment, R 13 is hydrogen, C1 to C 20 Alkyl groups, preferably C1-C 16 Alkyl groups, more preferably C1-C 10 is an alkyl group, or is optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, where the alkyl group is optionally substituted and can be selected from O, S, and NR 14 and R 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 13 is C1~C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 is an alkyl group, optionally interrupted by one or more O atoms, and the alkyl group is optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment, R 13 It is further preferred that R is a (poly)ethylene glycol chain containing a terminal -OH group. 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, and t-butyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, and i-propyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker, even more preferably selected from the group consisting of hydrogen, methyl, and ethyl, or optionally linked via a spacer moiety to further occurrences of D or elsewhere in the linker. Even more preferably, R 13 is hydrogen or is optionally linked via a spacer moiety to a further occurrence of D or elsewhere in the linker, and most preferably is 13 is hydrogen.

[0146] In a preferred embodiment, L 1 The structure (24): [ka] It is expressed as:

[0147] where a and R 13 is as defined above, and Sp 1 and Sp 2 is independently a spacer moiety, and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, more preferably b=0 and c=1. In one embodiment, the spacer Sp 1 and Sp 2 are independently linear or branched C1 to C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene groups and C9-C 200 alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups, which are optionally substituted and include O, S, and NR 16 and optionally interrupted by one or more heteroatoms selected from the group consisting of: 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24The alkyl, alkenyl, alkynyl, and cycloalkyl groups are optionally substituted. The alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, and preferably, the groups are interrupted by one or more O atoms and / or one or more S-S groups.

[0148] More preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylene group, C3-C 100 Cycloalkylene group, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkylarylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylalkenylene groups and C9-C 100 alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups, which are optionally substituted and include O, S, and NR 16 and optionally interrupted by one or more heteroatoms selected from the group consisting of: 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 It is selected from the group consisting of cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0149] Even more preferably, the spacer moiety Sp 1 and Sp 2 is, if present, independently a straight chain or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylene group, C3-C 50 Cycloalkylene group, C5-C 50 Cycloalkenylene group, C8-C 50 Cycloalkynylene group, C7-C 50 Alkylarylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene groups and C9-C 50 alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups, which are optionally substituted and include O, S, and NR 16 and optionally interrupted by one or more heteroatoms selected from the group consisting of: 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 It is selected from the group consisting of cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0150] Even more preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylene group, C3-C 20 Cycloalkylene group, C5-C 20 Cycloalkenylene group, C8-C 20 Cycloalkynylene group, C7-C20 Alkylarylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene groups and C9-C 20 alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups, which are optionally substituted and include O, S, and NR 16 and optionally interrupted by one or more heteroatoms selected from the group consisting of: 16 are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 It is selected from the group consisting of cycloalkyl groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, and cycloalkyl groups are optionally substituted.

[0151] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and are not substituted with O, S, or NR 16 optionally interrupted by one or more heteroatoms, preferably O, selected from the group 16 are independently selected from the group consisting of hydrogen and C1 to C4 alkyl groups, and are preferably hydrogen or methyl.

[0152] Most preferably, the spacer moiety Sp 1 and Sp 2 When present, each independently represents a linear or branched C1-C 20 alkylene groups, which are optionally substituted and selected from the group consisting of O, S, and NR 16 optionally interrupted by one or more heteroatoms selected from the group 16are independently hydrogen, C1 to C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 In this embodiment, the alkylene group is unsubstituted and is selected from the group consisting of O, S, and NR 16 optionally interrupted by one or more heteroatoms, preferably O and / or S-S, selected from the group 16 are independently selected from the group consisting of hydrogen and C1 to C4 alkyl groups, and are more preferably hydrogen or methyl.

[0153] Therefore, the preferred spacer moiety Sp 1 and Sp 2 As -(CH2) r -, -(CH2CH2) r -, -(CH2CH2O) r -, -(OCH2CH2) r -, -(CH2CH2O) r CH2CH2-, -CH2CH2(OCH2CH2) r -, -(CH2CH2CH2O) r -, -(OCH2CH2CH2) r -, -(CH2CH2CH2O) r CH2CH2CH2- and -CH2CH2CH2(OCH2CH2CH2) r wherein r is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, 4, 5, 6, 7, or 8, even more preferably 1, 2, 3, 4, 5, or 6, and even more preferably 1, 2, 3, or 4.

[0154] Alternatively, the preferred linker L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g - (In the formula: - d=0 or 1, preferably d=1; - e=an integer in the range of 0 to 10, preferably e=0, 1, 2, 3, 4, 5, or 6, preferably an integer in the range of 1 to 10, most preferably e=1, 2, 3, or 4; - f=0 or 1, preferably f=0; wherein d+e+f is at least 1, preferably in the range of 1 to 5; preferably d+f is at least 1, preferably d+f=1. - g=0 or 1, preferably g=1; - k=0 or 1, preferably k=1; A is a sulfamide group represented by structure (23); B is a —CH—CH—O— or —O—CH—CH— moiety, or (B) e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 a —CH2— moiety, where e1 is defined the same as e; - W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - C(O)(CH2) m C(O)-, -C(O)(CH2) m C(O)NH-, or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably W is -OC(O)NH-, -C(O)(CH2) m C(O)NH— or —C(O)NH—, where m is an integer ranging from 0 to 10, preferably m=0, 1, 2, 3, 4, 5, or 6, and most preferably m=2 or 3; - Preferably L 1 (W) k and (C(O)) g preferably via C(O), 2 , L 3 , or D, preferably L 2 (connected to It can be expressed as:

[0155]

[0154] In relation to this embodiment, the wavy line in structure (23) represents (W) k , (B) e , and (C(O)) g A represents a linkage to an adjacent group such as structure (23) where a=1 and R 13 =H or C1~C 20 is an alkyl group, and more preferably R 13 =H or methyl, most preferably R 13 =H).

[0156] Preferred linkers L 1 is the structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -, wherein (a) k=0; d=1; g=1; f=0; B=—CH—CH—O—; e=1, 2, 3, or 4, preferably e=2. (b) k = 1; W = -C(O)(CH2) m C(O)NH-; m=2; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; f=0; g=1; e1=1, 2, 3, or 4, preferably e=1. (c) k=1; W=-OC(O)NH-; d=0; B=-CH2-CH2-O-; g=1; f=0; e=1, 2, 3, or 4, preferably e=2. (d) k = 1; W = -C(O)(CH2) mC(O)NH-; m=2; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; f=0; g=1; e1=1, 2, 3 or 4, preferably e1=4. (e)k=1;W=-OC(O)NH-;d=0;(B) e =-(CH2-CH2-O) e1 -CH2-CH2-; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4. (f)k=1;W=-(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, m=3; d=0; (B) e =-(CH2-CH2-O) e1 -CH2-CH2-; g=1; f=0; e1=1, 2, 3 or 4, preferably e1=4. (g) k=0; d=0; g=1; f=0; B=-CH2-CH2-O-; e=1, 2, 3 or 4, preferably e=2. (h) k=1; W=-C(O)NH-; d=0; g=1; f=0; B=-CH2-CH2-O-; e=1, 2, 3 or 4, preferably e=2.

[0157]

[0156] In this specification, when d and / or f=1, a=1 and R 13 Preferably = H. Most preferably, the linker is structure (a).

[0158] In a preferred embodiment, the linker L 1 Q or Z and (L 2 ) o and a branched nitrogen atom located in the backbone between and containing a further moiety D as a substituent, the moiety D being preferably linked to the branched nitrogen atom via a linker. An example of a branched nitrogen atom is the nitrogen atom NR in structure (23). 13 where R 13 is linked to the second occurrence of D via a spacer moiety. Alternatively, the branched nitrogen atom can be linked to the structure -(W) k -(A) d -(B) e -(A)f -(C(O)) g -L 1 In one embodiment, L 1 is -(W) k -(A) d -(B) e -(A) f -(C(O)) g -BM[-(A) d -(B) e -(A) f -(C(O)) g -]2, where A, B, W, d, e, f, g, and k are as defined above and are individually selected at each occurrence; and B M is a branching moiety, preferably -(A) d -(B) e -(A) f -(C(O)) g - is a branched nitrogen atom where two instances of - are connected, where both (C(O)) g The part is -(L 2 ) o -(L 3 ) p -(L 4 ) q -D, wherein L 2 , L 3 , L 4 , o, p, q, and D are as defined above and are each individually selected. In a preferred embodiment, L 2 , L 3 , L 4 , o, p, q, and D are (C(O)) g is the same for both parts d connected to

[0159] Preferred linkers L containing a branched nitrogen atom 1 is the structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -N[-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, wherein (i) k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH-CH-O-; A is a compound of structure (23) (a = 0 and R 13 =H); e=1, 2, 3, or 4, preferably e=2. (j) k = d = g = e' = g' = 1; f = d' = 0; W = -C(O)-; B = B' = -CH-CH-O-; A is a compound of structure (23) (a = 0 and R 13 =H); e=1, 2, 3, or 4, preferably e=2.

[0160] [Linker L 2 ] Linker L 2 is a peptide spacer. Linker L 2 is absent (o=0) or present (o=1). Preferably, the linker L 2 is present and o=1. Peptide spacer L 2 and a cleavable linker L 3 Combinations of the linker L are well known in the art. 2 The L serves as a recognition and cleavage site for the cleavage enzyme, and more preferably, the peptide is recognized by a specific cleavage enzyme. This allows for cleavage in specific environments where these cleavage enzymes are expressed, such as in specific tumors. Since different peptide sequences are cleaved by different enzymes, the L 2 The groups also allow the conjugate to be customized for a particular therapy. The peptide sequence can be cleaved by intracellular and / or extracellular enzymes.

[0161] The peptide spacer can also be (NH-CR 17 -CO) n where R 17 represents an amino acid side chain known in the art. 17Also included within this definition is proline, in which R is bonded to the nitrogen atom to form a cyclic moiety. Here, the amino acids may be natural or synthetic. Preferably, all amino acids are in the L configuration. n is an integer ranging from 1 to 5, preferably from 2 to 4. Thus, the peptide spacer contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3), or tetrapeptide (n=4), and most preferably, the peptide spacer is a dipeptide. R 17 represents an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, and more preferably those derived from the side chains of Val, Cit, Ala, Glu, and Lys. In other words, R 17 is preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp), and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred embodiments of R are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). 17is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0162]

[0161] Any peptide spacer may be used, but preferably the peptide spacer is Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Glu-Gly-Cit, Glu-Gly-Val, Asp-Val-Cit, iGlu- Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Asn-Asn, Ala-Ala-Asn, Al a-Asn, Asn-Ala, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly, Leu-Gly, Tyr-Gly, Ala-Gly, Pro-Gly, P he-Gly, Phe-Gly, Ser-Gly, Gly-Phe-Gly, Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly, Phe-Gly-Gly-Gly, Gly-Gly-Gly-Phe, Phe-Phe-Gly-Gly, Gly-Gly-Phe-Phe, Gly-Gly-Gly-Phe-Gly, and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-

[0023] The amino acid sequence of the present invention is selected from Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala, Asn-Asn, Ala-Ala-Asn, Asn-Ala, and most preferably Glu-Val-Ala, Glu-Gly-Cit, Val-Cit, Val-Ala, or Asn-Ala, where AcLys is eN-acetyl lysine and iGlu is isoglutamate. In one embodiment, L 2 In another embodiment, L 2 In another embodiment, L2 In another embodiment, L 2 = Glu-Gly-Cit. In another embodiment, L 2 =Glu-Val-Ala.

[0163] In one embodiment, the R of the amino acid side chain 17 is preferably substituted with a polar group selected from an oxo group, a (poly)ethylene glycol diamine, a (poly)ethylene glycol or (poly)ethylene oxide chain, a (poly)propylene glycol or (poly)propylene oxide chain, a carboxylic acid group, a carbonate group, a carbamate group, a cyclodextrin, a crown ether, a sugar (e.g., monosaccharide, oligosaccharide), a phosphate or ester thereof, a phosphonic acid or ester, a phosphinic acid or ester, a sulfoxide, a sulfone, a sulfonic acid or ester, a sulfinic acid, or a sulfenic acid.

[0164] In a particularly preferred embodiment, L 2 comprises a peptide spacer represented by the general structure (25), preferably L 2 has the general structure (25): [ka] It is expressed as:

[0165]

[0164] where R 17 is as defined above, preferably R 17 is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates (L 1 ) n and(L 3 ) p and preferably L represented by structure (25): 2 is via NH (L 1 ) n is connected to (L 3 ) p is linked to.

[0166] [Linker L 3 ] Linker L 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is absent (p=0) or present (p=1). Preferably, the linker L 3 exists and p=1. L 2 The cleavage of the linker L 3 This results in 1,6-β elimination of L, resulting in decarboxylation and release of the payload, D. 2 This increases the probability of payload release in regions where enzymes capable of cleaving the linker L are overexpressed. Furthermore, payload release induces bystander death, which is advantageous for tumors where not all cancer cells overexpress the target receptor. Therefore, in this embodiment, the linker L 2 and L 3 It is preferable that both are present (o=p=1).

[0167] Preferably, L 3 is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably having the structure (26): [ka] It is a PABC derivative represented by the formula:

[0168]

[0167] Here, the wavy line indicates L 1 or L 2 , and L 4 or D. Typically, the PABC derivatives are linked to L via NH. 1 or L 2 Preferably, L 2 and through OC(O) 4 Or connected to D.

[0169]

[0168] Ring A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Preferred 5-membered rings are oxazole, thiazole, and furan. Preferred 6-membered rings are phenyl and pyridyl. Ring A may contain halogen, X 2 R 4 , N(R 4 )2, C 1~4 and optionally substituted with a substituent selected from alkyl, and NO2. 2 and R 4 is as defined above, including preferred embodiments thereof. In preferred embodiments, the optional substituents are F, Cl, Br, OH, OR 4 , SH, NH2, Et, Me, and NO2. In particularly preferred embodiments, ring A contains 0 to 2 substituents, more preferably 0 or 1 substituent, and most preferably ring A is unsubstituted. In preferred embodiments, ring A is 1,4-phenyl, 1,2-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0170]

[0169] R 21 H, R 26 , C(O)OH, and C(O)R 26 where R 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are optionally substituted and include O, S, and NR 28 and optionally interrupted by one or more heteroatoms selected from, where R 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10(hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3, or 4. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0171] In an alternative embodiment, the linker L 3 is present (p=1) and the linker L 2 does not exist (o=0), and L 3 is a para-glucuronide-meta-amido-benzyloxycarbonyl derivative, preferably having the structure (27): [ka] It is a glucuronide derivative represented by the formula:

[0172]

[0171] Here, the wavy line is L 1 , and L 4 or D. Typically, glucuronide derivatives are linked to L via NH. 1 is linked to L via O)CO 4 or D. Rings A and R 21is defined as in the PABC derivative represented by structure (26). Preferably, ring A is a 6-membered aromatic or heteroaromatic ring such as oxazole, thiazole, furan, phenyl, and pyridyl. In a preferred embodiment, ring A is 1,3,4-phenyl, 2,4,5-pyridyl, or 2,5,6-pyridyl. Most preferably, A is 1,3,4-phenyl. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0173]

[0172] A linker L represented by structure (27) 3 is cleavable by β-glucuronidase, which results in the self-immolation of the para-hydroxybenzyloxy group, decarboxylation, and release of the payload. ADCs containing the glucuronide derivative represented by structure (27) are particularly useful for treating cancers that overexpress β-glucuronidase. The concentration of β-glucuronidase in many solid tumors, including lung, breast, and gastrointestinal cancers, as well as in the tumor microenvironment, has been reported to be elevated compared to normal tissues, and this enzyme is not found in the systemic circulation. Therefore, conjugates according to the present invention containing the glucuronide derivative represented by structure (27) are preferably used to treat patients suffering from lung, breast, and gastrointestinal cancers.

[0174] [Linker L 4 ] Linker L 4 is absent (q=0) or present (q=1). Preferably, the linker L 4 is present and q=1. The linker L 4 teeth, - Structure-NR 22 -(C x -alkylene)-C(O)- (wherein x is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl; - Structure-NR 22-(CH2-CH2-O) e6 -(CH2) e7 -C(O)- (wherein e6 is an integer ranging from 1 to 10, e7 is an integer ranging from 1 to 3, and R 22 is H or C1-C4 alkyl; and - Structure-NR 22 -(C x -alkylene)-NR 22 -(C(O)) h (wherein h is 0 or 1, x is an integer ranging from 1 to 20, and R 22 is H or C1-C4 alkyl) is selected from.

[0175] Linker L 4 is an aminoalkanoic acid spacer, i.e., -NR 22 -(C x -alkylene)-C(O)-, where x is an integer ranging from 1 to 20, preferably from 1 to 10, and most preferably from 1 to 6. Here, the aminoalkanoic acid spacer is typically connected to L via a nitrogen atom. 3 and linked to D via a carbonyl moiety. 4 is selected from 6-aminohexanoic acid (Ahx, x=5), β-alanine (x=2), and glycine (Gly, x=1), and even more preferably 6-aminohexanoic acid or glycine. 4 = 6-aminohexanoic acid. 4 = glycine, where R 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0176] Alternatively, the linker L 4 is the structure -NR 22 -(CH2-CH2-O) e6 -(CH2) e7The spacer may be an ethylene glycol spacer represented by -(C(O)-, where e6 is an integer ranging from 1 to 10, preferably e6 is an integer ranging from 2 to 6, and e7 is an integer ranging from 1 to 3, preferably e7 is 2). 22 is H or C1-C4 alkyl, preferably R 22 is H or methyl, most preferably R 22 is H.

[0177] Alternatively, the linker L 4 is the structure -NR 22 -(C x -alkylene)-NR 22 -(C(O)) h - (wherein h is 0 or 1, and x is an integer ranging from 1 to 20, preferably an integer ranging from 2 to 6, even more preferably x=2 or 5, and most preferably x=2). 22 is H or C1-C4 alkyl, where R 22 is H or C1 to C4, preferably R 22 is H or methyl, most preferably R 22 is methyl, where h is preferably 1, in which case the linker L 4 is particularly suitable for conjugation via the phenolic hydroxyl groups present on payload D.

[0178] [Payload D] D, also referred to in the art as "payload," represents a compound that is or is to be linked to an antibody Ab. Payload molecules are well known in the art, particularly in the field of antibody-drug conjugates, as moieties that are covalently attached to an antibody and released therefrom upon incorporation of the conjugate and / or cleavage of the linker. In preferred embodiments, the payload is selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, a nanoparticle, a microparticle, and a biomolecule. Particularly preferred payloads are active substances and reporter molecules, particularly active substances.

[0179]

[0178] The term "active substance" as used herein relates to a pharmacological and / or biological substance, i.e., a biologically and / or pharmaceutically active substance, such as a drug, prodrug, cytotoxin, diagnostic agent, protein, peptide, polypeptide, peptide tag, amino acid, glycan, lipid, vitamin, steroid, nucleotide, nucleoside, polynucleotide, RNA, or DNA. Examples of peptide tags include human lactoferrin or cell-penetrating peptides such as polyarginine. An example of a glycan is oligomannose. An example of an amino acid is lysine.

[0180]

[0179] When the payload is an active substance, the active substance is preferably selected from the group consisting of drugs and prodrugs. More preferably, the active ingredient is selected from the group consisting of pharmaceutically active compounds, particularly low- to medium-molecular-weight compounds (e.g., about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In a further preferred embodiment, the active ingredient is selected from the group consisting of cytotoxins, antiviral agents, antibacterial agents, peptides, and oligonucleotides.

[0181]

[0180] Preferred cytotoxins are selected from the group consisting of nitrogen mustards, nitrosoureas, alkylsulfonates, triazenes, platinum-containing compounds, plant alkaloids, DNA topoisomerase inhibitors, antimetabolites, hormone therapy agents, kinase inhibitors, antibiotics, and further cytotoxins as defined herein below.

[0182] Suitable nitrogen mustards include chlorambucil, chlornaphazine, cyclophosphamide, dacarbazine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mannomustine, mitobronitol, melphalan, mitolactol, pipobroman, nobembine, phenesterine, prednimustine, thiotepa, trofosfamide, uracil mustard; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); duocarmycins (including synthetic analogs, KW-2189 and CBI-TMI); benzodiazepine monomers or dimers (e.g., pyrrolobenzodiazepines (PBDs), tomaymycin, indolinobenzodiazepines, isoindolinebenzodiazepines, imidazobenzothiadiazepines, and oxazolidinobenzodiazepines).

[0183]

[0182] Suitable nitrosoureas include carmustine, lomustine, chlorozotocin, fotemustine, nimustine, and ranimustine.

[0184]

[0183] Suitable alkyl sulfonates include busulfan, treosulfan, improsulfan, and piposulfan.

[0185]

[0184] Suitable triazenes include dacarbazine.

[0186]

[0185] Suitable platinum-containing compounds include carboplatin, cisplatin, oxaliplatin; aziridines such as benzodopa, carboquone, mesuredopa, and uredopa; ethyleneimines and methylameramines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine.

[0187] Suitable plant alkaloids include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine, navelbine), toxoids (e.g., paclitaxel, docetaxel, and their analogs), maytansinoids and their analogs (e.g., DM1, DM2, DM3, DM4, maytansine, and ansamitocins), cryptophycins (particularly cryptophycin 1 and cryptophycin 8), epothilones, eleutherobin, discodermolides, bryostatin, dolostatin, auristatin, tubulysin, cephalostatin, pancratistatin, sarcodictin, and spongistatin.

[0188]

[0187] Suitable DNA topoisomerase inhibitors include any camptothecin, such as 9-aminocamptothecin, exatecan, DXd (DX-8951 derivative), crisnatol, daunomycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novantrone, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin (RFS 2000); mitomycin, and mitomycin C.

[0189] Suitable antimetabolites include antifolates, DHFR inhibitors (e.g., methotrexate, trimetrexate, denopterin, pteropterin, aminopterin (4-aminopteroic acid), or other folic acid analogs), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea, deferoxamine), pyrimidine analogs (e.g., ancitabine, azacitidine, 6 - uracil analogues such as azauridine, capecitabine (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, 5-fluorouracil, floxuridine, latitrexed (Tomudex); cytosine analogues such as cytarabine, cytosine arabinoside, fludarabine; purine analogues such as azathioprine, fludarabine, mercaptopurine, thiamiprine, thioguanine), folic acid supplements (e.g., folinic acid).

[0190]

[0189] Suitable hormone therapy agents include receptor antagonists (e.g., antiestrogens such as LHRH agonists such as megestrol, raloxifene, tamoxifen, goserelin, leuprolide acetate, etc.; antiandrogens such as bicalutamide, flutamide, calsterone, dromostanolone propionate, epitiostanol, goserelin, leuprolide, mepitiostane, nilutamide, testolactone, trilostane, and other androgen inhibitors), retinoids / deltoids (e.g., vitamin D3 analogs such as CB 1093, E B1089 KH 1060, cholecalciferol, ergocalciferol), photodynamic therapy agents (e.g., verteporfin, phthalocyanines, photosensitizer Pc4, demethoxy-hypocrelin A).

[0191] Suitable kinase inhibitors include BIBW 2992 (anti-EGFR / Erb2), imatinib, gefitinib, pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, axitinib, pazopanib, vandetanib, E7080 (anti-VEGFR2), mubritinib, ponatinib (AP24534), bafetinib (INNO-406), bosutinib (SKI-606), cabozantinib, vismodegib, iniparib, ruxolitinib, CYT387, axitinib, tivozanib, sorafenib, and ispinesib.

[0192] Suitable antibiotics include enediyne antibiotics (e.g., calicheamicins, particularly calicheamicins γ1, δ1, α1, and β1), dynemycins (e.g., dynemycin A and deoxydynemycin), esperamicin, kedarcidin, C-1027, maduropeptin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin; chromomycin, dactinomycin, daunorubicin, nemorubicin , detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin.

[0193]

[0192] Suitable polyketides include acetogenins (in microparticles brilatacin and brilatacinone), gemcitabine, epoxomicin (e.g., carfilzomib), bortezomib, thalidomide, lenalidomide, pomalidomide, tosedostat, zibrestat, PLX4032, STA-9090, Stimuvax, allovectin 7, Xegeva, and Provenge.

