Anthracyclines and their conjugates

Abstract

The present invention relates to nemorubicin and analogs of PNU-159,682 bearing various substituents other than 2"-OMe on the morpholino ring that had a more beneficial effect on toxin toxicity than molecules bearing a 2"-OMe group. Furthermore, PNU variants bearing modified 2"-O-alkyl chains were found to exhibit enhanced tolerability in vivo. Thus, modification of the 2"-O-alkyl group has produced ADCs with carefully tailored potency and tolerability to improve patient dosing. Accordingly, the present invention relates to compounds represented by structure (1) and conjugates thereof, as well as pharmaceutical compositions and methods for targeting tumor cells and treating cancer. JPEG2025526869000141.jpg48149

Description

Detailed Description of the Invention

[0001] [Field of the Invention]

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to anthracyclines and antibody-drug conjugates prepared therewith, in particular antibody-drug conjugates using analogs of PNU-159,682 as cytotoxic payloads suitable for the treatment of cancer.

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

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

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

[0005]

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

[0006]

[0006] Acylation of the ε-amino group of lysine side chains is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, for example, SMCC, as applied in the production of Kadcyla®.

[0007]

[0007] Various reagents are known for alkylating thiol groups in cysteine side chains (see Figure 2). Among cysteine alkylation methods, most are based on the use of maleimide reagents, as applied, for example, in the production of Adcetris®. In addition to standard maleimide derivatives, various maleimide variants are also applied to more stable cysteine conjugation, as demonstrated, for example, by James Christie et al., J. Contr. Rel. 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol. 2014, 32, 1059-1062 (both incorporated by reference).Other approaches for cysteine alkylation include various approaches based on nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem. 2008, 19, 759-765, which are incorporated by reference), or nucleophilic addition at unsaturated bonds, such as reaction with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun. 2016, 7, 13128 and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both of which are incorporated by reference), reaction with phosphonamidates (see, e.g., Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, which are incorporated by reference), reaction with allenamides (see, e.g., Abbas et al., Bioconj. Chem. 2019, 58, 11625-11630, which are incorporated by reference), and the like. al., Angew. Chem. Int. Ed 2014, 53, 7491-7494), reaction with cyanoethynyl reagents (see, e.g., Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, which is incorporated by reference), reaction with vinyl sulfones (see, e.g., Gil de Montes et al., Chem. Sci. 2019, 10, 4515-4522, which is incorporated by reference), or reaction with vinyl pyridines (see, e.g., Seki et al., Chem. Sci., 2021, 12, 9060-9068 and https: / / iksuda.com / science / permalink / (accessed July 26, 2020)).An alternative approach to antibody conjugation without antibody re-engineering involves the reduction of interchain disulfide bridges followed by the use of bis-sulfone reagents (see, e.g., Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both of which are incorporated by reference), mono- or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., J. Am. Chem. Soc. 2010, 132, 1960-1965, both of which are incorporated by reference), or bis-bromomaleimides (see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., J. Am. Chem. Soc. 2010, 132, 1960-1965, both of which are incorporated by reference). al., Org. Biomol. Chem. 2014, 37, 7261-7269), bis-maleimide reagents (see, e.g., WO 2014114207), bis(phenylthio)maleimides (see, e.g., Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521, both of which are incorporated by reference), bis-bromopyridazinediones (see, e.g., Robinson et al., RSC Advances 2017, 7, 9073-9077, which are incorporated by reference), bis(halomethyl)benzenes (see, e.g., Ramos-Tomillero et al., RSC Advances 2017, 7, 9073-9077, which are incorporated by reference), and the like. These include the addition of payloads linked to cysteine cross-linking reagents such as cysteine cross-linkers (see, e.g., WO 2013173391), bis(halomethyl)aromatic compounds (see, e.g., WO 2013173391). Typically, ADCs prepared by cysteine cross-linking have a drug-antibody loading (DAR4) of about 4. Another useful technique for conjugation to cysteine side chains is via disulfide bonds, which are bioactivatable linkages that have been utilized to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci. 2017, 8, 366-370, which is incorporated by reference).

[0008] In addition to conjugation to the side chains of the naturally occurring amino acids lysine or cysteine, various other conjugation techniques have been explored based on a two-step approach involving (a) the introduction of a new reactive group F, followed by (b) reaction with another complementary reactive group Q. For example, one method can be used to introduce a given number of reactive moieties F, which can be two, four, or eight, into an antibody (see Figure 3).

