Antibody-exatecan conjugate

Abstract

The present invention relates to an antibody-drug conjugate having the structure (1) TIFF2023541637000112.tif64149 [wherein AB is an antibody and L 1 and L 2 is a linker, w is 0 or 1, Z is a connecting group obtained by metal-free click reaction or thiol ligation, R 17 are each independently an amino acid side chain, n is an integer ranging from 1 to 5, A is a 5- or 6-membered aromatic or heteroaromatic ring, x is an integer ranging from 1 to 8, and R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from R 22 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 23 and R is optionally interrupted by one or more heteroatoms selected from 23 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.

Description

[Technical Field]

[0001]

[0001] The present invention relates to an antibody-drug conjugate comprising an exatecan cytotoxic payload, which is suitable in the field of antibody-drug conjugates, particularly for the treatment of cancer. [Background technology]

[0002]

[0002] Antibody-drug conjugates (ADCs), considered highly effective therapeutic agents, consist of antibodies bound to a drug. Antibodies (also called ligands) can be in small protein formats (scFv, Fab fragments, DARPin, aphibodies, etc.), but are generally monoclonal antibodies (mAbs) selected based on high selectivity and affinity for a given antigen, a long circulating half-life, and little to no immunogenicity. Thus, mAbs as protein ligands for carefully selected biological receptors provide an ideal delivery platform for the selective targeting of pharmaceuticals. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used for the delivery of chemically conjugated cytotoxic substances to tumors via binding, internalization, intracellular processing, and finally the release of active catabolites. Cytotoxic substances can be in other formats such as small molecule toxins, protein toxins, or oligonucleotides. As a result, tumor cells can be selectively eradicated while preserving normal cells that were not targeted by the antibody. Similarly, while chemical conjugation of antibacterial drugs (antibiotics) to antibodies can be applied to the treatment of bacterial infections, the conjugation of anti-inflammatory drugs is under investigation for the treatment of autoimmune diseases, and, for example, the attachment of oligonucleotides to antibodies is a potentially promising technique for the treatment of neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to selected specific cellular sites is a powerful technique for the treatment of a wide range of diseases, offering many advantages over systemic delivery of the same drug.

[0003]

[0003] An alternative strategy for targeting and delivering specific protein substances using monoclonal antibodies is by gene fusion of the latter protein to one (or more) of the antibody's terminals, which may be the N-terminus or C-terminus of the light chain or heavy chain (or both). In this case, the bioactive protein of interest, such as a protein toxin like Pseudomonas exotoxin A (PE38) or an anti-CD3 single-chain variable fragment (scFv), is gene-encoded as a fusion to the antibody, though not necessarily, and possibly via a peptide spacer, and the antibody is expressed as a fusion protein. The peptide spacer may or may not contain a protease-sensitive cleavage site.

[0004]

[0004] In the field of ADCs, chemical linkers are typically employed to attach pharmaceuticals to antibodies. This linker needs to have several important attributes, including the need for plasma to be stable after long-term drug administration. A stable linker allows for the localization of the ADC to a planned site or cell in the body and prevents premature release of the payload in circulation. Its premature release indiscriminately induces all kinds of undesirable biological responses, thereby reducing the therapeutic index of the ADC. Once internalized, the ADC should be processed so that the payload is effectively released and therefore can bind to its target.

[0005]

[0005] Linkers belong to two families: non-cleavable linkers and cleavable linkers. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload and are sufficiently stable under physiological conditions regardless of the organ or biological compartment in which the antibody-drug conjugate resides. As a result, the release of the payload from an ADC containing a non-cleavable linker depends on the complete (lysosomal) degradation of the antibody after the ADC has been moved into the cell. As a result of this degradation, the payload (still containing the linker), as well as peptide fragments and / or amino acids, are released from the antibody to which the linker was originally attached. Cleavable linkers utilize the intrinsic properties of the cell or cellular compartment for the selective release of the payload from the ADC, thereby generally leaving no trace of linker after metabolic processing. There are three commonly used mechanisms for cleavable linkers: 1) sensitivity to specific enzymes, 2) pH sensitivity, and 3) sensitivity to the cellular redox state (or its microenvironment). The cleavable linker may also include self-destructive units based, for example, on para-aminobenzyl alcohol groups and their derivatives. The linker may also include additional non-functional elements, often called spacer or stretcher units, for connecting the linker to the reactive group for reaction with the antibody.

[0006]

[0006] Although ADCs have demonstrated clinical and preclinical activity, it remained unclear which factors, in addition to antigen expression in targeted tumor cells, determine such efficacy. For example, drug-to-antibody ratio (DAR), ADC binding affinity, payload potency, receptor expression level, internal transfer rate, transport, multidrug resistance (MDR) status, and other factors all played a role in influencing the outcomes of ADC treatment in vitro. In addition to the direct killing of antigen-positive tumor cells, ADCs also possess the ability to kill adjacent antigen-negative tumor cells, a so-called "bystander killing" effect, as reported by Sahin et al., Cancer Res., 1990, 50, 6944-6948 (incorporated by reference) and studied, for example, by Li et al., Cancer Res., 2016, 76, 2710-2719 (incorporated by reference). Generally, neutral cytotoxic payloads exhibit bystander death, while ionic (charged) payloads do not, as a result of the fact that ionic species do not readily permeate cellular membranes by passive diffusion. Established bystander effects include MMAE and DXd. Examples of payloads that do not exhibit bystander death include MMAF or the active catabolite (lysine-MCC-DM1) of Kadcyla.

[0007]

[0007] ADCs are prepared by a process called bioconjugation, which is the conjugation of a linker-drug and a protein. Many techniques for bioconjugation are known, as summarized in G. Thermanson, "Bioconjugate Techniques", Elsevier, 3rd edition, 2013, incorporated by reference. Two main techniques can be recognized for the preparation of ADCs by random conjugation: one based on acylation of the lysine side chain and the other on alkylation of the cysteine ​​side chain. Acylation of the ε-amino group in the lysine side chain is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, e.g., SMCC, which is used in the production of Kadcyla®. The main chemistry for alkylation of the thiol group in the cysteine ​​side chain is based on the use of maleimide reagents, e.g., as applied in the production of Adcetris®. In addition to standard maleimide derivatives, various maleimide variants can also be applied to more stable cysteine ​​conjugations, as is evident from James Christie et al., J. Contr. Rel., 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol., 2014, 32, 1059-1062, which are incorporated together by reference.Other methods for cysteine ​​alkylation include, for example, nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem., 2008, 19, 759-765, incorporated by reference), or various methods based on nucleophilic addition to unsaturated bonds, such as reactions with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun., 2016, 7, DOI:10.1038 / ncomms13128 and Ariyasu et al., Bioconj. Chem., 2017, 28, 897-9, both incorporated by reference). This includes reactions with phosphoamides (see, for example, Kasper et al., Angew. Chem. Int. Ed., 2019, 58, 11625-11630, incorporated by reference), reactions with allenamides (see, for example, Abbas et al., Angew. Chem. Int. Ed., 2014, 53, 7491-7494, incorporated by reference), reactions with cyanoethynyl reagents (see, for example, Kolodych et al., Bioconj. Chem., 2015, 26, 197-200, incorporated by reference), reactions with vinylsulfones (see, for example, Gil de Montes et al., Chem. Sci., 2019, 10, 4515-4522, incorporated by reference), or reactions with vinylpyridines (see, for example, https: / / iksuda.com / science / permalink / (accessed January 7, 2020)).An alternative to antibody conjugation that does not involve antibody reengineering involves reducing interchain disulfide crosslinks and subsequently using bis-sulfone reagents (see, for example, Balan et al., Bioconj. Chem., 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceuticals, 2015, 12, 1872-1879, both incorporated by reference), mono- or bis-bromomaleimides (see, for example, Smith et al., J.Am. Chem. Soc., 2010, 132, 1960-1965 and Schuma, both incorporated by reference). See cher et al., Org. Biomol. Chem., 2014, 37, 7261-7269), bis-maleimide reagent (see, e.g., International Publication No. 2014114207), bis(phenylthio)maleimide (see, e.g., both incorporated by reference, Schumacher et al., Org. Biomol. Chem., 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem., 2018, 29, 3516-3521), bis-bromopyridazinedione (see, e.g., Robinson et al., incorporated by reference, RSC This involves the addition of a payload attached to a cysteine ​​crosslinking reagent, such as Advances, 2017, 7, 9073-9077), bis(halomethyl)benzene (see, e.g., Ramos-Tomillero et al., Bioconj. Chem., 2018, 29, 1199-1208, incorporated by reference), or other bis(halomethyl) aromatic compounds (see, e.g., International Publication No. 2013173391). Typically, ADCs prepared by cysteine ​​crosslinking have approximately 4 drug-antibody loadings (DAR4). Another technique useful for conjugation to cysteine ​​side chains is the technique using disulfide bonds, which are bioactivatable links that have been used to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci., 2017, 8, 366-370, incorporated by reference).

[0008]

[0008] In addition to conjugation to lysine or cysteine, various other conjugation techniques have been explored over the past decade. One method is based on the gene encoding of non-natural amino acids, such as p-acetophenylalanine suitable for oximligation, or p-azidomethylphenylalanine or p-azidophenylalanine suitable for click chemistry conjugation, as is evident, for example, from Axup et al., Proc. Nat. Acad. Sci., 2012, 109, 16101-16106, incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem., 2014, 25, 351-361, incorporated by reference, introduced azidomethylphenylalanine (AzPhe) into a monoclonal antibody for conversion to ADC by metal-free click chemistry using a cell-free protein synthesis method. Furthermore, Nairn et al., Bioconj. Chem., 2012, 23, 2087-2097, incorporated by reference, demonstrated that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins by nutrient-dependent bacteria and further converted into protein conjugates by (copper-catalyzed) click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUA Gene encoding of aliphatic azides in recombinant proteins using pairs was shown by Nguyen et al., J.Am.Chem.Soc., 2009, 131, 8720-8721, via reference integration, and labeling was secured by click chemistry.

[0009]

[0009] Another method is based on the enzymatic introduction of non-natural functionalities. For example, Lhospice et al., Mol. Pharmaceut., 2015, 12, 1863-1871, incorporated by reference, employs the bacterial enzyme transglutaminase (BTG or TGase) for the introduction of the azide moiety into the antibody. A genetic method based on conversion in ADC using metal-free click chemistry following C-terminal TGase-mediated azide introduction was reported by Cheng et al., Mol. Cancer Therap., 2018, 17, 2665-2675, incorporated by reference.

[0010]

[0010] In International Publication No. 2014065661, all incorporated by reference, van Geel et al., Bioconj. Chem., 2015, 26, 2233-2242 and Verkade et al., Antibodies, 2018, 7, 12, it was shown that enzymatic remodeling of the natural antibody glycan at N297 allows for the introduction of azide-modified sugars suitable for attachment of cytotoxic payloads using click chemistry (see Figure 5A). Alternatively, the modified sugars are enzymatically incorporated in the form of disulfide bonds, and subsequently released after reduction to provide free thiol groups for conjugation by alkylation (see Figure 5B). Chemical methods for site-specific modification of antibodies without prior genetic modification have also been developed, as highlighted, for example, by Yamada and Ito, ChemBioChem., 2019, 20, 2729-2737.

[0011]

[0011] A common method for bioconjugating linker-drugs to azide-modified proteins is strain-enhanced alkyne-azide cycloaddition (SPAAC). In the SPAAC reaction, the linker-drug is functionalized with a cyclic alkyne, and the cycloaddition with the azide-modified antibody is driven by the release of ring strain. Conversely, the linker-drug is functionalized with an azide, and the antibody is functionalized with a cyclic alkyne. Various strained alkynes suitable for metal-free click chemistry are shown in Figure 1A.

[0012]

[0012] Furthermore, in the case of strained alkynes, the linker-drug bioconjugation to antibodies (and other biomolecules such as glycans and nucleic acids) can be achieved by various other metal-free click chemistry. See, for example, Nguyen and Presser, Nature rev. 2020, doi: 10.1038 / s41570-020-0205-0, incorporated by reference. For example, orthoquinones can be obtained by oxidation of specific tyrosines in proteins, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes. See, for example, Bruins et al., Chem. Eur. J., 2017, 24, 4749-4756, incorporated by reference. In addition to cyclooctin, several cycloheptins are also suitable for metal-free click chemistry, as reported by Wetering et al., Chem. Sci., 2020, doi: 10.1039 / d0sc03477k, incorporated by reference. The tetrazine moiety can also be introduced into proteins or glycans by various means, such as gene coding or chemical acylation, and may undergo cycloaddition with cyclic alkenes and alkynes. A list of functional group F and Q pairs for metal-free click chemistry is shown in Figure 2.

[0013]

[0013] Based on the above, a general method for preparing the protein conjugate exemplified for the monoclonal antibody in Figure 3 requires the reaction of a protein containing x reactive moieties F with a linker-drug construct containing a single molecule Q. A schematic diagram showing how the reactive molecule F can be introduced into the monoclonal antibody is shown in Figure 4.

[0014]

[0014] Conjugation of cytotoxic payloads to antibodies by any of the above methods is often challenging because the hydrophobicity of the payload, and possibly the hydrophobicity of the payload combined with the linker, interferes with its solubility in aqueous or buffer systems (preferred media for antibodies). As a result, conjugation of cytotoxic payloads is typically carried out in a medium consisting of water / buffer plus an organic cosolvent. Typical cosolvents for conjugation are DMSO, propylene glycol (PG), ethanol, DMF, DMA, and NMP, which promote linker-drug solubilization but are also well miscible with water. Typical amounts of cosolvent are 10-25% relative to the aqueous medium, but the cosolvent may be added up to 50% in some cases. Adding high amounts of cosolvent is particularly advantageous for conjugation processes where the payload is significantly hydrophobic (lipophilic), and where a large excess of linker-drug is required to achieve sufficient conversion to the desired product.

[0015]

[0015] Aside from the obvious benefits, a disadvantage of adding a substantial amount of organic co-solvent is that the antibody may be unstable in the solvent mixture and, as a result, may aggregate during the conjugation process. Typically, the level of aggregation is correlated with the amount of co-solvent, but this is also antibody-dependent. With particularly unstable antibodies, the level of aggregation can be significant, reaching 10% or even higher, resulting in reduced process yield. Furthermore, these levels of aggregates require additional processing steps (e.g., SEC or CHT) to remove the aggregates to an acceptable level. Another disadvantage of high co-solvent levels during conjugation is that, before size exclusion purification (SEC) can be performed, additional process steps must be introduced to remove the excess co-solvent, for example by dialysis, spin filtration or TFF.

[0016]

[0016] Currently, examples of cytotoxic payloads include microtubule disruptors [e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, tubulinin], DNA damaging agents [e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiapine dimers, duocalmycin, anthracyclines], topoisomerase inhibitors [e.g., DXd, SN-38], or RNA polymerase II inhibitors [e.g., amanitin]. ADCs that have reached market approval include, for example, payloads MMAE, MMAF, DM1, calicheamicin, SN-38, and DXd, and various primary studies have been conducted on ADCs based on duocalmycin, DM4, and PBD dimers. A wider range of payloads, such as eribulin, indolinobenzodiazepine dimers, PNU-159,682, hemiasterlin, doxorubicin, and vinca alkaloids, are also in the clinical evaluation stage or have already undergone clinical trials. Finally, various ADCs in the late preclinical stage are being conjugated with novel payloads, such as amanitin, KSP inhibitors, and MMADs.

[0017]

[0017] With the exception of sacituzumabgovetican (Trodelvy®), all clinical and commercial ADCs contain cytotoxic agents that are not suitable as standalone drugs. Trodelvy® is an exception as it features SN-38, which is also the active catabolite (SN-38 prodrug) of irinotecan, as its cytotoxic payload. Several other payloads now used in clinical ADCs were initially evaluated for chemotherapy as free drugs, such as calicheamicin, PBD dimers, and eribulin, but their extremely high cytotoxicity (picomolar to low nanomolar IC25) compared to the typically low micromolar concentrations of standard chemotherapy drugs such as paclitaxel and doxorubicin 50 The value was the reason for the failure.

[0018]

[0018] Currently, there is one commercially available ADC and five ADCs for various clinical stages, all of which are based on the payload DXd (structure shown in Figure 6), a synthetic derivative of exatecan, and are based on the linker-drug deruxtecan (Figure 8). Both DXd and exatecan are members of the camptothecin family (topoisomerase 1 inhibitors). Exatecan has been clinically evaluated as a standalone chemotherapy agent (exatecan mesylate, DX-8951f) because preclinical studies have shown it to be more potent than SN-38-based irinotecan (CPT-11) against various tumor xenograft models, including CPT-11-resistant tumors. Furthermore, it has been found that exatecan is not a substrate of the Pgp transporter that confers multidrug resistance, while SN-38 is a weak substrate of Pgp. However, clinical studies have shown only minimal efficacy of exatecan mesylate against various cancer types, summarized in Venditto and Simanek, Mol. Pharmaceut., 2010, 7, 307-349 (referenced), with neutropenia being the most common drug-related toxicity. A phase III trial comparing exatecan with gemcitabine did not demonstrate an extension of survival compared to gemcitabine monotherapy. As a result, development of exatecan mesylate as a free drug was discontinued.

[0019]

[0019] Subsequent exatecan-based research focused on the development of DE-310, a polymeric carrier system in which DX-8951f is covalently bonded to carboxymethyl dextranpolyalcohol (CM-Dex-PA) via a Gly-Gly-Phe-Gly (GGFG) tetrapeptide spacer. The GGFG peptide spacer of DE-310 was designed to be cleaved by a specific cysteine ​​protease, which was actually upregulated in preclinical studies in the tumor microenvironment, and it had been shown that a single dose of 11.4 mg / kg of DE-310 in mice exhibited stronger antitumor activity than repeated doses of DX-8951f alone (10 mg / kg per day for 5 days). However, the potential benefits of sustained release due to enhanced permeability and storage capacity (EPR) were not confirmed in Phase I trials for the treatment of human cancer, as reported by Wente et al., Invest New Drugs., 2005, 23, 339, incorporated by reference.

[0020]

[0020] Very recently, exatecan has been investigated as a payload in antibody-drug conjugates, as reported, for example, by Nakada et al., Bioorg. Med. Chem. Lett., 2016, 26, 1542-1545, incorporated by reference, and various linker formats have been screened. However, it has been observed that exatecan-based ADCs exhibit severe aggregation of up to 26%. Furthermore, it has been found that direct attachment of the GGFG linker to the exatecan amino group results in incomplete proteolytic removal of the GGFG peptide spacer, releasing a mixture of both free DX-8951 and G-DX-8951, i.e., N-glycyl-exatecan (see Figure 6). Indeed, as reported by Wente et al., Invest New Drugs., 2005, 23, 339 (incorporated by reference), previous clinical trials of DE-310 revealed that the concentration of G-DX-8951 in tumor tissue was approximately 10 times higher than that of DX-8951 itself, suggesting that G-DX-8951 may play a part in the cytotoxic activity of DE-310. At the same time, preclinical data, as reported by Shiose et al., Biol. Pharm. Bull., 2007, 30, 2365-2370 (incorporated by reference), showed that while the inhibitory activity of DX-8951 and G-DX-8951 on topoisomerase-I activity was similar, the in vitro cytotoxicity of DX-8951 was approximately 20 to 190 times stronger than that of G-DX-8951. Finally, PAMPA assays revealed that various N-aminoacylated derivatives exhibited significantly reduced membrane permeability compared to exatecan / DX-8951 itself.

[0021]

[0021] As a result of the reduced potency of G-DX-8951 compared to DX-8951, the insufficient cell membrane permeability of aminoacylated exatecan derivatives in general, and the significant aggregation potential of exatecan-based ADCs, a novel exatecan-based linker technology called deruxtecan (shown in Figure 8), based on an N-hydroxyacylated DX-8951 derivative (DXd) combined with a GGFG-aminal-based linker, was developed by Daiichi-Sankyo. The linker-payload is conjugated with an antibody via a cysteine ​​residue after the interchain disulfide bond is reduced with a reducing agent, thereby producing a DAR4 or DAR8 ADC (ADC4a or ADC4b, shown in Figure 10A, respectively) depending on the stoichiometry of the reducing agent. Alternatively, a cysteine-engineered antibody can be used to produce a DAR2 ADC (ADC5, shown in Figure 10B). The tetrapeptide is degraded by lysosomal enzymes such as cathepsin B and L, which are highly expressed in tumor cells, and it is thought that DXd is then released (accompanied by the release of formaldehyde) via the self-destruction of the free aminal portion. As expected, DXd was found to be highly permeable to cells, as reported by Ogitani et al., Cancer Sci., 2016, 107, 1039-1046, incorporated by reference, and consequently showed a significant bystander effect. Therefore, deruxtecan-based ADCs may be beneficial when treating tumors with heterogeneous targeting. Furthermore, ADCs induced by cysteine ​​conjugation with deruxtecan have been found to exhibit high stability and enable high drug loads of up to DAR8, as demonstrated in three ADC programs: DS-8201a (an ADC targeting HER2, currently marketed as Enhertu), U3-1402a (an ADC targeting HER3), and DS-6157a (an ADC targeting GRP20). In addition, two deruxtecan-based DAR4 ADCs, DS-1062a (an ADC targeting TROP-2) and DS-7300a (an ADC targeting B7-H3), are in various stages of clinical development.

[0022]

[0022] In addition to the deruxtecan-based program, three other ADCs with a camptothecin payload have reached clinical practice, two of which are SN-38-based ADCs, namely sacetizumab govetican (targeting TROP-2) and labetuzumab govetican (targeting CEACAM5), both based on SN-38. In fact, the first report on a camptothecin payload-based ADC by Walker et al., Bioorg. Med. Chem. Lett., 2002, 12, 217-219, incorporated by reference, contained SN-38 (conjugated with an anti-BR96 targeted antibody). Sacituzumab govetican, described by Sharkey et al., Clin. Cancer Res., 2015, 21, 5131-5138, incorporated by reference, was approved as Trodelvi® for the treatment of triple-negative breast cancer. A third ADC with a camptothecin payload (berotecan) that recently entered clinical practice is SKB264.