[0194] Cytotoxins further include isoprenylation inhibitors (such as lovastatin), dopaminergic neurotoxins (such as 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (such as staurosporine), actinomycins (such as actinomycin D and dactinomycin), bleomycins (bleomycin A2, bleomycin B2, peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca2+ ATPase inhibitors (e.g., thapsigargin), histone deacetylase inhibitors (e.g., vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat (MGCD010)), and the like. 3), belinstat, PCI-24781, entinostat, SB939, resminostat, gibinostat, AR-42, CUDC-101, sulforaphane, trichostatin A), thapsigargin, celecoxib, glitazones, epigallocatechin gallate, disulfiram, salinosporamide A; antiadrenal agents (e.g., aminoglutethimide, mitotane, trilostane; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; arabinoside; bestravcil; bissantrene, edatraxate, defofamine, demecolcine, diaziquone; eflornithine (DFMO), elfomithine; elliptinium acetate acetate), etoglucid; gallium nitrate; hydroxyurea; ibandronate, lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verrucarin A, roridin A, and anguidine).

[0195]

[0194] As used herein, the term "reporter molecule" refers to a molecule whose presence is easily detected, such as a diagnostic agent, dye, fluorophore, radioisotope label, contrast agent, magnetic resonance imaging agent, or mass label.

[0196] A wide variety of fluorophores, also called fluorescent probes, are known to those skilled in the art. Some fluorophores are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 10: "Fluorescent Probes", pp. 395-463 (incorporated by reference). Examples of fluorophores include all types of Alexa Fluor (e.g., Alexa Fluor 555), cyanine dyes (e.g., Cy3 or Cy5) and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthalimide derivatives, phycobiliprotein derivatives (e.g., allophycocyanin), chromomycin, lanthanide chelates, and nanocrystalline quantum dots.

[0197]

[0196] Examples of radioisotope labels include: 99m Tc, 111 In, 114m In, 115 In, 18 F, 14 C. 64 Cu, 131 I, 125 I, 123 I, 212 Bi, 88 Y, 90 Y, 67 Cu, 186 Rh, 188 Rh, 66 Ga, 67 Ga, and 10B includes, optionally, for example, DTPA (diethylenetriaminepentaacetic anhydride), DOTA (1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid), NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid), DTTA (N 1 -(p-Isothiocyanatobenzyl)-diethylenetriamine-N 1 , N 2 , N 3 , N 3 -tetraacetic acid), deferoxamine or DFA (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide), or HYNIC (hydrazinonicotinamide). Isotopic labeling techniques are known to those skilled in the art and are described, for example, in G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3 rd Ed. 2013, Chapter 12: “Isotopic labeling techniques”, pp. 507-534, (incorporated by reference).

[0198]

[0197] Polymers suitable for use as payload D in the compounds according to the invention are known to those skilled in the art, and some examples can be found, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3 rdEd. 2013, Chapter 18: "PEGylation and synthetic polymer modification", pp. 787-838 (incorporated by reference). When payload D is a polymer, payload D is preferably independently selected from the group consisting of poly(ethylene glycol) (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1,q-diaminoalkane polymers (where q is the number of carbon atoms in the alkane, and preferably q is an integer ranging from 2 to 200, preferably 2 to 10), (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (e.g., dextran), poly(amino acids) (e.g., poly(L-lysine)), and poly(vinyl alcohol).

[0199] Solid surfaces suitable for use as payload D are known to those skilled in the art. Solid surfaces include, for example, functional surfaces (e.g., the surface of a nanomaterial, a carbon nanotube, a fullerene, or a viral capsid), metal surfaces (e.g., a titanium, gold, silver, copper, nickel, tin, rhodium, or zinc surface), metal alloy surfaces (alloys made from, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin, uranium, zinc, and / or zirconium), polymer surfaces (wherein the polymer is, for example, polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly(dimethylsiloxane), or polymethyl methacrylate, polyacrylamide), glass surfaces, silicone surfaces, chromatographic support surfaces (wherein the chromatographic support is, for example, a silica support, an agarose support, a cellulose support, or an alumina support), and the like. When the payload D is a solid surface, D is preferably independently selected from the group consisting of a functional surface or a polymeric surface.

[0200]

[0199] Hydrogels are known to those skilled in the art. Hydrogels are water-swollen networks formed by cross-linking between polymer components. See, for example, A S Offman, Adv. Drug Delivery Rev. 2012, 64, 18 (incorporated by reference). When the payload is a hydrogel, the hydrogel is preferably composed of poly(ethylene) glycol (PEG) as the polymer base.

[0201]

[0200] Microparticles and nanoparticles suitable for use as payload D are known to those skilled in the art. A variety of suitable microparticles and nanoparticles are described, for example, in GT Hermanson, "Bioconjugate Techniques," Elsevier, 3 rd Ed. 2013 Chapter 14: "Microparticles and nanoparticles", pp. 549-587 (incorporated by reference). The microparticles or nanoparticles may be of any shape, such as spheres, rods, tubes, cubes, triangles, and cones. Preferably, the microparticles or nanoparticles are spherical. The chemical composition of the microparticles and nanoparticles may vary. When payload D is a microparticle or nanoparticle, the microparticle or nanoparticle may be, for example, a microparticle or nanoparticle made of a polymer, a microparticle or nanoparticle made of silica, or a microparticle or nanoparticle made of gold. When the particles are microparticles or nanoparticles made of a polymer, the polymer is preferably polystyrene or a copolymer of styrene (e.g., a copolymer of styrene with divinylbenzene, butadiene, acrylate, and / or vinyltoluene), polymethyl methacrylate (PMMA), polyvinyltoluene, poly(hydroxyethyl methacrylate (pHEMA), or poly(ethylene glycol dimethacrylate / 2-hydroxyethyl methacrylate) [poly(EDGMA / HEMA)]. Optionally, the surface of the microparticle or nanoparticle is modified, e.g., with a detergent, by graft polymerization of a secondary polymer, or by covalent attachment of another polymer or spacer moiety.

[0202]

[0201] Payload D may also be a biomolecule. Biomolecules and preferred embodiments thereof are described in more detail below. When payload D is a biomolecule, the biomolecule is selected from the group consisting of proteins (including glycoproteins such as antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides.

[0203]

[0202] In the context of the present invention, cytotoxic payloads are particularly preferred. Thus, D is preferably a cytotoxin, more preferably selected from the group consisting of colchicine, vinca alkaloids, anthracyclines, camptothecin, doxorubicin, daunorubicin, taxanes, calicheamicins, tubulysins, irinotecans, inhibitory peptides, amanitins, amatoxins, duocarmycins, epothilones, mitomycins, combretastatins, maytansines, auristatins, enediynes, pyrrolobenzodiazepines (PBDs), or indolinobenzodiazepine dimers (IGNs). The present invention is particularly suitable for cytotoxic payloads with moderate cytotoxicity. While conjugates of such payloads usually require administration at higher doses, considering the high DAR (high payload load) of the conjugates of the present invention, even conjugates of moderately toxic payloads can be administered at desirable low doses. In other words, D is preferably a payload that allows administration of medium to high doses (3-20 mg / kg) to patients, even at high DAR (DAR>>6). Considering the high payload loading in the conjugates of the present invention, the payload (in unconjugated form) should have an average cytotoxicity (IC) of >50 nM. 50 ) is particularly preferred. Thus, even payloads with lower cytotoxicity can be used in antibody-conjugates with high efficacy.

[0204] [Preferred linker L A ] Particularly preferred linkers L A is the structure -(L 11 )-BM(L 12 -)(L 13 -), where 11 is one of the following (i) to (x): (i)L 11 is *-C(O)-(CH2) a’ -C(O)-** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 1 to 5, preferably a'=3). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (ii)L 11 is -NH-C(O)-(CH2) a’ -** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 0 to 5, preferably a'=0). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (iii)L 11 is *=NC(O)-(CH2) a’ -** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 0 to 5, preferably a'=0). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (iv)L 11 is *-NH-C(O)-(CH2) a’ -C(O)-** (where the bond marked with * is Q 1 or Z 1and the bond marked with ** is connected to BM, where a' is an integer ranging from 1 to 5, preferably a'=3). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (v)L 11 is *=NC(O)-(CH2) a’ -C(O)-** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 1 to 5, preferably a'=3). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (vi)L 11 is *-C(O)-** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, which is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (vii)L 11 is *-(23)-(CH2CH2O) a’ -(C(O)) a’’ -** (where the bond marked with * is Q 1 or Z 1 The bond connected to (Y) and labeled with ** is connected to BM, and (23) refers to the group represented by structure (23). Furthermore, a' is an integer ranging from 0 to 5, preferably a'=1 to 4, most preferably a'=2, and a" is 0 or 1, preferably a" is 1. Furthermore, it is preferred that BM is a nitrogen atom (when y=2) or a carbon atom (when y=3). Also preferred is that BM=N. (viii)L 11 is *-(23)-(CH2CH2O) a’ -(23)-** (wherein the bond marked with * is Q 1 or Z 1The bond connected to y is connected to BM, and the bond marked with ** is connected to BM, and (23) refers to the group represented by structure (23). Furthermore, a' is an integer ranging from 0 to 5, preferably a'=1 to 4, and most preferably a'=2. Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (ix)L 11 is *-C(O)-NH-(CH2CH2O) a’ -C(O)-** (where the bond marked with * is Q 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 1 to 5, preferably a'=3). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N. (x)L 11 is *-C(O)-NH-(CH2CH2O) a’ -OC(O)-NH-** (where the bond marked with * is QQ 1 or Z 1 and the bond marked with ** is connected to BM, where a' is an integer ranging from 1 to 5, preferably a'=3). Furthermore, BM is preferably a nitrogen atom (when y=2) or a carbon atom (when y=3). Most preferably, BM=N.

[0205] Particularly preferred linkers L A is the structure -(L 11 )-BM(L 12 -)(L 13 -), where L 12 and L 13 is one of the following (xi) to (xv): (xi)L 12 and L 13 are both *-(CH2) a’ -OC(O)-NH)-(CH2CH2O) a’’ -CH2-CH2-NC(O)-** (where the bond marked with * is connected to BM and the bond marked with ** is connected to F2 wherein a' is an integer ranging from 0 to 4, preferably a'=2, and a'' is an integer ranging from 0 to 4, preferably a''=2. Furthermore, BM is preferably a nitrogen atom. (xii)L 12 and L 13 are both *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a’’ -Ph-** (where the bond labeled with * is connected to BM and the bond labeled with ** is connected to F 2 where a' is an integer ranging from 0 to 5, preferably a'=2, and a'' is an integer ranging from 0 to 4, preferably a''=1. Preferably, Ph is 1,4-Ph. Furthermore, BM is preferably a nitrogen atom. (xiii)L 12 and L 13 are both *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a’’ -NH-C(O)-Ph-** (where the bond marked with * is connected to BM and the bond marked with ** is connected to F 2 where a' is an integer ranging from 0 to 5, preferably a'=2, and a'' is an integer ranging from 0 to 4, preferably a'' is 2 or 3. Preferably, Ph is 1,4-Ph. Furthermore, BM is preferably a nitrogen atom. (xiv)L 12 and L 13 are both *-(CH2CH2O) a’ -CH2-CH2-C(O)-NH-(CH2) a’’ -O-Ph-** (where the bond labeled with * is connected to BM and the bond labeled with ** is connected to F 2 where a' is an integer ranging from 0 to 5, preferably a'=2, and a'' is an integer ranging from 0 to 4, preferably a'' is 2 or 3. Preferably, Ph is 1,4-Ph. Furthermore, BM is preferably a nitrogen atom. (xv)L12 and L 13 Both have the structure -(L 14 )-BM'(L 15 -)(L 16 -), wherein -L 14 L 11 and preferably according to any one of (i) to (x) as defined above, or L 14 is *-(CH2CH2O) a’ -CH2-CH2-(C(O)) a’’ -** (wherein the bond marked with * is connected to BM, the bond marked with ** is connected to BM', a' is an integer ranging from 0 to 5, preferably a'=1 to 4, most preferably a'=2, and a''hh are 0 or 1, preferably a'' is 1). - BM and BM' are branching moieties, preferably both nitrogen atoms. BM occurs once and BM' occurs twice (L 15 and L 16 ) and y=4. -L 15 and L 16 L 12 and L 13 Preferably according to (xiv) as defined above.

[0206] Preferably, the linker L 12 and L 13 Both have the same structure. A In a particularly preferred embodiment, the linker L 11 is a linker L according to any one of (i) to (x). 12 and L 13 follows one of (xi) to (xv).

[0207] Such a preferred linker L A may be used in the linker constructs according to the invention, the conjugates according to the invention, and the intermediate antibody-linker constructs according to the invention. 1is preferably represented by (Q26) or (Q37), and F 2 is preferably (F8a) (wherein R 29 is hydrogen or methyl). 1 is preferably represented by (Z26) or (Z37) (wherein ring Z is represented by (za)), and Z 2 is preferably (Z29) (wherein ring Z is (zj) (wherein R 29 is hydrogen or methyl).

[0208] [Method for synthesizing a conjugate represented by general structure (2)] In a further aspect, the present invention provides a process for preparing a conjugate according to the present invention, comprising: (a) Structure Ab(F 1 ) z where Ab is an antibody, z is 2 or 4, and F 1 is a click probe); (b) Add z equivalents of Q 1 L A (F 2 ) y (In the formula, Q 1 is F 1 is a click probe that reacts with L A is a heterobifunctional (y+1)valent linker, where y is 2, 3, or 4; F 2 Q 1 (a click probe that does not react with Z) to form the structure Ab(Z 1 L A (F 2 ) y ) z (In the formula, Z 1 is F 1 and Q 1 wherein the linker group is obtained by reaction of (c) dissolving the antibody-linker construct in z×y equivalents of Q 2 (L B )D x (In the formula, Q 2 is F 2 is a click probe that reacts with LB is an (x+1)-valent linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is a payload molecule) to form a compound of the structure Ab(Z 1 L A (Z 2 (L B )D x ) y ) z (In the formula, Z 2 is F 2 and Q 2 a step of obtaining a conjugate of The present invention relates to a process including:

[0209] The reaction carried out in steps (b) and (c) is a Click reaction, in which Click probe F reacts with Click probe Q to form linking group Z. This conjugation technique is known to those skilled in the art. The reaction occurs under conditions such that Q reacts with F to form a covalent bond. In the process according to the invention, Q reacts with F to form a covalent bond between the antibody and the payload. Complementary reactive groups Q and F are known to those skilled in the art and are described in more detail below.

[0210]

[0209] Thus, the reaction, conjugation, in the process according to the present invention is achieved via cycloaddition. Preferred cycloadditions are (4 + 2)-cycloadditions (e.g., Diels-Alder reaction) or (3 + 2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). The reaction in step (b) is preferably a [3 + 2]-cycloaddition, more preferably a 1,3-dipolar cycloaddition. The 1,3-dipolar cycloaddition is preferably an alkyne-azide cycloaddition, most preferably wherein Q is or contains an alkyne group and F is an azide group. The reaction in step (c) is a [4 + 2]-cycloaddition, preferably a Diels-Alder reaction. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. Cycloadditions such as the Diels-Alder reaction and the 1,3-dipolar cycloaddition are known in the art and one of ordinary skill in the art would know how to perform them.

[0211] [Process (a)] In step (a), the structure Ab(F 1 ) z Typically, two or four click probes F 1 is introduced into the antibody chemically or enzymatically. Preferably, z=2, and two click probes are introduced into the antibody.

[0212] In a preferred embodiment, an antibody containing two or four, preferably two, core N-acetylglucosamine moieties is reacted with a hydroxyl group of formula S(F 1 )-P(wherein S(F 1 ) is a reactive group Q 1 Two reactive groups F that can react with 1 wherein P is a nucleoside mono- or diphosphate, and the catalyst is S(F 1 The antibody is contacted with a compound capable of transferring a core-GlcNAc moiety to a core-GlcNAc moiety, wherein the antibody is typically an antibody that has been trimmed to the core-GlcNAc residue as further described below.

[0213] The starting material, i.e., an antibody containing two or four, preferably two, core-GlcNAc substituents, is known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the process according to the present invention further comprises deglycosylating the antibody glycan containing the core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody containing a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH, and / or a combination thereof, and the selection depends on the nature of the glycan. EndoSH is described in PCT / EP2017 / 052792, see Examples 1-3 and SEQ ID NO: 1, which is incorporated herein by reference.

[0214]

[0213] The structural feature S has been defined above for the conjugates according to the invention and this applies equally to this embodiment. When a nucleoside monophosphate or nucleoside diphosphate P is linked to a sugar derivative S(F 1 ) is connected to the expression S(F 1 )-P compounds are known in the art. For example, Wang et al., Chem. Eur. J. 2010, 16, 13343-13345; Piller et al., ACS Chem. Biol. 2012, 7, 753; Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462; and WO 2009 / 102820 (all incorporated herein by reference) disclose a number of compounds S(F 1 )-P and their synthesis. In a preferred embodiment, S(F 1The nucleoside monophosphate or diphosphate P in S(F)-P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP), and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP), most preferably P=UDP. Preferably, S(F)-P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), and cytidine diphosphate (CDP), most preferably P=UDP. 1 )-P is selected from the group consisting of GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetyl-galactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP, and 2-(but-3-ynoic acid amido)-2-deoxy-galactose-UDP. Most preferably, S(F 1 )-P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0215]

[0214] S(F 1 Suitable catalysts capable of transferring a nucleotide S(F) to a core-GlcNAc moiety are known in the art. Suitable catalysts are those capable of transferring a specific sugar derivative nucleotide S(F) to a core-GlcNAc moiety in that particular process. 1)-P is a catalyst for which the substrate is β(1,4)-glycosidic bond formation. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcTs) and β(1,4)-galactosyltransferases (GalTs), and most preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain. Suitable catalysts and variants thereof are disclosed in WO 2014 / 065661, WO 2016 / 022027, and WO 2016 / 170186, all of which are incorporated herein by reference. In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. The mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts can recognize internal sugars and sugar derivatives as acceptors. Thus, the sugar derivative S(F 1 ) is linked to the core-GlcNAc substituent in step (a), regardless of whether said GlcNAc is fucosylated or not.

[0216] Step (a) is preferably carried out in a suitable buffer solution, such as, for example, phosphate, buffered saline (e.g., phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris, and glycine. Suitable buffers are known in the art. Preferably, the buffer solution is phosphate-buffered saline (PBS) or Tris buffer. Step (a) is preferably carried out at a temperature in the range of about 4°C to about 50°C, more preferably in the range of about 10°C to about 45°C, even more preferably in the range of about 20°C to about 40°C, and most preferably in the range of about 30 to about 37°C. Step (a) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (a) is carried out at a pH in the range of about 7 to about 8.

[0217] [Step (b)] In step (b), the modified antibody Ab(F 1 ) z with a reactive group F 1 A reactive group Q capable of reacting with 1 and reacting Q with a linker construct according to the invention comprising 1 and F 1 The linking group Z resulting from the reaction between 1 Such a reaction results in an antibody-linker construct comprising the reactive group Q 1 is the reactive group F of the antibody 1 In step (b), z equivalents of Q are reacted with the antibody to covalently bond the antibody to the linker construct. 1 L A (F 2 ) y The reaction occurs at , but to ensure complete reaction, more equivalents of Q 1 L A (F 2 ) y may be present in the reaction mixture. Those skilled in the art can determine the optimum reaction conditions and reactant stoichiometry to obtain the optimum yield.

[0218] In a preferred embodiment, in step (b), the azide on the azide-modified antibody reacts with a benzocyclized or tetramethylated (hetero)cycloalkyne group by a cycloaddition reaction, preferably wherein Q 1 The cycloalkyne groups are represented by structures (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a), preferably structures (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a)d, and more preferably structures (Q40), (Q41), or (Q43), or structures (Q37) or (Q43). When using such benzo- or tetramethylated (hetero)cycloalkyne groups, the cycloaddition reaction can occur spontaneously without the need for a catalyst, via a reaction known as strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry."

[0219] [Process (c)] In step (c), the modified antibody-linker construct Ab(Z 1 L A (F 2 ) y ) z with a reactive group F 2 A reactive group Q capable of reacting with 2 and reacting with a payload linker containing Q 2 and F 2 The linking group Z resulting from the reaction with 2 Such a reaction results in an antibody conjugate containing the reactive group Q 2 is a reactive group F 2 Step (c) occurs under conditions such that the antibody reacts with z×y equivalents of Q to covalently bond the antibody to the payload. Step (c) may also be referred to as a conjugation reaction. In step (c), z×y equivalents of Q 2 (L B )D x The reaction occurs at , but to ensure complete reaction, more equivalents of Q 2 (L B )D xmay be present in the reaction mixture. Those skilled in the art can determine the optimum reaction conditions and reactant stoichiometry to obtain the optimum yield.

[0220] In step (c), the conjugate is formed by covalently linking multiple payloads to the antibody. This establishes the DAR of the conjugate in this step. The conjugation process of the present invention is particularly effective because it results in conjugates with an average DAR close to the theoretical value. Thus, the conjugates according to the present invention exhibit high homogeneity. The inventors further investigated the antibody side of the conjugation reaction, i.e., Ab(Z 1 L A (F 2 ) y ) z , typically L A It has been found that negative charges within the L can negatively affect the conjugation reaction, resulting in lower DAR values. However, the process according to the present invention can handle such negatively charged modified antibody-linkers and provide conjugates with unprecedented DAR values. A The negative charge of is present under the process conditions of step (c) and can result, for example, from deprotonation of a carboxylic acid group or a sulfamide group (e.g., according to structure (23)). Thus, in one embodiment, the linker L A is not negatively charged and preferably does not contain carboxylic acid and sulfamide groups. A is negatively charged and preferably contains a carboxylic acid group and / or a sulfamide group.

[0221] In a preferred embodiment, in step (c), a 1,2,4,5-tetrazine on the antibody-linker construct reacts with a bicyclononyne group via a cycloaddition reaction, wherein preferably Q 1is represented by structure (Q8), more preferably structure (Q29), and most preferably structure (Q42). When using such bicyclononyne groups, the cycloaddition reaction can occur spontaneously without the need for a catalyst via a process known as strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry."

[0222] [Reactive moiety Q] Reactive moiety Q 1 and Q 2 is a click probe. In the context of the present invention, Q is Q 1 and Q 2 In the context of the present invention, the term "reactive moiety" can refer to a chemical moiety containing a reactive group, but can also refer to the reactive group itself. For example, a cyclooctynyl group is a reactive group, i.e., a reactive group containing a CC triple bond. However, a reactive group, such as an azide reactive group, can be referred to as a reactive moiety herein.

[0223] Q is reactive and complementary to F. A reactive group is said to be "complementary" to a reactive group if it selectively reacts with said reactive group, optionally in the presence of other functional groups. Complementary reactive click probes are known to those skilled in the art and are described in more detail below. The exact nature of Q and F depends on the type of click reaction used. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, click probe Q comprises or is an alkene or alkyne moiety, more preferably the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne.

[0224]

[0223] Therefore, in a particularly preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, where the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group, or a (hetero)cyclodecynyl group. In the present specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, where the (hetero)cyclooctynyl group is optionally substituted.

[0225]

[0224] In particularly preferred embodiments, Q comprises a (hetero)cycloalkynyl group or a (hetero)cycloalkenyl group and has the structure (Q1): [ka] It is expressed as: where: - [ka] A bond shown as is a double or triple bond; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, - CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 , or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked via L; - u is 0, 1, 2, 3, 4, or 5; u' is 0, 1, 2, 3, 4, or 5, where u+u'=0, 1, 2, 3, 4, 5, 6, 7, or 8; - v=an integer in the range 0 to 16;

[0226] Typically, v = (u + u') × 2 (the connection to L indicated by the wavy bond is Y 2 through the carbon atoms of u and u') or [(u+u')×2]-1 (when the connection to L represented by the wavy bond is through one of the carbon atoms of u and u').

[0227] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and is represented by structure (Q1a): [ka] where: -R 15 and Y 2 is as defined above, - u is 0, 1, 2, 3, 4, or 5; u' is 0, 1, 2, 3, 4 or 5, where u+u'=4, 5, 6, 7 or 8; - v=an integer between 8 and 16.