[0009]

[0009] Examples of non-natural reactive functional groups F that can be used for linker-drug bioconjugation are oxime groups suitable for oxime ligation or azide groups suitable for click chemistry conjugation. Oxime or azide can be incorporated into antibodies by genetically encoding non-natural amino acids, such as p-acetophenylalanine, suitable for oxime ligation, or p-azidomethylphenylalanine or p-azidophenylalanine, suitable for click chemistry conjugation, as demonstrated by Axup et al. Proc. Nat. Acad. Sci. 2012, 109, 16101-16106, which is incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem. 2014, 25, 351-361, incorporated by reference, used cell-free protein synthesis to introduce azidomethylphenylalanine (AzPhe) into monoclonal antibodies for conversion to ADCs via metal-free click chemistry. Also, Nairn et al., Bioconj. Chem. 2012, 23, 2087-2097, incorporated by reference, have shown that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins using auxotrophic bacteria and further converted to protein conjugates via click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUAGenetic encoding of aliphatic azides in recombinant proteins using pairs was demonstrated by Nguyen et al., J. Am. Chem. Soc. 2009, 131, 8720-8721, which is incorporated by reference, and labeling was achieved by click chemistry via either copper-catalyzed alkyne azide cycloaddition (CuAAC) or strain-promoted alkyne azide cycloaddition (SPAAC). In addition to CuAAC and SPAAC, bioconjugation of linker-drugs to antibodies (and other biomolecules such as glycans and nucleic acids) can be achieved by a variety of other metal-free click chemistries; see, for example, Nguyen and Prescher, Nature Rev. Chem. 2020, 4, 476-489, which is incorporated by reference. For example, oxidation of certain tyrosines in proteins can yield ortho-quinones, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes; see, e.g., Bruins et al., Chem. Eur. J. 2017, 24, 4749-4756, incorporated by reference. In addition to cyclooctynes, certain cycloheptynes are also suitable for metal-free click chemistry, as reported by Wetering et al., Chem. Sci. 2020, 11, 9011-9016, incorporated by reference. Tetrazine moieties can also be introduced into proteins or glycans by various means, e.g., genetic encoding or chemical acylation, and can also 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 4.

[0010] In SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne, and cycloaddition with an azide-modified antibody is facilitated by the relief of ring strain. Conversely, the linker-drug can be functionalized with an azide, and the antibody can be functionalized with a cyclic alkyne. Various strained alkynes suitable for metal-free click chemistry are shown in Figure 5.

[0011]

[0011] A method gaining popularity in the field of ADCs is based on the enzymatic incorporation of a non-natural functional group F. For example, Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871, incorporated by reference, uses the bacterial enzyme transglutaminase (BTG or TGase) to incorporate an azide moiety onto an antibody. A genetic method based on C-terminal TGase-mediated azide introduction, followed by conversion in ADCs via metal-free click chemistry, was reported by Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675, incorporated by reference.

[0012]

[0012] In International Publication No. 2014065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12, and Wijdeven at al. MAbs 2022, 14, 2078466 (all incorporated by reference) have shown that enzymatic remodeling of the native antibody glycan at N297 allows for the introduction of azide-modified sugars suitable for the attachment of cytotoxic payloads using metal-free click chemistry (see Figure 6). Similarly, enzymatic glycan remodeling protocols can also be used to incorporate free thiol groups on antibodies for conjugation based on any of the methods described above for cysteine conjugation (see Figure 7).

[0013] Although ADCs have demonstrated clinical and preclinical activity, it has been unclear what factors determine such efficacy in addition to antigen expression on targeted tumor cells. For example, drug:antibody ratio (DAR), ADC binding affinity, payload potency, receptor expression level, internalization rate, transport, multidrug resistance (MDR) status, and other factors are all thought to affect the outcome of ADC treatment in vitro. In addition to directly killing antigen-positive tumor cells, ADCs also have the ability to kill adjacent antigen-negative tumor cells: the so-called "bystander killing" effect, as first 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 speaking, neutral cytotoxic payloads exhibit bystander killing, whereas ionic (charged) payloads do not, as a result of the inability of ionic species to readily cross cell membranes by passive diffusion. Payloads with established bystander effects include, for example, MMAE and DXd. An example of a payload that does not exhibit bystander killing is MMAF or the active catabolite (lysine-MCC-DM1) of Kadcyla®.