[0023]

[0023] Several preclinical studies using other variants of camptothecin-type payloads in the context of ADCs have also been recently disclosed. For example, Burke et al., Bioconj. Chem., 2009, 20, 1242-1250, incorporated by reference, describe the preparation of antibody-drug conjugates (ADCs) with novel camptothecin analogs that are 10 to 1000 times potent than camptothecin itself. ADCs with the potent camptothecin analog 7-butyl-9-amino-10,11-methylenedioxy-camptothecin are highly potent in vitro against a population of cancer cell lines, immunologically specific, and effective at well-tolerated doses in a renal cell carcinoma xenograft model. However, these ADCs have not been further developed. Finally, in 2019, Li et al., ACS Med. Chem. Lett., 2019, 10, 1386-1392, published a report on ADCs containing camptothecin induced by exatecan but lacking the F ring (see Figure 6, camptothecin ring numbering) in the payload. Since the chiral center of the F ring was inferred to complicate its synthesis and derivatization, novel camptothecins were synthesized that lacked the F ring in the payload but still behaved similarly to conjugates containing DXd in vitro and in vivo. In particular, ADCs with varying degrees of bystander killing, capable of releasing hydroxyl or thiol metabolites following peptide linker cleavage, were benchmarked against deruxtecan-based ADCs and found to have at least the same potency against target-positive cells and enhanced potency (bystander killing) against target-negative cells in co-culture. However, ADCs based on exatecan itself were not screened.

[0024]

[0024] A wide variety of camptothecin-based ADCs have been described, and several of these, all based on DXd, SN-38, or berotecan, are either in clinical evaluation or have reached commercial approval. However, to date, no ADCs based on a linker format designed to release exatecan as an active catabolite have been reported, possibly due to a tendency towards high aggregation reported by Nakada et al., Bioorg. Med. Chem. Lett., 2016, 26, 1542-1545, which may be incorporated by reference. As a solution, DXd was developed, whose significant bystander effect substantially contributes to the value of derutecan linker technology. [Overview of the project]

[0025]

[0025] To our surprise, the inventors found that linkers having a cleavable peptide-PABC system are highly suitable for metal-free click or thiol conjugation of exatecan to antibodies, producing antibody-exatecan conjugates that show no aggregation tendency at all or only a negligible aggregation tendency. The resulting ADCs were found to exhibit remarkable in vivo efficacy. Thus, for the first time, the inventors were able to prepare an ADC with an exatecan payload that shows no aggregation at all and exhibits the same level of efficacy as the most effective antibody-camptothecin conjugate known to date.

[0026]

[0026] The present invention relates first to an antibody-drug conjugate having structure (1). [ka] [In structure, AB is an antibody, L 1 and L 2 It is a linker, w is either 0 or 1. Z is a connecting group obtained by a metal-free click reaction or thiol ligation. R17 are each individually amino acid side chains, n is an integer in the range of 1 to 5, A is a 5- or 6-membered aromatic or heteroaromatic ring, x is an integer in the range of 1 to 8, R 21 is H, R 22 , C(O)OH and C(O)R 22 selected from, R 22 is C1 to C 24 (hetero)alkyl group, C3 to C 10 (hetero)cycloalkyl group, C2 to C 10 (hetero)aryl group, C3 to C 10 alkyl(hetero)aryl group and C3 to C 10 (hetero)arylalkyl group, which are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR 23 , and R 23 is independently selected from the group consisting of hydrogen and C1 to C4 alkyl groups.

[0027]

[0027] The present invention further relates to a method for synthesizing an antibody-drug conjugate according to the present invention, a linker-drug construct suitable for use in the method according to the present invention, a medical use of the antibody-drug conjugate according to the present invention, and a pharmaceutical composition comprising the antibody-drug conjugate according to the present invention.

Brief Description of the Drawings

[0028] [Figure 1A] It is a diagram showing a cyclic alkyne suitable for metal-free click chemistry and a preferred embodiment of the reactive moiety Q. This list is not exhaustive; for example, the alkyne can be further activated by fluorination, substitution of an aromatic ring or introduction of a heteroatom in an aromatic ring. [Figure 1B] It is a diagram showing various reagents suitable for reaction with cysteine side chains. The reagent can be of the monoalkylation type (A) or a crosslinking agent (B) for reaction with two cysteine side chains. [Figure 2] This figure shows a representative (but not exhaustive) group of functional groups (F) that can be introduced into antibodies by engineering, chemical modification, or enzymatic means, yielding a conjugate group Z after a metal-free click reaction with a complementary reactive group Q. Functional groups F can be artificially introduced (engineered) at any position of choice in the antibody. Some functional groups F (e.g., nitrile oxides, quinones) can react with strained alkenes as well as strained alkynes, for example, triazine or tetrazine are shown (bottom row). Pyridine or pyridazine conjugate groups are rearrangement products of the tetraazabicyclo[2.2.2]octane conjugate group, which are formed by losing N2 after the reaction of triazine or tetrazine with an alkyne (not an alkene), respectively. Conjugate group Z is a preferred conjugate group for use in the present invention. [Figure 3] This figure shows a general scheme for preparing antibody-drug conjugates by the reaction of a monoclonal antibody (usually a symmetric dimer) containing x functional groups F. The conjugate is obtained by incubation of antibody-(F)x with an excess of the linker-drug construct (Q-spacer-linker-payload), where F and Q react to form conjugate Z. [Figure 4] This figure shows the general process of non-genetic conversion of a monoclonal antibody to an antibody containing a probe (F) for click conjugation or thiol ligation. The click probe can be located at various positions on the antibody, depending on the technique employed. For example, the antibody may be converted to an antibody containing two click probes (left structure), four click probes (bottom structure), or eight probes (right structure) for click conjugation. [Figure 5A]This figure illustrates a specific example of site-specific conjugation of a payload based on azido-cyclooctin click chemistry, following glycan remodeling of full-length IgG. First, IgG is enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of azido sugars to the endoglycosidase-released core GlcNAc. In the next step, the azido-remodeled IgG is exposed to an immune cell-engaging polypeptide, modified with a single cyclooctin (SPAAC) for metal-free click chemistry, and yields a bispecific antibody in a 2:2 molecular format. It has also been shown that the cyclooctin-polypeptide construct may have specific spacers that allow for adjustment of the IgG-polypeptide distance between cyclooctin and polypeptide, or confer other properties to the resulting bispecific antibody. [Figure 5B] This figure illustrates a specific example of site-specific conjugation of a payload based on glycan remodeling of full-length IgG followed by thiol alkylation chemistry. First, IgG is enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms and subsequent glycosyltransferase-mediated transfer of thiol-modified (and disulfide-protected) sugar derivatives to the endoglycosidase-liberated core GlcNAc. In the next step, the remodeled IgG undergoes reduction (conversion of disulfides to thiols), possibly followed by oxidation, and then reaction with a payload modified with a suitable thiol-reactive reagent. [Figure 6] This figure shows the structures of the topoisomerase inhibitor DXd and exatecan, as well as N-glycyl-exatecan (G-DX-8951). Ring numbering is shown for exatecan. [Figure 7]This figure shows the structures of BCN-linker-drugs 1-3 suitable for application in ADCs by conjugation to azide-modified antibodies. Structures 1-3 contain a peptide-PABC cleavable linker and contain the Val-Ala (1a and 2a) or Val-Cit (1b or 2b) peptide. Structures 1 and 2 are designed for direct release of the payload, while structure 3 includes an additional portion (N,N'-dimethylethylenediaminocarbonyl) for payload release via cyclization. Structures 1 and 2 contain the cytotoxic payload exatecan, and structure 3 contains the cytotoxic payload SN-38. [Figure 8] This figure shows the structure of DXd-based maleimide-linker-drug 4 (also known as deruxtecan), which is suitable for application in ADCs by conjugation to free cysteine ​​side chains. [Figure 9] This figure shows the structures of ADCs obtained by enzymatic remodeling of N-glycans (for the introduction of azido sugars) followed by metal-free click conjugation with linker-drug 1 or 2. Remodeling and conjugation with 1 or 2 in native N297 glycan alone yields ADC1a or ADC2 of DAR4, respectively. Remodeling and conjugation with 1 in native N297 glycan plus an additional engineered N-glycosylation site (e.g., HC-L201N) yields ADC1b of DAR8. [Figure 10A] This figure shows the structure of ADC obtained by reducing the native disulfide bond and subsequently conjugating it with deruxtecan 4, yielding either ADC4a (average DAR4) or ADC4b (average DAR8) depending on the specific conditions adopted (reducing agent, e.g., stoichiometry of TCEP or DTT, and deruxtecan). [Figure 10B]This figure shows the structure of an ADC obtained from an antibody having a conjugation with deruxtecan 4, which is a natural amino acid variant, an engineered cysteine ​​that may be a cysteine ​​insertion or a cysteine ​​condensed at the N-terminus or C-terminus of an antibody, and a subsequent ADC5 (average DAR2). [Figure 11] This figure shows various antibody variants in which an azide group or thiol group can be used as a starting material for the subsequent conversion to an antibody conjugate. [Figure 12] This figure shows the HIC profiles of azido-trastuzumab (after enzyme remodeling), ADC1a (DAR4), and ADC4a (DAR4). Azido-trastuzumab shows a retention time of 9.2 minutes, and ADC1a according to the present invention shows a single peak with a retention time of 9.8 minutes, and therefore a relative retention time of 1.06. In contrast, the control ADC4a shows multiple peaks eluting from the HIC column (from 10.2 minutes to >12.3 minutes), and its relative retention time to azido-trastuzumab ranges from 1.11 to >1.33. [Figure 13] This figure shows the RP-HPLC trace of the conjugation of trastuzumab-(6-azideGalNAc)2 with 25a. [Figure 14] This figure shows the RP-UPLC trace under reducing conditions of the conjugation of trastuzumab S239C mutant trast-v3 with maleimide-exatecan variants 54a, 57b, 59a, or 60a. [Figure 15] This figure shows the RP-UPLC trace under reducing conditions of the conjugation of trastuzumab GalProSH trast-v2 with maleimide-exatecan variant 57b or 60a. [Figure 16] This figure shows the RP-UPLC trace under reducing conditions of the conjugation of trastuzumab trast-v4 with maleimide-exatecan variant 60a or 57b according to general procedure A. [Figure 17]This figure shows the RP-UPLC trace under reducing conditions of the conjugation of rituximab rit-v4 with maleimide-exatecan variant 60a or 57b according to general procedure A. [Figure 18] This figure shows the RP-UPLC trace under reducing conditions of the conjugation of trastuzumab trast-v4 with maleimide-exatecan variant 60a or 57b according to general procedure B. [Figure 19] This figure shows the results of an in vivo efficacy study monitoring of tumor volume in mice transplanted with the BT-474 cell line and administered DAR4 ADCs based on the vehicle, Kadcyla (T-DM1), and exatecan (ADC1a) and SN-38 (ADC3) (all as single doses). The ADC1a according to the present invention showed complete tumor resolution at a single dose of 12 mg / kg, which is a significant improvement compared to the control ADC3. [Figure 20A] This figure shows the results of an in vivo efficacy study monitoring of tumor volume in mice transplanted with the BT-474 cell line and administered a vehicle and DAR4 ADCs based on exatecan (ADC1a) and DXd (ADC4a) (all as single doses). [Figure 20B] This is a zoomed-in view of Figure 20A. ADC1a and control ADC4a according to the present invention showed no significant difference in efficacy, exhibiting a partial response at 4 mg / kg and complete tumor regression at 12 mg / kg once for both ADCs. [Figure 21] This figure shows the results of an in vivo efficacy study monitoring of tumor volume in mice transplanted with the BT-474 cell line and administered a vehicle and DAR4+DAR8 ADCs based on exatecan (ADC1a (DAR4) and ADC1b (DAR8)) and DXd (ADC4a (DAR4) and ADC4b (DAR4)) (all as single doses). [Figure 22]This figure shows the results of an in vivo efficacy study monitoring of tumor volume in mice transplanted with the BT-474 cell line and administered with an exatecan-based DAR4 ADC having a vehicle and either a short spacer (ADC1a(DAR4)) or a long spacer (ADC2(DAR4)). The ADC1a and ADC2 according to the present invention did not show a significant difference in efficacy. [Modes for carrying out the invention]

[0029] definition

[0052] As used herein and in the claims, the verb “to comprise” and its conjugations are used in their non-restrictive sense to mean that they include the matter following the word, but do not exclude matters that are not specifically stated. Furthermore, references to “elements” with the indefinite article “a” or “an” do not rule out the possibility that there may be more than one element unless the context explicitly requires that there be only one element; therefore, the indefinite article “a” or “an” usually means “at least one.”

[0030]

[0053] The compounds disclosed herein and in the claims may contain one or more chiral centers, and various diastereomers and / or enantiomers of the compounds may exist. Unless otherwise stated, any description of a compound herein and in the claims includes all diastereomers and mixtures thereof. Furthermore, unless otherwise stated, any description of a compound herein and in the claims includes individual enantiomers, any mixture of enantiomers, racemic or non-racemic. When the structure of a compound is shown as a specific enantiomer, it should be understood that the invention of this application is not limited to that specific enantiomer.

[0031]

[0054] Compounds may appear in various tautomer forms. Unless otherwise stated, the compounds according to this invention are intended to include all tautomer forms. When the structure of a compound is shown as a specific tautomer, it should be understood that the invention of this application is not limited to that specific tautomer.

[0032]

[0055] The compounds disclosed herein and in the claims may further exist as R and S stereoisomers. Unless otherwise stated, any description of a compound herein and in the claims includes both the individual R and individual S stereoisomers of the compound, as well as mixtures thereof. When the structure of a compound is shown as a stereoisomer of a particular S or R, it should be understood that the invention of this application is not limited to that particular S or R stereoisomer.

[0033]

[0056] The compounds disclosed herein and in the claims may further exist as R and S stereoisomers. Unless otherwise stated, any description of a compound herein and in the claims includes both the individual R and individual S stereoisomers of the compound, as well as mixtures thereof. When the structure of a compound is shown as a stereoisomer of a particular S or R, it should be understood that the invention of this application is not limited to that particular S or R stereoisomer.

[0034]

[0057] The compounds disclosed herein and in the claims may further exist as exo and endodiastereomers. Unless otherwise stated, any description of a compound herein and in the claims includes both the individual exodiastereomers and individual endodiastereomers of the compound, as well as mixtures thereof. When the structure of a compound is shown as an endo or exodiastereomer, it should be understood that the invention of this application is not limited to that particular endo or exodiastereomer.

[0035]

[0058] Compounds according to the present invention may exist in the form of salts, which are also encompassed by the present invention. A salt is a pharmaceutically acceptable salt that typically contains a pharmaceutically acceptable anion. The term “its salt” means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation. Where applicable, a salt is a pharmaceutically acceptable salt, but this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound, the compound can be protonated by an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid is the anionic component of the salt.

[0036]

[0059] The term “pharmaceutically acceptable” salt means a salt that is acceptable for administration to patients, such as mammals (a salt containing a counterion that has mammalian safety acceptable for a given administration regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt of a compound. Such salts are derived from a variety of organic and inorganic counterions known in the art, and include, for example, sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, tetraalkylammonium salts, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochlorides, hydrobromides, formates, tartrates, besilates, mesilates, acetates, maleates, and oxalates.

[0037]

[0060] In this specification, the term "protein" is used in its ordinary scientific sense. In this specification, polypeptides containing approximately 10 or more amino acids are considered proteins. Proteins may contain natural amino acids, but they may also contain non-natural amino acids.

[0038]

[0061] In this specification, the term “antibody” is used in its ordinary scientific sense. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Antibodies are an example of glycoproteins. In this specification, the term “antibody” is used in its broadest sense, specifically including monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. In this specification, the term “antibody” is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term “antibody” includes all immunoglobulins, but is also intended to include antigen-binding fragments of antibodies. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0039]

[0062] In this specification, “antibody fragment” is defined as a portion of an intact antibody that includes its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, minibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments(s), fragments(s) produced by Fab expression libraries, or any of the above epitope-binding fragments that bind immunospecifically to target antigens (e.g., cancer cell antigens, viral antigens, or microbial antigens).

[0040]

[0063] In this specification, "antigen" is defined as the unit to which an antibody specifically binds.

[0041]

[0064] In this specification, the terms “specific binding” and “specifically binding” are defined as a highly selective manner in which an antibody or antibody binds to its corresponding target antigen epitope and not to a large number of other antigens. Typically, an antibody or antibody derivative binds to at least about 1 × 10⁻¹⁶ antigens.-7 M, preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It binds with affinity M, and binds to a predetermined antigen with an affinity at least twice as high as the affinity it has for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).

[0042]

[0065] In this specification, the terms “substantial” or “substantially” are defined as the majority of a group of mixtures or samples, i.e., >50%, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the group.

[0043]

[0066] In this specification, “linker” is defined as a portion that connects two or more elements of a compound. For example, in an antibody conjugate, the antibody and payload are covalently bonded to each other via a linker. A linker may include one or more linkers and spacer portions that connect various parts within the linker.

[0044]

[0067] In this specification, a “spacer” or spacer portion is defined as a portion of a linker that provides a certain distance between two or more parts of the linker and covalently connects those parts. The linker may be, for example, a linker-structure portion, a linker conjugate, or a bioconjugate, as defined below.

[0045]

[0068] In this specification, “self-destructive group” is defined as a portion of a linker in an antibody-drug conjugate that has the function of conditionally releasing a free drug at a site targeted by a ligand unit. An activatable self-destructive moiety comprises an activatable group (AG) and a self-destructive spacer unit. Activation of the activatable group, for example, by enzymatic conversion of an amide group to an amino group or reduction of a disulfide to a free thiol group, initiates a self-destructive reaction sequence and causes the release of a free drug by one or more of various mechanisms, which may include a (transient) 1,6-elimination reaction of a p-aminobenzyl group to a p-quinone methide, and / or a subsequent second cyclization release mechanism, optionally accompanied by the release of carbon dioxide. The self-destructive assembly unit may be a portion of a chemical spacer connecting the antibody and payload (via a functional group). Alternatively, the self-destructive group may branch off from the chemical spacer connecting the antibody and payload, rather than being an inherent part of the chemical spacer.

[0046]

[0069] In this specification, “conjugate” is defined as a compound in which an antibody is covalently bound to a payload via a linker. A conjugate comprises one or more antibodies and / or one or more payloads.

[0047]

[0070] The term "payload" refers to a portion of a protein conjugate that is covalently bound to a targeting portion, such as an antibody, but is also covalently bound to molecules released from the conjugate after the incorporation of the protein conjugate and / or cleavage of the linker. Therefore, the payload also refers to a monovalent portion having one open end that is covalently bound to the targeting portion via a linker, and the molecules released therefrom. In the context of this invention, the payload is exatecan. This invention

[0048]

[0071] The inventors have developed an antibody-drug conjugate containing exatecan and a cleavable peptide-PABC system as a cytotoxic payload that exhibits no aggregation tendency or only a negligible aggregation tendency. The resulting ADC was found to exhibit remarkable in vivo efficacy.

[0049]

[0072] The present invention relates, firstly, to antibody-drug conjugates. In a second aspect, the present invention relates to a method for synthesizing antibody-drug conjugates according to the present invention. In a third aspect, the present invention relates to linker-drug constructs suitable for use in the methods according to the present invention. In a fourth aspect, the present invention relates to the medical use of antibody-drug conjugates according to the present invention, and to pharmaceutical compositions comprising antibody-drug conjugates according to the present invention. Those skilled in the art will understand that all aspects are related, and that everything stated about antibody-drug conjugates according to the present invention applies equally to the methods, linker-drug constructs, uses, and compositions according to the present invention, and vice versa.

[0050]

[0073] The present invention may be defined according to the following list of preferred embodiments. 1. Antibody-drug conjugate having structure (1) [ka] [In structure, AB is an antibody, L 1 and L 2 It is a linker, w is either 0 or 1. Z is a connecting group obtained by a metal-free click reaction or thiol ligation. R 17 These are each individual amino acid side chains, n is an integer in the range of 1 to 5. A is a 5-membered or 6-membered aromatic or heteroaromatic ring, x is an integer in the range of 1 to 8. R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from, R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 23 It is optionally interrupted by one or more heteroatoms selected from R 23 [These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.] 2. L 2 However, having structure (2) [ka] [In structure, The waveforms labeled with * are connected to Z, and the waveforms labeled with ** are connected to NH. Sp 1 and Sp 2 Each of these is a separate spacer part. n, A, R 17 and R 21 This is as described in Embodiment 1, The antibody-drug conjugate described in Embodiment 1. 3. Each Sp that appears 2 The same and each (NH-CR) that appears 17 -CO) n The same, each A that appears is the same, and each R that appears 21 The antibody-drug conjugate described in Embodiment 2 is the same as the one described in Embodiment 2. 4. L 2 Preferably Sp 1 However, it contains a sulfamide group represented by structure (3). [ka] [In structure, a = 0 or 1, R 13 is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted with O, S, and NR 14 [Here, R 14 [is optionally interrupted by one or more heteroatoms independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 This is the second appearance of C(O)X connected to N via the spacer portion. An antibody-drug conjugate according to any one of Embodiments 1 to 3. 5. L 2 Preferably Sp 1 However, the antibody-drug conjugate according to Embodiment 4 contains two groups of formula (3). 6. An antibody-drug conjugate according to any one embodiment of Embodiments 1 to 5, wherein each n that appears is 2. 7. Each (NH-CR) that appears 17 -CO) nThe antibody-drug conjugate according to any one embodiment of Embodiments 1 to 6, wherein the antibody-drug conjugate is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, and Lys, preferably from Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, and Ala-Ala-Asn, most preferably from Val-Cit or Val-Ala. 8. Z has a structure selected from (Z1) to (Z8) and (Z11) to (Z23). [ka] [In structure, The combination of waveforms marked with * is L 1 It is connected to A and B via optional selection, and the coupling of other waveforms is L 2 It is connected, The functional group R in (Z3), (Z7), and (Z8) is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (hetero)arylalkyl groups and C1-C 24 Selected from sulfonyl groups, each of which may be optionally substituted with O, S, and NR 32 [Here, R 32 [The group may be optionally interrupted by one or more heteroatoms independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.] R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 It is alkyl, R 29 C1~12 Alkyl, preferably C 1~4 Alkyl, most preferably ethyl, An antibody-drug conjugate according to any one embodiment of Embodiments 1 to 7. 9. Having structure (1a) [ka] [In structure, AB, L 1 , w, Z, A, R 21 And x are as described in Embodiment 1, R 17 It is CH3 or CH2CH2CH2NHC(O)NH2, m is an integer in the range of 1 to 10. q is an integer in the range of 0 to 10. p is either 0 or 1. An antibody-drug conjugate according to any one embodiment of Embodiments 1 to 8. 10. An antibody-drug conjugate according to any one embodiment of Embodiments 1 to 9, wherein w=0, the conjugation site is a cysteine ​​residue, preferably a cysteine ​​residue that is naturally present in the antibody after optional reduction of the disulfide bond, or the cysteine ​​residue is engineered, preferably by substitution of an amino acid with cysteine, cysteine ​​insertion, or introduction of a single cysteine ​​residue or a peptide fragment containing a cysteine ​​residue at its N-terminus or C-terminus. 11. Having structure (1b) [ka] [In structure, Z, L 2 , R 17 , A, R 21 n and x are as described in Embodiment 1, e is an integer in the range of 0 to 20. Su is a monosaccharide, G is the monosaccharide portion, GlcNAc is the N-acetylglucosamine moiety, Fuc is the fucose moiety, d is 0 or 1, The antibody-drug conjugate according to any one of Embodiments 1 to 9. 12. The antibody-drug conjugate according to any one of Embodiments 1 to 11, wherein the antibody AB targets HER2. 13. A method for synthesizing the antibody-drug conjugate according to any one of Embodiments 1 to 12, (i) A modified antibody of the structure AB-((L 1 )) w -F) x In the structure, AB is an antibody, L 1 is a linker, w is 0 or 1, F is a click probe that can react with Q in a metal-free click reaction, or a thiol or its precursor, x is an integer in the range of 1 to 8) (ii) A linker-drug construct represented by the structure (5)

Chemical formula

[0051]

[0074] The antibody-drug conjugate according to the present invention has structure (1) [ka] [In structure, AB is an antibody, L 1 and L 2 It is a linker, w is either 0 or 1. Z is a connecting group obtained by a metal-free click reaction or thiol ligation. R 17 These are each individual amino acid side chains, n is an integer in the range of 1 to 5. A is a 6-membered aromatic ring or heteroaromatic ring, x is an integer in the range of 1 to 8. R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from, R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 23 It is optionally interrupted by one or more heteroatoms selected from R 23 [These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.]