[0228]

[0227] In preferred embodiments, u+u'=4, 5, or 6, more preferably u+u'=5.

[0229]

[0228] In preferred embodiments, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0230]

[0229] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) to (Q20) shown herein below. [ka]

[0231]

[0230] Herein, the link to L, represented by a wavy bond, can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) may have a link to L, or may contain a hydrogen atom, or may be optionally functionalized. B (-) is preferably (-) OTf, Cl (-) , Br (-) , or I (-) and most preferably, B (-) teeth, (-) OTf. B (+)is a cation, preferably a pharmaceutically acceptable cation. (-) will be exchanged with anions present in the reaction mixture anyway, so B (-) does not need to be a pharmaceutically acceptable anion. When (Q19) is used for Q, it is preferred that the negatively charged counterion be pharmaceutically acceptable when the conjugate according to the invention is isolated, so that the conjugate can be readily used as a pharmaceutical.

[0232]

[0231] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38a) shown herein below. [ka]

[0233] In structure (Q38), B (-) is preferably (-) OTf, Cl (-) , Br (-) , or I (-) and most preferably, B (-) teeth, (-) It is OTf.

[0234] In preferred embodiments, Q comprises an optionally substituted (hetero)cyclooctyne or (hetero)cycloheptyne moiety, preferably represented by the structures (Q8), (Q26), (Q27), (Q28), (Q37), or (Q38a). Each of these preferred options for Q is further defined herein below.

[0235]

[0234] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety represented by structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety represented by structure (Q37) is unsubstituted.

[0236]

[0235] In an alternative preferred embodiment, Q comprises a cyclooctyne moiety represented by structure (Q8), more preferably represented by (Q29), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety represented by structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety represented by structure (Q39) shown below, where V is (CH2) l wherein l is an integer ranging from 0 to 10, preferably from 0 to 6. More preferably, l is 0, 1, 2, 3, or 4, more preferably, l is 0, 1, or 2, and most preferably, l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q has structure (Q42), further defined below.

[0237] In an alternative preferred embodiment, Q comprises a (hetero)cyclooctyne moiety represented by structure (Q26), (Q27), or (Q28), also referred to as a DIBO, DIBAC, DBCO, or ADIBO group, which is optionally substituted. In the context of this embodiment, Q preferably represents structure (Q40) or (Q41) shown below, where Y 1 is O or NR 11 and R 11 are independently hydrogen, straight-chain or branched C1-C 12 Alkyl group or C4-C 12 (Hetero)cyclooctyne moieties represented by structure (Q40) are optionally O-sulfonylated at one or more positions, while the ring of (Q41) can be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moieties represented by structure (Q40) or (Q41) are not further substituted. Most preferably, Q is represented by structure (Q43), further defined below.

[0238] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is represented by structure (Q37). [ka]

[0239] In a particularly preferred embodiment, Q comprises a cyclooctynyl group and has the structure (Q42): [ka] It is expressed as: where: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, - CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl group, C7-C 24 Alkyl (hetero)aryl groups and C7-C24 (hetero)arylalkyl groups; -R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)alkyl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is a second occurrence of Q or D linked via a spacer moiety; and - l is an integer ranging from 0 to 10.

[0240] In a preferred embodiment of the reactive group represented by structure (Q42), R 15 is hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the reactive group represented by structure (Q42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the reactive group represented by structure (Q42), R 19 is H. In preferred embodiments of the reactive group represented by structure (Q42), l is 0 or 1, and more preferably l is 1.

[0241] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and is represented by structure (Q43): [ka] where: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl groups are optionally substituted and include two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y is N or CR 15 and; - the carbon atoms in the fused aromatic ring may be replaced by nitrogen atoms as in (Q6a) to (Q6d), preferably Y is CR 15 is.

[0242] In a preferred embodiment of the group represented by structure (Q43), R 15 is hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group, wherein R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 is hydrogen and -S(O)3 (-)In preferred embodiments of the group represented by structure (Q43), Y is N or CH, more preferably Y=N.

[0243]

[0242] In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, wherein the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, and trans-(hetero)cyclooctenyl groups, where the (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, and trans-(hetero)cyclooctenyl groups are optionally substituted. Preferably, Q comprises a cyclopropenyl moiety represented by structure (Q44), a heterocyclobutene moiety represented by structure (Q45), a norbornene or norbornadiene group represented by structure (Q46), a trans-(hetero)cycloheptenyl moiety represented by structure (Q47), or a trans-(hetero)cyclooctenyl moiety represented by structure (Q48). Wherein, Y 3 is C(R 23 )2, NR 23 , or O, and each R 23 are individually hydrogen, C1-C6 alkyl, or optionally connected to L via a spacer, and [ka] is a single bond or a double bond. In a further preferred embodiment, the cyclopropenyl group is represented by structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is represented by structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group is represented by structure (Q52), (Q53), (Q54), (Q55), or (Q56). [ka]

[0244]

[0243] Here, the R group on Si in (Q50) and (Q51) is typically alkyl or aryl, preferably C1 to C6 alkyl.

[0245]

[0244] In a preferred embodiment, click probe Q comprises a moiety selected from (Q1) to (Q56), and more preferably is a moiety selected from (Q1) to (Q56).

[0246]

[0245] In the present invention, Q 1 is F 2 It is reactive to 2 is F 2 Because it is reactive to Q 1 and Q 2 The exact structure of the

[0247] In a preferred embodiment, F 1 is azide, and Q 1 is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F 2 is a tetrazine or nitrone, and Q 2 is a bicyclononyne or a cycloalkene, such as trans-cyclooctene or cyclopropene. More preferably, F 1 is azide, and Q 1 is a benzoannulated or tetramethylated (hetero)cycloalkyne, while F 2 is tetrazine, and Q 2is bicyclononyne, where Q 1 is preferably represented by structure (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), or (Q19a), more preferably represented by structure (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a), and most preferably represented by structure (Q40), (Q41), or (Q43), or represented by structure (Q37) or (Q43). 2 is preferably represented by structure (Q8), (Q44), (Q47), (Q48), (Q49), (Q54), (Q55), or (Q56), more preferably represented by structure (Q29), (Q48), or (Q49), and most preferably represented by structure (Q42). 2 is preferably represented by structure (Q8), more preferably represented by structure (Q29), and most preferably represented by structure (Q42).

[0248] [Reactive moiety F] Reactive moiety F 1 and F 2 is a click probe. In the context of the present invention, F is F 1 and F 2 where F is reactive and complementary to Q. Here, a reactive group is said to be "complementary" to a reactive group if the reactive group selectively reacts with the reactive group, optionally in the presence of other functional groups. Complementary reactive click probes are known to those skilled in the art and are described in more detail below. The exact nature of Q and F depends on the type of click reaction used. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, click probe F comprises or is an azide, nitrone, or tetrazine moiety.

[0249]

[0248] F is reactive to Q in a conjugation reaction as defined below, where preferably the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. As one skilled in the art will appreciate, the options for F are the same as the options for Q, except that F and Q are reactive with each other. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, or sydnones, and most preferably is an azide.

[0250] The reactive group F on the antibody is typically introduced by specific techniques, such as (bio)chemical or genetic techniques. The reactive group located in the antibody is prepared by chemical synthesis, for example, azide or terminal alkyne. Methods for preparing modified antibodies are known in the art, for example, from WO 2014 / 065661, WO 2016 / 170186, and WO 2016 / 053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between the modified antibody and the linker-toxin-construct is known to those skilled in the art.

[0251]

[0250] Preferably, F is a click probe reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, and sydnones. Preferred structures of the reactive groups are structures (F1) to (F10) shown herein below. [ka]

[0252]

[0251] Here, the wavy bond is Ab or L A In (F3), (F4), (F8) and (F9), the payload can be linked to any one of the wavy bonds. In this case, the other wavy bond is hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 sulfonyl groups, each of which (except hydrogen) is optionally substituted, and may be linked to an R group selected from O, S, and NR 32 and optionally interrupted by one or more heteroatoms selected from, where R 32 are independently hydrogen and The reactive group F is selected from the group consisting of C1-C4 alkyl groups. Those skilled in the art will understand which R groups can be applied to each of the groups F. For example, the R group linked to the nitrogen atom of (F3) can be selected from alkyl and aryl, and the R group linked to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl. Preferably, the reactive group F is selected from azide or tetrazine.

[0253] In a particularly preferred embodiment, F has the structure (F8a): [ka] It is a tetrazine represented by the formula:

[0254]

[0253] where R 29 is hydrogen, C 1~6 Alkyl, aryl, C(O)C 1~6 Alkyl, C(O)-aryl, C(O)-OC 1~6Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1~6 Alkyl, and C(O)-NR 33 -aryl, and R 33 is H or C 1~4 Preferably, R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. 29 It has been found that the optimum reactivity in the cycloaddition reaction is achieved when F is hydrogen. Therefore, in a preferred embodiment, F is 2 is (F8a), where R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl, more preferably R 29 is hydrogen or methyl, and most preferably R 29 is methyl.

[0255] In a preferred embodiment, click probe F 1 and F 2 is selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, dioxothiophenes, sydnones, iminosydnones, and catechols. Note that catechols can be oxidized in situ to form ortho-quinone groups, which react as click probes. Similarly, the term "tetrazine" also encompasses "hydrotetrazines," which are known precursors that form tetrazines upon in situ oxidation. Such precursors of click probes that form reactive groups in situ are also encompassed by the present invention. Figure 11 shows known examples of click probes formed by in situ oxidation. Preferred click probes F 1 and F 2 is selected from the group consisting of azides, tetrazines, ortho-quinones, and nitrones, more preferably azides, tetrazines, and nitrones. 1 is an azide or a nitrone, and F 2is an iminosydnone, a catechol that forms an ortho-quinone group in situ, or a tetrazine. More preferably, F 2 is an iminosydnone represented by structure (F7), a catechol which forms in situ structure (F10), or a tetrazine represented by structure (F8). Even more preferably, F 1 is an azide represented by structure (F1) or a nitro represented by structure (F3), and F 2 is a tetrazine represented by structure (F8a). Most preferably, F 1 is an azide represented by structure (F1), and F 2 is a tetrazine represented by structure (F8a).

[0256] [Linker construct represented by general structure (4)]

[0255] In a further aspect, the present invention relates to a linker construct. The linker construct according to the present invention has the structure (4): [ka] is a heterobifunctional (y+1)valent linker of formula (I). Here, Q 1 is the click probe, and L A is a linker, y is 2, 3, or 4, and F 2 Q 1 It is a click probe that has no reactivity to Q 1 , L A , y, and F 2 are defined elsewhere, and those definitions and their preferred embodiments are equally applicable to the linker constructs according to the invention.

[0257] In a preferred embodiment of the linker construct according to this aspect, Q 1 is a benzoannulated or tetramethylated (hetero)cycloalkyne. Such click probes Q 1 is preferably a click probe F 2 It is combined with tetrazine as Q 1and F 2 are click probes that are reactive in cycloaddition reactions, but do not react with each other.

[0258]

[0257] Q 1 is preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q11), (Q17), (Q18), (Q19), and (Q19a) as defined above, more preferably selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q18), and (Q19a), even more preferably represented by the structure (Q26), (Q27), (Q28), (Q32), (Q37), (Q38), or (Q38a), and most preferably represented by (Q37), (Q40), (Q41), or (Q43).

[0259]

[0258] F 2 is preferably a tetrazine represented by structure (F8), most preferably structure (F8a); [ka] It is a tetrazine represented by the formula: where R 29 is hydrogen, C 1~6 Alkyl, aryl, C(O)-C 1~6 Alkyl, C(O)-aryl, C(O)-OC 1~6 Alkyl, C(O)-O-aryl, C(O)-NR 33 -C 1~6 Alkyl, and C(O)-NR 33 -aryl, and R 33 is H or C 1~4 alkyl, preferably R 29 is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. In a preferred embodiment, R 33 is hydrogen or methyl, and most preferably R 33 is methyl.

[0260]

[0259] The integer y is 2, 3, or 4, preferably y is 2 or 3, and most preferably y is 2.

[0261] [Application] Conjugates of the present invention having a DAR of 6 or greater have high uniformity and a DAR value close to the theoretical DAR value. The conjugates of the present invention are further characterized by high stability, low aggregation tendency, and excellent therapeutic efficacy and tolerability. Therefore, the conjugates of the present invention are particularly suitable for the treatment of cancer. In this respect, the present invention further relates to a method for treating cancer, comprising administering a conjugate of the present invention to a subject in need thereof. The subject in need thereof is typically a cancer patient. The use of conjugates, such as antibody-drug conjugates, is well known in the field of cancer treatment, and the conjugates of the present invention are particularly suitable in this regard. The described method is typically suitable for the treatment of cancer. In the method according to this aspect, the antibody conjugate is typically administered in a therapeutically effective dose. This aspect of the present invention can also be described as a conjugate according to the present invention for use in the treatment of cancer. In other words, this aspect relates to the use of a conjugate according to the present invention for preparing a medicament or pharmaceutical composition for use in the treatment of cancer. In the present context, treating cancer is intended to include treating, imaging, diagnosing, preventing tumor growth, inhibiting tumors, and reducing tumors.

[0262] This aspect of the present invention can also be described as a method for targeting tumor cells expressing a specific extracellular receptor, comprising contacting a conjugate according to the present invention with cells likely to express the extracellular receptor, whereby the antibody specifically targets the extracellular receptor. Thus, the method according to this aspect is suitable for determining whether cells express a desired extracellular receptor. These tumor cells may be present in a subject, in which case the method comprises administering a conjugate according to the present invention to a subject in need thereof. Alternatively, the method is performed ex vivo or in vitro. In a preferred embodiment, the cells likely to express the extracellular receptor are cells that express the extracellular receptor. Targeting tumor cells preferably includes one or more of treating, imaging, diagnosing, preventing, inhibiting, and reducing the growth of tumor cells.

[0263] In the context of diagnosis, it is generally unclear whether the contacted cells actually express the specific extracellular receptor being investigated. For example, in the diagnosis of HER2-positive breast cancer, a conjugate containing an antibody targeting HER2, such as trastuzumab, can be contacted with the cells. If the tumor cells actually express HER2, the conjugate will target the cells, but if the tumor cells do not express HER2, the conjugate will not target the cells. Similarly, in the treatment of cancer cells that specifically express an extracellular receptor, those skilled in the art will understand that a cell binding agent, such as an antibody that targets that specific extracellular receptor, should be used.

[0264] In the method of the present invention, the extracellular receptor may be 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema Preferably, the extracellular receptor is selected from the group consisting of 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tenascin. Likewise, preferably, the tumor cells express an extracellular receptor selected from the same group.One skilled in the art can match a desired extracellular receptor with a suitable cell binding agent that can target that extracellular receptor.

[0265]

[0263] The conjugates of the present invention are also particularly suitable as antibiotics, antivirals, anti-inflammatory agents, and anti-autoimmune agents. Accordingly, in an alternative embodiment, the present invention relates to a method for treating inflammation of an infectious disease or autoimmune disease, comprising administering a conjugate according to the present invention to a subject in need thereof. In the method according to this aspect, the antibody conjugate is typically administered in a therapeutically effective dose. This aspect of the present invention may also be expressed as a conjugate according to the present invention for use in treating inflammation of an infectious disease or autoimmune disease. In other words, this aspect relates to the use of a conjugate according to the present invention for preparing a medicament or pharmaceutical composition for use in treating inflammation of an infectious disease or autoimmune disease. Here, the infection may be bacterial or viral.

[0266] In this embodiment, the antibody is preferably specific for an extracellular protein resulting from a viral infection and / or a tumor-associated carbohydrate antigen. Here, the extracellular protein resulting from a viral infection can be human poliovirus (HPV), human cytomegalovirus (HCMV), or human papillomavirus (HPV). Tumor-associated carbohydrate antigens (TACA) include Tn, STn, T-antigen, LDN, Lewis c (Le c ), Cialis-Lewis c (SLe c ), 6-Sialyl-Lewis c (6SLe c ), LN, alpha-Gal, 3SLN, 6SLN, H antigen, A antigen, B antigen, Lewis a (Le a ), Cialis-Lewis a (SLe a ), 6-Sialyl-Lewis a (6SLe a ), Lewis b (Le b ), Cialis-Lewis b (SLe b), 6-Sialyl-Lewis b (6SLe b ), Lewis x (Le x ), Cialis-Lewis x (SLe x ), 6-Sialyl-Lewis x (6SLe x ), Lewis y (Le y ), Cialis-Lewis y (SLe y ), 6-Sialyl-Lewis y (6SLe y ), and / or combinations thereof. The antibody may also be specific for both an extracellular protein and a TACA simultaneously.

[0267]

[0265] In this respect, the present invention also relates to a pharmaceutical composition comprising a conjugate according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains the conjugate according to the present invention in a pharmaceutically effective dose. [Brief explanation of the drawings]

[0268] [Drawing Description] [Figure 1] A general scheme for preparing antibody-drug conjugates by reaction of a monoclonal antibody (most often a symmetric dimer) containing x functional groups F is shown. The conjugate is obtained by incubating the antibody-(F)x with an excess of a linker-drug construct (Q-spacer-linker-payload) resulting in reaction of F with Q to form a linking group Z. [Figure 2] Various reagents suitable for reaction with cysteine ​​side chains are shown, which can be either mono-alkylating type (A) or cross-linking agents (B) for reaction with two cysteine ​​side chains. [Figure 3]Figure 1 shows a general process for non-genetic conversion of a monoclonal antibody (mAb) into an antibody containing probes for click conjugation (F). The click probes can be located at various positions in the antibody depending on the technique used. For example, an antibody can be converted into an antibody containing two click probes (structure on the left), four click probes (structure below), or eight probes (structure on the right) for click conjugation. [Figure 4] Figure 4 shows a representative (but not comprehensive) set of functional groups (F) that can be introduced into antibodies by genetic engineering, chemical modification, or enzymatic means, followed by a metal-free click reaction with a complementary reactive group Q to generate a linking group Z. Functional group F can be engineered into antibodies at any optimal position. Some functional groups F (e.g., nitrile oxides, quinones) can also react with strained alkenes in addition to strained alkynes, as shown for triazines or tetrazines (bottom row) as examples. Pyridine or pyridazine linking groups are the products of rearrangement of a tetrazabicyclo[2.2.2]octane linking group, formed by reaction of a triazine or tetrazine with an alkyne (but not an alkene), respectively, with loss of N. The linking group Z shown in Figure 4 is a preferred linking group for use in the present invention. [Figure 5] Preferred embodiments are presented for cyclic alkynes suitable for metal-free click chemistry, and reactive moieties Q. This list is not exhaustive, for example, alkynes can be further activated by fluorination, aromatic ring substitution, or introduction of heteroatoms in the aromatic ring. [Figure 6]We demonstrate the glycan remodeling of a full-length IgG followed by site-specific conjugation of a payload based on azido-cyclooctyne click chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all distinct glycoforms, followed by glycosyltransferase-mediated transfer of the azido sugar onto the core GlcNAc, which is released by the endoglycosidase. In the next step, the azido-remodeled IgG is subjected to a single cyclooctyne-modified immune cell-engaging polypeptide for metal-free click chemistry (SPAAC), resulting in a bispecific antibody in a 2:2 molecular format. We also demonstrate that the cyclooctyne-polypeptide construct has a specific spacer between the cyclooctyne and the polypeptide, which allows for tuning of the IgG polypeptide distance or imparts other properties to the resulting bispecific antibody. [Figure 7] The two-step process applied herein describes a glycan remodeling antibody bearing a functional group F1 reacts with a bifunctional linker Q1-L-F2 (where click probes Q1 and F2 are mutually unreactive), thereby undergoing a metal-free click chemistry reaction to form a bond Z1. The bifunctional linker can contain two to four occurrences of F2 (i.e., trivalent, tetravalent, or pentavalent linkers). In the second step, an antibody containing the reactive group F2 reacts with a click probe Q2. This click probe is a linker-drug moiety containing one to three drug occurrences. [Figure 8A] An example of a bifunctional linker 1-L-F2 is shown, where Q1 is DBCO (as in A) and F2 is a tetrazine analog (as in A), satisfying the condition that Q1 and F2 are not mutually reactive. [Figure 8B] Examples of bifunctional linkers 1-L-F2 are shown, where Q1 is TMTHSI (as in B and C) and F2 is a tetrazine analog (as in B and C), satisfying the condition that Q1 and F2 are not mutually reactive. [Figure 9]Two examples of bifunctional linkers Q1-L-F2 are shown, satisfying the condition that Q1 and F2 do not react with each other, where Q1 is TMTHSI (as in D) or DBCO (as in E), the tetrazine is alkyl (as in D), methyltetrazine) or phenyltetrazine (as in E), and the linker is tetravalent (as in D) or pentavalent (as in E). [Figure 10] An example of a bifunctional linker Q1-L-F2(F) is shown, where Q1 is DBCO and F2 is an iminosydnone variant known to react extremely slowly with DBCO (k<0.001 M-1 s-1). [Figure 11] Three examples of bifunctional linkers Q1-L-F2 are shown, where F2 is a potential click-reactive group (i.e., not reactive by itself but requires chemical or enzymatic conversion to a reactive click probe), such as phenol or catechol in compound G (which can be converted to an ortho-quinone upon treatment with tyrosinase or NaIO4, respectively), serine in compound H (which can be converted to a nitrone upon treatment with NaIO4 and then N-methylhydroxylamine), tetrazole in compound I (which can be converted to a nitrile imine upon treatment with 200 nm light), or dihydrotetrazine in compound M (which can be converted to a tetrazine upon treatment with horseradish peroxidase or 660 nm light). [Figure 12] Some examples of preferred linker constructs Q1-L-F2 are shown. [Figure 13] Some examples of preferred linker-payload constructs Q2(LB)Dx (where x=1) are shown. [Figure 14] Some examples of preferred linker-payload constructs Q2(LB)Dx (where x=2) are shown. [Figure 15] Some examples of preferred linker-payload constructs Q2(LB)Dx (where x=3) are shown. [Figure 16]The relative retention times (rrt) of various ADCs to trastuzumab measured by HIC-HPLC are shown, with rrt = 1.14 at DAR4 and rrt = 1.20 at DAR8, while Enhertu® as a comparative example shows RRT = 1.25. [Figure 17] Monomer levels of various ADCs under physiological conditions (PBS, 37°C) are shown. [Figure 18A] Figure 1 shows the RP-UPLC spectrum of intact trastuzumab-(tetrazine)4 [trastuzumab-(45)2]. [Figure 18B] Shifts in retention times of DAR2, 4 and 6 species when conjugated to 98 are shown. [Figure 19A] Figure 19A shows the stability of DAR8 ADCs over time in PBS at 37°C (percentages are relative to the DAR at t=0). Figure 19A shows that the decrease in DAR is equal for all ADCs, regardless of the linker used. The constructs are stable, with a very low decrease of <20% over 28 days. [Figure 19B] Figure 19B shows the stability of DAR8 ADC over time in PBS at 37°C (percentages are relative to the DAR at t=0). Figure 19B shows that no aggregation is observed over this 28 day period. [Figure 20A] Figure 1 shows the stability of the DAR8 ADC over time in human serum at 37° C. Again, all ADCs based on different linkers showed an equivalent decrease in DAR (percentage compared to the DAR at t=0). [Figure 20B] Figure 1 shows the stability of the DAR8 ADC over time in human serum at 37° C. Again, all ADCs based on different linkers showed equal aggregate formation. [Figure 21A] Figure 1 shows the stability of the DAR8 ADC over time in mouse serum at 37° C. Again, all ADCs based on different linkers showed equal DAR reductions (percentages compared to the DAR at t=0). [Figure 21B]Shown is the stability of the DAR8 ADC over time in mouse serum at 37° C. Again, all ADCs based on different linkers showed equal aggregate formation (which showed large variations over time, but the trend was the same). [Figure 22A]