[0014] Currently, cytotoxic payloads include, for example, microtubule-disrupting agents (e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, and tubulysin), DNA-damaging agents (e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, duocarmycins, anthracyclines, topoisomerase inhibitors (e.g., DXd, exatecan, and SN-38), or RNA polymerase II inhibitors (e.g., amanitin). While ADCs that have achieved market approval include, for example, payloads MMAE, MMAF, DM1, calicheamicin, SN-38, DXd, and PBD dimers, various pivotal trials are being conducted on ADCs based on duocarmycins or DM4. A wider variety of payloads are still in clinical evaluation or have been in clinical trials, such as eribulin, indolinobenzodiazepine dimers, PNU-159,682, amanitin, hemiasterlin, doxorubicin, vinca alkaloids, etc. Finally, various ADCs in the late preclinical stage are conjugated to novel payloads, such as KSP inhibitors, MMADs, and cryptophycins.

[0015] With the exception of sacituzumab govitecan (Trodelvy®), all clinical and commercially available ADCs contain cytotoxic drugs that are not suitable as stand-alone drugs. Trodelvy® is an exception because it features SN-38, which is also the active catabolite of irinotecan (the SN-38 prodrug), as its cytotoxic payload. Several other payloads currently used in clinical ADCs were initially evaluated for chemotherapy as free drugs, e.g., calicheamicin, PBD dimers, and eribulin, but have demonstrated extremely high cytotoxic potency (picomolar to low nanomolar IC ) compared to the typically low micromolar potency of standard chemotherapeutic agents such as paclitaxel and doxorubicin. 50 value).

[0016] Another cytotoxic agent that is gaining increasing interest for application in ADCs is PNU-159,682 (see Figure 9), an anthracycline derivative that is >1000 times more potent than doxorubicin. PNU-159,682 is one of the oxidative catabolites of nemorubicin (MMDX) and was developed as a synthetic analogue of doxorubicin, but without the cardiotoxicity associated with doxorubicin. PNU-159,682 is a bioactivation product formed from nemorubicin in the human liver under the action of CYP3A after oral administration. Interestingly, two other oxidative catabolites, nemorubicin N-oxide and PNU-159,682, have potencies similar to those of doxorubicin. Due to its high potency, PNU-159,682 is currently being investigated as a payload for ADCs, as reported, for example, by Dal Corso et al., J. Contr. Rel. 2017, 264, 211-218 (incorporated by reference). This paper reported that a non-internalizing antibody-drug conjugate based on an antibody specific for tenascin-C mediated potent therapeutic activity when equipped with PNU-159,682 linked to the antibody via a maleimide-based cysteine alkylation and a Val-Cit-PABC-based protease-sensitive cleavable linker, and a dimethylethylenediamine (DMEDA) cyclization cleavage element linked to the free 14-hydroxyl of PNU-159,682 via a carbamate group (Figure 9, top). The ADC was found to be stable in serum but could be efficiently cleaved in the subendothelial extracellular matrix by proteases released by dying tumor cells, resulting in successful tumor regression in various in vivo models. A similar PNU-159,682 ADC based on 14-OH acylation with Val-Cit-PABC-DMEDA was reported by Stefan et al., Mol. Cancer Ther. 2017, 16, 879-892, which is incorporated by reference, whereby linker-drugs were attached to the C-termini of various antibodies using sortase-mediated antibody conjugation (SMAC™) for the anti-HER2 antibody trastuzumab and the anti-CD30 antibody bretuximab (see Figure 10).In this study, the DMEDA-conjugated ADC was directly compared with another PNU-159,682 derivative prepared by oxidizing the hydroxy-ketone group to a carboxylic acid followed by amidation with a diglycyl-ethylenediamine (EDA) linker (Figure 9, bottom). Characterization of the resulting ADCs showed that they exhibited potency exceeding that of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla® and Adcetris®, which are based on the same antibody. However, the same report also showed that the cytotoxic selectivity of PNU-derived ADCs based on an EDA-amide linker was much more selective for target-positive cells than similar ADCs based on a DMEDA-carbamate linker, likely due to the specific release of PNU-159,682 from the latter ADCs. As a result, EDA-amide-based technology was selected for further development and is currently being applied in various clinical programs, including NBE-002 and SO-N102, for ADCs targeting ROR1 and Claudin18.2, respectively.