[0052]

[0075] The conjugate according to the present invention comprises a peptide spacer and a self-destructive group or a cleavable linker containing a para-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0053]

[0076] The peptide spacer is (NH-CR 17 -CO) n Defined by, where R 17 represents an amino acid side chain known in the art. In this specification, amino acids may be natural or synthetic amino acids. Preferably, all amino acids are in their L configuration. n is an integer in the range of 1 to 5, preferably in the range of 2 to 5. Thus, the peptide spacer contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2) or a tripeptide (n=3), and most preferably the peptide spacer is a dipeptide. Any peptide spacer can be used, but preferably the peptide spacer is selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, more preferably Val-Cit, Val-Ala, Ala-Ala-Asn, and most preferably Val-Cit or Val-Ala. In one embodiment, the peptide spacer is Val-Cit. In another embodiment, the peptide spacer is Val-Ala.

[0054]

[0077] R 17R represents an amino acid side chain, preferably selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolicine, 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 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-indole-3-yl)(Trp). 17 Particularly preferred embodiments include CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu), and CH(CH3)2(Val). Most preferably, R 17 These are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0055]

[0078] In a particularly preferred embodiment, the peptide spacer can be represented by a general structure (L3). [ka]

[0056]

[0079] In this specification, R 17The above is as described above, and preferably R 17 This is CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit).

[0057]

[0080] Para-aminobenzyloxycarbonyl (PABC) derivatives can be represented by the general structure (L4). [ka]

[0058]

[0081] A is a five-membered or six-membered aromatic or heteroaromatic ring, preferably a six-membered aromatic or heteroaromatic ring. Preferred five-membered rings are oxazole, thiazole, and furan. Preferred six-membered rings are phenyl and pyridyl. In preferred embodiments, A is 1,4-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0059]

[0082] R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from, R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 23 It is optionally interrupted by one or more heteroatoms selected from R 23 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, R 22 C3~C 10 It is a (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) sH or -(CH2CH2CH2O) s H is the most preferred polyalkylene glycol, preferably polyethylene glycol, preferably -(CH2CH2O) s H is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s = 1, 2, 3, or 4. More preferably R 21 is H or C(O)R 22 And R 22 =4-methylpiperazine or morpholine. Most preferably, R 21 H is H. Linker (L 1 , L 2 and L 5 )

[0060]

[0083] Linkers, also called linking units, are well known in the art, and any suitable linker can be used. In the context of the present invention, the exatecan payload is linker L 2 The linker L is chemically attached to the connecting group Z obtained by a metal-free click reaction or thiol ligation, and the connecting group Z is linked to the linker L. 1 It is chemically attached to the antibody via the linker L. The linking group Z is linked to the linker L. 5 via (O) a Chemically connected to a linker, especially linker L 2 It may include one or more branching points for multiple payloads to attach to a single linker. 1 It may exist (w=1) or not exist (w=0). Linker L 1 If present, F attaches directly to the antibody. Preferably, in the case of conjugation, w=0 is via thiol ligation. Preferably, in the case of conjugation, w=1 is via a click reaction. Linker L 5 The element may exist (r=1) or not exist (r=0).

[0061]

[0084] Linkers are, for example, linear or branched C1-C 200 Alkylene group, C2~C 200Alkenylene group, C2~C 200 Alkynylene group, C3~C 200 Cycloalkylene group, C5~C 200 Cycloalkenylene group, C8~C 200 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group, C9~C 200 The group can be selected from the group consisting of arylalkylene groups.Optionally, alkylene groups, alkenylene groups, alkylylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkylene groups may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, wherein the heteroatoms are preferably O, S(O) y and NR 12 Selected from the group consisting of, where y is 0, 1, or 2, preferably y=2, R 12 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 The linker is independently selected from the group consisting of (hetero)arylalkyl groups. The linker may include (poly)ethylene glycol diamine (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,z-diaminoalkanes, where z is the number of carbon atoms in the alkane, which can be, for example, in the range of 2 to 25.

[0062]

[0085] In a preferred embodiment, linker L 2 (especially Sp 1 If present, it contains a polar group. Such a polar group is -(O) a-C(O)-NH-S(O)2-NR 13 -(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 the following two structures can be selected. [ka] The polar groups may also include amino acids, preferably selected from Arg, Glu, Asp, Ser, and Thr. In this specification, a and R 13 This is further defined in structure (3) below. t is an integer in the range of 0 to 15, preferably 1 to 10, more preferably 2 to 5, and most preferably t = 2 or 4. R 30 H and C are separate terms. 1~12 Alkyl, C 1~12 Ariel, C 1~12 Alkaline or C 1~12 It is Aralkir. Linker L 2 It may contain more than one such polar group, such as at least two polar groups. The polar groups branch off from the linker L, which is a branched portion defined elsewhere. 2 (especially Sp 1 It can also be present in the branching of (if present). Preferably, nitrogen or carbon atoms are used as branching parts. -O(CH2CH2O) present in the branching t - Having a polar group is particularly preferable.

[0063]

[0086] In a preferred embodiment, linker L 2 This includes a sulfamide group, preferably a sulfamide group represented by structure (3). [ka]

[0064]

[0087] The wavy line represents the remainder of the conjugate, typically Z and (NH-CR). 17 -CO) n Represents a connection to the element via an optional spacer. Preferably, (O) a The C(O) portion is connected to Z, NR 13 The part is (NH-CR 17 -CO) n It connects to Linker L. 2 spacer part Sp 1 If it contains, the sulfamide group represented by structure (3) is the spacer portion Sp 1 It is preferable that it be included in

[0065]

[0088] In structure (3), a=0 or 1, preferably a=1, and R 13 is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are optionally substituted with O, S, and NR 14 [Here, R 14 [is optionally interrupted by one or more heteroatoms independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 This is a second appearance of the exatecan payload connected to N via a spacer portion (i.e., the nitrogen atom can be the branch portion).

[0066]

[0089] In a preferred embodiment, R 13 is hydrogen, C1~C 20 It is an alkyl group, or R 13This is a second appearance of the exatecan payload connected to N via a spacer portion. More preferably, R 13 is hydrogen, C1~C 10 This is a second appearance of the exatecan payload connected to N via an alkyl group or spacer portion. In this specification, the alkyl group is optionally substituted with O, S, and NR. 14 [Here, R 14 R is optionally interrupted by one or more heteroatoms, preferably O, selected independently from the group consisting of hydrogen and C1-C4 alkyl groups. In a preferred embodiment, R 13 is hydrogen. In another preferred embodiment, R 13 C1~C 20 Alkyl groups, fuaC1~C 16 Alkyl alkyl groups, more preferably C1-C 10 The alkyl group is optionally interrupted by one or more oxygen atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is more preferably a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 This is a second appearance of the exatecan payload connected to N via the spacer portion. In another preferred embodiment, R 13 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, and even more preferably from the group consisting of hydrogen, methyl and ethyl. Even more preferably, R 13 This is a second appearance of the exatecan payload connected to N via hydrogen or a spacer portion, most preferably R 13 It is hydrogen.

[0067]

[0090] The antibody-drug conjugate according to the present invention may contain two groups of formula (3), preferably both being L 2, more comfortably both Sp 1 It is included in. Typically, there is a spacer that connects the bases of both resulting equations (3), such as a linker as defined herein. Preferably, this is (CH2CH2O) m A PEG spacer is defined as follows, where m is an integer in the range of 1 to 10, preferably in the range of 2 to 6, and most preferably m is 2 or 4.

[0068]

[0091] In one embodiment, linker L 2 Preferably a spacer Sp 1 This includes structure (L3). (O) a C(O)NHS(O)2NH-(CH2CH2O) m -C(O)-(NHS(O)2) p N* (L3)

[0069]

[0092] In this specification, a and m are as described above, and p is 0 or 1. N* may represent the nitrogen atom or branch portion of the peptide spacer. branch part

[0070]

[0093] In a preferred embodiment, the linker of the conjugate according to the present invention includes a branched portion. In the context of the present invention, “branched portion” refers to a portion embedded in the linker that connects the three portions. In other words, the branched portion includes at least three bindings to the other portions, typically one binding to antibody AB connected to Z, one binding to the exatecan payload, and one binding to the second exatecan payload. The branched portion is preferably linked to linker L 2 It is embedded in. Any portion containing at least three bonds to other portions is suitable as a branched portion in the context of the present invention. In a preferred embodiment, the branched portion BM is selected from a carbon atom, a nitrogen atom, a phosphorus atom, a (hetero)aromatic ring, a (hetero)ring, or a polycyclic portion. Most preferably, the branched portion is a nitrogen atom.

[0071]

[0094] Therefore, Linker L 2 It is preferable that the branched portion preferably includes nitrogen atoms so that the two exatecan payloads are connected to a single portion Z. In a particularly preferred embodiment, L 2 It has structure (2). [ka]

[0072]

[0095] In this specification, the coupling of waveforms labeled with * is connected to Z, and the coupling of waveforms labeled with ** is connected to NH. 1 and Sp 2 These are each individual spacer parts. Variable elements n, A, R 17 and R 21 This is defined elsewhere and applies similarly to this embodiment. The two linker-exatecan portions connected to the branched nitrogen atom may be the same or different. Preferably, they are the same, i.e., each Sp 2 The same, and each (NH-CR) that appears 17 -CO) n The same, each A that appears is the same, and each R that appears is the same. 21 They are the same. Conjunction Z

[0073]

[0096] Z is a linker. The term “linker” refers to a structural element that connects one part of a conjugate to another part of the same bioconjugate. In (1), Z links antibody AB to the exatecan payload via a linker. The linker Z is a part that can be obtained by a metal-free click reaction or thiol ligation. As those skilled in the art will understand, the exact nature of Z depends on the nature of F and Q. Preferred embodiments for Q and F are further defined below.

[0074]

[0097] In a first preferred embodiment, the conjugation group Z may be obtained by a metal-free click reaction. Herein, the conjugation group Z may preferably include a triazole moiety, an isoxazole moiety, a dihydroisoxazole moiety, a bicyclo[2.2.2]octa-5,7-diene-2,3-dione moiety, a bicyclo[2.2.2]octa-5-ene-2,3-dione moiety, a 7-thiabicyclo[2.2.1]hepta-2,5-diene-7,7-dioxide moiety, a 7-thiabicyclo[2.2.1]hepta-2-ene-7,7-dioxide moiety, a pyrazole moiety, a pyridine moiety, a dihydropyridine moiety, a pyridazine moiety, or a dihydropyridazine moiety. Preferred structures for the conjugation group Z are shown below as (Z1) to (Z8). [ka]

[0075]

[0098] In this specification, the functional group R in (Z3), (Z7), and (Z8) is defined as hydrogen, C1-C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (hetero)arylalkyl groups and C1-C 24 The sulfonyl groups can be selected, and each of these (except hydrogen) may be optionally substituted with O, S, and NR. 32 [Here, R 32 The bonds in the waveform labeled with * are L 1 It is connected to, and the coupling of other waveforms is L 2It is connected to the atom. Those skilled in the art will understand which R groups can be applied to each of the connecting groups Z. For example, the R group connected to the nitrogen atom of (Z3) can be selected from the alkyl or aryl groups defined above, and the R group connected to the carbon atom of (Z3) can be selected from the hydrogen, alkyl, aryl, acyl, and sulfonyl groups defined above.

[0076]

[0099] In a particularly preferred embodiment, Q comprises a cyclic alkyne moiety, F is an azide, and Z comprises a triazole moiety formed by 1,3-dipolar addition cyclization of the alkyne moiety and the azide moiety. In another particularly preferred embodiment, Z comprises the structures (Z36) to (Z40) defined below [wherein the structure, the waveform coupling labeled with * is L 1 It is connected to, and the coupling of other waveforms is L 2 Represented by one of the following: [connected].

[0077]

[0100] In a particularly preferred embodiment, the connecting group Z is represented by structure (Z37). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y 2 C(R 31 )2, O, S or NR 31 And R 31 Each is individually, R 15 Alternatively, it is a second appearance of the exatecan payload connected via the spacer portion, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16.

[0078]

[0101] In a preferred embodiment, u+u'=4, 5, or 6, and more preferably u+u'=5. Typically, v=(u+u')×2 or [(u+u')×2]-1. In a preferred embodiment, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0079]

[0102] In a particularly preferred embodiment, the connecting group Z is represented by the structure (Z38). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) ,C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 18 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 19 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 This is the second appearance of the exatecan payload connected via the spacer portion. l is an integer in the range of 0 to 10.

[0080]

[0103] In a preferred embodiment of the reactive group represented by structure (Z38), R 15 is hydrogen, halogen, -OR16 [Here, R 16 [is hydrogen or a C1-C6 alkyl group], independently selected from the group consisting of a C1-C6 alkyl group and a C5-C6 (hetero)aryl group, more preferably R 15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 15 All are H. In a preferred embodiment of the reactive group represented by structure (Z38), R 18 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 18 Both are H. In a preferred embodiment of the reactive group represented by structure (Z38), R 19 is H. In a preferred embodiment of the reactive group represented by structure (Z38), l is 0 or 1, and more preferably l is 1.

[0081]

[0104] In a particularly preferred embodiment, the connecting group Z is represented by structure (Z39). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR 15 That is the case.

[0082]

[0105] In a preferred embodiment of the reactive group represented by structure (Z39), R 15 is hydrogen, halogen, -OR 16 [Here, R 16 [is hydrogen or C1-C6 alkyl], -S(O)3 (-) , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, more preferably R 15 It consists of hydrogen and -S(O)3 (-) It is independently selected from the group consisting of the following. In a preferred embodiment of the reactive group represented by structure (Z39), Y is N or CH, and more preferably Y=N.

[0083]

[0106] In a particularly preferred embodiment, the connecting group Z is represented by structure (Z36). [ka]

[0084]

[0107] Most preferably, the connecting group Z is structure (Z38) [in the structure, R 15 , R 18 and R 19 [All are H, and l is 1].

[0085]

[0108] In a second preferred embodiment, the connecting group Z may be obtained by thiol ligation. In this specification, the connecting group Z is probably L 1 Through L 2 It has one connection to and at least one connection to AB. A single connecting group Z may also have two connections to AB when a crosslinked thiol-reactive probe Q is used (see Figure 1B). Preferably, L1 There is no (w=0), and Z is directly connected to AB. In a preferred embodiment, the connecting group Z comprises a succinimidyl ring or a ring-opened succinic acid amide derivative thereof. Preferred choices of the connecting group Z, which include one connection to AB, include portions selected from (Z10) to (Z20) shown below. Preferred choices of the connecting group Z, which include two connections to AB, include portions selected from (Z11) to (Z23) shown below. [ka]

[0086]

[0109] In this specification, the combination(s) of waveforms marked with * are L 1 It is connected to A and B via optional selection, and the coupling of other waveforms is L 2 It is connected to [the specified location]. Furthermore, the following applies: R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 It is alkyl, R 29 C 1~12 Alkyl, preferably C 1~4 Alkyl, most preferably ethyl. Conjugation style

[0087]

[0110] The conjugate according to the present invention can be prepared by any form of conjugation. The conjugation reaction is thiol ligation or a metal-free click reaction. Modified antibody AB-((L 1 ) w -F) x The properties of L 1 The properties of w and x depend on the type of conjugation adopted. x is (L) on the antibody. 1 ) w This refers to the number of attachment sites (conjugation sites), which are integers in the range of 1 to 8. If Z (in the conjugate) or F (in the modified antibody) is directly attached to the antibody, then L 1It does not exist, and w=0. In this embodiment, the modified antibody is AB-(F) x It is sometimes called [this].

[0088]

[0111] x is linker L 1 x represents the amount of probe F present on the antibody via the optionally selected linker L, and is an integer in the range of 1 to 8. Preferably, x is an integer in the range of 1 to 6, more preferably x=1, 2, 3, or 4, even more preferably x=1 or 2, and most preferably x=2. Typically, x refers to the average number of portion Z attached to the antibody. In preferred embodiments, particularly when conjugation is performed by a click reaction, the conjugate according to the present invention usually has the same amount of portion Z attached to the antibody, although the conjugation reaction may sometimes be slightly incomplete. Notably, linker L 2 These are Linker L 2 Each can contain more than one exatecan payload, such as one or two payload molecules.

[0089]

[0113] In a preferred embodiment, w=1, and the mode of conjugation involves conjugation via the antibody glycan, preferably via the N-glycosylation site. Preferably, the modified antibody has the structure GlcNAc(Fuc) d -(G) e -Su-F [In the structure, e is an integer in the range of 0 to 20, Su is a monosaccharide, G is a monosaccharide moiety, GlcNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, and d is 0 or 1] contains one or more glycans. In other words, L 1 Preferably, -GlcNAc(Fuc) d -(G) e -Su-[Here, GlcNAc(Fuc) d-This is represented by [where is attached to the peptide portion of the antibody, and Su is attached to Z or F]. In this specification, GlcNAc is the core N-acetylglucosamine moiety typically present in the glycan structure of an antibody. In this specification, the core N-acetylglucosamine moiety refers to the N-acetylglucosamine moiety directly attached to the peptide chain of the antibody. This core N-acetylglucosamine moiety is optionally fucosylated (where d is 0 or 1), which is a common feature of antibodies.

[0090]

[0114] (G) e (G) represents the glycoform of the antibody. The present invention can be applied to antibodies of any glycoform. Typical monosaccharides present in glycans from which G can be selected include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and xylose. Therefore, (G) e The glycan can be a linear or branched oligosaccharide containing an e monosaccharide moiety. Typical glycans have e in the range of 4 to 16, preferably 6 to 10. In preferred embodiments, the antibody is pruned to e=0. Such pruning can be carried out by an endoglycosidase enzyme such as EndoS. Conjugation via the glycan preferably employs an N-glycosylation site connected to an N-glycosylation site, more preferably an asparagine amino acid of the antibody, and most preferably a conserved glycosylation site at amino acid N297 of the antibody.

[0091]

[0115] Su is a monosaccharide containing F. Su is preferably selected from the group consisting of galactose (Gal), mannose (Man), glucose (Glc), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid or sialic acid (Sial), and fucose (Fuc). Su is preferably glucose or a galactose derivative, more preferably a galactose derivative, and most preferably N-acetylgalactosamine.

[0092]

[0116] In an alternative preferred embodiment, w=0, and the conjugation site in the antibody is a cysteine ​​residue, preferably one that is naturally present in the antibody after optional reduction of the disulfide bond, as known in the art, or the cysteine ​​residue is engineered, preferably by substitution of an amino acid with cysteine, cysteine ​​insertion, or introduction of a single cysteine ​​residue or a peptide fragment containing a cysteine ​​residue at the N-terminus or C-terminus.

[0093]

[0117] AB represents an antibody. Those skilled in the art will understand that the present invention can be applied to any antibody. Preferably, the antibody targets HER2. Preferred conjugate

[0094]

[0118] A preferred conjugate according to the present invention has structure (1f). [ka]

[0095]

[0119] In this specification, L 2 The precise properties of are further specified, including the branched portion N. AB, L 1 Z, A, a, R 13 , R 21 x, n, R 17 m and p are defined elsewhere in this specification, L 5 is the linker, r is 0 or 1, and q is an integer in the range of 0 to 10. Preferably, linker L 5 It does not exist or is CH2. Preferably, m is an integer in the range of 1 to 10, q is an integer in the range of 1 to 4, and p is 1. Preferably, n is 1, 2, or 3. More preferably, AB is an antibody, L 1 It is a linker, Z is a connecting group represented by the structure (Z38), preferably R in the structure. 15 , R18 and R 19 All are H, and l is 1. a=1, R 13 It is hydrogen, r=0, L 5 It does not exist. (NH-CH(R 17 )-C(O)) n These are Val-Ala or Val-Cit, respectively. m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, x is an integer in the range of 2 to 4, preferably x = 2. R 21 These are H.