[0023] Figure 1 shows in vitro efficacy data for NCI-N87 cells treated with increasing concentrations of the ADCs trastuzumab-(3)2-(59)4, trastuzumab-(3)2-(63)4, and the corresponding free payloads mitomycin and gefitinib. Both DAR8 ADCs demonstrate improved N87 cell killing with IC50 values ​​of 0.2 nM and 0.5 nM, respectively, compared to 94 nM and 997 nM for the corresponding free payloads. [Figure 22B] Figure 1 shows in vitro efficacy data for BT-474 cells treated with increasing concentrations of the ADCs trastuzumab-(3)2-(59)4 and trastuzumab-(3)2-(63)4 and the corresponding free payloads mitomycin and gefitinib. A similar trend was observed in BT-474 cells, with IC50 values ​​of 0.2 nM and 0.5 nM, respectively, compared to 905 nM and 410 nM for the corresponding free payloads. [Figure 22C] Figure 1 shows in vitro efficacy data for MDA-MB231 cells treated with increasing concentrations of the ADCs trastuzumab-(3)2-(59)4, trastuzumab-(3)2-(63)4, and the corresponding free payloads mitomycin and gefitinib. The negative control cell line, MDA-MB-231, showed no efficacy, and no differentiation was observed between the ADC and the free payload. [Figure 23A] In vitro efficacy data for NCI-N87 cells treated with increasing concentrations of DAR8 ADCs with various payloads are shown. All ADCs demonstrated similar cell killing against NCI-N87 cells, with IC50 values ​​ranging from 0.08 to 0.34 nM. [Figure 23B]Figure 1 shows in vitro efficacy data for MDA-MB-231 cells treated with increasing concentrations of DAR8 ADCs with various payloads. The negative control cell line, MDA-MB-231, showed no cell killing, with no differentiation between the various ADCs. [Figure 24A] Figure 24 shows a comparison of in vitro DAR between mitomycin ADCs. Figure 24A shows the change in efficacy of high DAR species compared to low DAR species. [Figure 24B] Figure 24B shows a comparison of in vitro DAR between mitomycin ADCs. Figure 24B shows the negative control cell line, MDA-MB-231, and no significant differences were observed between the ADCs. [Figure 25A]

[0023] Figure 1 shows in vivo efficacy data over time for the EMT6-hHER2 expressing model. The anti-tumor response of DAR4 compared to DAR8 is similar with dose level adjustment for payload. [Figure 25B] The graph shows changes in mouse body weight over time. [Figure 26A] Figure 1 shows in vivo efficacy data over time of low dose treatment (2 mg / kg) on ​​the EMT6-hHER2 expressing model. All DAR8 ADCs according to the invention show similar anti-tumor responses, with improvements compared to the benchmark Enhertu at the same dose level. [Figure 26B] 1 shows the time course of body weight in mice treated with low doses. [Figure 26C] Plots from high dose treatment (6 mg / kg) of three ADCs are shown. All three DAR8 ADCs according to the invention tested show complete responses by day 30. [Figure 26D] 1 shows the time course of body weight in mice treated with high doses. [Figure 26E] Kaplan-Meier plot of survival over time for test articles at both low and high dose treatments. A clear benefit is observed when comparing the benchmark Enhertu with the novel DAR8 ADC of the present invention. [Figure 27]In vivo tolerability data over time in female CD-1 mice are shown. Only one mouse at the highest concentration was sacrificed due to severe weight loss (>20%). Therefore, the maximum tolerated dose is at least 80 mg / kg. [Figure 28A] Figure 1 shows the RP-UPLC spectrum of intact trastuzumab-(tetrazine)4 (trastuzumab-(3)2). [Figure 28B] Figure 28B shows the RP-UPLC spectrum of the same construct conjugated to 7. The spectrum in Figure 28B was generated before purification and contained some residual compound 3. The spectrum in Figure 28B shows that the DAR8 construct was formed almost exclusively, with only very minor peaks for the others. [Figure 28C] RP-UPLC spectra of the same constructs conjugated to 9 are shown. The spectrum in Figure 28C shows that the DAR8 construct is formed almost exclusively, with only very minor peaks for the others.

[0269] [Example]

[0282] The invention is illustrated by the following examples.

[0270] [General Reagents and Analyses]

[0292] General procedure for analytical RP-UPLC (DTT-treated samples): Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL in PBS pH 7.4) was added to 12.5 mM DTT, 100 mM Tris-HCl pH 8.0 (40 μL) and incubated at 37 °C for 15 min. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on a Waters Acquity UPLC-SQD column. Samples (5 μL) were injected at 0.4 mL / min onto a BioResolve RP mAb 2.1 × 150 mm, 2.7 μm column (Waters) at a column temperature of 70 °C. A linear gradient was applied over 9 min, from 30 to 54% acetonitrile in 0.1% TFA and water. The absorbance of the eluted peak was measured at 215 nm, followed by automatic integration (MassLynx, Waters) to determine the reaction conversion.

[0271]

[0293] General procedure for mass spectrometry analysis of (modified) monoclonal antibodies: Prior to mass spectrometry analysis, IgG was treated with IdeS, which allows for analysis of the Fc / 2 fragment. For analysis of both the light and heavy chains, a solution of 20 μg of (modified) IgG was incubated with 100 mM DTT in a total volume of 4 μL at 37°C for 5 min. Azide functional groups, if present, are reduced to amines under these conditions. For analysis of the Fc / 2 fragment, a solution of 20 μg of (modified) IgG was incubated with IdeS / Fabricator™ (1.25 U / μL) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL at 37°C for 1 h. After diluting the sample to 80 μL, electrospray ionization time-of-flight (ESI-TOF) analysis was performed on a JEOL AccuTOF. Deconvoluted spectra were obtained using Magtran software.

[0272]

[0294] General procedure for SEC analysis: HPLC-SEC analysis was performed on an Agilent 1100 series (Hewlett Packard) using an Xbridge BEH200A (3.5 μM, 7.8 × 300 mm, PN 186007640 Waters) column. Samples were diluted to 1 mg / mL in PBS and run at 0.86 mL / min isocratic flow (100 mM sodium phosphate (NaHPO / NaPO), 200 mM NaCl, pH 6.8, containing 10% isopropanol) for 17 min.

[0273]

[0295] General procedure for enzymatic remodeling of IgG to mAb-(6-N3-GalNAc)2: IgG (15 mg / mL) was incubated with 1 wt% EndoSH (as described in PCT / EP2017 / 052792, see Examples 1-3 and SEQ ID NO: 1, which is incorporated herein by reference), 3 wt% His-TnGalNAcT (as described in PCT / EP2016 / 059194, see Examples 3 and 4 and SEQ ID NO: 49, which is incorporated herein by reference), 0.01% AP (Roche), and UDP6-N3-GalNAc (compound 2d in Figure 3, 10 equivalents relative to IgG) in 6 mM MnCl2 and TBS for 16 hours at 30°C. The functionalized IgG was then purified using a HiTrap MabSelect sure 5 mL column. After loading the reaction mixture, the column was washed with TBS + 0.2% Triton and TBS. IgG was eluted with 0.1 M AcOH (pH 2.7) and neutralized with 2.5 M Tris-HCl (pH 8.8). After dialysis three times against 20 mM histidine, 150 mM NaCl (pH 7.5), IgG was concentrated to 15–20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0274] [Examples 1 to 49: Synthesis of click probes and linker conjugates] Example 1: Preparation of Compound 3

[0296] To a solution of compound 1 (10.0 mg, 1 eq., 12.2 umol) in dry DMF (850 μL) was added compound 2 (10.3 mg, 3 eq., 36.6 μmol), followed by triethylamine (6.18 mg, 8.51 μL, 5 eq., 61.1 μmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by preparative HPLC (30% to 100%, MeCN / water + 10 mM NH4HCO3, run time 21 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 3 (5 mg, 4.7 μmol, 38%) as a pink solid. 57 H 67 N 12 O 10 + (M+H) + LCMS (ESI+) calculated for 1079.5, found 1079.9. [ka]

[0275] Example 2: Preparation of Compound 5

[0297] Compound 4 (163 mg, 240 μmol) was added to a mixture of exatecan mesylate (125 mg, 235 μmol) and DIPEA (61 mg, 82 μL, 0.47 mmol) in dry DMF (0.9 mL). After 20 h, the reaction mixture was diluted with 9 mL of DCM and purified by silica gel flash column chromatography (0→40% MeOH / DCM) to give 5 (155 mg, 159 μmol, 68%). 55 H 54 FN6O 10 + (M+H) + LCMS (ESI+) calculated for 977.39, found 977.72. In addition to 5, exatecan free base (82.4 mg, 189 μmol, 20%) was recovered. 24 H 23 FN3O4 + (M+H) + LCMS (ESI+) calculated for 436.5, found 436.5. [ka]

[0276] Example 3. Preparation of Compound 7

[0298] The synthesis of BCN-HS-PEG2-HS-(va-PABC-Ex)2 (7) is also described in PCT / EP2021 / 075401 (Example 4), which is incorporated herein. To a solution of compound 5 (155 mg, 159 μmol) in DMF (1.6 mL) was added Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 6 (65 mg, 72 μmol) in DMF (1.4 mL). The reaction mixture was stirred for 18 h, diluted with DCM (20 mL), and purified by silica gel flash column chromatography (0→40% MeOH / DCM) to give 7 (94 mg, 44 μmol, 28%) as a pale yellow solid. 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + LCMS (ESI+) calculated for 1066.9, found 1067.1. [ka]

[0277] Example 4. Preparation of Compound 9

[0299] The synthesis of BCN-HS-PEG-(eva-PABC-Ex) (9) is also described in PCT / EP2021 / 075401 (Example 11), which is incorporated herein by reference. A solution of BCN-HS-PEG-b-(Glu(OFm)-OH) (8, 12.1 mg, 10 μmol, 1.0 equiv.) dissolved in anhydrous DMF (180 μL) was added to a solution of NH-Val-Ala-PABC-exatecan (5b, Fmoc-deprotected 5, 19 mg, 25 μmol, 2.5 equiv.) in anhydrous DCM (180 μL), DIPEA (11 μL, 63 μmol, 6.2 equiv.), and HATU (8.9 mg, 23 μmol, 2.3 equiv.). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with DCM (800 μL) and purified by silica gel flash column chromatography (0% → 20% MeOH / DCM) to give the product as a clear oil (yield determination difficult due to DMF content). 140 H 150 F2N 17 O 33 S +2 (M / 2+H) + LCMS (ESI+) calculated for 1334.0, found 1334.8.

[0278]

[0300] This compound was dissolved in DMF (300 μL) and triethylamine (21 μL, 150 μmol, 15 eq) was added. After 17 h at room temperature, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel flash column chromatography (0% → 45% MeOH / DCM) to give compound 9 (10.2 mg, 4.4 μmol, 44%) as a yellow solid. 112 H 130 F2N 17 O 33 S +2 (M / 2+H) + LCMS (ESI+) calculated for 1156.2, found 1156.7. [ka]

[0279] Example 5. Preparation of Compound 11

[0301] Diethanolamine (894.1 mg, 816.5 L, 1.1 equivalents, 8.504 mmol) and sodium carbonate (1.188 g, 1.45 equivalents, 11.21 mmol) were added to 1,4-dioxane (20 mL) and water (20 mL). The mixture was stirred at room temperature to form a suspension. (9H-Fluoren-9-yl)methyl carbonochloridate (2000 mg, 1 equivalent, 7.731 mmol) was dissolved in toluene (5 mL) and added dropwise to the reaction solution (the internal temperature was controlled not to exceed 25°C during the addition), and stirring was continued for 2 hours. The pH was adjusted to 3 with 4 M HCl (aqueous), and the aqueous phase was extracted twice with ethyl acetate (50 mL). The organic phase was washed with saturated brine (2 x 50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give compound 10 (3.13 g, 7.6 mmol, 99%, 80% pure) as an opaque oil. 19 H 22 No. 4 + (M+H) + LCMS (ESI+) calculated for 328.2, found 928.3.

[0280]

[0302] Compound 10 (1260 mg, 1 eq, 3.849 mmol) was dissolved in dry DCM (50 mL). Bis(perfluorophenyl)carbonate (3.185 g, 2.1 eq, 8.082 mmol) was then added, followed by DIPEA (1.492 g, 2.01 mL, 3 eq, 11.55 mmol), and stirring was continued for 3 h. The residue was then purified by silica gel flash column chromatography (0→10% acetone / DCM) to give compound 11 (2.5 g, 3.3 mmol, 87%, 100% pure) as a clear oil. 33 H 20 F 10 No.8 + (M+H) + LCMS (ESI+) calculated for 748.1, found 748.4. [ka]

[0281] Example 6. Preparation of Compound 13

[0303] A solution of 2,2'-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine) (3.40 g, 3.35 mL, 10 equiv., 22.9 mmol) in DCM (40 mL) was cooled to 0 °C. To this was added di-tert-butyl dicarbonate (500 mg, 1 equiv., 2.29 mmol), and the mixture was stirred at room temperature for 18 h. The organic phase was washed with water (3 × 40 mL) until all unreacted 2,2'-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine) was extracted. The organic phase was dried (NaSO) and concentrated in vacuo to give 12 (361 mg, 1.3 mmol, 57%, 90% pure) as a yellow oil. 11 H 25 N2O4 + (M+H) + LCMS (ESI+) calculated for 249.2, found 249.4.

[0282]

[0304] To a solution of amine 12 (150.0 mg, 1 equiv., 200.7 μmol) in dry DCM (5.00 mL) was added compound 11 (109.6 mg, 2.2 equiv., 441.5 μmol), followed by DIPEA (129.7 mg, 175 μL, 5 equiv., 1.003 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo. The resulting crude product was dissolved in DCM (1 mL) and loaded onto a column for purification by silica gel flash column chromatography (0→20% MeOH / DCM) to afford the Boc-protected intermediate (218 mg, 0.20 mmol, 99%, 80% purity) as a colorless oil. 43 H 66 N5O 14 + (M+H) + LCMS (ESI+) calculated for 876.5, found 876.7.

[0283]

[0305] To a solution of the intermediate (176 mg, 1 equiv., 201 μmol) in dry DCM (5.00 mL) was added HCl in dioxane (147 mg, 1.00 mL, 4 mol, 20 equiv., 4.02 mmol). The reaction mixture was stirred at room temperature for 18 h and then concentrated in vacuo to give the HCl salt 13 (150 mg, 200 μmol, 99%) as an off-white foam. 33 H 50 N5O 10 + (M+H) + LCMS (ESI+) calculated for 676.4, found 676.6. [ka]

[0284] Example 7. Preparation of Compound 15

[0306] To a solution of sulfo-D-alanine hydrate (554 mg, 1 equiv., 2.96 mmol) and triethylamine (599 mg, 825 μL, 2 equiv., 5.92 mmol) in DMF (15 mL) was added di-tert-butyl dicarbonate (775 mg, 1.2 equiv., 3.55 mmol). The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated in vacuo. The residue was redissolved in CHCl (10 mL) and treated with EtO (30 mL) with vigorous stirring. The organic solvent was decanted and discarded. The oily residue was washed with EtO (20 mL) and dried in vacuo to give compound 14 (1134 mg, 2.9 mmol, 100%, 70% purity) as a colorless oil. 14 NO7S - (MH) - LCMS (ESI-) calculated for 268.1, found 268.3.

[0285]

[0307] To a solution of N-Boc-cysteic acid 14 (54 mg, 3 equiv., 0.20 mmol) in DMF (1 mL), compound 13 (50 mg, 1 equiv., 67 μmol), HATU (76 mg, 3 equiv., 0.20 mmol), and DIPEA (52 mg, 70 μL, 6 equiv., 0.40 mmol) were added and stirring was continued for 2 h. The mixture was concentrated in vacuo, and the crude product was dissolved in DCM (10 mL). The mixture was purified by silica gel flash column chromatography (0% to 70% MeOH / DCM) to give the Boc-protected intermediate (50 mg, 36 μmol, 53%, 84% purity) as a colorless oil. 49 H 75 N7O 22 S2 + (M+H) + LCMS (ESI+) calculated for 1178.4, found 1178.7.

[0286]

[0308] To a solution of the Boc-protected intermediate (50 mg, 1 equiv., 42 μmol) in dry DCM (2.00 mL) and MeOH (1.00 mL) was added HCl in 1,4-dioxane (46 mg, 0.32 mL, 4 mol, 30 equiv., 1.3 mmol). The reaction mixture was stirred at room temperature for 26 hours, after which additional HCl in 1,4-dioxane (46 mg, 0.32 mL, 4 mol, 30 equiv., 1.3 mmol) was added to the reaction mixture. Stirring was continued for an additional hour, after which the reaction mixture was concentrated in vacuo to give compound 15 (45 mg, 43 μmol, 100%) as an off-white solid. 39 H 60 N7O 18 S2 + (M+H) + LCMS (ESI+) calculated for 978.3, found 978.6. [ka]

[0287] Example 8. Preparation of Compound 17

[0288]

[0309] To a solution of 4-(6-methyl-1,2,4,5-tetrazin-3-yl)butanoic acid (4.3 mg, 2.5 equiv., 24 μmol) in DMF (1 mL) was added HATU (11 mg, 3 equiv., 29 μmol) and DIPEA (7.4 mg, 9.9 μL, 6 equiv., 57 μmol). After 10 min, the HCl salt 15 (10 mg, 1 equiv., 9.5 mol) was added and stirring was continued for 1 h. The reaction mixture was concentrated in vacuo, and the crude product was dissolved in MeCN (1 mL). The solution was purified by reverse-phase chromatography (10% to 100%, MeCN / water) to give 16 (7 mg, 5 μmol, 50%, 95% pure) as a pink solid. 53 H 76 N 15 O 20 S2 + (M+H) + LCMS (ESI+) calculated for 1306.5, found 1306.7.

[0289]

[0310] Triethylamine (11 mg, 15 μL, 20 equiv., 0.11 mmol) was added to a solution of compound 16 (7.0 mg, 1 equiv., 5.4 μmol) in DMF (0.200 mL) and HO (10 μL). The reaction mixture was stirred for 18 h until complete Fmoc deprotection was observed. TMTHSI-OSU (2.7 mg, 1.5 equiv., 8.0 mol) was added to the reaction mixture, and stirring was continued for 4 days. The reaction mixture was purified by preparative HPLC (30% to 100%, MeCN / water + 1% AcOH, run time 21 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 17 (2.5 mg, 1.9 μmol, 36%) as a pink solid. 49 H 78 N 16 O 20 S3 -2 (M / 2-H) - LCMS (ESI-) calculated for 653.2, found 653.6. [ka]

[0290] Example 9. Preparation of Compound 19

[0311] To a solution of compound 11 (50.0 mg, 1 equiv., 66.9 μmol) in dry DMF (1.00 mL) was added 3-(2-(2-aminoethoxy)ethoxy)propanoic acid (26.1 mg, 2.2 equiv., 147 μmol), followed by DIPEA (43.2 mg, 334 μL, 5 equiv., 334 μmol). After stirring for 18 h, the reaction mixture was concentrated in vacuo. The resulting crude product was dissolved in DCM (3 mL) and loaded onto a column for purification by silica gel flash column chromatography (0-10% MeOH / DCM (containing 1% AcOH)) to afford 18 (55.3 mg, 75.4 μmol, 113%) as a colorless oil. 35 H 48 N3O 14 + (M+H) + LCMS (ESI+) calculated for 734.3, found 734.6.

[0291]

[0312] To a solution of compound 18 (50.0 mg, 1 eq, 68.1 μmol) dissolved in DCM (2 mL) and DMF (2 mL) was added DCC (32.3 mg, 2.3 eq, 157 μmol) and 1-hydroxypyrrolidine-2,5-dione (18.0 mg, 2.3 eq, 157 μmol). The reaction mixture was stirred at room temperature for 18 hours. Sulfo-L-alanine (34.6 mg, 3 eq, 204 μmol) and DIPEA (105.6 mg, 142.4 μL, 12 eq, 818 μmol) were added to the reaction mixture, and stirring was continued for 24 hours. The mixture was filtered, and the filtrate was concentrated in vacuo. The resulting crude product was dissolved in water and MeCN and purified by reverse-phase chromatography (0→100% MeCN / HO+1% AcOH) to give compound 19 (73.1 mg, 70.6 μmol, 104%) as a colorless oil. 41 H 56 N5O 22 S2 - (MH) - LCMS (ESI-) calculated for 1034.3, found 1034.7. [ka]

[0292] Example 10: Preparation of Compound 21

[0313] DIPEA (7.49 mg, 10.1 μL, 6 eq., 57.9 μmol) and HATU (9.18 mg, 2.5 eq., 24.1 mol) were added to compound 19 (10.0 mg, 1 eq., 9.65 mol) in DMF (0.5 mL). Subsequently, (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (5.05 mg, 2.2 eq., 21.2 μmol) was added. The mixture was stirred for 1 hour, concentrated in vacuo, and the resulting crude product was dissolved in DCM (10 mL). This solution was purified by silica gel flash column chromatography (0% to 30% MeOH / DCM) to give compound 20 (2.4 mg, 1.7 μmol, 18%) as a pink solid. 61 H 74 N 15 O 20 S2 - (MH) - LCMS (ESI-) calculated for 1400.5, found 1400.7.

[0293]

[0314] Triethylamine (1.7 mg, 2.4 μL, 10 equiv., 17 μmol) was added to compound 20 (2.4 mg, 1.0 equiv., 1.7 μmol) in DMF (0.1 mL). The reaction mixture was stirred for 42 h until Fmoc deprotection was observed. TMTH-SI-OSU (0.70 mg, 1.2 equiv., 2.1 μmol) was added to the reaction mixture, and stirring was continued for 92 h. LCMS analysis indicated the formation of 21. 57 H 79 N 16 O 20 S3 - (MH) - LCMS (ESI-) calculated for 1403.5, found 1403.6. [ka]

[0294] Example 11: Preparation of Compound 23

[0315] To a solution of 4-(6-methyl-1,2,4,5-tetrazin-3-yl)butanoic acid (24 mg, 2.5 equiv., 0.13 mmol) in DMF (1 mL) was added HATU (61 mg, 3 equiv., 0.16 mmol) and DIPEA (55 mg, 74 μL, 8 equiv., 0.43 mmol). After 10 min, compound 13 (40 mg, 1 equiv., 53 μmol) was added and stirring was continued for 1 h. The reaction mixture was purified by silica gel flash column chromatography (0%→10% MeOH / DCM) to give the Fmoc-protected intermediate (82 mg, 53 μmol, 97%, 65% purity) as a pink solid. 47 H 66 N 13 O 12 + (M+H) + LCMS (ESI+) calculated for 1004.5, found 1004.8.

[0295]

[0316] To a solution of the Fmoc-protected intermediate (82 mg, 65 wt%, 1 equiv, 53 μmol) in DCM (1.0 mL) was added piperidine (0.18 g, 0.21 mL, 40 equiv, 2.1 mmol) and stirring was continued for 22 h. The reaction mixture was washed with saturated NaHCO3, dried, and concentrated in vacuo to give 42 mg of a pink crude product. The crude product was dissolved in DCM (1 mL) and purified by silica gel flash column chromatography (0% to 15% MeOH / DCM) to give compound 22 (11.7 mg, 15.0 μmol, 28%) as a pink solid. 32 H 56 N 13 O 10 + (M+H) + LCMS (ESI+) calculated for 782.4, found 782.7.

[0296]

[0317] To a solution of compound 22 (5.8 mg, 1 equiv., 7.4 μmol) in DCM (0.50 mL) was added DBCO-C6-OSu (4.8 mg, 1.5 equiv., 11 μmol), HOBt (1.7 mg, 1.5 equiv., 11 μmol), and triethylamine (3.8 mg, 5.2 μL, 5 equiv., 37 μmol). The resulting reaction mixture was allowed to stand for 3 days, then diluted with 550 μL of DMF and purified by preparative HPLC (600 μL injection volume of DMF; 5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 21 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 23 (1.6 mg, 1.5 μmol, 20%) as a pink oil. 53 H 73 N 14 O 12 + (M+H) + LCMS (ESI+) calculated for 1097.5, found 1097.8. [ka]

[0297] Example 12: Preparation of Compound 25

[0318] 2-(2-aminoethoxy)ethan-1-ol (185.9 mg, 177.4 μL, 3 equiv., 1.768 mmol) was added to a solution of TMTHSI-OSu (200.6 mg, 1 equiv., 589.3 μmol) in DCM (3 mL). The reaction mixture was stirred for 21 h and then purified by silica gel flash column chromatography (0% to 60% acetone / DCM) to give the inactive alcohol (168.2 mg, 509.0 μmol, 86.37%) as a colorless oil. 15 H 27 N2O4S + (M+H) + LCMS (ESI+) calculated for 331.2, found 331.1.