[0017]

[0017] A similar method for producing ADCs based on oxidation of the hydroxy-ketone of PNU-159,682 followed by coupling of the resulting acid has been disclosed (see WO2016127081). Various derivatives of PNU include amides, hydrazides, and acylhydroxylamine derivatives.

[0018] Besides the modification and covalent attachment of PNU-159,682 via the hydroxyketone moiety, there are surprisingly few reports detailing the use of the methoxy-morpholino group for attachment to antibodies. WO2009099741 shows how PNU-159,682 can be conjugated via the hydroxy-ketone moiety to an antibody with an engineered cysteine, and although suggestions are made for preparing conjugates by attachment at various positions of the morpholine group, including replacement of 2"-OMe with a carbamate linker, none of the morpholine-linked structures were possible.

[0019]

[0019] Details of the tolerability of NBE-002 in cynomolgus monkeys have been disclosed (AACR2018, Abstract #737), indicating an MTD of approximately 3 mg / kg on a qw3x3 dosing schedule; however, it should be noted that one monkey developed an immune response after the third dose. An ongoing Phase 1 study (clinical trial NCT04441099) remains uncertain as to what the MTD will be in humans. Given the extremely high potency of PNU in preclinical models (MED of approximately 0.033 mg / kg), it is possible that the MTD in humans could be (substantially) less than 1 mg / kg. As a result, in vivo receptor saturation is likely not achieved after administration (typically intravenously), leading to suboptimal tumor uptake and increased clearance of the ADC.

[0020] One approach to increasing the administered dose of ADCs in patients is to reduce the drug load of the antibody; for example, a DAR1 format with the same payload may be preferred because it may have a two-fold higher MTD than a similar DAR2 format. Ruddle et al., ChemMedChem 2019, 14, 1185-1195, recently demonstrated that DAR1 conjugates can be prepared from antibody Fab fragments. The resulting DAR1-type Fab fragments were shown to be highly homogeneous, stable in serum, and exhibit excellent cytotoxicity. Subsequent publications, White et al., MAbs 2019, 11, 500-515, and further in WO2019034764 (incorporated by reference), demonstrated that DAR1 conjugates can also be prepared from full IgG antibodies using Flexmab technology. We demonstrated that the Flexmab-derived DAR1 ADC was highly resistant to payload loss in serum and exhibited potent antitumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, this ADC was well tolerated in rats at twice the dose compared to a site-specific DAR2 ADC prepared using a single maleimide-containing PBD dimer.

[0021] Although it would be advantageous to have an ADC in the DAR1 format, no DAR1 technology has been reported to date that improves the therapeutic index compared to DAR2 ADCs. Also, no technology has been reported for generating DAR1 ADCs from whole antibodies without the need for monoclonal antibody reengineering, which would inherently make DAR2 ADCs easier to generate.

[0022] Another approach to increasing the dose level of ADCs, particularly PNU-based ADCs, would involve generating analogs with reduced potency. For example, Holte et al., Bioorg.Med.Chem.Lett. 2020, 30, 127640, incorporated by reference, generated various PNU analogs with a wide range of cytotoxic activity by oxidation-modification of the hydroxyketone moiety of the molecule. Structure-activity relationships were investigated, leading to the development of six linker-drug conjugations to antibodies, resulting in ADCs that showed an increased MED of approximately 1 mg / kg compared to conventional PNU-159,682 in various preclinical models.

[0023]

[0023] A final approach to modulating the potency of PNU-159,682 requires modification of the morpholino group, particularly the 2"-OMe group. As a single example, International Publication No. 2012073217 reports the preparation and in vitro evaluation of 2"-OEt analogs of PNU-159,682, which exhibited 3- to 8-fold higher in vitro potency compared to the OME variant in two different cell lines (A2780 and MCF7).