[0096]

[0120] A preferred conjugate according to the present invention has structure (1a). [ka]

[0097]

[0121] In this specification, L 2 The precise properties of are further specified, including the branched portion N. AB, L 1 Z, A, R 21 x, R 17 m and p are defined elsewhere in this specification, and q is an integer in the range of 0 to 10. Preferably, R 17 is CH3 or CH2CH2CH2NHC(O)NH2, m is an integer in the range of 1 to 10, q is an integer in the range of 1 to 4, and p is 1. More preferably, AB is an antibody, L 1 It is a linker, Z is a connecting group represented by the structure (Z38), preferably R in the structure. 15 , R 18and R 19 All are H, and l is 1. R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, x is an integer in the range of 2 to 4, preferably x = 2. R 21 These are H.

[0098]

[0122] A preferred conjugate according to the present invention has structure (1b). [ka]

[0099]

[0123] In this specification, L 2 The precise properties of are further specified, including the branched portion N. AB, L 1 , w, Z, A, R 21 x, R 17 m and p are defined elsewhere in this specification, and q is an integer in the range of 0 to 10. Preferably, R 17 is CH3 or CH2CH2CH2NHC(O)NH2, m is an integer in the range of 1 to 10, q is an integer in the range of 1 to 4, and p is 1. More preferably, AB is an antibody, L 1 It is a linker, w is either 0 or 1. Z is a connecting group represented by structure (Z1), R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is 2 or 4, preferably q=2. A is 1,4-phenyl, x is an integer in the range of 2 to 4, preferably x = 2. R 21 These are H.

[0100]

[0124] Another preferred conjugate according to the present invention has structure (1c). [ka]

[0101]

[0125] In this specification, L 1 The precise properties and mode of conjugation to antibodies are further specified. Z, L 2 , A, R 21 , x and R 17 The terms are defined elsewhere in this specification. Additionally, e is an integer in the range of 0 to 20, Su is a monosaccharide, G is a monosaccharide moiety, GlcNAc is an N-acetylglucosamine moiety, Fuc is a fucose moiety, and d is 0 or 1.

[0102]

[0126] A particularly preferred conjugate according to the present invention is the form and structure of the conjugation (1c) L 1 L of Definition and Structure (1f) 2 The definitions are combined, and therefore the structure (1g) is formed. [ka]

[0103]

[0127] In this specification, e, Su, G, GlcNAc, Fuc, d, Z, L 5 ,r,m,p,q,A,a,R 13 , R 21 , n, R 17All of , and x, including their preferred embodiments, are as described above.

[0104]

[0128] In a particularly preferred embodiment, the conjugate according to the present invention has structure (1d) [in the structure, d is either 0 or 1. e is 0, and G does not exist. Su is GalNAc, Z is a connecting group represented by the structure (Z38), preferably R in the structure. 15 , R 18 and R 19 All are H, and l is 1. a=1, R 13 It is hydrogen, r=0, L 5 It does not exist. (NH-CH(R 17 )-C(O)) n These are Val-Ala or Val-Cit, respectively. m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, x is an integer in the range of 2 to 4, preferably x = 2. R 21 [Each of these is H].

[0105]

[0129] A particularly preferred conjugate according to the present invention is the form and structure of the conjugation (1c) L 1 L of Definition and Structure (1a) 2 The definitions are combined, and therefore have structure (1d). [ka]

[0106]

[0130] In this specification, e, Su, G, GlcNAc, Fuc, d, Z, m, p, q, R 17 , A, R 21 All of x and x, including their preferred embodiments, are as described above.

[0107]

[0131] In a particularly preferred embodiment, the conjugate according to the present invention has structure (1d) [in the structure, d is either 0 or 1. e is 0, and G does not exist. Su is GalNAc, Z is a connecting group represented by the structure (Z38), preferably R in the structure. 15 , R 18 and R 19 All are H, and l is 1. R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl; x is an integer in the range of 2 to 4, preferably x = 2. R 21 [Each of these is H].

[0108]

[0132] A particularly preferred conjugate according to the present invention is the form and structure of the conjugation (1c) L 1 L of Definition and Structure (1b) 2 The definitions are combined, and therefore have structure (1e). [ka]

[0109]

[0133] In this specification, e, Su, G, GlcNAc, Fuc, d, Z, m, p, q, R17 , A, R 21 All of x and x, including their preferred embodiments, are as described above.

[0110]

[0134] In a particularly preferred embodiment, the conjugate according to the present invention has structure (1e) [in the structure, d is either 0 or 1. e is 0, and G does not exist. Su is GalNAc, Z is a connecting group represented by structure (Z1), R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is 2 or 4, preferably q=2. A is 1,4-phenyl, x is an integer in the range of 2 to 4, preferably x = 2. R 21 [Each of these is H].

[0111]

[0135] In a particularly preferred embodiment, the conjugate according to the present invention has structure (1h). [ka]

[0112]

[0136] In this specification, e, Su, G, GlcNAc, Fuc, and d are as described above, including in their preferred embodiments, and n=0 or 1. Structure (1h) corresponds to a conjugate of compound (31a) with n=0 and compound (32a) with n=1 in the examples. Most preferably, e=0 and Su is N-acetylgalactosamine. Method for synthesizing antibody-drug conjugates

[0113]

[0137] In a second aspect, the present invention relates to a method for preparing an antibody-drug conjugate according to the present invention. The method according to the present invention is (i) structure AB-((L 1 ) w -F) x The process includes (ii) reacting a modified antibody with a linker-drug construct represented by structure (5). The reaction is a conjugation reaction that forms a covalent bond between the exatecan payload and the antibody. The reaction is a metal-free click reaction or thiol ligation, forming an antibody-drug conjugate in which the drug is covalently bound to the antibody via a connecting group Z formed by the metal-free click reaction between Q and F or the thiol ligation between Q and F.

[0114]

[0138] In the method according to the present invention, structure AB-((L 1 ) w -F) x Modified antibody [In the structure, AB is antibody, L 1 [wherein is a linker, w is 0 or 1, F is a click probe that can react with Q in a metal-free click reaction or a thiol or precursor thereof that can react with Q in thiol ligation, and x is an integer in the range of 1 to 8]. When F is reacted with Q of the linker-drug construct represented by structure (5) via a metal-free click reaction or thiol ligation, a connecting group Z is formed. In one embodiment, the modified antibody is AB-(F) x It is sometimes referred to as [modified antibody]. Methods for preparing modified antibodies are known in the art from, for example, International Publications 2014 / 065661, 2016 / 170186, and 2016 / 053107, which are incorporated herein by reference. From the same literature, conjugation reactions between modified glycoproteins and linker-drug constructs containing cytotoxicities and click probes are known to those skilled in the art.

[0115]

[0139] In the method according to the present invention, the linker-drug construct has structure (5) [ka] [In structure, L 2 It is a linker, Q is a click probe or thiol-reactive probe capable of performing a metal-free click reaction. R 17 These are each individual amino acid side chains, n is an integer in the range of 1 to 5. A is a 5-membered or 6-membered aromatic or heteroaromatic ring, x is an integer in the range of 1 to 8. R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from, R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 23 It is optionally interrupted by one or more heteroatoms selected from R 23 [These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.] Metal-free click reaction

[0116]

[0140] Metal-free click reactions are well known in the art (see, for example, International Publication No. 2014 / 065661, incorporated by reference, and Nguyen and Presser, Nature rev., 2020, doi:10.1038 / s41570-020-0205-0), and can typically take the form of 1,3-dipolar cycloaddition or (4+2) cycloaddition. Alkyne-azide cycloaddition can be strain-enhanced (e.g., strain-enhanced alkyne-azide cycloaddition, SPAAC). In preferred embodiments, the bioconjugation reaction is a metal-free strain-enhanced cycloaddition, most preferably a metal-free strain-enhanced alkyne-azide cycloaddition. In preferred embodiments, conjugation is achieved via cycloaddition such as (4+2) cycloaddition or 1,3-dipolar cycloaddition, preferably 1,3-dipolar cycloaddition.

[0117]

[0141] A typical (4+2) cycloaddition is the Diels-Alder reaction, where Q is a diene or dienophile. As will be understood by those skilled in the art, in the context of the Diels-Alder reaction, the term “diene” refers to a 1,3-(hetero)diene, encompassing conjugated dienes (R2C=CR-CR=CR2), imines (e.g., R2C=CR-N=CR2 or R2C=CR-CR=NR, R2C=NN=CR2) and carbonyls (e.g., R2C=CR-CR=O or O=CR-CR=O). Hetero-Diels-Alder reactions with N-containing and O-containing dienes are known in the art. Any diene known in the art to be suitable for (4+2) cycloaddition can be used as the reactive group Q. Preferred dienes include tetrazine, 1,2-quinone, and triazine. Any dienophile known to be suitable for (4+2) cycloaddition in the art can be used as the reactive group Q, but the dienophile is preferably an alkene or alkyne group, most preferably an alkyne group. In the conjugation via (4+2) cycloaddition, Q is preferably a dienophile (F is a diene), and more preferably Q is an alkynyl group or contains an alkynyl group.

[0118]

[0142] In 1,3-dipolar cycloaddition, Q is either a 1,3-dipole or a parent dipole. Any 1,3-dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q. Preferred 1,3-dipoles include azide groups, nitrone groups, nitrile oxide groups, nitrile imine groups, and diazo groups. Any parent dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q, but the parent dipole is preferably an alkene or alkyne group, most preferably an alkyne group. In conjugation via 1,3-dipolar cycloaddition, it is preferable that Q is a parent dipole (F is a 1,3-dipole), and more preferably that Q is an alkynyl group or contains an alkynyl group.

[0119]

[0143] Therefore, in a preferred embodiment, Q is selected from a parent dipole and a dienophile. Click probeQ

[0120]

[0144] Click probe Q is used in conjugation reactions to convert the linker-drug construct into structure AB-(F). x Q is connected to the antibody. Q is reactive to click probe F in a metal-free click reaction. Such click probes are known in the art and include cyclic alkenes, cyclic alkynes, azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, and cydonones. Preferably, Q is a cyclic alkene or cyclic alkyne moiety, and most preferably, Q is a cyclic alkyne moiety.

[0121]

[0145] In a particularly preferred embodiment, click probe Q includes a cyclic alkyne moiety. The alkynyl group may also be called a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, and the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctinyl group, a (hetero)cyclononinyl group, or a (hetero)cyclodecynyl group. In this specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is optionally substituted with a (hetero)cycloheptynyl group or an optionally substituted with a (hetero)cyclooctinyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctinyl group, and the (hetero)cyclooctinyl group is optionally substituted. Preferably, Q includes a (hetero)cyclooctinyl moiety represented by the following structure (Q1). In another preferred embodiment, the (hetero)cyclooctinyl group is represented by the structures (Q37), (Q38), or (Q39) as further defined below. Preferred examples of the (hetero)cyclooctinyl group include the structure also called the DIBO group (Q2), the structure also called the DIBAC group (Q3), or the structure also called the BARAC group (Q4), the structure also called the COMBO group (Q5), and the structure also called the BCN group (Q6), all of which are shown below, in which Y 1 is O or NR 11 And R 11 These are hydrogen, linear or branched C1-C 12 Alkyl alkyl group or C4-C 12 The group is independently selected from the group consisting of (hetero)aryl groups. The aromatic ring in (Q2) is optionally O-sulfonylated at one or more positions, and the rings of (Q3) and (Q4) may be halogenated at one or more positions. Particularly preferred cycloalkynyl groups are optionally substituted bicyclo[6.1.0]nona-4-in-9-yl] groups (BCN groups). Preferably, the bicyclo[6.1.0]nona-4-in-9-yl] group is given by the following formula (Q6) [wherein V is (CH2)] l(Q6) In the context of the base, l is most preferably 1. [ka]

[0122]

[0146] In another preferred embodiment, the click probe Q is selected from the group consisting of (Q7) to (Q21) shown below. [ka]

[0123]

[0147] In this specification, L is represented by the combination of waveforms. 2 The connection to Q can be to any available carbon or nitrogen atom. (-) is an anion, preferably selected from OTf, Cl, Br or I, most preferably B (-) teeth, (-) OTf. In the conjugation reaction, B (-) Q does not need to be a pharmaceutically acceptable anion, as it can be exchanged with any anion present in the reaction mixture. When (Q21b) is used as Q, the negatively charged counterion is preferably pharmaceutically acceptable at the time of isolation of the conjugate, so that the conjugate according to the present invention can be readily used as a pharmaceutical.

[0124]

[0148] In another preferred embodiment, the click probe Q is selected from the group consisting of (Q22) to (Q36) shown below. [ka]

[0125]

[0149] In structure (Q36b), B (-)is an anion, preferably selected from OTf, Cl, Br, or I, with B being most preferably OTf.

[0126]

[0150] In a particularly preferred embodiment, the click probe Q comprises a (hetero)cycloalkynyl group and is represented by the structure (Q37). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y 2 C(R 31 )2, O, S or NR 31 And R 31 Each is individually, R 15 Alternatively, it is a second appearance of the exatecan payload connected via the spacer portion, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16.

[0127]

[0151] In a preferred embodiment, u+u'=4, 5, or 6, and more preferably u+u'=5. Typically, v=(u+u')×2 or [(u+u')×2]-1. In a preferred embodiment, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0128]

[0152] In a particularly preferred embodiment, the click probe Q comprises a cyclooctinyl group and is represented by the structure (Q38). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) ,C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R18 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 19 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Selected from the group consisting of (hetero)arylalkyl groups, the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 This is the second appearance of the exatecan payload connected via the spacer portion. l is an integer in the range of 0 to 10.

[0129]

[0153] In a preferred embodiment of the reactive group represented by structure (Q38), R 15 is hydrogen, halogen, -OR 16 , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 15 All are H. In a preferred embodiment of the reactive group represented by structure (Q38), R 18 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 18 Both are H. In a preferred embodiment of the reactive group represented by structure (Q38), R 19is H. In a preferred embodiment of the reactive group represented by structure (Q38), l is 0 or 1, and more preferably l is 1. A particularly preferred embodiment of the reactive group represented by structure (Q38) is the reactive group represented by structure (Q30).

[0130]

[0154] In a particularly preferred embodiment, the click probe Q comprises a cyclooctinyl group and is represented by the structure (Q39). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR 15 That is the case.

[0131]

[0155] In a preferred embodiment of the reactive group represented by structure (Q39), R 15 is hydrogen, halogen, -OR 16 -S(O)3(-) , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 It consists of hydrogen and -S(O)3 (-) It is independently selected from the group consisting of the following. In a preferred embodiment of the reactive group represented by structure (Q39), Y is N or CH, and more preferably Y=N.

[0132]

[0156] In a particularly preferred embodiment, the click probe Q comprises a heterocycloheptynyl group and is represented by the structure (Q36a). [ka]

[0133]

[0157] In an alternative preferred embodiment, the click probe Q includes a cyclic alkene moiety. The alkenyl group Q may also be called a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, and the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecynyl group, all of which may be optionally substituted. Particularly preferred are a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctenyl group, and the (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctinyl group is optionally substituted. Preferably, Q includes a cyclopropenyl moiety represented by structure (Q40), a trans-(hetero)cycloheptenyl moiety represented by structure (Q41), or a trans-(hetero)cyclooctenyl moiety represented by structure (Q42). In another preferred embodiment, the cyclopropenyl group is represented by structure (Q43). In another preferred embodiment, the trans-(hetero)cycloheptene group is represented by structure (Q44) or (Q45). In another preferred embodiment, the trans-(hetero)cyclooctene group is represented by structure (Q46), (Q47), (Q48), (Q49), or (Q50). [ka]

[0134]

[0158] In this specification, the R group(s) of Si in (Q44) and (Q45) are typically alkyl or aryl, preferably C1-C6 alkyl. Thiol ligation

[0135]

[0159] Thiol ligation for antibody-drug conjugate preparation is well known in the art and is sometimes called alkylation of thiol groups. It typically takes the form of nucleophilic reactions such as nucleophilic substitution or Michael reactions. A preferred Michael reaction is the maleimide-thiol reaction, which is widely used in bioconjugation. Therefore, in preferred embodiments, Q is reactive in nucleophilic reactions, preferably nucleophilic substitution or Michael reactions. In this specification, Q preferably comprises a maleimide moiety, a haloacetamide moiety, an allenamide moiety, a phosphoamidite moiety, a cyanoethynyl moiety, a vinylsulfone, a vinylpyridine moiety, or a methylsulfonylphenyloxadiazole moiety, most preferably a maleimide moiety. Particularly preferred options for Q are shown in Figure 1B. Thiol-reactive probe Q

[0136]

[0160] Thiol-reactive probe Q is used in conjugation reactions to form a linker-drug construct with structure AB-((L 1 ) w -F) x It is bound to the antibody. Q is reactive to thiols or their precursor F in thiol ligation. Such probes are known in the art and can be selected from the group consisting of maleimide moieties, haloacetamide moieties, allenamide moieties, phosphoamidite moieties, cyanoethynyl moieties, vinylsulfone, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, Q contains a maleimide moiety or is a maleimide moiety.

[0137]

[0161] In another preferred embodiment, probe Q is selected from the group consisting of (Q51) to (Q65) shown below. [ka] [In structure, X 6 This is a halogen such as H, a halogen, PhS, MeS, preferably Cl, Br, I, etc. X 7 This is a halogen such as a halogen, PhS, MeS, preferably Cl, Br, I, etc. R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 It is alkyl, R 25 H, C 1~12 Alkyl, C 1~12 Ariel, C 1~12 Alkaline or C 1~12 Aralkyl, preferably H or paramethylphenyl, The aromatic rings in (Q55) and (Q57) can be optionally selected to be heteroaromatic rings such as phenyl or pyridine rings.

[0138]

[0162] In a preferred embodiment of the thiol-reactive probe (Q51), probe Q is selected from the group consisting of (Q66) to (Q68) shown below. [ka] [In structure, R 27 C 1~12 Alkyl, C 1~12 Ariel, C 1~12 Alkaline or C 1~12 It is Aralkir, t is an integer in the range of 0 to 15, preferably 1 to 10. Preferred linker-drug construct

[0139]

[0163] In a particularly preferred embodiment, the linker-drug construct according to the present invention has structure (5c) [ka] [In structure, Q, m, p, q, R 17 , A and R 21 Each of these is as described above.

[0140]

[0164] Most preferably, the linker-drug construct according to the present invention has structure (5a) [in the structure, Q is a connecting group represented by the structure (Q38), preferably R in the structure. 15 , R 18 and R 19 All are H, and l is 1. R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, R 21 [Each of these is H].

[0141]

[0165] In a particularly preferred embodiment, the linker-drug construct according to the present invention has structure (5a) [ka] [In structure, Q, m, p, q, R 17 , A and R 21 Each of these is as described above.

[0142]

[0166] Most preferably, the linker-drug construct according to the present invention has structure (5a) [in the structure, Q is a connecting group represented by the structure (Q38), preferably R in the structure. 15 , R 18 and R 19 All are H, and l is 1. R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, R 21 [Each of these is H].

[0143]

[0167] In a particularly preferred embodiment, the linker-drug construct according to the present invention has structure (5b) [ka] [In structure, Q, m, p, q, R 17 , A and R 21 Each of these is as described above.

[0144]

[0168] Most preferably, the linker-drug construct according to the present invention has structure (5a) [in the structure, Q is a connecting group represented by structure (Q1), R 17 Each of these is individually CH3 or CH2CH2CH2NHC(O)NH2, preferably R 17 These are CH3, m is 2 or 4, preferably m=2. p is 0 or 1, preferably p=1. q is either 2 or 4, preferably q=2 in each case. A is 1,4-phenyl, R 21 [Each of these is H].

[0145]

[0169] In a particularly preferred embodiment, the linker-drug construct according to the present invention has structure (5d) [ka] [In the structure, n=0 or 1].

[0146]

[0170] Structure (5d) corresponds to compound (31a) with n=0 and compound (32a) with n=1 in the examples. Reactive group F

[0147]

[0171] The reactive group F is a click probe or a thiol or its precursor. Therefore, in a first preferred embodiment, F is a click probe. Click probe F is used in a conjugation reaction to link a linker-drug construct to a modified antibody. F is reactive to click probe Q in a metal-free click reaction. Such click probes are known in the art and include cyclic alkenes, cyclic alkynes, azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, and cydonones.

[0148]

[0172] Preferably, F is reactive to cyclic alkenes and cyclic alkynes and is typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, and cydonones. Preferred structures for the reactive group are shown below as (F1) to (F10). [ka]

[0149]

[0173] In this specification, a waveform conjugate represents a connection to an antibody. For (F3), (F4), (F8), and (F9), the antibody can be connected to any one of the waveform conjugates. Then, the other waveform conjugates can be connected to hydrogen, C1-C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (hetero)arylalkyl groups and C1-C 24It can be attached to an R group selected from sulfonyl groups, and each of these (except hydrogen) may be optionally substituted with O, S and NR. 32 [Here, R 32 The R group may be optionally interrupted by one or more heteroatoms independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Those skilled in the art will understand which R groups can be applied to each of the click probes F. For example, the R group connected to the nitrogen atom of (F3) can be selected from alkyl and aryl groups, and the R group connected to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl and sulfonyl groups. Preferably, the click probe F is selected from azide or tetrazine. Most preferably, the click probe F is azide.

[0150]

[0174] In a second preferred embodiment, F is a thiol or its precursor. The thiol or its precursor F is used in a conjugation reaction to link a linker-drug construct to a modified antibody. F is reactive to the thiol-reactive probe Q in thiol ligation. Thiol precursors in the context of bioconjugation are known in the art and include disulfides. These may be naturally occurring cross-linked disulfides present in antibodies or synthetically introduced disulfides, which are reduced as known in the art. Preferably, F is a thiol group. Linker - Drug construct

[0151]

[0175] In a third aspect, the present invention relates to a linker-drug construct represented by structure (5). [ka] [In structure, L 2 It is a linker, Q is a click probe or thiol-reactive probe capable of performing a metal-free click reaction. R17 These are each individual amino acid side chains, n is an integer in the range of 1 to 5. A is a 5-membered or 6-membered aromatic or heteroaromatic ring, x is an integer in the range of 1 to 8. R 21 H, R 22 , C(O)OH and C(O)R 22 Selected from, R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, which are optionally substituted with O, S, and NR. 23 It is optionally interrupted by one or more heteroatoms selected from R 23 [These are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups.]