[0298]

[0319] The inert alcohol (168.2 mg, 1.0 equiv., 509.0 μmol) was dissolved in DCM (3.00 mL), followed by the addition of bis(4-nitrophenyl)carbonate (309.7 mg, 2 equiv., 1.018 mmol) and triethylamine (154.5 mg, 213 μL, 3.0 equiv., 1.527 mmol). The solution was stirred for 2 hours and then purified by silica gel flash column chromatography (0% to 30% acetone / acetone) to give compound 24 (262.5 mg, 0.41 mmol, 80%, 77% pure). 22 H 30 N3O8S + (M+H) + LCMS (ESI+) calculated for 496.2, found 496.2.

[0299]

[0320] To a solution of compound 22 (5.8 mg, 1 eq., 7.4 μmol) in DCM (0.50 mL) was added compound 24 (5.5 mg, 1.5 eq., 11 μmol), HOBt (1.1 mg, 1 eq., 7.4 μmol), and triethylamine (3.8 mg, 5.2 μL, 5 eq., 37 μmol). The resulting reaction mixture was stirred for 18 hours and then purified by preparative HPLC (600 μL injection volume of DMF; 30% to 100% MeCN / water + 10 mM NH4HCO3, run time 21 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 25 (3.9 mg, 3.4 μmol, 46%) as a pink oil. 48 H 80 N 15 O 15 S + (M+H) + LCMS (ESI+) calculated for 1138.6, found 1138.8. [ka]

[0300] Example 13: Preparation of Compound 26

[0321] To a solution of compound 1 (9.35 mg, 1 equiv., 11.4 μmol) in anhydrous DMF (500 μL) was added the HCl salt of methyltetrazine-PEG4-amine (9.8 mg, 2.1 equiv., 25 μmol), followed by triethylamine (5.2 mg, 7.2 μL, 4.5 equiv., 52 μmol). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with 200 μL of DMF and purified by preparative HPLC (30% to 70%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 26 (9.1 mg, 6.9 μmol, 61%) as a pink oil. 67 H 87 N 12 O 16 + (M+H) + LCMS (ESI+) calculated for 1315.6, found 1315.8. [ka]

[0301] Example 14: Preparation of Compound 27

[0322] To a solution of compound 1 (8.5 mg, 1.0 equiv., 10 μmol) in dry DMF (500 μL) was added the HCl salt of methyltetrazine-benzylamine (7.4 mg, 3.0 equiv., 31 μmol), followed by triethylamine (5.3 mg, 7.2 μL, 5.0 equiv., 52 μmol). The reaction mixture was stirred at room temperature for 75 minutes and then purified by preparative HPLC (30% to 95% MeCN / water + 10 mM NH4HCO3, run time 12 minutes, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 27 (6.1 mg, 5.9 μmol, 57%, purity 97%) as a pink solid. 53 H 59 N 12 O8 + (M+H) + LCMS (ESI+) calculated for 991.5, found 991.7. [ka]

[0302] Example 15: Preparation of Compound 29

[0323] To a solution of compound 24 (49.4 mg, 80 wt%, 1 equiv., 79.7 μmol) in anhydrous DCM (500 μL) was added diethanolamine (8.91 mg, 1.06 equiv., 84.7 μmol) and triethylamine (49 mg, 67 μL, 6.0 equiv., 0.48 mmol). After stirring at room temperature for 21 h, bis(4-nitrophenyl)carbonate (65.4 mg, 2.70 equiv., 215 μmol) was added to the reaction mixture. After stirring for an additional 2 h, the reaction mixture was purified by silica gel flash column chromatography (0% to 20% acetone / DCM) to give compound 28 (40.9 mg, 35 μmol, 43%, 67% purity) as a yellow oil. 34 H 42 N5O 15 S + (M+H) + LCMS (ESI+) calculated for 792.2, found 792.5.

[0303]

[0324] Compound 28 (7.9 mg, 67% wt, 1 eq, 6.7 μmol) was dissolved in anhydrous DMF, followed by the addition of compound 2 (4.5 mg, 2.4 eq, 16 μmol) and triethylamine (3.4 mg, 4.7 μL, 5 eq, 33 μmol). After stirring at room temperature for 24 hours, the reaction mixture was purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 29 (5.3 mg, 5.3 μmol, 79%) as a pink oil. 46 H 62 N 13 O 11 + (M+H) + LCMS (ESI+) calculated for 1004.4, found 1004.6. [ka]

[0304] Example 16: Preparation of Compound 31

[0325] A solution of Boc-Glu(OFm)-OH (639 mg, 1 equiv., 1.50 mmol) in DCM (5 mL) was cooled to 0 °C, followed by the addition of trifluoroacetic acid (3.08 g, 2.08 mL, 18 equiv., 27.0 mmol). The reaction mixture was stirred for 19 h, then concentrated in vacuo and coevaporated with DCM (6 × 5 mL) to give the TFA salt of NH-Glu(Fm)-OH (569.9 mg, 1.30 mmol, 86.6%) as an off-white solid. 19 H 20 No. 4 + (M+H) + LCMS (ESI+) calculated for 326.2, found 326.3.

[0305]

[0326] To a solution of compound 28 (10.0 mg, 1 equiv., 13 μmol) in anhydrous DMF (250 μL) and anhydrous DCM (250 μL) was added the TFA salt of NH-Glu(Fm)-OH (14 mg, 2.5 equiv., 32 μmol) and DIPEA (9.8 mg, 13 μL, 6 equiv., 76 μmol). After stirring at room temperature for 15 hours, the reaction mixture was purified by silica gel flash column chromatography (0% to 30% MeOH / DCM) to give compound 30 (14.2 mg, 12 μmol, 95%, 98% pure). 60 H 70 N5O 17 S + (M+H) + LCMS (ESI+) calculated for 1164.5, found 1164.7.

[0306]

[0327] HATU (5.1 mg, 2.1 equiv., 13 μmol) and DIPEA (4.9 mg, 6.6 μL, 6 equiv., 38 μmol) were added to a solution of compound 30 (7.3 mg, 1 equiv., 6.3 μmol) in DCM (500 μL). After stirring at room temperature for 5 minutes, the HCl salt of methyltetrazine-benzylamine (3.4 mg, 2.3 equiv., 14 μmol) and anhydrous DMF (200 μL) were added to the reaction mixture. After stirring at room temperature for 3 hours, the reaction mixture was purified by silica gel flash column chromatography (0→70% EtOAc / heptane, followed by 0%→10% MeOH / DCM) to give the Fmoc-protected tetrazine intermediate (3.6 mg, 1.5 μmol, 23%, 62% pure). 80 H 88 N 15 O 15 S + (M+H) + LCMS (ESI+) calculated for 1530.6, found 1530.8.

[0307]

[0328] The Fmoc-protected tetrazine intermediate (3.6 mg, 1.5 μmol, 23%, 62% purity) was dissolved in anhydrous DMF (300 μL), followed by the addition of piperidine (8.0 mg, 40 equiv., 94 μmol). After stirring at room temperature for 2 hours, the reaction mixture was further diluted with DMF (200 μL) and purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 31 (0.6 mg, 0.5 μmol, 20%, 95% purity) as a pink oil. 52 H 68 N 15 O 15 S + (M+H) + LCMS (ESI+) calculated for 1174.5, found 1174.6. [ka]

[0308] Example 17: Preparation of Compound 33

[0329] A. To a solution of compound 14 (11.4 mg, 1.7 equiv., 29 μmol) in dry DMF (200 μL) was added HATU (22 mg, 3.4 equiv., 58 μmol), followed by DIPEA (17 mg, 24 μL, 8.0 equiv., 134 μmol). After 30 min, methyltetrazine-benzylamine HCl salt (4.0 mg, 1.0 equiv., 17 μmol) was added. The reaction mixture was stirred at room temperature for 5 h, concentrated in vacuo, and purified by silica gel flash column chromatography (0% to 20% MeOH / DCM) to give the Boc-protected compound (4.5 mg, 9.9 μmol, 59%, 97% purity) as a pink solid. C. 18 H 23 N6O6S - (MH) - LCMS (ESI-) calculated for 451.1, found 451.1.

[0309]

[0330] The Boc-protected compound (2.2 mg, 97% wt, 1.0 equiv, 4.9 μmol) was dissolved in DCM (200 μL) and TFA (22 mg, 15 μL, 40 equiv, 190 μmol) was added. The reaction mixture was stirred at room temperature for 1 hour and concentrated in vacuo to give compound 32 (2.3 mg, 4.9 μmol, 100% purity) as a pink oil. The yield was assumed to be quantitative, and compound 32 was used in the next step without further purification. 13 H 15 N6O4S - (MH) - LCMS (ESI-) calculated for 351.1, found 351.0.

[0310]

[0331] To a solution of compound 28 (3.5 mg, 1.0 equiv., 4.4 μmol) in dry DMF (300 μL) was added compound 32 (3.8 mg, 1.8 equiv., 8.1 μmol), followed by EtN (8.8 mg, 12 μL, 20 equiv., 20 μmol) and HOBt (0.7 mg, 1.0 equiv., 4.4 μmol). The reaction mixture was stirred at room temperature for 20 hours and then purified by preparative HPLC (30% → 95% MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 33 (0.5 mg, 0.4 μmol, 9%, 100% pure) as a pink solid. 48 H 62 N 15 O 17 S3 - (MH) - LCMS (ESI-) calculated for 1216.4, found 1216.7. [ka]

[0311] Example 18: Preparation of Compound 35

[0332] N-Boc-ethylenediamine (10.3 mg, 10.2 μL, 6.85 equiv., 64.4 μmol) was added to a solution of compound 28 (10.2 mg, 73 wt.%, 1 equiv., 9.40 μmol) in anhydrous DCM (500 μL). After stirring at room temperature for 1 hour, the reaction mixture was purified by silica gel flash column chromatography (0%→30% EtOAc / DCM, followed by 0%→10% MeOH / DCM) to give the Boc-protected intermediate (7.7 mg, 8.7 μmol, 92%, 93.7% purity). 36 H 64 N7O 13 S + (M+H) + LCMS (ESI+) calculated for 834.4, found 834.3.

[0312]

[0333] The Boc-protected intermediate (7.2 mg, 1 equiv., 8.6 μmol) was dissolved in anhydrous DCM (300 μL), followed by the addition of TFA (20 mg, 13 μL, 20 equiv., 0.17 mmol). The mixture was stirred at room temperature for 1 hour, then concentrated in vacuo and coevaporated with toluene to give compound 34 (7.2 mg, 8.7 μmol, 100%). The yield was assumed to be quantitative, and the product was used in the next step without further purification. 26 H 48 N7O9S + (M+H) + LCMS (ESI+) calculated for 634.3, found 634.3.

[0313]

[0334] HATU (4.4 mg, 2.7 equivalents, 12 μmol) and DIPEA (3.4 mg, 4.5 μL, 6 equivalents, 26 μmol) were added to a solution of 4-(6-methyl-1,2,4,5-tetrazin-3-yl)butanoic acid (2.0 mg, 2.5 equivalents, 11 μmol) in anhydrous DMF (150 μL). After stirring at room temperature for 30 minutes, a solution of compound 34 (3.6 mg, 1 equivalent, 4.3 μmol) in anhydrous DMF (150 μL) was added. After an additional 45 min, the reaction mixture was purified by preparative HPLC (5% to 95% MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 35 (2.2 mg, 1.7 μmol, 39%, purity 74%) as a pink oil. 40 H 64 N 15 O 11 S + (M+H) + LCMS (ESI+) calculated for 962.5, found 962.7. [ka]

[0314] Example 19: Preparation of Compound 37

[0335] The HCl salt of EDC (77.1 mg, 2.1 equiv., 338 μmol) and HOBt (49.3 mg, 2 equiv., 322 μmol) were added to a solution of Fmoc-Ser-OH (52.7 mg, 1 equiv., 161 μmol) in anhydrous DMF (1 mL). After stirring at room temperature for 10 minutes, N-Boc-ethylenediamine (4.0 mg, 3.9 μL, 1.2 equiv., 25 μmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then purified by silica gel flash column chromatography (0%→5% MeOH / DCM). The residue was triturated with DCM to give the Boc-protected intermediate (53.7 mg, 85.2 μmol, 52.9%, 74.5% purity) as a white solid. 25 H 32 N3O6 + (M+H) + LCMS (ESI+) calculated for 470.2, found 470.5.

[0315]

[0336] The Boc-protected intermediate (53.7 mg, 74.5 wt%, 1 equiv., 85.2 μmol) was dissolved in anhydrous DCM (2 mL), followed by the addition of trifluoroacetic acid (194 mg, 131 μL, 20 equiv., 1.70 mmol). The mixture was stirred at room temperature for 2.5 h, then concentrated in vacuo and coevaporated with toluene to give compound 36 (39.8 mg, 85.1 μmol, 100%). The yield was assumed to be quantitative, and the product was used in the next step without further purification. 20 H 24 N3O4 + (M+H) + LCMS (ESI+) calculated for 370.2, found 370.2.

[0316]

[0337] To a solution of compound 36 (9.5 mg, 2.5 equiv., 20 μmol) in anhydrous DCM (580 μL) was added compound 1 (12.8 mg, 1.9 equiv., 15.7 μmol) and DIPEA (5.2 mg, 7.1 μL, 5 equiv., 41 μmol). After stirring at room temperature for 75 min, complete conversion to the Fmoc-protected DBCO intermediate was observed, at which point piperidine (14 mg, 16 μL, 20 equiv., 0.16 mmol) was added to the reaction mixture. After stirring for 4 h, the reaction mixture was concentrated in vacuo, and the residue was partitioned between water and DCM. The aqueous layer was evaporated in vacuo, and the crude product was transferred to an Eppendorf tube. The residue was dissolved in water (500 μL) and DMF (400 μL), the resulting solution was centrifuged, and the supernatant was purified by preparative HPLC (5% to 50% MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 37 (1.4 mg, 1.4 μmol, 17%, purity 86%). 43 H 61 N8O 12 - (MH) - LCMS (ESI-) calculated for 881.4, found 881.3. [ka]

[0317] Example 20: Preparation of Compound 38

[0338] To a solution of compound 28 (17 mg, 61 wt%, 1 equiv., 13 μmol) in anhydrous DCM (500 μL) was added a solution of compound 36 (22.5 mg, 3.75 equiv., 47.6 μmol) in anhydrous DCM (750 μL), followed by DIPEA (14.7 mg, 20 μL, 8.75 equiv., 0.11 mmol). After stirring at room temperature for 26 hours, the solution was concentrated in vacuo, and the residue was dissolved in DMF (600 μL) and purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give the Fmoc-protected TMTHSI intermediate (8.5 mg, 6.8 μmol, 53%).62 H 78 N9O 17 S + (M+H) + LCMS (ESI+) calculated for 1252.5, found 1252.8.

[0318]

[0339] The Fmoc-protected TMTHSI intermediate (8.5 mg, 1 equiv. 6.8 μmol) was dissolved in anhydrous DMF (450 μL), followed by the addition of piperidine (23 mg, 27 μL, 40 equiv., 0.27 mmol). After stirring for 2 h, the reaction mixture was purified by reverse-phase chromatography (10% to 100%, MeCN / water + 10 mM NH4HCO3) to give compound 38 (1.8 mg, 2.2 μmol, 33%). 32 H 58 N9O 13 S + (M+H) + LCMS (ESI+) calculated for 808.4, found 808.7. [ka]

[0319] Example 21: Preparation of Compound 41

[0340] To a solution of tert-butyl (R)-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-yl)carbamate (509 mg, 1.0 equiv., 1.67 mmol) in dry DCM (10 mL) was added CSI (260 mg, 159 μL, 1.1 equiv., 1.83 mmol), and the reaction mixture was stirred at room temperature for 10 min. EtN (506 mg, 697 μL, 3.0 equiv., 5.00 mmol) was added, followed by m-PEG-NH (380 mg, 1.1 equiv., 1.83 mmol). The reaction mixture was stirred at room temperature for 3 h and then purified by silica gel flash column chromatography (0%→25% MeOH / DCM) to give the Boc- and TBS-protected compound (1.01 g, 1.5 mmol, 90%, 92% purity) as a clear oil. 19 H 44 N3O 11 SSi +(M+H-Boc) + LCMS (ESI+) calculated for 518.3, found 518.5.

[0320]

[0341] MeOH (8.7 g, 11 mL, 5.10 2 After cooling on ice, acetyl chloride (1.28 g, 1.16 mL, 30 equiv., 16.3 mmol) was added dropwise. The reaction mixture was stirred at 0° C. for 5 minutes. The ice bath was removed, and a solution of the Boc- and TBS-protected compound (337 mg, 92% wt., 1.0 equiv., 545 μmol) in dry DCM (1.5 mL) was added. The reaction mixture was stirred at room temperature for 18 hours and concentrated in vacuo to give compound 39 (196 mg, 446 μmol, 82% wt., 100% pure). 13 H 30 N3O9S + (M+H) + LCMS (ESI+) calculated for 404.1, found 404.4.

[0321]

[0342] To a solution of compound 11 (60 mg, 1.0 equiv., 80 μmol) in dry DCM (3000 μL) was added compound 39 (177 mg, 5.0 equiv., 402 μmol), followed by DIPEA (62 mg, 84 μL, 6.0 equiv., 482 μmol). The reaction mixture was stirred at room temperature for 22 hours and then purified by silica gel flash column chromatography (0% to 10% MeOH / DCM) to give the diol compound (72 mg, 53 μmol, 66%, 87% pure). 47 H 76 N7O 24 S2 + (M+H) + LCMS (ESI+) calculated for 1186.4, found 1186.7.

[0322]

[0343] To a solution of the diol compound (5.0 mg, 87 wt%, 1.0 equiv, 3.7 μmol) in dry DCM (200 μL) was added bis-PNP carbonate (3.3 mg, 3.0 equiv, 11 μmol), followed by DIPEA (2.8 mg, 3.8 μL, 6.0 equiv, 22 μmol). The reaction mixture was stirred at room temperature for 19 hours. Methyltetrazine-benzylamine HCl salt (2.4 mg, 2.8 equiv, 10 μmol) was added. The reaction mixture was stirred at room temperature for 3 hours and then purified by silica gel flash column chromatography (0%→15% MeOH / DCM) to give compound 40 (2.6 mg, 1.6 μmol, 43%, 100% pure). 69 H 97 N 18 O 26 S2 + (M+NH4) + LCMS (ESI+) calculated for 1657.6, found 1658.0.

[0323]

[0344] To a solution of compound 40 (2.6 mg, 1.0 equiv., 1.6 μmol) in DCM (200 μL) was added piperidine (5.4 mg, 6.3 μL, 40 equiv., 63 μmol). The reaction mixture was stirred at room temperature for 4 hours. This was purified by silica gel flash column chromatography (0% to 20% MeOH / DCM) to give the Fmoc-deprotected compound (2.5 mg, 1.6 μmol, 100%, 92% purity). 54 H 97 N 17 O 24 S2 + (M+H) + LCMS (ESI+) calculated for 1418.5, found 1419.0.

[0324]

[0345] To a solution of the Fmoc-deprotected compound (2.5 mg, 1.0 equiv., 1.8 μmol) in DMF (200 μL) was added compound 24 (1.3 mg, 1.5 equiv., 2.6 μmol), followed by EtN (1.8 mg, 2.4 μL, 10 equiv., 18 μmol) and HOBt (0.4 mg, 1.5 equiv., 2.6 μmol). The reaction mixture was stirred at room temperature for 23 hours and then purified by preparative HPLC (5% to 95% MeCN / water + 10 mm NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 41 (0.6 mg, 0.3 μmol, 20%, 100% pure) as a pink solid. 70 H 109 N 19 O 29 S3 +2 (M / 2+H) + LCMS (ESI+) calculated for 887.8, found 887.9. [ka]

[0325] Example 22: Preparation of Compound 45

[0346] The HCl salt of EDC (228.7 mg, 1.2 equiv., 1.002 mmol) and HOAt (34.10 mg, 250.5 μL, 1 mol, 0.3 equiv., 250.5 μmol) were added to a solution of Fmoc-NH-propionic acid (400.3 mg, 1.2 equiv., 1.002 mmol) in anhydrous DCM (5 mL). After stirring at room temperature for 30 min, a solution of tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (400.3 mg, 1.2 equiv., 1.002 mmol) in anhydrous DMF (1 mL) was added. The reaction mixture was stirred at room temperature for 75 min, after which the DCM was evaporated and the residue purified by reverse-phase chromatography (10% to 100%, MeCN / water + 10 mM NH4HCO3) to give compound 42 (585.4 mg, 628 μmol, 75.2%, purity 95.2%) as a colorless oil. 47 H 71 N2O 14 + (M+H) +LCMS (ESI+) calculated for 887.5, found 887.8.

[0326]

[0347] Compound 42 (585.4 mg, 95% wt, 1 eq, 626.9 μmol) was dissolved in anhydrous DCM (1.5 mL), followed by the addition of trifluoroacetic acid (2.2 g, 1.5 mL, 31 eq, 19 mmol). After stirring at room temperature for 2 hours, DCM (10 mL) was added, and the reaction mixture was extracted with 1 M HCl (3 × 10 mL). The combined aqueous layers were extracted with DCM (2 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give compound 43 (327.7 mg, 0.41 mmol, 65%, 90% pure) as a pale yellow gel. 35 H 47 N2O 14 + (M+H) + LCMS (ESI+) calculated for 719.3, found 719.7.

[0327]

[0348] The HCl salt of EDC (24.8 mg, 4.09 equiv., 109 μmol) and HOBt (16.7 mg, 4.11 equiv., 109 μmol) were added to a solution of compound 43 (21.2 mg, 90% wt., 1.0 equiv., 26.5 μmol) in anhydrous DCM (400 μL). After stirring at room temperature for 90 minutes, the HCl salt of methyltetrazine-benzylamine (25.2 mg, 3.99 equiv., 106 μmol) was added. The reaction mixture was stirred for 15 minutes and then purified by silica gel flash column chromatography (0%→15% MeOH / DCM) to give the Fmoc-protected thorazine intermediate (15.5 mg, 11 μmol, 43%, 94% purity) as a pink oil. 65 H 74 N 17 O 11 + (M+H) + LCMS (ESI+) calculated for 1268.6, found 1269.1.

[0328]

[0349] The Fmoc-protected thorazine intermediate (15.5 mg, 95.0 wt%, 1 equiv, 11.6 μmol) was dissolved in anhydrous DCM (200 μL), followed by the addition of piperidine (43 mg, 50 μL, 44 equiv, 0.51 mmol). After stirring at room temperature for 90 minutes, the reaction mixture was purified by silica gel flash column chromatography (0% to 35% MeOH / DCM) to afford compound 44 (11.8 mg, 9.9 μmol, 86%, 88% pure) as a pink solid. 50 H 64 N 17 O9 + (M+H) + LCMS (ESI+) calculated for 1046.5, found 1046.9.

[0329]

[0350] To a solution of compound 44 (15 mg, 92 wt%, 1 equiv., 13 μmol) in anhydrous DMF (450 μL) was added DBCO-NHS ester (8.0 mg, 1.5 equiv., 20 μmol), followed by DIPEA (8.5 mg, 11 μL, 5 equiv., 66 μmol). After stirring at room temperature for 1 h, the reaction mixture was further diluted with 250 μL of DMF and purified by preparative HPLC (30% to 95%, MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column). The resulting residue was dissolved in DCM (1 mL) and precipitated by the addition of diethyl ether (15 mL). The precipitate was washed with diethyl ether (3 × 5 mL) and concentrated in vacuo to give compound 45 (9.5 mg, 7.1 μmol, 54%) as a pink solid. 69 H 77 N 18 O 11 + (M+H) + LCMS (ESI+) calculated for 1333.6, found 1333.7. [ka]

[0330] Example 23: Preparation of Compound 46

[0351] To a solution of compound 44 (3.0 mg, 1.0 equiv., 2.7 μmol) in dry DMF (200 μL) was added TMTHSI-OSu (3.3 mg, 3.6 equiv., 9.6 μmol), followed by triethylamine (2.6 mg, 3.8 μL, 10 equiv., 26 μmol) and HOBt (0.5 mg, 1.2 equiv., 3.2 μmol). The reaction mixture was stirred at room temperature for 26 hours and then purified by preparative HPLC (30% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 46 (0.6 mg, 0.4 μmol, 20%, purity 93%) as a pink solid. 61 H 79 N 18 O 11 S + (M+H) + LCMS (ESI+) calculated for 1271.6, found 1271.8. [ka]

[0331] Example 24: Preparation of Compound 49

[0352] To a solution of compound 47 (25.4 mg, 1 equiv., 24.0 μmol, commercially available from GenScript Biotech) in anhydrous DMF (500 μL) was added methyltetrazine-NHS ester (20.3 mg, 2.59 equiv., 62.0 μmol), followed by DIPEA (21.5 mg, 29.0 μL, 6.95 equiv., 166 μmol). After stirring for 30 minutes, the desired product was precipitated by the addition of diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 × 1.5 mL) and concentrated in vacuo to give the Alloc-protected tetrazine intermediate (57.0 mg, 21.9 μmol, 91.2%, 49.4% pure). 63 H 70 N 17 O 14 + (M+H) + LCMS (ESI+) calculated for 1288.5, found 1288.9.

[0332]

[0353] The Alloc-protected tetrazine intermediate (22.9 mg, 49.4 wt%, 1 equiv., 8.78 μmol) was dissolved in anhydrous DMF (500 μL), followed by the addition of tetrakis(triphenylphosphine)palladium(0) (1.26 mg, 179 μL, 6.1 mmol, 0.124 equiv., 1.09 μmol) and pyrrolidine (2.0 mg, 2.3 μL, 3.2 equiv., 28 μmol). After 10 min, the reaction mixture was purified by reverse-phase chromatography (10% to 100%, MeCN / water + 10 mM NH4HCO3) to give compound 48 (13.2 mg, 5.9 μmol, 67%, 54% pure) as a pink solid. 57 H 61 N 17 O 14 + (M+H) + LCMS (ESI+) calculated for 1208.5, found 1209.0.

[0333]

[0354] Compound 48 (13.2 mg, 54% wt, 1 eq, 5.90 μmol) was dissolved in anhydrous DMF (350 μL), followed by the addition of piperidine (10.0 mg, 11.7 μL, 20 eq, 118 μmol). After 35 min, the reaction mixture was purified by reverse-phase chromatography (10% to 100%, MeCN / water + 10 mM NH4HCO3) to afford the Fmoc-deprotected intermediate (3.4 mg, 3.3 μmol, 56%) as a pink solid. 42 H 52 N 17 O 12 + (M+H) + LCMS (ESI+) calculated for 986.4, found 986.7.

[0334]

[0355] To a solution of the Fmoc-deprotected intermediate (3.4 mg, 1 equiv., 3.3 μmol) in anhydrous DMF (200 μL) was added DBCO-NHS ester (3.1 mg, 2.3 equiv., 7.7 μmol) and DIPEA (2.1 mg, 2.9 μL, 5 equiv., 17 μmol). After 1 h, the reaction mixture was purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 49 (0.7 mg, 0.5 μmol, 20%, purity 94%) as a pink oil. 61 H 65 N 18 O 14 + (M+H) + LCMS (ESI+) calculated for 1273.5, found 1273.8. [ka]

[0335] Example 25: Preparation of Compound 51

[0356] To a solution of compound 48 (7.1 mg, 1 equiv., 5.9 μmol) in anhydrous DMF (300 μL) was added amino-PEG-OH (6.5 mg, 3 equiv., 18 μmol), followed by DIPEA (4.6 mg, 6.1 μL, 6 equiv., 35 μmol) and HATU (4.9 mg, 2.2 equiv., 13 μmol). After 45 min, the desired product was precipitated by the addition of diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 × 1.5 mL) and concentrated in vacuo to give compound 50 (31.4 mg, 10 μmol, 170%, 62% pure) as a pink solid. The yield was assumed to be quantitative, and the product was used in the next step without further purification. 89 H 126 N 19 O 28 - (MH) - LCMS (ESI-) calculated for 1908.9, found 1908.6.

[0336]

[0357] Compound 50 (11 mg, 1 equiv., 5.8 μmol) was dissolved in anhydrous DMF (300 μL), followed by the addition of piperidine (9.8 mg, 11 μL, 20 equiv., 0.12 mmol). After 2.5 h, the desired product was precipitated by the addition of diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 × 1.5 mL) and concentrated in vacuo to give the Fmoc-deprotected intermediate (8.0 mg, 3.2 μmol, 55%, 67% purity) as a pink solid. 74 H 116 N 19 O 26 - (MH) - LCMS (ESI-) calculated for 1686.8, found 1687.2.

[0337]

[0358] To a solution of the Fmoc-deprotected intermediate (8.0 mg, 1 equiv., 4.7 μmol) in anhydrous DMF (350 μL) was added DBCO-NHS ester (2.9 mg, 1.5 equiv., 7.1 μmol) and DIPEA (3.1 mg, 4.1 μL, 5 equiv., 24 μmol). After 1 h, the reaction mixture was purified by preparative HPLC (30% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm). The residue was dissolved in DMF (500 μL) and precipitated by the addition of diethyl ether (1 mL). The precipitate was washed with diethyl ether (3 × 1.5 mL) and concentrated in vacuo to give compound 51 (2.8 mg, 1.3 μmol, 27%, purity 90%) as a purple solid. 93 H 132 N 20 O 28 +2 (M / 2+H) + LCMS (ESI+) calculated for 988.5, found 988.7. [ka]

[0338] Example 26: Preparation of Compound 54

[0359] To a solution of compound 52 (25.0 mg, 1 equiv., 17.0 μmol, commercially available from GenScript Biotech) in anhydrous DMF (500 μL) was added methyltetrazine-NHS ester (19.6 mg, 3.52 equiv., 59.9 μmol), followed by DIPEA (15.4 mg, 20.7 μL, 7 equiv., 119 μmol). After stirring for 2 h, the reaction mixture was diluted with DMF (4 mL), followed by the addition of diethyl ether (10 mL) to precipitate the desired product. The precipitate was washed with diethyl ether (3 × 5 mL) and concentrated in vacuo to afford the Alloc-protected tetrazine intermediate (73.9 mg, 29 μmol, 170%, 70% purity) as a pink solid. 87 H 98 N 25 O 20 + (M+H) + LCMS (ESI+) calculated for 1812.7, found 1813.0.

[0339]

[0360] The Alloc-protected tetrazine intermediate (30.8 mg, 1 equiv., 17.0 μmol) was dissolved in anhydrous DMF (3 mL), followed by the addition of tetrakis(triphenylphosphine)palladium(0) (1.96 mg, 218 μL, 7.8 mmol, 0.1 equiv., 1.70 μmol) and pyrrolidine (3.62 mg, 4.19 μL, 3 equiv., 51.0 μmol). After 10 min, the reaction mixture was purified by reverse-phase chromatography (10% to 100%, MeCN / water + 10 mM NH4HCO3) to give compound 53 (21.0 mg, 11 μmol, 63%, 86% pure) as a pink solid. 78 H 86 N 25 O 20 + (M+H) + LCMS (ESI+) calculated for 1692.6, found 1692.9.

[0340]

[0361] Compound 53 (21 mg, 86% wt, 1 equiv, 11 μmol) was dissolved in anhydrous DMF (2 mL), followed by the addition of piperidine (18 mg, 21 μL, 20 equiv, 0.21 mmol). After 10 min, the desired product was precipitated by the addition of diethyl ether (10 mL). The precipitate was washed with diethyl ether (3 × 5 mL) and concentrated in vacuo to give the Fmoc-deprotected intermediate (24.9 mg, 15 μmol, 140%, 89% purity) as a pink solid. The yield was assumed to be quantitative, and the product was used in the next step without further purification. 63 H 76 N 25 O 18 + (M+H) + LCMS (ESI+) calculated for 1470.6, found 1470.9.

[0341]

[0362] To a solution of the Fmoc-deprotected intermediate (8.2 mg, 89% wt, 1 equiv, 5.0 μmol) in anhydrous DMF (500 μL) was added DBCO-NHS ester (6.0 mg, 3 equiv, 15 μmol) and DIPEA (1.9 mg, 2.6 μL, 3 equiv, 15 μmol). After 45 min, the reaction mixture was purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm). The product-containing fractions were lyophilized to give compound 54 (4.1 mg, 1.8 μmol, 36%, purity 76%) as a pink solid. 82 H 87 N 26 O 20 - (MH) - LCMS (ESI+) calculated for 1755.7, found 1755.2. [ka]

[0342] Example 27: Preparation of Compound 55

[0363] To a solution of compound 54 (8.00 mg, 1 equiv., 4.55 μmol) in anhydrous DMF (500 μL) was added 1-aminooctaethylene glycol (8.1 mg, 4.8 equiv., 22 μmol), followed by HATU (6.5 mg, 3.8 equiv., 17 μmol) and DIPEA (3.53 mg, 4.76 μL, 6 equiv., 27.3 μmol). After 10 min, the desired product was precipitated by the addition of diethyl ether (10 mL). The precipitate was washed with diethyl ether (3 × 5 mL) and diluted with DMF (600 μL) and DMSO (300 μL). The reaction mixture was purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) and the product-containing fractions were lyophilized to give compound 55 (0.6 mg 0.2 μmol, 5%). 130 H 189 N 29 O 41 2+ (M / 2+H) + LCMS (ESI+) calculated for 1406.7, found 1406.8. [ka]

[0343] Example 28: Preparation of Compound 57

[0364] To a solution of compound 6 (500 mg, 5.55 mL, 100 mmol, 1 equiv., 555 μmol) in DMF (5 mL) was added triethylamine (281 mg, 387 μL, 5 equiv., 2.78 mmol), followed by Val-Ala-PABC (375 mg, 2.3 equiv., 1.28 mmol). The resulting solution was stirred for 4 h and then purified by silica gel flash column chromatography (0% to 20% MeOH / DCM) to afford the diol intermediate (431 mg, 356 μmol, 64.2%) as a white solid. 52 H 77 N 10 O 19 S2 + (M+H) +LCMS (ESI+) calculated for 1209.5, found 1210.1.

[0344]

[0365] To a solution of the diol intermediate (157 mg, 1 equiv., 130 μmol) in DMF (2 mL) was added bis(4-nitrophenyl)carbonate (118 mg, 3 equiv., 389 μmol), followed by DIPEA (83.9 mg, 113 μL, 5 equiv., 649 μmol). After stirring for 2 h, additional bis(4-nitrophenyl)carbonate (118 mg, 3 equiv., 389 μmol) was added to the reaction mixture. After stirring for a total of 4 h, the reaction mixture was purified by silica gel flash column chromatography (0%→10% MeOH / DCM) to afford compound 56 (178 mg, 116 μmol, 89.1%) as a white solid. 66 H 83 N 12 O 27 S2 + (M+H) + LCMS (ESI+) calculated for 1539.5, found 1540.0.

[0345]

[0366] To a stock solution of compound 56 (4.10 mg, 26.6 μL, 100 mmol, 1 eq, 2.66 μmol) in DMF was added triethylamine (1.35 mg, 1.85 μL, 5 eq, 13.3 μmol) and DMAP (650 μg, 5.32 μL, 1000 mmol, 2 eq, 5.32 μmol), followed by a stock solution of paclitaxel (5.00 mg, 58.6 μL, 100 mmol, 2.2 eq, 5.86 μmol) in DMF. The resulting solution was allowed to stand at room temperature for 25 hours and then purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 minutes, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 57 (1.4 mg, 0.41 μmol, 16%, 88% purity) as a white solid. 148 H 176 N 12 O 49 S2 +2 (M / 2+H) + LCMS (ESI+) calculated for 1485.0, found 1485.5. [ka]

[0346] Example 29: Preparation of Compound 59

[0367] To a solution of Fmoc-Val-Ala-PABC-PNP 4 (152.7 mg, 1.5 equiv., 224.3 μmol) in dry (500 μL) water was added a stock solution of mitomycin C in DMF (50.00 mg, 1.496 mL, 100 mmol, 1 equiv., 149.6 μmol), DIPEA (57.99 mg, 78.2 μL, 3 eq., 448.7 μmol), and HOBt (34.35 mg, 224.3 μL, 1000 mmol, 1.5 equiv., 224.3 μmol). After standing for 5 h, the reaction mixture was purified by silica gel flash column chromatography (0% → 10% MeOH / DCM) to give the Fmoc-protected intermediate (115 mg, 91.9 μmol, 62%, 70% pure) as a brown solid. 46 H 53 N8O 11 + (M+NH4) + LCMS (ESI+) calculated for 893.4, found 894.0.

[0347]

[0368] The Fmoc-protected intermediate (115 mg, 70 wt%, 1 equiv., 91.9 μmol) was dissolved in DMF (500 μL) and piperidine (78.3 mg, 90.8 μL, 10 equiv., 919 μmol) was added. The resulting solution was stirred for 1 h and then purified by silica gel flash column chromatography (0% to 30% MeOH / DCM) to give compound 58 (72.9 mg, 112 μmol, 121%) as a brown solid. 31 H 40 N7O9 + (M+H) + LCMS (ESI+) calculated for 654.3, found 654.8.

[0348]

[0369] Compound 58 (5.0 mg, 70% wt, 3 equiv, 5.4 μmol) was dissolved in DCM (100 μL), followed by the addition of compound 6 (1.6 mg, 18 μL, 100 mmol, 1 equiv, 1.8 μmol) and DIPEA (3.0 mg, 4.0 μL, 13 equiv, 23 μmol). The resulting solution was allowed to stand for 4 days and purified by silica gel flash column chromatography (0% → 10% MeOH / DCM). The crude product was then purified by preparative HPLC (5% → 95% MeCN / water, run time 12 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 59 (3.4 mg, 1.7 μmol, 93%, purity 94%) as a brown solid. 84 H 108 N 18 O 31 S2 - M *- LCMS (ESI+) calculated for 1928.7, found 1928.5. [ka]

[0349] Example 30: Preparation of Compound 61

[0370] To a solution of Fmoc-Val-Ala-PABC-PNP 4 (155.2 mg, 1.2 equiv., 228.0 μmol) in dry DCM (500 μL) was added a stock solution of gemcitabine in DMF (50.00 mg, 1.900 μL, 100 mmol, 1 equiv., 190.0 μmol), DIPEA (73.66 mg, 99.3 μL, 3 equiv., 569.9 μmol), and DMAP (11.60 mg, 94.98 μL, 1000 mmol, 0.5 equiv., 94.98 mol). After standing for 3 h, the reaction mixture was purified by silica gel flash column chromatography (0%→20% MeOH / DCM), followed by preparative HPLC of the resulting crude product (5%→95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give the Fmoc-protected intermediate (15 mg, 19 μmol, 9.8%) as a white solid. 40 H43 F2N6O 10 + (M+H) + LCMS (ESI+) calculated for 805.7, found 805.3.

[0350]

[0371] The Fmoc-protected intermediate (15 mg, 1 equiv., 19 μmol) was dissolved in DMF (200 μL) and triethylamine (57 mg, 78 μL, 30 equiv., 0.56 mmol) was added. The resulting solution was allowed to stand for 22 h and then purified by silica gel flash column chromatography (0% to 100% MeOH / DCM) to afford compound 60 (8.9 mg, 15 μmol, 82%) as a white solid. 25 H 33 F2N6O8 + (M+H) + LCMS (ESI+) calculated for 583.2, found 583.6.

[0351]

[0372] Compound 60 (3.8 mg, 2.1 μmol, 42%) was dissolved in DMF (250 μL), followed by the addition of compound 6 (4.6 mg, 51 μL, 100 mmol, 1 equiv., 5.1 μmol) and triethylamine (5.2 mg, 7.1 μL, 10 equiv., 51 μmol). The resulting solution was allowed to stand for 22 h and then purified by preparative HPLC (5% to 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 61 (3.8 mg, 2.1 μmol, 42%) as a white solid. 72 H 96 F4N 16 O 29 S2 +2 (M / 2+H) + LCMS (ESI+) calculated for 894.3, found 895.0. [ka]

[0352] Example 31: Preparation of Compound 63

[0373] To a solution of gefitinib (50.0 mg, 1.0 equiv., 112 μmol) in dry DMF (1.1 mL) and dry DCM (2.5 mL) cooled to 0° C., sodium hydride (9.0 mg, 60% by weight, 2.0 equiv., 224 μmol) was added. The reaction mixture was stirred at 0° C. for 1 hour and then warmed to room temperature, after which compound 4 (114 mg, 1.5 equiv., 168 μmol) was added. The reaction mixture was stirred at room temperature for 21 hours and then purified by silica gel flash column chromatography (0→10% MeOH / DCM) to give the Fmoc-protected intermediate (16.0 mg, 15 μmol, 14%, 95% purity). 53 H 56 ClFN7O9 - (M-H+HCOOH) - LCMS (ESI-) calculated for 988.4, found 988.8.

[0353]

[0374] To a solution of the Fmoc-protected intermediate (16.0 mg, 95% wt, 1.0 equiv, 15.4 μmol) in dry DMF (200 μL) was added EtN (46.7 mg, 64.3 μL, 30 equiv, 461 μmol). The reaction mixture was stirred at room temperature for 60 h and purified by silica gel flash column chromatography (0→40% MeOH / DCM) to give compound 62 (11.4 mg, 14 μmol, 93%, 96% purity). 38 H 46 ClFN7O7 - (M-H+HCOOH) - LCMS (ESI-) calculated for 766.3, found 766.7.

[0354]

[0375] To a solution of compound 62 (11.4 mg, 96 wt%, 3.0 equiv., 14.3 μmol) in dry DMF (200 μL) was added compound 6 (4.3 mg, 1.0 equiv., 4.8 mol), followed by DIPEA (6.15 mg, 8.3 μL, 10 equiv., 47.6 mol). The reaction mixture was stirred at room temperature for 20 hours and then purified by preparative HPLC (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 63 (4.4 mg, 1.8 μmol, 39%, purity 90%). 96 H 122 Cl2F2N 18 O 23 S2 2+ (M / 2+H) + LCMS (ESI+) calculated for 1033.9, found 1034.4. [ka]

[0355] Example 32: Preparation of Compound 67

[0376] To a solution of paclitaxel (50.0 mg, 1.0 equiv., 58.6 μmol) in dry DMF (586 μL) was added bis(4-nitrophenyl)carbonate (35.6 mg, 2.0 equiv., 117 μmol), followed by triethylamine (17.8 mg, 24.5 μL, 3.0 equiv., 176 μmol). The reaction mixture was stirred at 0° C. for 3 h and then purified by silica gel flash column chromatography (0→100% EtOAc / heptane) to give compound 64 (32.9 mg, 30 μmol, 50%, 90% purity) as a white powder. 54 H 55 N2O 18 + (M+H) + LCMS (ESI+) calculated for 1019.3, found 1019.7.

[0356]

[0377] To a solution of compound 4 (750 mg, 1.0 equiv., 1.10 mmol) in dry THF (5.51 mL) was added tert-butyl (S)-methyl(pyrrolidin-2-ylmethyl)carbamate (394 mg, 1.7 equiv., 1.84 mmol), followed by DIPEA (427 mg, 576 μL, 3.0 equiv., 3.31 mmol). The reaction mixture was stirred at room temperature for 3 hours and then purified by silica gel flash column chromatography (0→10% MeOH / DCM) to give the Boc-protected intermediate (861 mg, 1.14 mmol, 88%, 85% purity) as a pale yellow solid. 42 H 57 N6O8 + (M+NH4) + LCMS (ESI+) calculated for 773.4, found 773.7.

[0357]

[0378] To a solution of the Boc-protected intermediate (833 mg, 85 wt%, 1.0 equiv, 937 μmol) in dry DCM (2.8 mL) was added TFA (2.14 g, 1.43 mL, 20 equiv, 18.7 mmol) at −15° C. The reaction mixture was stirred at −15° C. for 4 h and then precipitated with EtO. The solid was collected by filtration, washed with additional EtO, and concentrated in vacuo to give compound 65 (737 mg, 947 μmol, 101%, 99% pure) as a brown solid. 37 H 46 N5O6 + (M+H) + LCMS (ESI+) calculated for 656.3, found 656.6.

[0358]

[0379] To a solution of compound 65 (20.2 mg, 99% by weight, 1.0 equiv., 19.9 μmol) in dry DMF (100 μL) and dry DCM (200 μL) was added compound 64 (29.3 mg, 90% by weight, 1.3 equiv., 25.9 μmol), followed by DIPEA (7.7 mg, 10.4 μL, 3.0 equiv., 59.7 mol). The reaction mixture was stirred at room temperature for 1 hour and purified by silica gel flash column chromatography (0→5% MeOH / DCM) to give the Fmoc-protected intermediate (26.5 mg, 16.0 μmol, 78%, 90% pure) as a pale yellow solid.85 H 95 NO 21 + (M+H) + LCMS (ESI+) calculated for 1535.7, found 1536.4.

[0359]

[0380] To a solution of the Fmoc-protected intermediate (26.5 mg, 90% wt, 1.0 equiv, 15.5 μmol) in DMF (200 μL) was added piperidine (4.0 mg, 4.6 μL, 3.0 equiv, 46.6 μmol). The reaction mixture was stirred at room temperature for 1 hour and then purified by silica gel flash column chromatography (0→20% MeOH / DCM) to give compound 66 (18.9 mg, 14.4 μmol, 93%, 100% pure) as a white solid. 70 H 85 NO 19 + (M+H) + LCMS (ESI+) calculated for 1313.6, found 1314.1.

[0360]

[0381] To a solution of compound 66 (5.0 mg, 2.5 equiv., 3.8 μmol) in dry DMF (90 μL) was added compound 6 (1.4 mg, 15 μL, 1.0 equiv., 1.5 μmol), followed by EtN (1.7 mg, 2.4 μL, 11.0 equiv., 17 μmol). The reaction mixture was stirred at room temperature for 30 hours and then purified by preparative HPLC (5% to 95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 67 (2.6 mg, 0.8 μmol, 53%, 100% pure). 162 H 196 N 16 O 51 S2 2- (M / 2-H) - LCMS (ESI-) calculated for 1623.1, found 1623.5. [ka]

[0361] Example 33: Preparation of Compound 70

[0382] A solution of nemorubicin (150.0 mg, 1 equiv., 233.0 μmol) in DMF (1.2 mL) and DCM (2.0 mL) was cooled to 0° C., followed by the addition of bis(4-nitrophenyl)carbonate (286.6 mg, 4.04 equiv., 942.1 μmol) and triethylamine (94.33 mg, 130 μL, 4 equiv., 932.2 μmol). After stirring at 0° C. for 4.5 h, the reaction mixture was purified by silica gel flash column chromatography (0% to 7% MeOH / DCM) to give compound 68 (170.1 mg, 0.16 mmol, 68%, 75% purity) as a red oil. 39 H 41 N2O 17 + (M+H) + LCMS (ESI+) calculated for 809.2, found 809.5.

[0362]

[0383] To a solution of compound 68 (67.9 mg, 75 wt%, 1 eq, 63.0 μmol) in anhydrous DMF (200 μL) and anhydrous DCM (500 μL) was added compound 65 (63.3 mg, 76 wt%, 0.992 eq, 62.5 μmol) and DIPEA (48.8 mg, 65.8 μL, 6 eq, 378 μmol). After stirring the reaction for 17 hours, piperidine (53.6 mg, 62.2 μL, 10 eq, 630 μmol) was added. The reaction mixture was stirred at room temperature for 1 hour and then purified by silica gel flash column chromatography (0% to 30% MeOH / DCM) to give compound 69 (48.2 mg, 43.7 μmol, 69.4%) as a red oil. 55 H 71 NO 18 + (M+H) + LCMS (ESI+) calculated for 1103.5, found 1103.9.