[0152]

[0176] The definitions and preferred embodiments of linker-drug constructs described in the context of the first and second aspects of the present invention, and L 2 Q, R 17 , n, A, x and R 21 This also applies to a third aspect of the present invention. The linker-drug construct according to this aspect is ideally suited as an intermediate in the preparation of the antibody-drug conjugate according to the present invention. Therefore, the present invention relates to the structure AB-((L 1 ) w -F) x The present invention also relates to the use of the linker-drug construct in metal-free click conjugation reactions or thiol ligation conjugation reactions with modified antibodies. application

[0153]

[0177] The conjugate according to the present invention is particularly suitable for the treatment of cancer. Accordingly, the present invention further relates to the use of the conjugate according to the present invention in pharmaceuticals. In another embodiment, the present invention also relates to a method for treating a subject in need thereof, comprising the step of administering the conjugate according to the present invention to the subject. A method according to this embodiment may also be referred to as the conjugate according to the present invention for therapeutic use, particularly for use in the treatment of a subject in need thereof. A method according to this embodiment may also be referred to as the use of the conjugate according to the present invention for the manufacture of pharmaceuticals. In this specification, administration is typically carried out using a therapeutically effective amount of the conjugate according to the present invention.

[0154]

[0178] The present invention further relates to a method for treating a particular disease in a subject requiring its use, comprising the administration of a conjugate according to the present invention as defined above. The particular disease can be selected from cancer and autoimmune diseases, preferably the disease is cancer. The subject requiring its use is typically cancer patients. The use of antibody-drug conjugates is well known in the field of such treatment, particularly cancer treatment, and the conjugate according to the present invention is particularly suited in this respect. In this embodiment of the method, the conjugate is typically administered in a therapeutically effective dose. This embodiment of the present invention may also be referred to as the conjugate according to the present invention for use in the treatment of a particular disease in a subject requiring its use, preferably for the treatment of cancer. In other words, this embodiment relates to the use of the conjugate according to the present invention for the preparation of a pharmaceutical or pharmaceutical composition for use in the treatment of a particular disease in a subject requiring its use, preferably for use in the treatment of cancer. In one embodiment, the cancer is a HER2-positive cancer, such as HER2-positive breast cancer, HER2-positive gastric cancer, HER2-positive colon cancer, HER2-positive lung cancer, HER2-positive pancreatic cancer, HER2-positive urothelial carcinoma, HER2-positive brain cancer, HER2-positive ovarian cancer, etc. Therefore, in one embodiment, the patient is HER2-positive.

[0155]

[0179] In the context of this invention, administration refers to systemic administration. Therefore, in one embodiment, the method defined herein is a method for systemic administration of a conjugate. Given the specificity of the conjugates, they can be administered systemically, yet they still exert their activity in or near the target tissue (e.g., a tumor). Systemic administration offers significant advantages over local administration, as it allows the drug to reach tumor metastases undetectable by imaging techniques and can be applicable to hematological malignancies.

[0156]

[0180] The present invention further relates to a pharmaceutical composition comprising an antibody-payload conjugate according to the present invention and a pharmaceutically acceptable carrier. [Examples]

[0157]

[0181] The present invention will be explained by the following examples. General procedure for analytical RP-HPLC (DTT reduction)

[0158]

[0182] Prior to RP-HPLC analysis, IgG (10 μL, 1 mg / mL in PBS, pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 μL) and incubated at 37°C for 15 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, and 2% formic acid (50 μL). RP-HPLC analysis was performed using an Agilent 1100 series (Hewlett Packard). The sample (10 μL) was injected at 0.5 mL / min into a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm (Waters) column at a column temperature of 70°C. A linear gradient of 0.1% TFA and acetonitrile in water from 30% to 54% was applied over 16.8 minutes. General Procedure for Analytical RP-UPLC

[0159]

[0183] 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 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, and 2% formic acid (50 μL). RP-UPLC analysis was performed using a Waters Acquity UPLC-SQD. The sample (5 μL) was injected at 0.4 mL / min into a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm column (Waters) at a column temperature of 70°C. A linear gradient of 0.1% TFA and acetonitrile in water from 30% to 54% was applied over 9 minutes. General Procedures for Analytical SEC

[0160]

[0184] HPLC-SEC analysis was performed using an Agilent 1100 series HPLC system (Hewlett Packard) with an Xbridge BEH200A column (3.5 μM, 7.8 × 300 mM, PN 186007640 Waters). Samples were diluted to 1 mg / mL in PBS and measured for 16 minutes using the isocratic method at 0.86 mL / min (0.1 M sodium phosphate buffer containing 10% isopropanol, pH 6.9 (NaHPO4 / Na2PO4)). General procedure for analytical HPLC-MS (IdeS digestion)

[0161]

[0185] Prior to mass spectrometry, IgG was treated with IdeS. This enabled the analysis of Fc / 2 fragments. For Fc / 2 fragment analysis, a solution of 20 μg of (modified) IgG was incubated in 10 μL total volume with IdeS / Fabricator™ (1.25 U / μL) pH 6.6 in PBS at 37°C for 1 hour. The sample was diluted to 80 μL and then subjected to electrospray ionization time-of-flight (ESI-TOF) analysis using JEOL AccuTOF. Deconvolution spectra were obtained using Magtran software.

[0162]

[0186] Compound 3 (see Figure 7) was prepared according to International Publication No. 2020 / 094670 (linker-drug compound 42). Compound 4 (deruxtecan, see Figure 8) was obtained from MedChemExpress. The structures of various antibody formats for metal-free click conjugation (v1 and v5) or thiol ligation (v2, v3, v4) are shown in Figure 11. Example 1. Preparation of Compound 6 [ka]

[0163]

[0187] To a solution of BCN-OH (5, 1.5 g, 10 mmol) in DCM (150 mL), CSI (0.87 mL, 1.4 g, 10 mmol), Et3N (2.8 mL, 2.0 g, 20 mmol), and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol) were added under an N2 atmosphere. The mixture was stirred for 10 minutes and quenched by adding saturated NH4Cl aqueous solution (150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na2SO4) and concentrated. The residue was purified by column chromatography. Product 6 was obtained as a slightly yellow, concentrated oil (2.06 g, 5.72 mmol, 57%). 1H NMR (400 MHz, CDCl3) δ (ppm)6.0 (bs, 1H), 4.28 (d, J = 8.2 Hz, 2H), 3.78-3.73 (m, 2H), 3.66-3.61 (m, 2H),3.61-3.55 (m, 2H), 3.34 (t, J = 4.9 Hz, 2H), 2.37-2.15 (m, 6H), 1.64-1.48 (m,2H), 1.40 (quintet, J = 8.7 Hz, 1H), 1.05-0.92 (m, 2H). Example 2. Preparation of Compound 7 [ka]

[0164]

[0188] To a stirred solution of 6 (47 mg, 0.13 mmol) in DCM (10 mL), CSI (11 μL, 18 mg, 0.13 mmol) was added. After 30 minutes, Et3N (91 μL, 66 mg, 0.65 mmol) and a solution of diethanolamine (16 mg, 0.16 mmol) in DMF (0.5 mL) were added. After 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et3N (54 μL, 39 mg, 0.39 mmol) were added. After a further 4.5 hours, the reaction mixture was concentrated, and the residue was purified by gradient column chromatography (33 → 66% Â / heptane (1% AcOH)) to obtain 7 as a colorless oil (88 mg, 0.098 mmol, 75%). 1 H NMR (400 MHz, CDCl3) δ (ppm) 8.28-8.23 (m, 4H), 7.42-7.35 (m, 4H), 4.52 (t, J = 5.4 Hz,4H), 4.30 (d, J = 8.3 Hz, 2H), 4.27-4.22 (m, 2H), 3.86 (t, J = 5.3 Hz, 4H),3.69-3.65 (m, 2H), 3.64-3.59 (m, 2H), 3.30-3.22 (m, 2H), 2.34-2.14 (m, 6H),1.62-1.46 (m, 2H), 1.38 (quintet, J = 8.7 Hz, 1H), 1.04-0.92 (m, 2H). Example 3. Preparation of compounds 9a and 9b [ka]

[0165]

[0189] Compound 8a (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 hours, the reaction mixture was diluted in 9 mL of DCM and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain 9a (155 mg, 159 μmol, 68%). LCMS (ESI+) C 55 H 54 FN6O10 + (M+H) + The calculated value was 977.39, and the measured value was 977.72. In addition to 9a, free base of exatecan (82.4 mg, 189 μmol, 20%) was recovered. LCMS (ESI+) C 24 H 23 FN3O4 + (M+H) + The calculated value is 436.46, and the measured value is 436.54.

[0166]

[0190] Compound 8b (29.1 mg, 38 μmol) was added to a mixture of exatecan mesylate (19.8 mg, 37.2 μmol) and DIPEA (9.6 mg, 13 μL, 74.5 μmol) in dry DMF (150 μL). After 5 hours, the reaction mixture was diluted in 3 mL of DCM and purified by automated gradient silica gel column chromatography (0 → 10% MeOH / DCM) to obtain 9b (28.2 mg, 26.5 μmol, 74%) as a pale yellow solid. LCMS (ESI+) C 58 H 60 FN8O 11 + (M+H) + The calculated value is 1063.44, and the measured value is 1063.72. Example 4. Preparation of compounds 1a and 1b (See Figure 7 for their structures)

[0167]

[0191] To a solution of compound 9a (155 mg, 159 μmol) in DMF (1.6 mL), Et3N (73 mg, 101 μL, 0.72 mmol) and a solution of compound 7 (65 mg, 72 μmol) in DMF (1.4 mL) were added. The reaction mixture was stirred for 18 hours, diluted with DCM (20 mL), and purified by gradient column chromatography (0 → 40% MeOH / DCM) to obtain 1a as a pale yellow solid (94 mg, 44 μmol, 28%). LCMS (ESI+) C 102 H 118 F2N 16 O 29 S2 2+ (M / 2+H) + The calculated value is 1066.88, and the measured value is 1067.12.

[0192] Compound 7 (3.5 mg, 3.9 μmol) in DMF (78 μL) was mixed with compound 9b (8.2 mg, 9.7 μmol) in DMF (97 μL) and Et3N (2.4 mg, 3.3 μL, 23.4 μmol). The reaction mixture was allowed to stand for 20 hours. The reaction mixture was diluted with DCM (2 mL) and purified by automated gradient silica gel column chromatography (0 → 30% MeOH / DCM). A portion of the obtained material was re-purified by preparative RP-HPLC (XBridge prep C18 5 μm OBD, 30 × 100 mm, 30 → 90% MeCN / H2O + 1% AcOH) to obtain compound 1b as a white solid (1.0 mg, 0.43 μmol, 11%). LCMS (ESI+) C 108 H 130 F2N 20 O 31 S2 2+ (M / 2+H) + The calculated value is 1152.93, and the measured value is 1152.58. Example 5. Preparation of compounds 10a and 10b [ka]

[0168]

[0193] Compound 9a (32 mg, 33 μmol) was dissolved in DMF (1 mL), and piperidine (28 mg, 33 μL, 0.33 mmol) was added. The mixture was stirred for 5.5 hours, concentrated, dissolved in DCM (2 mL), and purified by gradient column chromatography (0 → 30% MeOH / DCM (1% AcOH)) to obtain 10a (14.8 mg, 19.6 μmol, 59%). LCMS (ESI+) C 40 H 44 FN6O8 + (M+H) + The calculated value is 755.32, and the measured value is 755.46.

[0169]

[0194] To a solution of compound 9b (28.2 mg, 26.5 μmol) in DMF (200 μL), piperidine (22.6 mg, 26.2 μL, 265 μmol) was added. The reaction mixture was allowed to stand for 5.5 hours. The reaction mixture was diluted with DCM (2 mL) and purified by gradient column chromatography (0 → 30% MeOH / DCM) to obtain 10b as a pale yellow solid (16.3 mg, 19.4 μmol, 73%). LCMS (ESI+) C 43 H 50 FN8O9 + (M+H) + The calculated value is 841.37, and the measured value is 841.53. Example 6. Preparation of Compound 2a [ka]

[0170]

[0195] To a stirred solution of 6 (172 mg, 0.48 mmol) in DCM (20 mL), CSI (42 μL, 67 mg, 0.48 mmol) was added. After 20 minutes, Et3N (331 μL, 240 mg, 2.38 mmol) and a solution of 11 (250 mg, 0.55 mmol) in DMF (1 mL) were added. After 60 minutes, p-nitrophenyl chloroformate (242 mg, 1.20 mmol) and Et3N (643 μL, 467 mg, 1.43 mmol) were added. After 16 hours, an additional p-nitrophenyl chloroformate (50 mg, 0.25 mmol) was added, and stirring was continued for 4 hours. The reaction mixture was concentrated, and the residue was purified by gradient column chromatography (20 → 100% RINKAN / heptane (1% AcOH)) to obtain compound 12 as a colorless oil (318 mg, 0.25 mmol, 53%). LCMS (ESI+) C 50 H 73 N6O 27 S2 + (M+H + The calculated value is 1253.40, and the measured value is 1253.49.

[0171]

[0196] Compound 10 (14.8 mg, 19.6 μmol) in DMF (200 μL) solution and Et3N (4.0 mg, 5.6 μL, 40 μmol) were added to a solution of compound 12 (10.0 mg, 8.0 μmol) in DMF (200 μL). The reaction mixture was left to stand for 64 hours. The reaction mixture was purified by preparative HPLC (XBridge prep C18 5 μm OBD, 30 × 100 mm, 30 → 100% CH3CN / H2O (containing 1% AcOH)) to obtain 2a as a pale yellow film (4.3 mg, 1.73 μmol, 22%). LCMS (ESI+) C 118 H 150 F2N 16 O 37 S2 2+ (M / 2+H) + The calculated value is 1242.99, and the measured value is 1243.13. Example 7. Preparation of Compound 14 [ka]

[0172]

[0197] Preparation of 13: To a solution of 6 (3.62 g, 10 mmol) in DCM (200 mL), 4-nitrophenyl chloroformate (2.02 g, 10 mmol) and Et3N (4.2 mL, 3.04 g, 30 mmol) were added. After stirring at ambient temperature for 1.5 hours, the reaction mixture was concentrated and purified by automated gradient silica gel column chromatography (20% → 70% siRNA / heptane + 1% AcOH) to obtain 13 as a white foam (4.07 g, 11.7 mmol, 77%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.32 - 8.27 (m, 2H), 7.46 - 7.40 (m, 2H), 5.56 (t, J = 5.4Hz, 1H), 4.49 - 4.42 (m, 2H), 4.28 (d, J = 8.2 Hz, 2H), 3.80 - 3.76 (m, 2H),3.70 - 3.65 (m, 2H), 3.39-3.30 (m, 2H), 2.36 - 2.16 (m, 6H), 1.62-1.46 (m, 2H),1.38 (quintet, J = 8.7 Hz, 1H), 1.05-0.92 (m, 2H).

[0173]

[0198] Preparation of 14: To a mixture of 13 (2.61 g, purity 86.8% by 1H-NMR, 4.62 mmol, 1.0 equivalent) in DCM (80.0 mL), 4-nitrophenyl chloroformate (2.14 g, 10.6 mmol, 2.30 equivalents) was added, followed by the addition of Et3N (3.70 mL, 2.68 g, 26.5 mmol, 5.75 equivalents). The reaction mixture was stirred at room temperature for 4.5 hours, then concentrated under vacuum to obtain a yellow oil. The residue was purified by gradient column chromatography (50 → 100% siRNA / heptane + 1% AcOH), followed by a second gradient column chromatography (50 → 100% siRNA / heptane + 1% AcOH) to obtain 14 as a white solid (2.1 g, purity 88% by 1H-NMR, 2.25 μmol, 49%). 1H NMR (400 MHz, DMSO-d6) δ (ppm) 8.33-8.20 (m, 4H), 7.40 (d, J = 8.4 Hz, 4H), 5.74 (t, J = 5.8Hz, 1H), 4.56-4.42 (m, 4H), 4.40-4.31 (m, 2H), 4.26 (d, J = 8.3 Hz, 2H),3.83-3.72 (m, 4H), 3.71-3.64 (m, 2H), 3.60 (t, J = 4.9 Hz, 2H), 3.34-3.23 (m,2H), 2.36-2.14 (m. 6H), 1.82-1.44 (m, 4H), 1.44-1.31 (m, 1H), 0.98-0.92 (m,2H). Example 8: Preparation of compound 17b [ka]

[0174]

[0199] Preparation of 16b: To a stirred solution of 14 (62 mg, 75 μmol) in DMF (500 μL), 15b (60 mg, 158 μmol) and Et3N (63 μL, 46 mg, 450 μmol) were added. After 3.5 hours, bis(4-nitrophenyl) carbonate (137 mg, 450 μmol) and Et3N (63 μL, 46 mg, 450 μmol) were added. The reaction mixture was diluted with DMF (500 μL) and stirred for 1.5 hours. The reaction mixture was concentrated, and the residue was purified by automated gradient silica gel column chromatography (0 → 15% MeOH / DCM) to obtain 16b as a white solid (86 mg, 52.7 μmol, 70%). LCMS (ESI+) C 72 H 94 N 15 O 27 S + (M+H) + The calculated value is 1632.62, and the measured value is 1632.70.

[0175]

[0200] Preparation of 17b: To a mixture of exatecan mesylate (2.0 mg, 3.7 μmol) in DMF (40 μL), Et3N (0.9 mg, 1.2 μL, 8.9 μmol) and a solution of compound 16b (2.9 mg, 1.78 μmol) in DMF (25 μL) were added. The reaction mixture was allowed to stand for 19 hours. The reaction mixture was diluted with DMF (100 μL) and purified by preparative HPLC (XBridge prep C18 5 μm OBD, 30 × 100 mm, 30 → 100% MeCN / H2O (containing 1% AcOH)) to obtain 17b as a white solid (2.5 mg, 1.1 μmol, 62%). LCMS (ESI+) C 108 H 129 F2N 19 O 29 S2 2+ (M / 2+H) + The calculated value is 1113.45, and the measured value is 1113.80. Example 9: Preparation of compound 21b [ka]

[0176]

[0201] Preparation of 19: Under an N2 atmosphere, CSI (597 μL, 0.94 g, 6.7 mmol) was added to a refrigerated solution (0°C) of BCN-OH (5, 1.0 g, 6.7 mmol) in DCM (50 mL). After 7 minutes, stirring was continued at ambient temperature, and an additional CSI (58 μL, 0.1 g, 0.67 mmol) was added. After stirring for 6 minutes, Et3N (1.86 mL, 1.35 g, 13.3 mmol) was added, followed by stirring for 3 minutes, and then 2-[2-(1-piperazinyl)ethoxy]ethanol (18, 1.42 mL, 1.51 g, 8.65 mmol) was added. The mixture was stirred for 17 hours. The reaction mixture was concentrated under vacuum, and the residue was purified by automated gradient silica gel column chromatography (0→10% MeOH / DCM) to obtain compound 19 as a bright yellow foam (2.05 g, 3.91 mmol, 58%). 1H NMR (400 MHz, CDCl3) δ (ppm) 4.21 (d, J = 8.2 Hz, 2H), 3.72-3.63 (m, 4H), 3.62-3.56 (m,2H), 3.47-3.36 (m, 4H), 2.70-2.55 (m, 6H), 2.37-2.15 (m, 6H), 1.66-1.48 (m,2H), 1.39 (quintet, J = 8.7 Hz, 1H), 1.03-0.89 (m, 2H).LCMS (ESI+) C 19 H 32 N8O6S + (M+H + The calculated value was 430.20, and the measured value was 430.43.

[0177]

[0202] Preparation of 20: To a stirred refrigerated suspension (0°C) of 19 (750 mg, 1.75 mmol) in DCM (18 mL), CSI (167 μL, 272 mg, 1.92 mmol) was added. After stirring at 0°C for 2 minutes, stirring was continued at ambient temperature for 28 minutes. Next, Et3N (1.22 mL, 886 mg, 8.75 mmol) was added, and after stirring for 3 minutes, a solution of diethanolamine (229 mg, 209 μL, 2.18 mmol) in DMF (0.5 mL) was added. After 40 minutes, bis(4-nitrophenyl) carbonate (1.33 g, 4.38 mmol) and Et3N (725 μL, 526 mg, 5.20 mmol) were added. After 18 hours, an additional bis(4-nitrophenyl) carbonate (266 mg) was added, and stirring was continued for 25 hours. The reaction mixture was concentrated, and the residue was purified by automated gradient silica gel column chromatography (0 → 20% MeOH / Â, followed by 50% MeOH / DCM). After storage and concentration of the fraction containing the product, the residue was re-purified twice by preparative RP-HPLC (XBridge prep C18 5μm OBD, 30×100mm, 30 → 90% CH3CN / H2O + 1% AcOH) to obtain 20 as a film (10.5 mg, 10.8 μmol, 0.6%). LCMS (ESI+) C 38 H 48 N7O 19 S2 + (M+H +The calculated value was 970.24, and the measured value was 970.39.

[0178]

[0203] Preparation of 21b: Compound 10b (20.4 mg, 27.1 μmol) in DMF (175 μL) and Et3N (6.6 mg, 9.1 μL, 65 μmol) were added to a solution of compound 20 (10.5 mg, 10.8 μmol) in DMF (270 μL). The reaction mixture was allowed to stand for 17.5 hours. The reaction mixture was diluted with DCM (2 mL) and purified by automated gradient silica gel column chromatography (0 → 20% MeOH / DCM) to obtain 21b as a pale yellow solid (13.5 mg, 6.13 μmol, 57%). LCMS (ESI+) C 106 H 125 F2N7O 19 S2 2+ (M / 2+2H) + The calculated value is 1101.41, and the measured value is 1101.61. Example 10: Preparation of compound 25a [ka]

[0179]

[0204] Preparation of 23a: To a solution of 10a (21.7 mg, 28.7 μmol, 1.0 equivalent) and Fmoc-Gly-Gly-OH (12.6 mg, 36.6 μmol, 1.24 equivalents) in dry DMF, HBTU (18.5 mg, 48.9 μmol, 1.7 equivalents) was added, followed by the addition of DiPEA (12.3 mg, 16.5 μL, 94.9 μmol, 3.3 equivalents). The resulting mixture was mixed, left at room temperature for 110 minutes, then diluted with DCM, and the resulting mixture was purified by gradient column chromatography (0 → 12% MeOH / DCM) to obtain 23a as a bright yellow solid (36 mg, HPLC purity 48%, 16 μmol, 55.0%). LCMS (ESI+) C 59 H 60 FN8O 12 + (M+H + The calculated value is 1091.43, and the measured value is 1091.61.

[0180]

[0205] Preparation of 24a: Compound 23a (36 mg, 48 wt%, 16 μmol, 1.0 equivalent) was dissolved in a mixture of DMF (250 μL) and H2O (1.25 μL). To the resulting mixture, Et3N (8.0 mg, 11 μL, 79 μmol, 5.0 equivalents) was added, and the resulting solution was left at room temperature for approximately 5 hours. Next, an additional Et3N (8.0 mg, 11 μL, 79 μmol, 5.0 equivalents) was added to the reaction mixture, and the mixture was left at room temperature for 18 hours, then stored in a freezer for another day. The reaction mixture was removed from the freezer, concentrated under vacuum, and then co-evaporated (three times) with dry DMF to obtain 24a as a brown oil. It was used without further purification. LCMS (ESI+) C 44 H 50 FN8O 10 + (M+H + The calculated value was 869.36, and the measured value was 869.53.

[0181]

[0206] Preparation of 25a: A vial containing 24a (14 mg, 16 μmol, 3.2 equivalents) was mixed with a 100 mM compound 7 (4.5 mg, 50 μL, 5.0 μmol, 1.0 equivalent) DMF solution, followed by the addition of Et3N (2.5 mg, 3.5 μL, 25 μmol, 5.0 equivalents). An additional 50 μL of dry DMF was added to the resulting mixture, and the reaction mixture was left at room temperature for 4.5 hours. Next, the reaction mixture was diluted with dry DMF and purified by RP-HPLC (C18, 30% → 90% MeCN (containing 1% AcOH) / water (containing 1% AcOH)) to obtain 25a (3.6 mg, 1.5 μmol, yield 31%). LCMS (ESI+) C 110 H 130 F2N 20 O 33 S 2+ (M+2H + Calculated value: 1180.93. Measured value: 1180.94. Example 11: Preparation of compound 26a [ka]

[0182]

[0207] To a solution of 10a (18.9 mg, 0.025 mmol) in anhydrous DMF (500 μL), 13 (16 mg, 0.03 mmol) and Et3N (11 μL, 0.075 mmol) were added. After stirring at ambient temperature for 23 hours, the reaction mixture was diluted with DMF until the total volume reached 700 μL, and purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN / H2O + 1% AcOH). Product 26a was obtained as a grayish-white solid (5.3 mg, 4.6 μmol, 18.6%). LCMS (ESI+) C 56 H 66 FN8O 15 S + (M+H) + The calculated value is 1142.23, and the measured value is 1142.54. Example 12: Preparation of compounds 31a and 32a [ka]

[0183]

[0208] Preparation of 27a: A vial containing Boc-Glu(OtBu)-OH (11.1 mg, 36.6 μmol, 1.32 equivalents) was mixed with a 148.4 mM 10a (21.0 mg, 187.5 μL, 27.83 μmol, 1.0 equivalent) dry DMF solution, followed by the addition of HBTU (18.6 mg, 49.0 μmol, 1.76 equivalents) and an additional 75 μL of dry DMF. Finally, DiPEA (12.2 mg, 16.5 μL, 94.7 μmol, 3.40 equivalents) was added, and the reaction mixture was mixed until a brown solution was obtained. The reaction mixture was left at room temperature for approximately 35 minutes, then diluted with DCM (2.8 mL). The resulting solution was then purified by gradient column chromatography (0 → 12% MeOH / DCM) to obtain 27a as a white residue (30.8 mg, 29.6 μmol, quantitative yield). LCMS (ESI+) C 54 H 67 FN7O 13 + (M+H + The calculated value is 1040.48, and the measured value is 1040.70.

[0184]

[0209] Preparation of 28a: Fmoc-Glu(OFm)-OH (418.3 mg, 0.93 equivalents, 763.8 μmol) was added to a 10 mL container containing 10a (619.9 mg, 1 equivalent, 821.3 μmol) in 2.5 mL of dry DMF solution. Additional dry DMF (1.0 mL) was added to the resulting white suspension, followed by the addition of DIPEA (318.5 mg, 429 μL, 3 equivalents, 2.464 mmol). HBTU (289.7 mg, 0.93 equivalents, 763.8 μmol) was added to this suspension along with additional dry DMF (0.5 mL). The reaction mixture was mixed for 5 minutes to produce a solution, which was then left at room temperature for another 40 minutes. The reaction mixture was diluted with DCM (40 mL), and the resulting mixture was purified by gradient column chromatography (0 → 10% MeOH / DCM) to obtain 28a as a white solid (617.5 mg, 481 μmol, 63.0%). LCMS (ESI+) C 74 H 71 FN7O 13 + (M+H + The calculated value is 1284.51, and the measured value is 1284.91.

[0185]

[0210] Preparation of 29a: DMF (7.5 mL) and Et3N (670 μL, 486.5 mg, 4.81 mmol, 10 equivalents) were added to a vial containing 28a (617.5 mg, 481 μmol, 1.0 equivalent). The resulting mixture was heated in a water bath at 40°C for 5 minutes to produce a brown solution, which was left at room temperature for 18 hours. The reaction mixture was then maintained in a freezer for 3 days. Next, the reaction mixture was removed from the freezer and concentrated under vacuum to obtain 29a as a brown oil. It was used without further purification. LCMS (ESI+) C 45 H 51 FN7O 11 + (M+H + The calculated value was 884.36, and the measured value was 884.70.

[0186]

[0211] Preparation of 30a: A vial containing 27a (10.8 mg, 10.4 μmol, 1.0 equivalent) was placed in an ice bath, followed by the addition of ice-cold TFA (1.04 g, 700 μL, 9.15 mmol, 881 equivalents). The resulting solution was mixed and then left in an ice bath for 70 minutes. The reaction mixture was then concentrated under vacuum (in a 34°C water bath), the residue was dissolved in a 1:1 mixture of MeCN and DMSO, and then purified by RP-HPLC (C18, 5% → 90% MeCN (+1% AcOH) / water (+1% AcOH)) to obtain 30a as ammonium acetate salt (4.5 mg, 5.1 μmol, yield 49%). LCMS (ESI+) C 45 H 51 FN7O 11 + (M+H + The calculated value was 884.36, and the measured value was 884.55.

[0187]

[0212] Preparation of 31a: Crude 29a (386 mg, 437 μmol, 3.86 equivalents) was dissolved in dry DMF (300 μL), then 14 (105.6 mg, 88 wt% by 1H-qNMR, 113.1 μmol, 1.0 equivalent), additional dry DMF (200 μL), and Et3N (78.8 μL, 57.2 mg, 565 μmol, 5.0 equivalents) were added. The resulting mixture was mixed to produce a brown solution. It was left at room temperature for 85 minutes. Next, the reaction mixture was stored in a freezer for 18.5 hours, then removed from the freezer and left at room temperature for an additional 6 hours, after which the reaction mixture was stored in the freezer for another 18 hours. Finally, the reaction mixture was removed from the freezer and purified by RP-HPLC (C18, 50% → 100% MeCN (1% AcOH) / water (1% AcOH)). The pure fraction was combined with a pure fraction obtained from a smaller-scale reaction at room temperature for 5 hours using 29a (38.9 mg, 44.0 μmol, 3.88 equivalents), 14 (10.6 mg, 88 wt% by 1H-q NMR, 11.4 μmol, 1.0 equivalent) and Et3N (7.91 μL, 5.74 mg, 56.8 μmol, 5.0 equivalents) in dry DMF (53 μL). The pure fraction was concentrated in vacuum to obtain 31a as a grayish-white residue (86.6 mg, 37.5 μmol, 33.1%). LCMS (ESI+) C112 H 131 F2N 17 O 33 S 2+ (M + 2H + ) calculated value 1156.44, measured value 1157.03.

[0188]

[0213] Preparation of 32a: To a vial containing 30a (4.50 mg, 5.09 μmol, 3.28 equivalents), 100 mM dry DMA solution of compound 7 (1.40 mg, 15.5 μL, 1.55 μmol, 1.0 equivalent) was added, followed by addition of Et3N (1.26 mg, 1.73 μL, 12.4 μmol, 8.00 equivalents). The resulting mixture was vortexed and heated at 43 °C for about 10 minutes to obtain an orange solution. Then, the reaction mixture was left in the dark at room temperature for 2 hours and 45 minutes, and then stored in the refrigerator for 16 hours. Next, the reaction mixture was taken out of the refrigerator and left at room temperature for 40 minutes, and then the reaction mixture was diluted with DMF. The resulting solution was purified by RP - HPLC (C18, 30%→90% MeCN(+1%AcOH) / water(+1%AcOH)) to obtain 32a as a white solid (1.0 mg, 0.42 μmol, 27%). LCMS (ESI+) C 112 H 132 F2N 18 O 35 S2 + (M + 2H + ) calculated value 1195.93, measured value 1196.33. Example 13: Preparation of Compound 35a

Chemical formula

[0189]

[0214] Preparation of compound 33: Compound 5 (101 mg, 0.67 mmol) was dissolved in DCM (800 μL), and chlorosulfonyl isocyanate (CSI, 64.0 μL, 0.74 mmol) was added, resulting in a brown solution. After stirring at ambient temperature for 17 minutes, Et3N (187.0 μL, 1.34 mmol) was added (the mixture turned yellow), followed by the dissolution of N,N,-(2-hydroxyethyl)ethylenediamine (110.9 mg, 0.748 mmol) in DCM (1.0 mL). After stirring at ambient temperature for a further 18 hours, the crude mixture was concentrated under vacuum and purified by automated gradient silica gel column chromatography (0% → 30% MeOH / DCM) to obtain compound 33 as a white waxy solid (33.5 mg, 0.083 mmol, 12%). LCMS (ESI+) C 17 H 30 N3O6S + (M+H) + The calculated value was 404.50, and the measured value was 404.42.

[0190]

[0215] Preparation of 34: To a solution of 33 (16.5 mg, 0.041 mmol) in DCM (900 μL), bis(4-nitrophenyl) carbonate (29.9 mg, 0.098 mmol) and Et3N (17.0 μL, 0.012 mmol) were added. After stirring at ambient temperature for 96 hours, the crude mixture was concentrated under vacuum and purified by automated gradient silica gel column chromatography (10% → 100% siRNA / heptane) to obtain 34 as a clear oil (2.5 mg, 0.003 mmol, 8%). LCMS (ESI+) C 31 H 36 N5O 14 S + (M+H) + The calculated value is 734.71, and the measured value is 734.48.

[0191]

[0216] Preparation of 35a: To a solution of 34 (2.5 mg, 0.003 mmol) in DMF (110 μL), stock 200 mM 10a (41.0 μL, 6.2 mg, 0.008 mmol) and Et3N (3.0 μL, 0.02 mmol) were added. After 4.5 hours at ambient temperature, the reaction mixture was purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 5% → 90% MeCN / H2O + 1% AcOH). Product 35a was obtained as a colorless film (1.0 mg, 0.5 μmol, 10%). LCMS (ESI+) C 99 H 112 F2N 15 O 24 S + (M+H) + The calculated value is 1966.10, and the measured value is 1966.85. Example 14: Preparation of Compound 39a [ka]

[0192]

[0217] Preparation of 37a: To a solution of DBCO-PEG4-OSu (36 mg, 63.0 mg, 0.097 mmol) in DMF (2.0 mL), Val-Ala-PAB (15a, 26.0 mg, 0.108 mmol) was added, followed by the addition of Et3N (41.0 μL, 0.28 mmol). After stirring at ambient temperature for 2 hours, Val-Ala-PAB (15a, 7.8 mg, 0.03 mmol) dissolved in DMF (150 μL) was further added. After another 45 minutes, the reaction mixture was concentrated to a volume of 0.5 mL and purified by automated gradient silica gel column chromatography (0% → 15% MeOH / DCM) to obtain 37a as a clear oil (94 mg, 0.113 mmol). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.72 - 7.61 (m, 3H), 7.44 - 7.21 (m, 9H), 5.12 (d, J = 14.0Hz, 1H), 4.74 - 4.56 (m, 3H), 4.24 - 4.14 (m, 1H), 3.83 - 3.73 (m, 1H), 3.70(dd, J = 14.0 Hz; J = 1.8 Hz, 1H), 3.66 - 3.36 (m, 16H), 3.35 - 3.14 (m, 2H),2.70 - 1.80 (m, 6H), 1.49 - 1.36 (m, 3H), 1.05 - 0.90 (m, 6H).

[0193]

[0218] Preparation of 38a: To a solution of 37a (94.0 mg, 0.113 mmol) in DMF (1.5 mL), bis(4-nitrophenyl) carbonate (38.0 mg, 0.12 mmol) and Et3N (46.0 μL, 0.33 mmol) were added. After stirring for 2 hours, the excess bis(4-nitrophenyl) carbonate (10.0 mg, 0.03 mmol) was added. After a further 1 hour, the reaction mixture was concentrated until 1 mL of solvent remained, and purified by automated gradient silica gel column chromatography (100% DCM, followed by 0% → 10% MeOH / DCM) to obtain 38a as a clear oil (79 mg, 0.095 mmol). LCMS (ESI+) C 52 H 60 N6O 14 + (M+H) + The calculated value is 994.07, and the measured value is 994.74.

[0194]

[0219] Preparation of 39a: To a solution of exatecan free base (7.28 mg, 0.0167 mmol, recovered in the preparation of 9a; see Example 3) in DMF (214 μL), 38a (15.1 mg, 0.015 mmol) and Et3N (6.0 μL, 0.04 mmol) were added. After 16.5 hours at ambient temperature, the reaction mixture was diluted with DMF to 600 μL and purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN / H2O + 1% AcOH). Product 39a was obtained as a colorless film (8.3 mg, 15.2 μmol, 42%). LCMS (ESI+) C 70 H 78 FN8O 15 + (M+H) + The calculated value is 1290.41, and the measured value is 1290.02. Example 15: Preparation of Compound 43a [ka]

[0195]

[0220] Preparation of 41: To a solution of DIBO (40 mg, 220 mg, 1.0 mmol) in anhydrous DCM (15 mL), chlorosulfonyl isocyanate (CSI, 88.1 μL, 1.0 mmol) was added to form a white suspension. After stirring at ambient temperature for 20 minutes, Et3N (282 μL, 2.0 mmol) was added, followed by 2-amino-ethoxyethanol (117 mg, 1.11 mmol). After stirring for another 20 minutes, the reaction mixture was quenched by adding saturated aqueous NH4Cl solution (30 mL). After separation, the aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by automated gradient silica gel column chromatography (0% → 7% MeOH / DCM) to obtain 41 as a light yellow oil (498 mg, 1.15 mmol). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.52 - 7.42 (m, 1H), 7.35 - 7.29 (m, 2H), 7.28 - 7.19 (m,5H), 5.42 - 5.39 (bs, 1H), 3.48 - 3.43 (m, 4H), 3.36 - 3.32 (m, 2H), 3.23 -3.18 (m, 2H), 3.17 - 3.12 (m, 2H).

[0196]

[0221] Preparation of 42: To a solution of 41 (192 mg, 0.44 mmol) in DCM (30 mL), chlorosulfonyl isocyanate (CSI, 38.8 μL, 0.44 mmol) was added dropwise under an N2 atmosphere. After stirring at ambient temperature for 20 minutes, Et3N (311 μL, 2.23 mmol) was added, and the mixture was stirred for 10 minutes. Then, a solution of diethanolamine (51.6 μL, 0.53 mmol) in DMF (1.5 mL) was added. After stirring the reaction for 1 hour, 4-nitrophenyl chloroformate (180 mg, 0.89 mmol) and Et3N (187 μL, 1.34 mmol) were added. After stirring at ambient temperature for 18 hours, the reaction mixture was purified by automated gradient silica gel column chromatography (0% → 6% MeOH / DCM) to obtain 42 as a white solid (187 mg, 0.19 mmol). LCMS (ESI+) C 40 H 39 N6O 19 S2 + (M+H) + The calculated value is 971.90, and the measured value is 971.26.

[0197]

[0222] Preparation of 43a: To a solution of 10a (15 mg, 0.02 mmol) in anhydrous DMF (110 μL), 42 (7.9 mg, 0.008 mmol) and Et3N (5.0 μL, 0.04 mmol) were added. After 6 hours at ambient temperature, the reaction mixture was purified by automated gradient silica gel column chromatography (0% → 15% MeOH / DCM) to obtain 43a as a grayish-white solid (10.1 mg, 0.0046 mmol, 55%). LCMS (ESI+) C 108 H 115F2N 16 O 29 S2 + (M / 2+H) + The calculated value is 1101.64, and the measured value is 1101.97. Example 16: Preparation of compound 49a [ka]

[0198]

[0223] Preparation of 45: To a solution of (9H-fluoren-9-yl)methyl(5-hydroxypentyl)carbamate (397 mg, 1.22 mmol, 1.0 equivalent) in DCM (55 mL), CSI (106 μL, 1.22 mmol, 1.0 equivalent) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 20 minutes, after which Et3N (850 μL, 6.10 mmol, 5.0 equivalent) was added. The resulting mixture was stirred at room temperature for 10 minutes, after which a solution of diethanolamine (144 mg, 1.37 mmol, 1.11 equivalent) in DMF (1.7 mL) was added. The reaction mixture was stirred for 2 hours, after which 4-nitrophenyl chloroformate (492 mg, 2.44 mmol, 2.0 equivalent) was added together with additional Et3N (340 μL, 2.44 mmol, 2.0 equivalent). The reaction mixture was stirred for 18 hours, then concentrated under vacuum. The resulting residue was dissolved in DCM (100 mL) and washed with saturated NH4Cl aqueous solution (50 mL). The aqueous layer was extracted with DCM (50 mL), and the combined organic layers were dehydrated with MgSO4 and concentrated under vacuum. The residue was then purified by gradient column chromatography (0 → 5% MeOH / DCM), followed by a second column chromatography (65% Â / heptane) to obtain 45 as a white powder (342 mg, 395 μmol, yield 32.3%). 1H NMR (400 MHz, CDCl3) δ (ppm) 8.24 (d, J = 9.2 Hz, 4H), 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d,J = 7.5 Hz, 2H), 7.42-7.33 (m, 6H), 7.33-7.27 (m, 2H), 4.86 (t, J = 5.5 Hz,1H), 4.51 (t, J = 5.4 Hz, 4H), 4.43 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.8 Hz,1H), 4.17-4.09 (m, 2H), 3.84 (t, J = 4.4Hz, 4H), 3.24-3.14 (m, 2H), 1.72-1.62(m, 2H), 1.62-1.48 (m, 3H), 1.48-1.36 (m, 2H).

[0199]

[0224] Preparation of 46a: A 25 mM crude dry DMF solution (25 mg, 1.1 mL, 28 μmol, 4.0 equivalents) containing Et3N (37.7 μL, 27.3 mg, 270 μmol, 10 equivalents) was concentrated under vacuum. To the resulting residue, a dry DMF solution (150 μL) containing 45 (6.1 mg, 7.1 μmol, 1.0 equivalent) was added, followed by the addition of Et3N (6.0 μL, 4.2 mg, 42 μmol, 6.0 equivalents). The resulting mixture was heated at 45°C for 5 minutes, then left at room temperature for 2.5 hours, and then stored in a refrigerator for 17 hours. Next, the reaction mixture was removed from the freezer, diluted with dry DMF, filtered through a membrane filter, and then purified by RP-HPLC (C18, 50% → 100% MeCN (+1% AcOH) / water (+1% AcOH)) to obtain 46a (7.4 mg, 3.1 μmol, yield 44%). LCMS (ESI+) C 117 H 131 F2N 17 O 32 S 2+ (M+2H + The calculated value was 1178.44, and the measured value was 1178.79.

[0200]

[0225] Preparation of 49a: DMF (200 μL) was added to a vial containing 46a (7.4 mg, 3.1 μmol, 1.0 equivalent), followed by the addition of Et3N (4.4 μL, 3.2 mg, 31.6 μmol, 10 equivalents). The resulting mixture was left at room temperature for 2 hours, followed by the addition of Et3N (10 μL, 7.3 mg, 71.7 μmol, 23 equivalents). The reaction mixture was mixed and left at room temperature for 21 hours. Next, DBCO-NHS ester 48 (2.1 mg, 5.2 μmol, 1.7 equivalents) in DMF (5 μL) was added. The resulting brown solution was left at room temperature for 1 hour, followed by storage in a refrigerator for 18 hours. The following day, the reaction mixture was removed from the freezer and then purified by RP-HPLC (C18, 50% → 100% MeCN (+1% AcOH) / water (+1% AcOH)) to obtain an impure product. This was further purified by gradient column chromatography (0 → 30% MeOH / DCM) to obtain 49a as a white solid (2.5 mg, 1.0 μmol, yield 33%). LCMS (ESI+) C 121 H 134 F2N 18 O 32 S 2+ (M+2H + The calculated value is 1210.96, and the measured value is 1211.20. Example 17: Preparation of Compound 52a [ka]

[0201]

[0226] Preparation of 50a: Compound 45 (16.2 mg, 18.7 μmol, 1.0 equivalent) was added to a vial containing compound 10a (87.3 μmol, 1.71 equivalents), followed by the addition of dried DMF (200 μL) and Et3N (9.5 mg, 13 μL, 94 μmol, 5.0 equivalents). The resulting brown solution was left at room temperature for 50 minutes, and then transferred to a vial containing additional intermediate 10a (55.3 μmol, 3.0 equivalents). The resulting reaction mixture was left at room temperature for 40 minutes, followed by the addition of dried DMF (150 μL). The reaction mixture was left at room temperature for 20 hours, and then stored in a refrigerator for 3 days. Next, the reaction mixture was removed from the freezer, and 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-amine) (5.0 μL) was added. The reaction mixture was left at room temperature for 20 minutes, then diluted with dry DMF, filtered through a 0.2 μm nylon syringe filter, and then purified by RP-HPLC (C18, 50% → 100% MeCN (+1% AcOH) / water (+1% AcOH)) to obtain an impure product. This was further purified by gradient column chromatography (0 → 30% MeOH / DCM) to obtain 50a as a white residue (6.5 mg, 3.06 μmol, yield 16%). LCMS (ESI+) C 107 H 117 F2N 15 O 26 S 2+ (M+2H + The calculated value was 1049.40, and the measured value was 1049.28.