[0363]

[0384] To a solution of compound 69 (6.9 mg, 3 equiv., 6.3 μmol) in anhydrous DMF (150 μL) was added a solution of compound 6 in DMF (1.9 mg, 21 μL, 100 mmol, 1.0 equiv., 2.1 μmol), followed by triethylamine (1.1 mg, 1.5 μL, 5 equiv., 10 μmol). After stirring at room temperature for 22 h, N,N-dimethyl-1,2-ethanediamine (0.08 mg, 0.1 μL, 0.4 equiv., 0.9 μmol) was added to quench unreacted and unreacted compound 6. The reaction mixture was stirred for 15 min and then purified by silica gel flash column chromatography (0% to 8% MeOH / DCM) to give compound 70 (3.4 mg, 1.2 μmol, 58%) as a red oil. 132 H 172 N 16 O 49 S2 2+ (M / 2+H) + LCMS (ESI+) calculated for 1415.0, found 1415.4. [ka]

[0364] Example 34: Preparation of Compound 72

[0385] The synthesis of compound 71 is described in PCT / EP2021 / 075401 (Example 7), which is incorporated herein. Compound 58 (18.2 mg, 1 equivalent, 27.8 μmol) was dissolved in DMF (150 μL) and compound 71 (20 mg, 91.5 wt%, 1.3 equivalents, 35 μmol) (dissolved in 50 μL of dry DMF) was added, followed by triethylamine (7.0 mg, 9.6 μL, 2.5 equivalents, 69 μmol). The reaction mixture was allowed to stand for 20 hours and then purified by preparative HPLC (5% to 95%, MeCN / water + 1% AcOH, run time 12 minutes, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 72 (24.6 mg, 23.3 μmol, 83.7%, purity 98.5%) as a brown solid. 47 H 65 N 10 O 16 S+ (M+NH4) + LCMS (ESI+) calculated for 1057.4, found 1057.8. [ka]

[0365] Example 35: Preparation of Compound 75

[0386] To a solution of β-lapachone (100 mg, 1.0 equiv., 405 μmol) in THF (3000 μL) and water (3000 μL) was added NaSO (356 mg, 4.3 equiv., 1.74 mmol), followed by TBAB (659 mg, 5.0 equiv., 2.02 mmol). The reaction mixture was heated to 80 °C. A solution of KOH (504 mg, 20 equiv., 8.09 mmol) in water (800 μL) was added, followed by a solution of Boc-para-aminobenzyl bromide (656 mg, 5.5 equiv., 2.23 mmol). The reaction mixture was refluxed for 150 min, after which ethyl acetate (10 mL) was added. The organic layer was washed with water (3 × 10 mL), dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was then dissolved in heptane (5 mL) and purified by silica gel flash column chromatography (0→20% EtOAc / heptane) to afford the Boc-protected intermediate (123 mg, 203 μmol, 50%, 74% purity) as a pale yellow oil. 27 H 32 No. 5 + (M+H) + LCMS (ESI+) calculated for 450.2, found 450.3.

[0366]

[0387] To a solution of the Boc-protected intermediate (123 mg, 74 wt%, 1.0 equiv, 203 μmol) in dry DCM (2000 μL) cooled to 0° C., TFA (500 μL) was added. The reaction mixture was stirred at 0° C. for 3 h and then concentrated in vacuo to give compound 73 (94 mg, 203 μmol, 100%, 100% pure). The yield was assumed to be quantitative. 22 H 24 No. 3 - (M+H) -LCMS (ESI-) calculated for 350.1, found 350.1.

[0367]

[0388] To a solution of Fmoc-Val-Ala-OH (117 mg, 1.5 equiv., 284 μmol) in dry DMF (350 μL) and dry DCM (2400 μL) was added DIPEA (74 mg, 99 μL, 3.0 equiv., 568 μmol) and HATU (108 mg, 1.5 equiv., 284 μmol). The reaction mixture was stirred for 30 minutes, after which a solution of compound 73 (88 mg, 1.0 equiv., 189 μmol) in dry DMF (750 μL) was added. The reaction mixture was stirred for 10 minutes and then purified by silica gel flash column chromatography (0→3% MeOH / DCM) to give compound 74 (193 mg, 0.2 mmol, 110%, 79% pure) as a pale yellow oil. 45 H 48 N3O7 + (M+H) + LCMS (ESI+) calculated for 742.4, found 742.6.

[0368]

[0389] To a solution of compound 74 (193 mg, 79 wt%, 1.0 equiv, 205 μmol) in dry DMF (2500 μL) was added piperidine (52 mg, 61 μL, 3.0 equiv, 616 μmol). The reaction mixture was stirred for 1 h and purified by silica gel flash column chromatography (0→15% MeOH / DCM) to give the Fmoc-deprotected intermediate (64 mg, 98 μmol, 47%, 78% purity). 30 H 36 N3O5 - (MH) - LCMS (ESI-) calculated for 518.3, found 518.3.

[0369]

[0390] To a solution of the Fmoc-deprotected intermediate (31.2 mg, 78% wt, 4.9 equiv, 48.6 μmol) in dry DMF (200 μL) was added compound 6 (8.9 mg, 1.0 equiv, 9.9 μmol), followed by EtN (15.0 mg, 20.6 μL, 15 equiv, 148 μmol). The reaction mixture was stirred at room temperature for 49 hours and then purified by preparative HPLC (30% to 95% MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 75 (5.2 mg, 3.1 μmol, 32%, 100% pure). 82 H 105 N 10 O 23 S2 + (M+H) + LCMS (ESI+) calculated for 1661.7, found 1662.1. [ka]

[0370] Example 36: Preparation of Compound 77

[0391] A solution of midostaurin (22.4 mg, 1 equiv., 39.3 μmol) in anhydrous THF (200 μL) was cooled to 0° C., followed by the addition of sodium hydride (3.3 mg, 60 wt.%, 2.1 equiv., 83 μmol). After stirring at 0° C. for 20 minutes, compound 4 (53.6 mg, 2.01 equiv., 78.7 mol) was added. The reaction mixture was stirred for 10 minutes, then quenched with water (50 μL), and stirring was continued at 0° C. for 1 hour. Piperidine (66.8 mg, 77.6 μL, 20 equiv., 785 μmol) was then added to the reaction mixture. After stirring at 0° C. for 2.5 hours, the reaction mixture was diluted with DCM (700 μL) and purified by silica gel flash column chromatography (0%→35% MeOH / DCM). The residue was dissolved in DCM (500 μL) and precipitated by the addition of cold diethyl ether (1.5 mL). The precipitate was washed with cold diethyl ether (3×1.5 mL) and concentrated in vacuo to give compound 76 (23.2 mg, 25 μmol, 64%, purity 96%) as a white solid. 51 H 52N7O8 + (M+H) + LCMS (ESI+) calculated for 890.4, found 890.8.

[0371]

[0392] To a solution of compound 76 (11.6 mg, 3 equiv., 13.0 μmol) in anhydrous DMF (250 μL) was added compound 6 (4.1 mg, 95% wt., 1.0 equiv., 4.3 μmol) and triethylamine (2.20 mg, 3.03 μL, 5 equiv., 21.7 μmol). After stirring at room temperature for 18 h, the reaction mixture was diluted with DCM (2 mL) and purified by silica gel flash column chromatography (0% to 10% MeOH / DCM) to give compound 77 (6.4 mg, 2.3 μmol, 53%, 86% pure). 124 H 137 N 19 O 29 S2 +2 ((M+NH4+H) / 2) + LCMS (ESI+) calculated for 1210.5, found 1210.9. [ka]

[0372] Example 37: Preparation of Compound 79

[0393] Fmoc-Val-Ala-OH (40.0 mg, 1.5 equiv., 97.4 μmol) was dissolved in dry DMF (0.500 mL) and added to a stock solution of lenalidomide in DMF (16.8 mg, 325 μL, 200 mmol, 1 equiv., 65.0 μmol), followed by the addition of DIPEA (33.6 mg, 45.3 μL, 4 equiv., 260 μmol) and HATU (49.4 mg, 2 equiv., 130 μmol). The reaction mixture was stirred for 20 hours and then purified by silica gel flash column chromatography (0→10% MeOH / DCM) to give compound 78 (23 mg, 35 μmol, 54%) as a colorless oil. 36 H 38 N5O7 + (M+H) + LCMS (ESI+) calculated for 652.3, found 652.6.

[0373]

[0394] To a solution of compound 78 (23 mg, 1 equiv., 35 μmol) in DCM (1.0 mL), piperidine (60 mg, 70 μL, 20 equiv., 0.71 mmol) was added and stirring was continued for 1 h. The reaction mixture was purified by silica gel flash column chromatography (0→30% MeOH / DCM) to afford the Fmoc-deprotected intermediate (18 mg, 34 μmol, 95%, 80% purity) as a white solid. 21 H 26 N5O5 - (MH) - LCMS (ESI+) calculated for 428.2, found 428.3.

[0374]

[0395] To a solution of the Fmoc-deprotected intermediate (12.00 mg, 3 equiv., 27.94 μmol) in anhydrous DMF (279.4 μL) was added compound 6 (8.390 mg, 1 equiv., 9.314 μmol) and triethylamine (9.424 mg, 13.0 μL, 10 equiv., 93.14 μmol). After stirring at room temperature for 16 hours, the Fmoc-deprotected intermediate (6.000 mg, 1.5 equiv., 13.97 μmol) and triethylamine (4.712 mg, 6.49 μL, 5 equiv., 46.57 mol) were added. After stirring at room temperature for 3 h, the reaction mixture was purified by prep HPLC; (5% to 95%, MeCN / water + 10 mM NH4HCO3, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 79 (6 mg, 3 μmol, 30%, purity 80%). 64 H 85 N 14 O 23 S2 + (M+H) + LCMS (ESI+) calculated for 1481.5, found 1481.9. [ka]

[0375] Example 38: Preparation of Compound 82

[0396] A solution of gemcitabine (50 mg, 2 equiv., 0.19 mmol) in DMF (0.95 mL) was cooled to -15 °C (ice-salt bath). To this was added bis(4-nitrophenyl)carbonate (29 mg, 1 equiv., 95 μmol) and DIPEA (37 mg, 50 μL, 3 equiv., 0.28 mmol). After stirring at -15 °C for 3 h, compound 65 (88 mg, 1.2 equiv., 0.11 mmol) and DIPEA (37 mg, 50 μL, 3 equiv., 0.28 mmol) were added. The ice bath was removed, and after stirring at room temperature for 15 min, the reaction mixture was purified by silica gel flash column chromatography (0→20% MeOH / DCM) to give compound 80 (57.4 mg, 59.0 μmol, 62%, 97% purity) as a white solid. 47 H 55 F2N8O 11 + (M+H) + LCMS (ESI+) calculated for 945.4, found 945.8.

[0376]

[0397] To a solution of compound 80 (57.4 mg, 1 eq., 60.7 umol) in anhydrous DMF (607 uL) was added piperidine (15.5 mg, 18.0 uL, 3 eq., 182 umol). After stirring at room temperature for 1 hour, the reaction mixture was purified by preparative HPLC (10% to 60% MeCN / water + 10 mM NH4HCO3, run time 23 min, column Xbridge prep C18 5 um OBD, 30 x 100 mm) to give compound 81 (13.2 mg, 18.3 umol, 30%, purity 100%) as a white solid. 32 H 45 F2N8O9 + (M+H) + LCMS (ESI+) calculated for 723.3, found 723.6.

[0377]

[0398] To a solution of compound 81 (8.00 mg, 3 equiv., 11.1 μmol) in anhydrous DMF (221 mL) was added compound 6 (3.32 mg, 1 equiv., 3.69 μmol) and triethylamine (3.73 mg, 5.14 μL, 10 equiv., 36.9 μmol). After stirring at room temperature for 18 hours, the reaction mixture was purified by preparative HPLC (20% to 70% MeCN / water + 10 mM NH4HCO3, run time 23 minutes, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 82 (2.8 mg, 1.3 μmol, 35%, purity 95%). 86 H 120 F4N 20 O 31 S2 +2 (M / 2+H) + LCMS (ESI+) calculated for 1034.4, found 1034.7. [ka]

[0378] Example 39: Preparation of Compound 86

[0399] To a solution of gemcitabine (96 mg, 1 eq., 0.36 mmol) in 1,4-dioxane (6.65 mL) and water (1.65 mL) was added sodium carbonate (0.15 g, 4 eq., 1.5 mmol) and di-tert-butyl dicarbonate (88 mg, 89 μL, 1.1 eq., 0.40 mmol). After stirring at room temperature for 24 h, additional di-tert-butyl dicarbonate (40 mg, 41 μL, 0.5 eq., 0.18 mmol) was added. After stirring at room temperature for 18 h, additional di-tert-butyl dicarbonate (40 mg, 41 μL, 0.5 eq., 0.18 mmol) was added. After stirring at room temperature for 6 h, water (20 mL) was added, and the reaction mixture was extracted with EtOAc (2×25 mL). The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel flash column chromatography (20→100% acetone / DCM) to give the Boc-protected intermediate (83 mg, 0.23 mmol, 62%, 99% purity) as a white solid. 14 H 20F2N3O6 + (M+H) + LCMS (ESI+) calculated for 364.1, found 364.4.

[0379]

[0400] To a solution of the Boc-protected intermediate (83 mg, 1 equiv., 0.23 mmol) in 1,4-dioxane (4.6 mL) was added di-tert-butyl dicarbonate (640 mg, 0.67 mL, 13 equiv., 2.9 mmol). After stirring at 37 °C for 24 h, the reaction mixture was allowed to reach room temperature and stirred for an additional 72 h. Water (20 mL) was added, and the reaction mixture was extracted with EtOAc (2 × 25 mL). The combined organic layers were washed with brine, dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel flash column chromatography (0 → 20% acetone / DCM) to give compound 83 (93.3 mg, 201 μmol, 88%, 100% purity) as a white solid. 19 H 28 F2N3O8 + (M+H) + LCMS (ESI+) calculated for 464.2, found 464.5.

[0380]

[0401] To a solution of compound 83 (21 mg, 1 equiv., 45 μmol) in MeCN (0.45 mL) was added triethylamine (14 mg, 19 μL, 3 equiv., 0.14 mmol) and bis(4-nitrophenyl)carbonate (28 mg, 2 equiv., 91 μmol). After stirring at room temperature for 18 h, the reaction mixture was purified by silica gel flash column chromatography (0→10% MeOH / DCM) to give compound 84 (28 mg, 43 μmol, 95%, 97% purity) as a yellow solid. 26 H 31 F2N4O 12 + (M+H) + LCMS (ESI+) calculated for 629.2, found 629.5.

[0381]

[0402] To a solution of compound 84 (14 mg, 1.3 equiv., 22 μmol) in anhydrous DCM (0.13 mL) was added a solution of compound 65 (18 mg, 73 wt%, 1.0 equiv., 17 μmol) in anhydrous DMF (66 μL) and DIPEA (6.6 mg, 8.9 μL, 3.0 equiv., 51 μmol). After stirring at room temperature for 1 hour, the reaction mixture was purified by silica gel flash column chromatography (0→20% MeOH / DCM) to afford the Fmoc-protected intermediate (25 mg, 18 μmol, 99%, 81% purity) as a white solid. 57 H 71 F2N8O 15 + (M+H) + LCMS (ESI+) calculated for 1145.5, found 1145.9.

[0382]

[0403] To a solution of the Fmoc-protected intermediate (21 mg, 1 equiv., 18 μmol) in DMF (0.2 mL) was added piperidine (4.6 mg, 5.3 μL, 3 equiv., 54 μmol). After stirring at room temperature for 4 hours, the reaction mixture was purified by silica gel flash column chromatography (0→20% MeOH / DCM) to give compound 85 (9.6 mg, 10 μmol, 57%, 98% purity). 42 H 61 F2N8O 13 + (M+H) + LCMS (ESI+) calculated for 923.4, found 923.8.

[0383]

[0404] A solution of compound 85 (9.6 mg, 1 equiv., 10 μmol) in DCM (0.16 mL) was cooled to 0° C., and TFA (52 μL, 68 equiv., 0.68 mmol) was added. After stirring at 0° C. for 3 h, the reaction mixture was concentrated in vacuo to give the Boc-deprotected intermediate (12.9 mg, 7.9 μmol, 76%, 58% purity) as an off-white solid. 32 H 45 F2N8O9 + (M+H) + LCMS (ESI+) calculated for 723.3, found 723.6.

[0384]

[0405] To a solution of the Boc-deprotected intermediate (9.9 mg, 1 equiv., 10 μmol) in anhydrous DMF (0.21 mL) was added triethylamine (11 mg, 15 μL, 10 equiv., 0.10 mmol) and compound 6 (8.2 mg, 1.5 equiv., 16 μmol). After stirring at room temperature for 18 hours, triethylamine (11 mg, 15 μL, 10 equiv., 0.10 mmol) was added. After stirring at room temperature for 6 hours, HOBt (1.6 mg, 1 equiv., 10 μmol) was added. After stirring at room temperature for 18 hours, DMAP (1.3 mg, 1 equiv., 10 μmol) was added and the reaction mixture was heated to 40°C. After stirring at 40 °C for 6 h, the reaction mixture was purified by preparative HPLC (5% to 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) and a second time by preparative HPLC (10% to 60% MeCN / water + 10 mM NH4HCO3, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 86 (1.2 mg, 1.1 μmol, 10%, 100% pure). 48 H 67 F2N 10 O 16 S + (M+H) + LCMS (ESI+) calculated for 1109.4, found 1109.8. [ka]

[0385] Example 40: Preparation of Compound 87

[0406] To a solution of compound 56 (10.5 mg, 1.0 equiv., 6.8 μmol) in dry DMF (270 μL) was added the HCl salt of daunorubicin (9.6 mg, 170 μL, 2.5 equiv., 17 μmol), followed by EtN (6.9 mg, 9.5 μL, 10 equiv., 68 μmol) and DMAP (0.8 mg, 1.0 equiv., 6.8 μmol). The reaction mixture was stirred at room temperature for 22 hours and then purified by preparative HPLC (5% to 95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 87 (5.7 mg, 2.5 μmol, 36%, 100% pure). 108 H 128 N 12 O 41 S2 2- (M / 2-H) - LCMS (ESI-) calculated for 1156.4, found 1156.8. [ka]

[0386] Example 41: Preparation of Compound 88

[0407] To a solution of compound 71 (4.79 mg, 1.3 equiv., 8.34 μmol) in dry DMF (100 mL) was added compound 66 (8.43 mg, 1.0 equiv., 6.42 μmol) and triethylamine (1.95 mg, 2.68 μL, 3.0 equiv., 19.3 μmol). The reaction mixture was allowed to stand at room temperature for 8 h, then diluted with DMF (500 μL) and purified by preparative HPLC (650 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column). The resulting crude product was redissolved in 400 μL of DMF and purified again by preparative HPLC (450 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 23 min, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 88 (3.5 mg, 2.0 μmol, 31%, purity 97.77%). 86 H 107 N8O26 S + (M+H) + LCMS (ESI+) calculated for 1699.7, found 1700.2. [ka]

[0387] Example 42: Preparation of Compound 91

[0408] To a solution of Fmoc-Val-Ala-PABC-OPNP 4 (35.0 mg, 1.0 equiv., 51.4 μmol) in dry THF (2.57 mL) was added 3-[(2R)-pyrrolidin-2-yl]propanoic acid hydrochloride (23.1 mg, 2.5 equiv., 129 μmol) and DIPEA (19.9 mg, 26.9 μL, 3.0 equiv., 154 μmol). The reaction mixture was stirred at room temperature under a N atmosphere. After 40 min, 300 μL of dry DMF was added to aid solubility. After 6.5 h, the reaction mixture was concentrated in vacuo to give a clear, pale yellow oil. The crude product was redissolved in DCM (3 ml) and the solution was purified by silica gel flash column chromatography (0% to 20% MeOH / DCM) to give the unactivated acid intermediate (47.4 mg, 60.2 μmol, 117%, 86.93% purity) as a pale yellow solid. 38 H 45 N4O8 + (M+H) + LCMS (ESI+) calculated for 685.3, found 685.8.

[0388]

[0409] To a solution of the unactivated acid intermediate (47.4 mg, 1.0 equiv., 60.2 μmol) in dry DCM (661 μL) was added 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (15.0 mg, 1.3 equiv., 78.2 μmol) and 2,3,5,6-tetrafluorophenol (13.0 mg, 1.3 equiv., 78.2 μmol). 50 μL of dry DMF was added to aid solubility. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo. The crude product was redissolved in DCM (1 mL) and purified by silica gel flash column chromatography (0% to 30% acetone / DCM) to give compound 89 (60 mg, 31 μmol, 52%, 43.64% purity). 44 H 48 F4N5O8 + (M+NH4) + LCMS (ESI+) calculated for 850.3, found 850.7.

[0389]

[0410] To a solution of compound 89 (60.0 mg, 1.0 equiv., 31 μmol) in a mixture of dry DCM (187 μL) and anhydrous DMF (100 μL) was added paclitaxel (34.0 mg, 1.25 equiv., 39.0 μmol). The reaction mixture was stirred for 50 minutes, after which DMAP (4.2 mg, 1.1 equiv., 35.0 μmol) was added. After 30 minutes, the reaction mixture was diluted with DCM (3 mL) and purified by silica gel flash column chromatography (0%→20% MeOH / DCM) to give the Fmoc-protected intermediate (49.3 mg, 26.5 μmol, 84%, 81.77% purity). 85 H 94 N5O 21 + (M+H) + LCMS (ESI+) calculated for 1520.6, found 1521.2.

[0390]

[0411] To a solution of the Fmoc-protected intermediate (49.3 mg, 1.0 equiv., 26.5 μmol) in DMF (200 μL) was added piperidine (6.77 mg, 7.86 μL, 3.0 equiv., 79.5 μmol). The reaction mixture was stirred at room temperature for 30 minutes and then diluted with DCM (4 mL). The solution was then purified by silica gel flash column chromatography (0%→15% MeOH / DCM) to give compound 90 (15.7 mg, 11.9 μmol, 45%, 98.65% purity). 70 H 84 N5O 19 + (M+H) + LCMS (ESI+) calculated for 1298.6, found 1299.0.

[0391]

[0412] To a solution of compound 90 (7.8 mg, 3.0 equiv., 5.93 μmol) in dry DCM (100 μL) was added compound 6 (1.78 mg, 1.0 equiv., 1.98 μmol) and triethylamine (1.0 mg, 1.38 μL, 5.0 equiv., 9.88 μmol). The reaction mixture was allowed to stand at room temperature for 7.5 hours, then maintained at -20°C for 17 hours. The reaction mixture was allowed to warm to room temperature for an additional 5 hours before being diluted with DMF (500 μL) and subsequently purified by preparative HPLC (550 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 23 minutes, Xbridge prep C18 5 μm OBD, 30 × 100 mm column) to give compound 91 (3.3 mg, 1.0 μmol, 52%, 100% pure). 162 H 198 N 14 O 51 S2 +2 (M / 2+H) + LCMS (ESI+) calculated for 1609.1, found 1609.6. [ka]

[0392] Example 43: Preparation of Compound 92

[0413] To a solution of compound 71 (4.45 mg, 1.3 equiv., 7.75 μmol) in dry DMF (30 μL) was added compound 90 (7.85 mg, 1.0 equiv., 5.96 μmol) dissolved in dry DMF (70 μL), followed by triethylamine (1.81 mg, 2.49 μL, 3.0 equiv., 17.9 μmol). The reaction mixture was allowed to stand at room temperature for 7 hours and then cooled to −20° C. for 17 hours. The reaction was then allowed to warm to room temperature for an additional 5 hours before dilution with DMF (400 μL) and subsequent purification by preparative HPLC (550 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 23 minutes, column bridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 92 (6.6 mg, 3.8 μmol, 64%, 97.87% purity). 86 H 106 N7O 26 S + (M+H) + LCMS (ESI+) calculated for 1684.7, found 1685.1. [ka]

[0393] Example 44: Preparation of Compound 96

[0414] To a solution of 2-(Fmoc-amino)ethanol (250 mg, 1.0 equiv., 882 μmol) in dry DCM (20 mL) cooled to −35° C., CSI (147 mg, 90 μL, 1.2 equiv., 1.1 mmol) was added. After stirring at −35° C. for 20 min, EtN (268 mg, 369 μL, 3.0 equiv., 2.7 mmol) was added, followed by 2-(2-aminoethoxy)ethan-1-ol (111 mg, 106 μL, 1.2 equiv., 1.1 mmol). The reaction mixture was warmed to room temperature and stirred for 1 h. PNP chloroformate (267 mg, 1.5 equiv., 1.3 mmol) was added, followed by additional EtN (268 mg, 369 μL, 3.0 equiv., 2.7 mmol). The reaction mixture was stirred at room temperature for 1 hour, concentrated in vacuo, dissolved in DCM (6 mL), and purified by silica gel flash column chromatography (0% to 3% MeOH / DCM) to give compound 93 (387 mg, 560 μmol, 63%, 95% purity). 29 H 31 N4O 12 S + (M+H) + LCMS (ESI+) calculated for 659.2, found 659.3.