[0202]

[0227] Preparation of 52a: DMF (150 μL) was added to a vial containing 50a (1.6 mg, 0.76 μmol, 1.0 equivalent), followed by the addition of Et3N (1.5 mg, 2.1 μL, 15 μmol, 20 equivalents). The resulting light brown solution was left at room temperature for 50 minutes, followed by the addition of an additional Et3N (3.5 μL). The reaction mixture was left at room temperature for another 18 hours and then concentrated in vacuo. To this crude residue was added a solution of ((1R,8S,9s)-bicyclo[6.1.0]nona-4-en-9-yl)methyl (4-nitrophenyl) carbonate (BCN-OPNP, 1.2 mg, 3.8 μmol, 5.0 equivalents) in dry DMF (10 μL), followed by the addition of Et3N (0.35 mg, 0.48 μL, 3.4 μmol, 4.5 equivalents). The resulting brown solution was vortexed and left at room temperature for 140 minutes. The reaction mixture was then diluted with DCM and the resulting mixture was purified by gradient column chromatography (0→10% MeOH / DCM) to afford 52a as a white solid (1.7 mg, purity by HPLC 86%, 0.71 μmol, 94%). LCMS (ESI+) C 103 H 119 F2N 15 O 26 S<{0000971}>(M + 2H + ) calculated value 1026.41, found 1026.11.<{0002481}><{0002482}><{0002483}><{0002484}>

Chemical Structure

[0203] <{0002489}><{0002490}> It should be noted that there might be some unclear or potentially incorrect notations in the original text (such as 103 etc. which seem to be some kind of chemical structure or formula notations that are not fully clear without more context). The translation tries to be as literal as possible while maintaining the integrity of these notations.To a solution of 10a (15 mg, 0.020 mmol) in anhydrous DMF (150 μL), 2,5-dioxypyrrolidine-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoate (53 mg, 5.5 mg, 0.018 mmol) and DIPEA (10 μL, 0.060 mmol) were added. After 35 minutes at ambient temperature, the reaction mixture was purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN / H2O + 1% AcOH). Product 54a was obtained as a clear oil (2.4 mg, 2.1 μmol, 11%). LCMS (ESI+) C 50 H 55 FN7O 11 + (M+H) + The calculated value is 949.01, and the measured value is 949.82. Example 19: Preparation of Compound 57b [ka]

[0204]

[0229] Preparation of 55b: H-Val-Cit-PAB-OH (15b, 259 mg, 684 μmol, 1.0 equivalent) was added to a vial containing compound 53 (684 μmol) in dry DMF (2 mL), and the resulting solution was stirred at room temperature for 17 hours. Next, the reaction mixture was diluted with additional DMF (2.0 mL), then poured into Et2O (80 mL), and filtered. The filtered solid was washed with Et2O (25 mL, twice), and then concentrated under vacuum to obtain 55b as a white solid (378 mg, 1H-NMR purity 95.3%, 629 μmol, yield 91.9%). 1H NMR (400 MHz, DMSO-d6) δ (ppm) 9.91 (s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.82 (d, J = 8.4 Hz,1H), 7.56 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 7.02 (s, 2H),6.06-5.90 (m, 1H), 5.42 (s, 2H), 5.11 (t, J = 5.6 Hz, 1H), 4.52-4.33 (m, 3H),4.20 (t, J = 7.6 Hz, 1H), 3.09-2.93 (m, 2H), 2.26-2.06 (m, 2H), 2.05-1.91 (m,1H), 1.77-1.65 (m, 1H), 1.65-1.56 (m, 1H), 1.56-1.30 (m, 6H), 1.28-1.15 (m,2H), 0.93-0.76 (m, 6H).

[0205]

[0230] Preparation of 56b: To a solution of 55b (214 mg, 95.3% 1H-NMR purity, 0.357 mmol) and bis(4-nitrophenyl) carbonate (217 mg, 0.714 mmol) in DMF (2.0 mL), DIPEA (88 μL, 0.535 mmol) was added. After stirring at ambient temperature for 2 hours, the reaction mixture was poured into diethyl ether (80 mL) and filtered. The residue was suspended in diethyl ether (2 × 50 mL) and filtered again. After concentration under vacuum, 56b was obtained as a bright yellow solid (251.0 mg, 0.34 mmol, 95%). LCMS (ESI+) C 35 H 44 N7O 11 + (M+H) + The calculated value is 738.76, and the measured value is 738.25. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 10.08 (s, 1H), 8.40-8.28 (m, 2H), 8.12 (d, J = 7.6 Hz, 1H),7.82 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 8.5, 2H), 7.62-7.53 (m, 2H), 7.42 (d, J= 8.4 Hz, 2H), 7.02 (s, 2H), 5.99 (t, J = 5.4 Hz, 1H), 5.43 (s, 2H), 5.26 (s,2H), 4.46-4.34 (m, 1H), 4.21 (t, J = 7.4 Hz, 1H), 3.12-2.88 (m, 2H), 2.26-2.04(m, 2H), 2.04-1.89 (m, 1H), 1.77-1.67 (m, 1H), 1.67-1.56 (m, 1H), 1.56 (m, 6H),1.27-1.14 (m, 2H), 0.94-0.78 (m, 6H).

[0206]

[0231] Preparation of 57b: To a solution of exatecan mesylate (20 mg, 0.038 mmol) in anhydrous DMF (220 μL), DIPEA (33.0 μL, 0.19 mmol) and 56b (25 mg, 0.034 mmol) were added. After 2 hours at ambient temperature, the crude reaction mixture was directly purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 90% MeCN / H2O + 1% AcOH). Product 57b was obtained as a clear oil (8.1 mg, 7.8 μmol, 21%). LCMS (ESI+) C 53 H 61 FN9O 12 + (M+H) + The calculated value is 1035.10, and the measured value is 1035.82. Example 20: Preparation of compound 59b [ka]

[0207]

[0232] To a solution of 10a (15 mg, 0.020 mmol) in anhydrous DMF (150 μL), maleimide-PEG1-OPNP (58 mg, 7.7 mg, 0.022 mmol) and DIPEA (10 μL, 0.060 mmol) were added. After 2.5 hours at ambient temperature, the crude reaction mixture was directly purified by preparative RP-HPLC (Column Xbridge prep C18 5 μm OBD, 30 × 100 mm, 30% → 100% MeCN / H2O + 1% AcOH). Product 59a was obtained as a clear oil (3.6 mg, 3.4 μmol, 17%). LCMS (ESI+) C 49 H 53 FN7O 13 + (M+H) + The calculated value is 966.98, and the measured value is 966.77. Example 21: Preparation of compound 60a [ka]

[0208]

[0233] A vial containing maleimidocaproate NHS ester (53 mg, 5.2 mg, 17 μmol, 1.0 equivalent) was mixed with a 25 mM crude 29a dry DMF solution (15 mg, 0.68 mL, 17 μmol, 1.0 equivalent) containing Et3N (23.3 μL, 16.9 mg, 167 μmol, 10 equivalents). The resulting brown solution was left at room temperature for 40 minutes, and then additional maleimidocaproate NHS ester (2.1 mg, 6.8 μmol, 0.4 equivalents) in DMF (20 μL) was added. The RM was left at room temperature for another 24 minutes, and then purified by RP-HPLC (C18, 40% → 100% MeCN (1% AcOH) / water (1% AcOH)) to obtain 60a as a white residue (3.3 mg, 2.5 μmol, HPLC purity 80%, yield 14%). LCMS (ESI+) C 55 H 62 FN8O 14 + (M+H + The calculated value was 1077.44, and the measured value was 1077.78. Example 22. Preparation of ADC1a-DAR4 (See Figure 9 for structure)

[0209]

[0234] The bioconjugate according to the present invention was prepared by conjugation of compound 1a, a linker conjugate, to the biomolecule azide-modified trastuzumab. Accordingly, to a solution of trastuzumab-(6-N3-GalNAc)2 (604 μL, 20.6 mg, 34.1 mg / ml in PBS pH 7.4, trst-v1) prepared according to International Publication No. 2016170186, PBS pH 7.4 (63 μL), 1,2-propylene glycol (587 μL), and compound 1a (80 μL, 10 mM solution in DMF) were added. The reaction was incubated overnight at room temperature, followed by dialyzing to PBS pH 7.4. The residual free payload was removed by adding charcoal (1.2 mg of charcoal per 1 mg of ADC), followed by overnight rotation at room temperature. Charcoal was removed by centrifugation and filtration, and ADCs were purified using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectral analysis of the IdeS digestion sample showed one major product corresponding to the conjugated Fc / 2 fragment (two ring-closed lactone forms of the payload) (observed mass 26494 Da, approximately 60% of the total Fc / 2 fragment, calculated mass 26495 Da), and one minor product corresponding to the conjugated Fc / 2 fragment (one ring-closed lactone form and one ring-open carboxylate form of the payload) (observed mass 26510 Da, approximately 30% of the total Fc / 2 fragment, calculated mass 26513 Da).

[0210]

[0235] ADC1a-DAR8 (see Figure 9 for structure) was similarly prepared by conjugation of compound 1a to trastuzumab-(HC-L196N mutant)-(6-N3-GalNAc)4(trast-v5). Example 23. Preparation of ADC2(DAR4)

[0211]

[0236] The bioconjugate according to the present invention was prepared by conjugation of compound 2, a linker conjugate, to the biomolecule azide-modified trastuzumab. Accordingly, to a solution of trastuzumab-(6-N3-GalNAc)2 (363 μL, 12.0 mg, 33.1 mg / ml in PBS pH 7.4, trst-v1) prepared according to International Publication No. 2016170186, PBS pH 7.4 (37 μL), 1,2-propylene glycol (336 μL), and compound 2 (64 μL, 10 mM solution in DMF) were added. After incubation of the reaction overnight at room temperature, an additional 2 (16 μL, 10 mM solution in DMF) was added and incubated overnight at room temperature. The reaction was dialyzed to PBS pH 7.4, and the residual free payload was removed by adding charcoal (4.8 mg of charcoal per 1 mg of ADC) followed by rotation at room temperature for 4 hours. The charcoal was removed by centrifugation and filtration, and the ADC was purified using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectral analysis of the IdeS digested sample showed one major product corresponding to the conjugated Fc / 2 fragment (two ring-closed lactone forms of the payload) (observed mass 26847 Da, approximately 60% of the total Fc / 2 fragment, calculated mass 26847 Da), and one minor product corresponding to the conjugated Fc / 2 fragment (one ring-closed lactone form and one ring-open carboxylate form of the payload) (observed mass 26865 Da, approximately 30% of the total Fc / 2 fragment, calculated mass 26865 Da). Example 24. Preparation of ADC3 (DAR4)

[0212]

[0237] To a solution of trastuzumab-(6-azide GalNAc)2 (333 μL, 11 mg, 33 mg / mL in PBS pH 7.4, trast-v1), trastuzumab-3 (350 μL, 2 mM solution in PG, 10 equivalents compared to IgG) was added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-3 (ADC-3) (calculated mass 26732 Da, observed mass 26733 Da). Example 25. Preparation of ADC4a and ADC4b (DAR4 and DAR8)

[0213]

[0238] Trastuzumab (290 mg, 18 mg / mL in formulation buffer + 25 mM EDTA + 25 mM Tris pH 8.5, trust-v4) was incubated with TCEP (386 μL, 2.05 equivalents μL, 10 mM in MQ) for 90 minutes. Deruxtecan (4, 1.6 mL, 6 equivalents, 7.1 mM in DMA) was added to the reaction, followed by incubation at room temperature for 90 minutes. The reaction was then quenched with an excess of N-acetylcysteine ​​and purified by TFF. Analysis by RP-HPLC showed the formation of the product trust-v4-4a (ADC4a) with a mean DAR of 3.94.

[0214]

[0239] After reducing trast-v4 with 5-6 equivalents of TCEP, ADC4b was similarly prepared by conjugation with compound 4. Analysis by RP-HPLC showed the formation of the product trast-v4-4b (ADC4b) with an average DAR of 7.0. Example 26. Trastuzumab-(HC-L196N mutant)-(6-N 3 -GalNAc) 4 Conjugation of trast-v5 to 17b

[0215]

[0240] To a solution of trastuzumab-(HC-L196N variant)-(6-N3-GalNAc)4 (6.8 μL, 151 μg, 22.2 mg / ml in PBS pH 7.4, trst-v5), PBS pH 7.4 (0.7 μL) and 17b (2.5 μL, 8 mM solution in DMF, 20 equivalents compared to IgG) were added. The reaction was incubated at room temperature for 18 hours. Mass spectral analysis of the reduced sample showed three heavy chain products corresponding to the non-conjugate heavy chain (observed mass 50317 Da, approximately 70% of the total heavy chain product), the single-conjugate heavy chain (observed mass 52544 Da, approximately 25% of the total heavy chain product), and the double-conjugate heavy chain (observed mass 54768 Da, approximately 5% of the total heavy chain product). Example 27. Trastuzumab-(6-azidoGalNAc) with 1a 2 trast-v1 conjugation

[0216]

[0241] To a solution of trastuzumab-(6-azide GalNAc)2 (167 μL, 5 mg, 30 mg / mL in PBS pH 7.4, trast-v1), 1a (167 μL, 1.2 mM solution in PG, 6 equivalents compared to IgG) was added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-1a (calculated mass 26496 Da, observed mass 26498 Da). Example 28. Trastuzumab-(6-azideGalNAc) with 2a 2 trast-v1 conjugation

[0217]

[0242] To a solution of trastuzumab-(6-azide GalNAc)2 (400 μL, 12 mg, 30 mg / mL in PBS pH 7.4, trast-v1), 2a (400 μL, 1.6 mM solution in PG, 8 equivalents compared to IgG) was added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-2a (calculated mass 26847 Da, observed mass 26847 Da). Example 29. Trastuzumab-(6-azideGalNAc) with 21b 2 trast-v1 conjugation

[0218]

[0243] To a solution of trastuzumab-(6-azide GalNAc)2 (167 μL, 5 mg, 30 mg / mL in PBS pH 7.4), 21b (167 μL, 1.2 mM solution in PG, 6 equivalents compared to IgG) was added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-21b (calculated mass 26565 Da, observed mass 26567 Da). Example 30. Trastuzumab-(6-azideGalNAc) with 1b 2 trast-v1 conjugation

[0219]

[0244] To a solution of trastuzumab-(6-azide GalNAc)2 (167 μL, 5 mg, 30 mg / mL in PBS pH 7.4, trast-v1), 1b (167 μL, 1.2 mM solution in PG, 6 equivalents compared to IgG) was added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-1b (calculated mass 26668 Da, observed mass 26671 Da). Example 31. Trastuzumab-(6-azideGalNAc) with 25a 2 trast-v1 conjugation

[0220]

[0245] To a solution of trastuzumab-(6-azide GalNAc)2 (300 μL, 5 mg, 16.7 mg / mL in PBS pH 7.4, trast-v1), 50 μL of sodium deoxycholate (110 mM in MQ) and 25a (150 μL, 1.33 mM solution in PG, 6 equivalents compared to IgG) were added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. RP-HPLC analysis of the DTT-reduced sample (see Figure 13) showed a clear shift of HC after conjugation (RT). HC-0 =8.7 minutes, RT HC-25a This showed a response time of 10.5 minutes. This corresponds to a mean drug-antibody ratio (DAR) of 3.66, suggesting the formation of trust-v1-25a. Example 32. Trastuzumab-(6-azideGalNAc) with 26a 2 trast-v1 conjugation

[0221]

[0246] To a solution of trastuzumab-(6-azide GalNAc)2 (20.9 μL, 0.5 mg, 23.92 mg / mL in PBS pH 7.4, trast-v1), PBS pH 7.4 (9.1 μL), sodium deoxycholate (5 μL, 110 mM), and compound 26a (15 μL, 1.3 mM solution in PG, 6 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-26a (calculated mass 25504 Da, observed mass 25505 Da). Example 33. Trastuzumab-(6-azideGalNAc) with 32a 2 trast-v1 conjugation

[0222]

[0247] To a solution of trastuzumab-(6-azide GalNAc)2 (12.54 μL, 0.3 mg, 23.92 mg / mL in PBS pH 7.4, trast-v1), PBS pH 7.4 (5.46 μL), sodium deoxycholate (3 μL, 110 mM), and compound 32a (9 μL, 0.8 mM solution in PG, 7 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-32a (calculated mass 26753 Da, observed mass 26752 Da). Example 34. Trastuzumab-(6-azideGalNAc) with 35a 2 trast-v1 conjugation

[0223]

[0248] To a solution of trastuzumab-(6-azide GalNAc)2 (12.54 μL, 0.3 mg, 23.92 mg / mL in PBS pH 7.4, trast-v1), sodium deoxycholate (3 μL, 110 mM) and compound 35a (15 μL, 1.2 mM solution in PG, 9 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-35a (calculated mass 26328 Da, observed mass 26329 Da). Example 35. Trastuzumab-(6-azideGalNAc) with 31a 2 trast-v1 conjugation

[0224]

[0249] To a solution of trastuzumab-(6-azide GalNAc)2 (15 mL, 250 mg, 16.67 mg / mL in TBS pH 7.4, trast-v1), 2.5 mL of sodium deoxycholate (110 mM in MQ) and compound 31a (7.5 mL, 0.88 mM solution in PG, 4 equivalents compared to IgG) were added. After incubation at room temperature for 18 hours, the reaction was followed by purification using an AKTA Purifier-10 (GE Healthcare) column with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column. Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-31a (calculated mass 26675 Da, observed mass 26672 Da). Example 36. Rituximab-(6-azideGalNAc) with 39a 2 rit-v1 conjugation

[0225]

[0250] Enzymatic remodeling of rituximab to rituximab-(6-N3-GalNAc)2 was performed by incubating rituximab (15 mg / mL) with EndoSH (1 wt / wt%) described in international application PCT / EP2017 / 052792, His-TnGalNAcT described in international application PCT / EP2016 / 059194 (5 wt / wt%), and UDP 6-N3-GalNAc (25 equivalents compared to IgG) prepared according to international application PCT / EP2016 / 059194 in TBS containing 10 mM MnCl2 at 30°C for 16 hours. Next, the functionalized IgG was 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 glycine-HCl pH 2.7 and neutralized with 1 M Tris-HCl pH 8.8. After three dialyze passes to PBS, the IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0226]

[0251] Conjugation: Compound 39a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of rituximab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in PBS pH 7.4, rit-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC rit-v1-39a (calculated mass 25619.6 Da, observed mass 25618.1 Da). Example 37. Trastuzumab-(6-azideGalNAc) with 39a 2 trast-v1 conjugation

[0227]

[0252] Compound 39a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of trastuzumab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in TBS pH 7.4, trast-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC rit-v1-39a (calculated mass 25619.6 Da, observed mass 25618.1 Da). Example 38. Rituximab-(6-azideGalNAc) with 43a 2 rit-v1 conjugation

[0228]

[0253] Compound 43a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of rituximab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in PBS pH 7.4, rit-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC rit-v1-43a (calculated mass 26532.5 Da, observed mass 26531.0 Da). Example 39. Trastuzumab-(6-azideGalNAc) with 43a 2 trast-v1 conjugation

[0229]

[0254] Compound 43a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of trastuzumab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in TBS pH 7.4, trast-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-43a (calculated mass 26566.7 Da, observed mass 26564.5 Da). Example 40. Rituximab-(6-azideGalNAc) with 49a 2 rit-v1 conjugation

[0230]

[0255] Compound 49a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of rituximab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in PBS pH 7.4, rit-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC rit-v1-49a (calculated mass 26750.7 Da, observed mass 26751.1 Da). Example 41. Trastuzumab-(6-azideGalNAc) with 49a 2 trast-v1 conjugation

[0231]

[0256] Compound 49a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of trastuzumab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in TBS pH 7.4, trast-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-49a (calculated mass 26784.9 Da, observed mass 26783.8 Da). Example 42. Rituximab-(6-azideGalNAc) with 52a 2 rit-v1 conjugation

[0232]

[0257] Compound 52a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of rituximab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in PBS pH 7.4, rit-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC rit-v1-52a (calculated mass 26381.4 Da, observed mass 26381.9 Da). Example 43. Trastuzumab-(6-azideGalNAc) with 52a 2 trast-v1 conjugation

[0233]

[0258] Compound 52a (15 μl; 0.53 mM solution in PG, 4 equivalents compared to IgG) was added to a solution of trastuzumab-(6-azide GalNAc)2 (15 μl; 20 mg / ml in TBS pH 7.4, trast-v1). The reaction was incubated overnight at room temperature, followed by buffer exchange to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed a single main product corresponding to the conjugated ADC trast-v1-52a (calculated mass 26415.6, observed mass 26414.2 Da). Example 44. Rituximab-(6-azideGalNAc) with 1a 2 rit-v1 conjugation

[0234]

[0259] To a solution of rituximab-(6-azide GalNAc)2 (12.24 μL, 0.3 mg, 24.5 mg / ml in PBS pH 7.4, rit-v1), sodium deoxycholate (3 μL, 110 mM) and compound 1a (9 μL, 1.33 mM solution in PG, 6 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed the formation of a product corresponding to the conjugated ADC rit-v1-1a (calculated mass 26463 Da, observed mass 26461 Da). Example 45. Rituximab-(6-azideGalNAc) with 32a 2 rit-v1 conjugation

[0235]

[0260] To a solution of rituximab-(6-azide GalNAc)2 (12.24 μL, 0.3 mg, 24.5 mg / ml in PBS pH 7.4, rit-v1), sodium deoxycholate (3 μL, 110 mM) and compound 32a (9 μL, 0.89 mM solution in PG, 4 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed the formation of a product corresponding to the conjugated ADC rit-v1-32a (calculated mass 26721 Da, observed mass 26720 Da). Example 46. Rituximab-(6-azideGalNAc) with 31a 2 rit-v1 conjugation

[0236]

[0261] To a solution of rituximab-(6-azide GalNAc)2 (12.24 μL, 0.3 mg, 24.5 mg / ml in PBS pH 7.4, rit-v1), sodium deoxycholate (3 μL, 110 mM) and compound 31a (9 μL, 0.89 mM solution in PG, 4 equivalents compared to IgG) were added. After incubating the reaction at room temperature for 18 hours, the buffer was changed to PBS pH 7.4 using a centrifugal filter (Amicon Ultra 0.5 ml MWCO 10 kDa, Merck Millipore). Mass spectrometry of the IdeS digested sample showed the formation of a product corresponding to the conjugated ADC rit-v1-31a (calculated mass 26642 Da, observed mass 26640 Da). Example 47. Trastuzumab-(GalNProSSMe) 2 Enzyme remodeling to (trast-v2b)

[0237]

[0262] Trastuzumab (5 mg, 22.7 mg / ml) was incubated with EndoSH (1 wt / wt%) as described in international application PCT / EP2017 / 052792 for 1 hour, followed by the addition of TnGalNAcT (expressed in CHO) (10 wt / wt%) and UDP-GalNProSSMe (40 equivalents compared to IgG). The mixture was then incubated in 10 mM MnCl2 and TBS at 30°C for 16 hours. After the addition of the components, the final concentration of trastuzumab was 12.5 mg / ml. Functionalized IgG was purified using a protA column (5 mL, MabSelect Sure, Cytiva). After loading the reaction mixture, the column was washed with TBS. IgG was eluted with 0.1 M NaOAc pH 3.5 and neutralized with 2.5 M Tris-HCl pH 7.2. After three dialyze passes to PBS, functionalized trastuzumab was concentrated to 17.4 mg / ml using a Vivaspin Turbo 4 ultrafiltration unit (Sartorius). Mass spectral analysis of the sample after IdeS treatment showed one main Fc / 2 product (observed mass 24430 Da) corresponding to the expected product (trast-v2b). Example 48. Conjugation of trastuzumab S239C mutant trast-v3 with maleimide-exatecan variant 54a, 57b, 59a, or 60a

[0238]

[0263] Trastuzumab S239C mutant (transient expression in CHO by Evitria, heavy chain mutation S239C) (1 mg, 10 mg / ml in PBS + 10 mM EDTA, trast-v3) was incubated with TCEP (6.5 μL, 10 mM in MQ) at 37°C for 2 hours. The reductive antibody was spin-filtered with PBS + 10 mM EDTA using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore) and subsequently diluted to 100 μL. DHA (6.5 μL, 10 mM in MQ) was then added, and the reaction was incubated at room temperature for 3 hours. The reaction was divided into four parts (20 μL, 0.2 mg of antibody each), and maleimide-exatecan variant (54a, 57b, 59a, or 60a) (2.3 μL, 5 mM in DMF) was added to each part, followed by incubation at room temperature for 1 hour. The conjugates were spin-filtered into PBS using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore) and subsequently analyzed by RP-UPLC (see Figure 14). [Table 1] Example 49. Conjugation of trastuzumab GalProSH trast-v2 with maleimide-exatecan variant 60a

[0239]

[0264] Trastuzumab GalProSSMe (0.5 mg, 10 mg / ml in PBS + 10 mM EDTA, trast-v2b) was incubated with TCEP (3.3 μL, 10 mM in MQ) at 37°C for 2 hours. The reductive antibody was spin-filtered with PBS + 10 mM EDTA using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Subsequently, dehydroascorbic acid (DHA 3.3 μL, 10 mM in MQ) was added, and the reaction was incubated at room temperature for 3 hours. Maleimide-exatecan 60a (1.3 μL, 5 mM in DMF) was added to a portion of the reaction (10 μL, 0.1 mg of antibody), and the mixture was then incubated at room temperature for 1 hour. The conjugates were spin-filtered into PBS using a centrifugal filter (Amicon Ultra-0.5mL MWCO 10kDa, Merck Millipore) and subsequently analyzed by RP-UPLC. RP-UPLC analysis of the DTT-treated conjugates showed conversion to the conjugate trast-v2-60a with a DAR of 1.15 (see Figure 15). Example 50. Conjugation of trastuzumab GalProSH trast-v2 with maleimide-exatecan variant 57b

[0240]

[0265] Trastuzumab GalProSSMe (0.5 mg, 10 mg / ml in PBS + 10 mM EDTA, trast-v2b) was incubated with TCEP (3.3 μL, 10 mM in MQ) at 37°C for 2 hours. The reductive antibody was spin-filtered with PBS + 10 mM EDTA using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore). Subsequently, DHA (3.3 μL, 10 mM in MQ) was added, and the reaction was incubated at room temperature for 3 hours. Maleimide-exatecan 57b (1.3 μL, 5 mM in DMF) was added to a portion of the reaction (10 μL, 0.1 mg of antibody), and the mixture was then incubated at room temperature for 1 hour. The conjugates were spin-filtered into PBS using a centrifugal filter (Amicon Ultra-0.5mL MWCO 10kDa, Merck Millipore) and subsequently analyzed by RP-UPLC. RP-UPLC analysis of the DTT-treated conjugates showed conversion to the conjugate trast-v2-57b with a DAR of 1.37 (see Figure 15). General experiment: Interchain maleimide conjugation after TCEP reduction step

[0241]

[0266] General Experiment A-3. Conjugation using 3.5 equivalents of TCEP: mAb-v4 (10 mg / ml in PBS + 10 mM EDTA) was incubated with TCEP (3.5 equivalents, 10 mM in MQ) at 37°C for 1 hour. Maleimide-exatecan (2.3 μL, 5 mM in DMF) was added to a portion of the reaction (20 μL, 0.2 mg antibody), and the mixture was subsequently incubated at room temperature for 1 hour. The conjugate was spin-filtered into PBS using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore) and subsequently analyzed by RP-UPLC. DAR was calculated according to the following formula.

[0242]

[0267] General Experiment B - Conjugation using 7 equivalents of TCEP: mAb-v4 (10 mg / ml in PBS + 10 mM EDTA) was incubated with TCEP (7 equivalents, 10 mM in MQ) at 37°C for 1 hour. Maleimide-exatecan (10-35 equivalents in DMF) was added to a portion of the reaction (20 μL, 0.2 mg antibody), and then incubated at room temperature for 1 hour. The conjugate was spin-filtered into PBS using a centrifugal filter (Amicon Ultra-0.5 mL MWCO 10 kDa, Merck Millipore) and subsequently analyzed by RP-UPLC. DAR was calculated according to the following formula.

[0243]

[0268] Average DAR calculation formula:

number

[0244]

[0269] Trastuzumab was conjugated to maleimide-exatecan 60a according to General Experiment A. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-60a conjugate with a DAR of 4.6 (see Figure 16). Example 52. Conjugation of trastuzumab trast-v4 and maleimide-exatecan 57b

[0245]

[0270] Following General Experiment A, trastuzumab was conjugated to maleimide-exatecan 57b. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-57b conjugate with a DAR of 5.6 (see Figure 16). Example 53. Conjugation of rituximab rit-v4 and maleimide-exatecan 60a

[0246]

[0271] Rituximab was conjugated to maleimide-exatecan 60a according to General Experiment A. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the rit-v4-60a conjugate with a DAR of 2.5 (see Figure 17). Example 54. Conjugation of rituximab rit-v4 and maleimide-exatecan 57b

[0247]

[0272] Rituximab was conjugated to maleimide-exatecan 57b according to General Experiment A. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the rit-v4-57b conjugate with a DAR of 3.4 (see Figure 17). Example 55. Conjugation of trastuzumab trast-v4 and maleimide-exatecan 59a

[0248]

[0273] Following General Experiment A, trastuzumab was conjugated to maleimide-exatecan 59a. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-59a conjugate with a DAR of 2.2. Example 56. Conjugation of rituximab rit-v4 and maleimide-exatecan 59a

[0249]

[0274] Rituximab was conjugated to maleimide-exatecan 59a according to General Experiment A. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the rit-v4-59a conjugate with a DAR of 3.6. Example 57. Conjugation of trastuzumab trast-v4 and maleimide-exatecan 60a

[0250]

[0275] Trastuzumab was conjugated to maleimide-exatecan 60a according to General Experiment B. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-60a conjugate with a DAR of 5.7 (see Figure 18). Example 58. Conjugation of trastuzumab trast-v4 and maleimide-exatecan 57b

[0251]

[0276] Following General Experiment B, trastuzumab was conjugated to maleimide-exatecan 57b. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-57b conjugate with a DAR of 6.2 (see Figure 18). Example 59. Conjugation of rituximab rit-v4 and maleimide-exatecan 60a

[0252]

[0277] Rituximab was conjugated to maleimide-exatecan 60a according to General Experiment B. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the rit-v4-60a conjugate with a DAR of 5.8. Example 60. Conjugation of trastuzumab trast-v4 and maleimide-exatecan 59a

[0253]

[0278] Following General Experiment B, trastuzumab was conjugated to maleimide-exatecan 59a. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the trast-v4-59a conjugate with a DAR of 2.6. Example 61. Conjugation of rituximab rit-v4 and maleimide-exatecan 59a

[0254]

[0279] Rituximab was conjugated to maleimide-exatecan 59a according to General Experiment B. RP-UPLC analysis of the DTT-treated conjugate showed conversion to the rit-v4-59a conjugate with a DAR of 4.4. Example 62. Aggregation study

[0255]

[0280] ADC (1 mg / mL in PBS pH 7.4) was incubated at 37°C. Aggregation levels were measured at 0, 1, 4, 7, 14, and 21 days using an Agilent 1100 series HPLC (Hewlett Packard). A sample (3 μL, 1 mg / mL) was injected at 0.86 mL / min into an Xbridge BEH 200 Å column (3.5 μM, 7.8 × 300 mm, Waters). A fixed composition elution using 0.1 M sodium phosphate buffer pH 6.9 (NaH2PO4 / Na2HPO4) was performed for 16 minutes. Aggregation levels are shown in Table 1. [Table 2] Example 63: Analytical HIC

[0256] HIC analysis was performed using an Agilent 1100 series HPLC (Hewlett Packard). The sample (3 μL, 1 mg / ml in PBS) was injected at 0.8 mL / min into a TSKgel® butyl-NPR HPLC column (3.5 cm × 4.6 mm, 2.5 μm, Tosoh Bioscience). A linear gradient from 2M ammonium sulfate / 50 mM potassium phosphate pH 6.0 to 20% isopropanol / 50 mM potassium phosphate pH 6.0 was applied over 13 minutes. The results of the HIC analysis are shown in Figure 12. Example 64: BT-474 Efficacy Study

[0257]

[0281] At the beginning of the experimental phase, 8-12 week old CR female CB.17 SCID mice (obtained from Charles River Laboratories, USA) were given 1 × 10⁶ of 50% Matrigel. 7 Individual BT-474 tumor cells were injected subcutaneously into the flank (BT-474 cell xenograft model). Tumor volume was 100-150 mm². 3 When the range was within the specified range, a single dose was intravenously injected into 7 mice / group on day 1. The test items and dose levels are shown below. Tumors were measured twice a week for 43 days. The results for tumor volume (mean) are shown in Figures 19-22. [Table 3]

Claims

1. Antibody-drug conjugate having structure (1) 【Chemistry 1】 [In the structure, AB is an antibody, L １ and L ２ It is a linker, w is either 0 or 1, Z is a conjugation group obtained by a metal-free click reaction. R １７ These are each individual amino acid side chains, n is an integer in the range of 1 to 5. (NH-CR １７ -CO) ｎ This is a peptide spacer selected from Val-Ala, Glu-Val-Ala, AcLys-Val-Ala, and Asp-Val-Ala. A is a five-membered or six-membered aromatic or heteroaromatic ring, x is an integer in the range of 1 to 8. R ２１ is selected from H, R ２２ , COOH and COR ２２ , and R ２２ is C １ to C ２４ -(hetero)alkyl group, C ３ to C １０ -(hetero)cycloalkyl group, C ２ to C １０ -(hetero)aryl group, C ３ to C １０ alkyl(hetero)aryl group and C ３ to C １０ (hetero)arylalkyl group, and they are optionally substituted and are optionally interrupted by one or more heteroatoms selected from O, S and NR ２３ , and R ２３ is independently selected from the group consisting of hydrogen and C １ to C ４ alkyl group].

2. L ２ However, it has structure (2) 【Chemistry 2】 [In the structure, The waveforms marked with * are connected to Z, and the waveforms marked with ** are connected to NH. Sp １ and Sp ２ Each of these is a separate spacer part. n, A, R １７ and R ２１ The antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate is as described in claim 1.

3. Each Sp that appears ２ The same and each (NH-CR) that appears １７ -CO) ｎ The same, each appearing A is the same, and each appearing R ２１ The antibody-drug conjugate according to claim 2, wherein the two are the same.

4. L ２ However, it contains a sulfamide group represented by structure (3), 【Transformation 3】 [In the structure, a = 0 or 1, R １３ is hydrogen, C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ Selected from the group consisting of (hetero)arylalkyl groups, the C １ ~C ２４ alkyl group, C ３ ~C ２４ Cycloalkyl groups, 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 with O, S, and NR １４ [Here, R １４ is hydrogen and C １ ~C ４ [Independently selected from the group consisting of alkyl groups] is optionally interrupted by one or more heteroatoms selected from, or R １３ This is the second appearance of C(O)X connected to N via the spacer portion. The antibody-drug conjugate according to claim 1.

5. L ２ The antibody-drug conjugate according to claim 4, wherein the group comprises two of the groups of formula (3).

6. Each peptide that appears (NH-CR １７ -CO) ｎ The antibody-drug conjugate according to any one of claims 1 to 5, wherein the conjugate is Val-Ala.

7. Z has a structure selected from (Z1) to (Z8). 【Chemistry 4】 [In the structure, The combination of waveforms marked with * is L １ It is connected to A and B via an optional connection, and the coupling of other waveforms is L ２ It is connected, In (Z3), (Z7), and (Z8), the functional group R is hydrogen, C １ ~C ２４ alkyl group, C ２ ~C ２４ Acyl group, C ３ ~C ２４ Cycloalkyl groups, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl group, C ３ ~C ２４ (Hetero)arylalkyl groups and C １ ~C ２４ Selected from sulfonyl groups, each of which may be optionally substituted, O, S and NR ３２ [Here, R ３２ is hydrogen and C １ ~C ４ [Independently selected from the group consisting of alkyl groups] [May be optionally interrupted by one or more heteroatoms selected from the group consisting of alkyl groups] The antibody-drug conjugate according to any one of claims 1 to 6.

8. Having structure (1f) or (1b) 【Transformation 5】 [In the structure, AB, L １ Z, A, R ２１ , n, R １７ And x are as described in claim 1, a and R １３ This is as described in claim 5, L ５ It is a linker, r is either 0 or 1. m is an integer in the range of 1 to 10. q is an integer in the range of 0 to 10. p is either 0 or 1. 【Transformation 6】 [In the structure, Z, L ２ , R １７ , A, R ２１ , n and x are as described in claim 1, e is an integer in the range of 0 to 20. Su is a monosaccharide, G is the monosaccharide portion, GlcNAc is the N-acetylglucosamine portion, Fuc is the fucose portion, d is either 0 or 1. The antibody-drug conjugate according to any one of claims 1 to 7.

9. Having structure (1h) 【Transformation 7】 [In the structure, e, Su, G, GlcNAc, Fuc and d are as described in claim 8, n is either 0 or 1. The antibody-drug conjugate according to claim 8.

10. The antibody-drug conjugate according to claim 9, wherein n is 0.

11. Having structure (1i) 【Transformation 8】 [In the structure, e, Su, G, GlcNAc, Fuc and d are as described in claim 8, n is either 0 or 1. The antibody-drug conjugate according to claim 8.

12. The antibody-drug conjugate according to claim 11, wherein n is 1.

13. A method for synthesizing an antibody-drug conjugate according to any one of claims 1 to 12, (i) Structure AB-((L １ ) ｗ -F) ｘ Modified antibodies [In the structure, AB is an antibody, L １ It is a linker, w is either 0 or 1, F is a click probe that can react with Q in a metal-free click reaction. x is an integer in the range of 1 to 8. (ii) Linker-drug construct represented by structure (5) 【Chemistry 9】 [In the structure, L ２ It is a linker, Q is a click probe capable of performing a metal-free click reaction. R １７ These are each individual amino acid side chains, n is an integer in the range of 1 to 5. (NH-CR １７ -CO) ｎ is a peptide spacer selected from Val-Ala, Glu-Val-Ala, AcLys-Val-Ala and Asp-Val-Ala, A is a five-membered or six-membered aromatic or heteroaromatic ring, R ２１ H, R ２２ , C(O)OH and C(O)R ２２ Selected from, R ２２ 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 with O, S and NR ２３ It is optionally interrupted by one or more heteroatoms selected from R ２３ is hydrogen and C １ ~C ４ Reacting with [independently selected from the group consisting of alkyl groups] A method comprising the step of forming an antibody-drug conjugate in which a drug is covalently bound to the antibody via a conjugate group Z formed by a metal-free click reaction between Q and F.

14. The method according to claim 13, wherein the click probe Q includes a cyclic alkyne portion or a cyclic alkene portion.

15. (a) Whether the click probe Q is selected from the group consisting of (Q22) to (Q36) 【Chemistry 10】 [In the structure, B is a pharmaceutically acceptable anion.] Or the (hetero)cycloalkynyl moiety Q is represented by structure (Q37) 【Chemistry 11】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, Y ２ C(R) ３１ ) ２ , O, S or NR ３１ And R ３１ Each of them individually, R １５ Alternatively, it is a second appearance of the exatecan payload connected via the spacer portion, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16. Or the cyclooctinyl moiety Q is represented by structure (Q38) 【Chemistry 12】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, R １８ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, R １９ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Selected from the group consisting of (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 the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R １９ This is the second appearance of the exatecan payload connected via the spacer portion, l is an integer in the range of 0 to 10. Or the (hetero)cyclooctinyl moiety Q is represented by structure (Q39) 【Chemistry 13】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR １５ [is] Alternatively, the heterocycloheptinyl moiety Q may be represented by the structure (Q36a), 【Chemistry 14】 Alternatively, (b) the click probe Q is selected from the group consisting of an optionally substituted (hetero)cyclopropenyl group, (hetero)cyclobutenyl group, trans-(hetero)cycloheptenyl group, trans-(hetero)cyclooctenyl group, trans-(hetero)cyclononenyl group or trans-(hetero)cyclodecynyl group, or the click probe Q is selected from the group consisting of (Q40) to (Q50), 【Chemistry 15】 The method according to claim 13, wherein the R group(s) of Si in (Q44) and (Q45) is alkyl or aryl.

16. The click probe F is selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, and cydonones, and / or the click reaction is a 1,3-dipolar cycloaddition or a (4+2) cycloaddition. The method according to any one of claims 13 to 15.

17. The method according to claim 16, wherein the click probe F is an azide portion.

18. AB-((L １ ) ｗ -F) ｘ However, it is represented by structure (6). 【Chemistry 16】 [In the structure, e is an integer in the range of 0 to 10. Su is a monosaccharide, G is the monosaccharide portion, GlcNAc is the N-acetylglucosamine portion, Fuc is the fucose portion, d is either 0 or 1. The method according to any one of claims 13 to 17.

19. Linker-drug construct represented by structure (5) 【Chemistry 17】 [In the structure, L ２ It is a linker, Q is a click probe capable of performing a metal-free click reaction. R １７ These are each individual amino acid side chains, n is an integer in the range of 1 to 5. (NH-CR １７ -CO) ｎ This is a peptide spacer selected from Val-Ala, Glu-Val-Ala, AcLys-Val-Ala, and Asp-Val-Ala. A is a five-membered or six-membered aromatic or heteroaromatic ring, R ２１ H, R ２２ , C(O)OH and C(O)R ２２ Selected from, R ２２ 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 with O, S and NR ２３ It is optionally interrupted by one or more heteroatoms selected from R ２３ is hydrogen and C １ ~C ４ [Independently selected from the group consisting of alkyl groups].

20. The linker-drug construct according to claim 19, wherein the click probe Q comprises a cyclic alkyne moiety or a cyclic alkene moiety.

21. (a) Whether the click probe Q is selected from the group consisting of (Q22) to (Q36) [Chemistry 18] [In the structure, B is a pharmaceutically acceptable anion.] Or the (hetero)cycloalkynyl moiety Q is represented by structure (Q37) 【Chemistry 19】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, Y ２ C(R) ３１ ) ２ , O, S or NR ３１ And R ３１ Each of them individually, R １５ Alternatively, it is a second appearance of the exatecan payload connected via the spacer portion, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16. Or the cyclooctinyl moiety Q is represented by structure (Q38) 【Chemistry 20】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, R １８ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, R １９ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Selected from the group consisting of (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 the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R １９ This is the second appearance of the exatecan payload connected via the spacer portion, l is an integer in the range of 0 to 10. Or the (hetero)cyclooctinyl moiety Q is represented by structure (Q39) 【Chemistry 21】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR １５ [is] Alternatively, the heterocycloheptinyl moiety Q may be represented by the structure (Q36a), 【Chemistry 22】 Alternatively, (b) the click probe Q is selected from the group consisting of an optionally substituted (hetero)cyclopropenyl group, (hetero)cyclobutenyl group, trans-(hetero)cycloheptenyl group, trans-(hetero)cyclooctenyl group, trans-(hetero)cyclononenyl group or trans-(hetero)cyclodecynyl group, or the click probe Q is selected from the group consisting of (Q40) to (Q50), 【Chemistry 23】 The linker-drug construct according to claim 20, wherein the R group(s) of Si in (Q44) and (Q45) is alkyl or aryl.

22. Having structure (5d) 【Chemistry 24】 [In the structure, n = 0 or 1] The linker-drug construct according to claim 19.

23. The linker-drug construct according to claim 22, wherein n is 0.

24. Having structure (5e) 【Chemistry 25】 [In the structure, n = 0 or 1] The linker-drug construct according to claim 19.

25. The linker-drug construct according to claim 24, wherein n is 1.

26. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

27. An antibody-drug conjugate according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 26, for use in the treatment of subjects requiring treatment.

28. An antibody-drug conjugate according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 26 for use in the treatment of cancer.

29. The antibody-drug conjugate or pharmaceutical composition for use according to claim 28, wherein the cancer is HER2-positive cancer.

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