[0394]

[0415] To a solution of compound 93 (20.6 mg, 95% wt, 1.5 equiv, 31.3 μmol) in dry DCM (400 μL) and dry DMF (200 μL) was added compound 94 (50.0 mg, 1.0 equiv, 20.8 μmol, commercially available from Levena Biopharma), followed by DIPEA (26.9 mg, 36.2 μL, 10 equiv, 208 μmol) and HOBt (3.2 mg, 1.0 equiv, 20.8 μmol). After stirring at room temperature for 20 hours, piperidine (17.7 mg, 20.5 μL, 10 equiv, 208 μmol) was added. The reaction mixture was stirred for 3 hours and purified by silica gel flash column chromatography (0% to 30% MeOH / DCM) to give compound 95 (48.8 mg, 18.1 μmol, 87%, 100% pure) as a white solid. 130 H 212 N24O 35 S 2+ (M / 2+H) +LCMS (ESI+) calculated for 1350.8, found 1351.2.

[0395]

[0416] To a solution of compound 95 (12.2 mg, 1.0 equiv., 4.5 μmol) in dry DMF (100 μL) was added methylcyclopropene-PNP (1.3 mg, 1.2 equiv., 5.4 μmol), followed by DIPEA (2.9 mg, 3.9 μL, 5.0 equiv., 22.6 μmol). The reaction mixture was stirred at room temperature for 2 hours and then purified by preparative HPLC (30% to 95% MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 96 (4.9 mg, 1.7 μmol, 39%, 100% pure). 136 H 218 N 24 O 37 S 2+ (M / 2+H) + LCMS (ESI+) calculated for 1406.8, found 1406.7. [ka]

[0396] Example 45: Preparation of Compound 97

[0417] To a solution of compound 95 (12.2 mg, 1.0 equiv., 4.5 μmol) in dry DMF (100 μL) was added TCO-NHS ester (1.5 mg, 1.2 equiv., 5.4 μmol), followed by DIPEA (2.9 mg, 3.9 μL, 5.0 equiv., 22.6 μmol). The reaction mixture was stirred at room temperature for 2 hours and then purified by preparative HPLC (30% to 95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 97 (5.5 mg, 1.9 μmol, 43%, 100% pure). 139 H 224 N 24 O 37 S 2+ (M / 2+H) + LCMS (ESI+) calculated for 1427.8, found 1427.7. [ka]

[0397] Example 46: Preparation of Compound 98

[0418] To a solution of compound 76 (1.4 mg, 1.0 equiv., 1.6 μmol) in anhydrous DMF (80 μL) was added compound 71 (0.83 mg, 1.0 equiv., 22 μL, 71 mM, 1.6 μmol) and triethylamine (0.48 mg, 0.7 μL, 3.0 equiv., 4.7 μmol). After standing at room temperature for 46 hours, the reaction mixture was diluted with DCM (500 μL) and purified by silica gel flash column chromatography (0% → 10% MeOH / DCM). The combined impure batches were repurified by preparative HPLC (110 μL injection volume of DMF; 30% → 95%, MeCN / water + 1% AcOH, run time 12 min, Xbridge prep C18 5 μm OBD, 10 × 100 mm column) to give compound 98 (0.7 mg, 0.5 μmol, 30%). 67 H 77 N 10 O 15 S + (M+NH4) + LCMS (ESI+) calculated for 1293.5, found 1293.9. [ka]

[0398] Example 47: Preparation of Compound 100

[0419] To a solution of UCN-01 (10 mg, 1.0 equiv., 21 μmol) in anhydrous DMF (60 μL) and DCM (200 μL) was added compound 4 (21 mg, 1.5 equiv., 31 μmol), DIPEA (8.0 mg, 11 μL, 3.0 equiv., 62 μmol), and HOBt (2.8 mg, 43 μL, 447 mM, 1.0 equiv., 21 μmol). After stirring at room temperature for 22 hours, the reaction mixture was purified by silica gel flash column chromatography (0% to 15% MeOH / DCM) to afford the Fmoc-protected intermediate (16.0 mg, 15 μmol, 73%, 97% purity) as an off-white solid. 59 H 56 N7O9 + (M- - OH) + LCMS (ESI+) calculated for 1006.41, found 1006.75.

[0399]

[0420] To a solution of the Fmoc-protected intermediate (8.4 mg, 1.0 equiv., 8.2 μmol) in DMF (200 μL) was added triethylamine (8.3 mg, 11 μL, 10 equiv., 82 μmol). After 21 h at room temperature, the reaction mixture was concentrated in vacuo to give compound 99 in quantitative yield (6.6 mg, 8.2 μmol). 44 H 46 N7O7 + (M- - OH) + LCMS (ESI+) calculated for 784.4, found 784.6.

[0400]

[0421] To a solution of compound 99 (6.6 mg, 1.0 equiv., 8.2 μmol) in anhydrous DMF (200 μL) was added compound 71 (4.8 mg, 1.1 equiv., 9.1 μmol) and triethylamine (2.5 mg, 3.4 μL, 3.0 equiv., 25 μmol). After 23 h at room temperature, the reaction mixture was diluted with DCM (1.0 mL) and purified by silica gel flash column chromatography (0% to 15% MeOH / DCM) to give compound 100 (9.0 mg, 8.0 μmol, 90%). 60 H 68 N9O 14 S +(M-OH - ) + LCMS (ESI+) calculated for 1170.5, found 1170.8. [ka]

[0401] Example 48: Preparation of Compound 102

[0422] To a solution of staurosporine (21.3 mg, 1.0 equiv., 45.7 μmol) in anhydrous DMF (450 μL) was added Fmoc-Val-Ala-PABC-OPFP (36.4 mg, 1.1 equiv., 50.2 μmol) and DIPEA (17.7 mg, 23.9 μL, 3.0 equiv., 137 μmol). After 6.5 h at room temperature, the reaction mixture was diluted with DCM (1 mL) and purified by silica gel flash column chromatography (0% to 10% MeOH / DCM) to give the Fmoc-protected intermediate (44.2 mg, 43.0 μmol, 93%). 59 H 58 N7O9S + (M+H) + LCMS (ESI+) calculated for 1008.4, found 1008.7.

[0402]

[0423] To a solution of the Fmoc-protected intermediate (44.2 mg, 1.0 equiv., 42.5 μmol) in DMF (500 μL) was added triethylamine (43.0 mg, 59.3 μL, 10.0 equiv., 425 μmol). After 22 h at room temperature, the reaction mixture was concentrated in vacuo to give compound 101 in quantitative yield. 44 H 48 N7O7 + (M+H) + LCMS (ESI+) calculated for 786.4, found 786.6.

[0403] To a solution of compound 101 (12.8 mg, 3.0 equiv., 16.3 μmol) in anhydrous DMF (150 μL) was added compound 6 (5.0 mg, 1.0 equiv., 5.0 μmol) and triethylamine (3.0 mg, 4 μL, 5.0 equiv., 30 μmol). After 20.5 h at room temperature, the reaction mixture was diluted with DCM (1.0 mL) and purified by silica gel flash column chromatography (0% to 20% MeOH / DCM) to give compound 102 (10.7 mg, 4.88 μmol, 90%). 110 H 126 N 18 O 27 S2 2+ (M / 2+H) + LCMS (ESI+) calculated for 1098.4, found 1098.4. [ka]

[0404] Example 49: Preparation of Compound 106

[0424] To TMTHSI-OSu (200.0 mg, 1 equiv., 587.6 μmol) was added a solution of 2-aminoethan-1-ol (107.7 mg, 106.4 μL, 3 equiv., 1.763 mmol) in dry DCM (5 mL). The resulting suspension was stirred for 18 hours, after which the reaction mixture was purified by silica gel flash column chromatography (0% to 20% acetone / DCM) to give compound 103 (175 mg, 0.46 mmol, 79%, 76% purity) as a white solid. 13 H 23 N2O3S + (M+H) + LCMS (ESI+) calculated for 287.1, found 286.9.

[0405]

[0425] Compound 103 (100.0 mg, 80% wt, 1 eq, 279.3 μmol) was dissolved in dry DCM (8 mL) and cooled to -35°C. CSI (57.7 mg, 35.5 μL, 1.46 eq, 407.7 μmol) was then added. After stirring for 10 min, triethylamine (84.80 mg, 117 μL, 3 eq, 838.0 μmol) was added, followed by 2-(2-aminoethoxy)ethan-1-ol (44.05 mg, 42.04 μL, 1.5 eq, 419.0 μmol). The ice bath was removed, and stirring was continued at room temperature for 3 h. The reaction mixture was then purified by silica gel flash column chromatography (0%→10% MeOH / DCM) to give compound 104 (61 mg, 0.12 mmol, 42%, 95% purity) as a white solid. 18 H 33 N4O8S2 + (M+H) + LCMS (ESI+) calculated for 497.2, found 497.5.

[0406]

[0426] Compound 104 (60.0 mg, 1 equiv., 121 μmol) was dissolved in dry DCM (4.00 mL) and cooled to -40 °C. Next, CSI (17.1 mg, 10.5 μL, 1 equiv., 121 μmol) was added. After 5 min, triethylamine (61.1 mg, 84.2 μL, 5 equiv., 604 μmol) was added, followed by a stock solution of diethanolamine (14.6 mg, 13.3 μL, 1.15 equiv., 139 mol) in dry THF (130 μL). After 5 h, additional triethylamine (36.7 mg, 50.5 μL, 3 equiv., 362 μmol) was added, followed by PNP chloroformate (60.9 mg, 2.5 equiv., 302 μmol). The reaction mixture was stirred at room temperature for a total of 42 h and purified by silica gel flash column chromatography (0% to 80% acetone / DCM). The resulting impure batch was repurified by preparative HPLC (750 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 105 (8.3 mg, 7.2 μmol, 6.0%, 90% purity) as a white solid. 37 H49 N8O 21 S3 + (M+H) + LCMS (ESI+) calculated for 1037.2, found 1037.6.

[0407]

[0427] To compound 105 (8.30 mg, 1 equiv., 8.00 μmol), crude compound 5b (13.3 mg, 176 μL, 100 mmol, 2.2 equiv., 17.6 μmol) in DMF was added, followed by triethylamine (4.05 mg, 5.58 μL, 5 equiv., 40.0 μmol). The reaction mixture was allowed to stand for 18 hours and then purified by preparative HPLC (550 μL injection volume of DMF; 30% to 95%, MeCN / water + 1% AcOH, run time 12 min, column Xbridge prep C18 5 μm OBD, 30 × 100 mm) to give compound 106 (6.0 mg, 2.1 μmol, 26%, purity 80%) as a white solid. 105 H 126 F2N 18 O 31 S3 +2 (M / 2+H) + LCMS (ESI+) calculated for 1134.9, found 1135.2. [ka]

[0408] [Examples 50 to 71: Preparation of click probe-functionalized antibodies] Ex...

Claims

1. 1. A process for preparing an antibody-payload conjugate having a DAR of 6 or greater, comprising: (a) Structure Ab(F １ ) ｚ where Ab is an antibody, z is 2 or 4, and F １ is a click probe; (b) dipping the modified antibody in z equivalents of Q １ L Ａ (F ２ ) ｙ (In the formula, Q １ is F １ is a click probe that reacts with L Ａ is a heterobifunctional (y+1)valent linker, where y is 2, 3, or 4; F ２ Is Q １ ) to give a click probe of the structure Ab (Z １ L Ａ (F ２ ) ｙ ) ｚ (In the formula, Z １ is F １ and Q １ wherein the linking group is obtained by reaction of (c) sequestering the antibody-linker construct with z×y equivalents of Q ２ (L Ｂ ) D ｘ (In the formula, Q ２ is F ２ is a click probe that reacts with L Ｂ is an (x+1)-valent linker, where x is 1, 2, 3, or 4, provided that x+y is at least 4, and D is a payload molecule, to form a compound of the structure Ab(Z １ L Ａ (Z ２ (L Ｂ ) D ｘ ) ｙ ) ｚ (In the formula, Z ２ is F ２ and Q ２ a step of obtaining a conjugate of A process involving:

2. y is 2 and L Ａ is the structure -(L １１ )-BM(L １２ -)(L １３ -) (In the formula, - L １１ Is, Q １ or Z １ is connected to L １２ and L １３ Is F ２ or Z ２ is connected to L １１ , L １２ , and L １３ are individually, CH ２ , CH=CH, C≡C, C(O), NR １３ ,O,S,S(O),S(O) ２ , P.R. １３ , and P(O)R １３ Preferably, the building blocks are selected from one or more building blocks selected from CH ２ , CH=CH, C(O), NR １３ , O, S, S(O), and S(O) ２ is selected from - BM is preferably a branched moiety selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)cyclic ring, or a polycyclic moiety, more preferably BM is a nitrogen atom 2. The process of claim 1, wherein the heterobifunctional trivalent linker is

3. X is 2 and L Ｂ is the structure - (8) １ )-[(L ２ ) ｏ - (8) ３ ) ｐ - (8) ４ ) ｑ -] ２ [In the formula, - L １ Is, Q ２ or Z ２ is connected to L ４ Both occurrences of are linked to D; - L １ , L ２ , L ３ , and L ４ are individually, Q ２ or Z ２ is a linker linking D together; o, p, and q are each independently 0 or 1, preferably o=p=1; Preferably, (a) linker L １ teeth, -(W) ｋ -(A) ｄ -(B) ｅ -(A) ｆ -(C(O)) ｇ -BM[-(A) ｄ’ -(B) ｅ’ -(A) ｆ’ -(W) ｇ’ -] ２ (In the formula, d and d' are each 0 or 1; e and e' are each an integer ranging from 1 to 10; f and f′ are individually 0 or 1; g and g' are each an integer ranging from 0 to 10; k=0 or 1, provided that if k=1, then d=0; A is a carbamoyl or acylsulfamido group represented by structure (23) 【Chemistry 1】 (wherein a=0 or 1, R １３ is hydrogen, C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups, and C ３ ~C ２４ (hetero)arylalkyl groups, １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups, and C ３ ~C ２４ (Hetero)arylalkyl groups are optionally substituted and include O, S, and NR １４ and R １４ are independently hydrogen and C １ ~C ４ alkyl groups, or R １３ is D linked to N via a spacer moiety, preferably said spacer moiety is -(B) ｇ -(C(O)) ｇ - (L ２ ) ｏ - (L ３ ) ｐ - (L ４ ) ｑ - is); - W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH ２ ) ｍ C(O)-, -C(O)(CH ２ ) ｍ C(O)NH- or -(4-Ph)CH ２ NHC(O)(CH ２ ) ｍ C(O)NH—, where m is an integer ranging from 0 to 10; - B is -CH ２ -CH ２ —O— or —O—CH ２ -CH ２ - moiety, or (B) ｅ is -(CH ２ -CH ２ -O) ｅ１ -CH ２ -CH ２ - moiety, where e1 is an integer ranging from 1 to 10; - BM is a branching moiety, to which - (A) ｄ -(B) ｅ -(A) ｆ -(C(O)) ｇ - Two instances of are concatenated, and both (C(O)) ｇ’ The part is -(L ２ ) ｏ - (L ３ ) ｐ - (L ４ ) ｑ -D, wherein L ２ , L ３ , L ４ , o, p, q, and D are each selected individually); and / or (b) linker L ２ is preferably a peptide spacer comprising 1 to 5 amino acids, more preferably a dipeptide, tripeptide, or tetrapeptide spacer; and / or (c) linker L ３ is an autoimmune spacer, preferably: - if o=1, L ３ is a para-aminobenzyloxycarbonyl (PABC) derivative represented by structure (26): 【Chemistry 2】 - if o=1, L ３ is a glucuronide derivative represented by structure (27); 【Transformation 3】 In the formula, R ２１ is H, R ２６ or C(O)R ２６ and R ２６ is C １ ~C ２４ (hetero)alkyl group, C ３ ~C １０ (hetero)cycloalkyl group, C ２ ~C １０ (hetero)aryl group, C ３ ~C １０ Alkyl (hetero)aryl groups, and C ３ ~C １０ (hetero)arylalkyl groups, which are optionally substituted and include O, S, and NR ２８ and R ２８ are independently hydrogen and C １ ~C ４ alkyl groups, preferably R ２１ is H or C(O)R ２６ and R ２６ = 4-N-methyl-piperazine or morpholine, most preferably R ２１ is H; and / or (d) linker L ４ teeth, - Structure-NR ２２ -(C ｘ -alkylene)-C(O)- (wherein x is an integer ranging from 1 to 20, and R ２２ is H or C １ ~C ４ an aminoalkanoic acid spacer represented by the formula: - Structure-NR ２２ - (CH ２ -CH ２ -O) ｅ６ - (CH ２ ) ｅ７ -C(O)- (wherein e6 is an integer ranging from 1 to 10, e7 is an integer ranging from 1 to 3, and R ２２ is H or C １ ~C ４ an ethylene glycol spacer represented by the formula: - Structure-NR ２２ -(C ｘ -alkylene)-NR ２２ -(C(O)) ｈ - (wherein h is 0 or 1, x is an integer ranging from 1 to 10, and R ２２ is H or C １ ~C ４ a diamine spacer represented by is a spacer selected from 3. The process of claim 1 or 2, wherein the heterobifunctional trivalent linker is

4. The process according to any one of claims 1 to 3, wherein the reaction in step (b) and the reaction in step (c) are both cycloadditions, preferably the reaction in step (b) is a 1,3-dipolar cycloaddition and the reaction in step (c) is an inverse electron demand Diels-Alder cycloaddition.

5. - Q １ and Q ２ are click probes each individually comprising a (hetero)cycloalkyne moiety or a (hetero)cyclo-E-alkene moiety, and preferably the click probes are selected from the group consisting of (Q2) to (Q20) represented by the following structures: 【Chemistry 4】 (In the formula, - wavy bond is L Ａ or L Ｂ represents a linkage to any available carbon or nitrogen atom; - The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) are L Ａ Or L Ｂ may have a linkage to, contain hydrogen atoms, or be substituted; - B （－） is an anion; - B （＋） is a cation) and / or -F １ and F ２ are each individually click probes selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, dioxothiophenes, sydnones, iminosydnones, and catechols, and preferably the click probes are selected from the group consisting of (F1) to (F10) represented by the following structures: 【Transformation 5】 (In the formula, - Wavy bonds are Ab or L Ａ In (F3), (F4), (F8), and (F9), the connection can be through any one of the wavy bonds, and the other bond can be hydrogen, C １ ~C ２４ Alkyl group, C ２ ~C ２４ Acyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl(hetero)aryl groups, C ３ ~C ２４ (hetero)arylalkyl groups, and C １ ~C ２４ sulfonyl groups, each of which may be optionally substituted, and O, S, and NR ３２ and R ３２ are independently hydrogen and C １ ~C ４ alkyl groups) The process according to any one of claims 1 to 4.

6. -F １ is an azide or a nitrone, and Q １ is preferably a benzoannulated or tetramethylated (hetero)cycloalkyne selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q17), (Q18), (Q19) and (Q19a) as defined in claim 5; -F ２ is tetrazine and Q ２ is bicyclononyne or cycloalkene, preferably Q ２ is (Q8), (Q44), (Q47), (Q48), (Q49), (Q54), (Q55), or (Q56) as defined in claim 5, and F ２ is represented by (F8a): 【Transformation 6】 (In the formula, R ２９ is hydrogen, C １～６ Alkyl, aryl, C(O)-C １～６ Alkyl, C(O)-aryl, C(O)—O—C １～６ Alkyl, C(O)—O-aryl, C(O)—NR ３３ -C １～６ Alkyl, and C(O)—NR ３３ -aryl, R ３３ is H or C １～４ alkyl, preferably R ２９ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl The process according to any one of claims 1 to 5.

7. The process according to any one of claims 1 to 6, wherein the payload D is a pharmaceutically active compound, preferably a cytotoxin.

8. The antibody provided in step (a) has the structure: Ab[(L ６ )S(F １ )] ｚ (In the formula, Z is 2 or 4; - L ６ -GlcNAc(Fuc) ｗ - (G) ｊ -S-(L ７ ) ｗ’ -, G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0 or 1, and L ７ -N(H)C(O)CH ２ -, -N(H)C(O)CF ２ - or -CH ２ - is) The process according to any one of claims 1 to 7, wherein

9. A conjugate obtainable by the process according to any one of claims 1 to 8.

10. Structure Ab(Z １ L Ａ (Z ２ (L Ｂ ) D ｘ ) ｙ ) ｚ A conjugate having the formula: AB is an antibody; -Z １ and Z ２ is a linking group obtained by reaction between two click probes; - L Ａ is a heterobifunctional (y+1)valent linker; - L Ｂ is an (x+1)-valent linker; x is 1, 2, 3 or 4; y is 2, 3, or 4, provided that x+y is at least 4; Z is 2 or 4; D is the payload, Conjugates.

11. Z １ and Z ２ has the structures (Z2) to (Z38a): 【Transformation 7】 (In the formula, - wavy bond is L Ａ or L Ｂ represents a linkage to any available carbon or nitrogen atom; Ring Z is Ab or L Ａ is linked to, preferably selected from triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, oxazoline, isoxazolidine, pyrazoline, piperazine, or pyridazine; - The nitrogen atoms of (Z10), (Z13), (Z14), and (Z15) are L Ａ Or L Ｂ may have a linkage to, or may contain a hydrogen atom, or may be substituted; - B （－） is an anion; - B （＋） is a cation) 11. The conjugate of claim 10, wherein the conjugate is individually selected from:

12. A pharmaceutical composition comprising the conjugate of any one of claims 9 to 11 and a pharmaceutically acceptable carrier.

13. Structure Q １ L Ａ (F ２ ) ｙ A linker construct having - Q １ is a click probe; - L Ａ is a heterobifunctional (y+1)valent linker; -F ２ Is, Q １ a click probe that does not react with; y is 2, 3 or 4; Linker constructs.

14. - Q １ is preferably a benzoannulated or tetramethylated (hetero)cycloalkyne selected from (Q5), (Q6), (Q6a), (Q6b), (Q6c), (Q6d), (Q7), (Q17), (Q18), (Q19) and (Q19a) as defined in claim 5; -F ２ is preferably (F8a): 【Transformation 8】 (In the formula, -R ２９ is hydrogen, C １～６ Alkyl, aryl, C(O)-C １～６ Alkyl, C(O)-aryl, C(O)—O—C １～６ Alkyl, C(O)—O-aryl, C(O)—NR ３３ -C １～６ Alkyl, and C(O)—NR ３３ -aryl, wherein R ３３ is H or C １～４ alkyl, preferably R ２９ is selected from hydrogen, methyl, phenyl, pyridyl, pyridinyl, and pyrimidinyl. It is a tetrazine represented by the formula: y is 2, 3 or 4, preferably y is 2 or 3, most preferably y is 2; The linker construct of claim 13.

15. - a method for targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting a conjugate according to any one of claims 9 to 11 with cells capable of expressing said extracellular receptor, wherein said antibody specifically targets said extracellular receptor; and / or - a method for treating cancer, comprising administering to a subject in need thereof a conjugate according to any one of claims 9 to 11, wherein the cancer cells specifically express an extracellular receptor, Preferably, the extracellular receptor is 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD45, CD 46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, Nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tenascin, or the antibody is specific to an extracellular protein resulting from a viral infection and / or a tumor-associated carbohydrate antigen (TACA).

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