[0024] [Summary of the Invention] The present inventors have surprisingly found that analogs of nemorubicin and PNU-159,682 having various substituents other than 2"-OMe on the morpholino ring are compatible with molecules having a 2"-OMe group (i.e., R 1We found that 2"-O-alkyl derivatives of nemorubicin or PNU-159,682 exhibited significantly lower in vitro potency than 2"-O-alkyl derivatives of nemorubicin or PNU-159,682 (where 2"-O-alkyl is methyl ketone). A similar decrease in potency was observed for various 2"-O-alkyl derivatives of nemorubicin or PNU-159,682 covalently attached to monoclonal antibodies in the form of antibody-drug conjugates (ADCs), whereby covalent attachment could be ensured by carbamoylation of the hydroxyketone group or oxidation of the hydroxyketone group followed by coupling of the resulting carboxylic acid. Furthermore, we found that covalent attachment to antibodies could also be achieved by incorporating chemoselective handles, including but not limited to amino, thiol, or hydroxy groups, into the 2"-O-alkyl chain, leaving either the hydroxyketone group (as in doxorubicin) or the methylketone group (as in daunorubicin) intact. Furthermore, we found that PNU variants with modified 2"-O-alkyl chains exhibited improved in vivo tolerability. Thus, modification of the 2"-O-alkyl group has yielded ADCs with carefully tuned potency and tolerability to improve patient dosing.

[0025] The present invention relates to a novel toxin represented by structure (1) and its conjugate represented by structure (2). In this regard, the present invention relates to a method for preparing the conjugate according to the invention. In a further aspect, the present invention relates to a method for targeting tumor cells. Related thereto is a first medical use of the conjugate according to the invention and a second medical use for the treatment of cancer.

[0026] [Detailed explanation] [Definition]

[0026] As used in this specification and claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but items not specifically mentioned are not excluded.

[0027]

[0027] Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0028] A linker is defined herein as a moiety that connects (covalently bonds) two or more elements of a compound. A linker may include one or more spacer moieties. A spacer moiety is defined herein as a moiety that covalently bonds two (or more) parts of the linker with a certain spacing (i.e., providing a distance between them). A linker can be, for example, part of a linker-construct, linker-conjugate, linker-payload (e.g., linker-drug), or antibody-conjugate, as defined below.

[0029]

[0029] A "hydrophilic group" or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that impart improved polarity and therefore improved aqueous solubility to the attached molecule. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonate groups, phosphate groups, acylsulfamide groups, or carbamoylsulfamide groups. In addition to higher solubility, other effects of hydrophilic groups include improved click conjugation efficiency, and less aggregation when incorporated into antibody-drug conjugates, improved pharmacokinetics, thereby resulting in higher efficacy and in vivo tolerability.

[0030] The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced with a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt. The term "pharmaceutically acceptable" salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that has acceptable mammalian safety for a given administration regimen). Such salts may be derived from a pharmaceutically acceptable inorganic or organic base and a pharmaceutically acceptable inorganic or organic acid. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counterions known in the art, such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0031]

[0031] The term "enediyne", or "enediyne antibiotic", or "enediyne-containing cytotoxin" refers to any cytotoxin characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule known in the art, including neocarzinostatin (NCS), C-1027, kedarcidin (KED), maduropeptin (MDP), N1999A2, sporolide (SPO), cyanosporacid (CYA and CYN), and phyziolide, calicheamicin (CAL), esperamicin (ESP), dynemicin (DYN), namenamemicin, shishijimicin, and uncialamicin (UCM).

[0032] As used herein, the term "alkylaminosugar" refers to a tetrahydropyranyl moiety linked via its 2-position to an alcohol functional group, thereby forming an acetal functional group, and further substituted with (at least) one N-alkylamino group at the 3-, 4-, or 5-position. In this context, an "N-alkylamino group" refers to an amino group bearing one methyl, ethyl, or 2-propyl group.

[0033] The term "click probe" refers to a functional moiety that can undergo a click reaction, i.e., two compatible click probes click with each other so that they become covalently linked in the product. Compatible probes for click reactions are known in the art and preferably include (cyclic) alkynes and azides. In the context of the present invention, click probe Q in a compound according to the invention can react with click probe F on a (modified) protein such that, upon click reaction, a conjugate is formed, resulting in the protein being conjugated to the compound according to the invention. Here, F and Q are compatible click probes.

[0034]

[0034] The term "(hetero)alkyl" refers to alkyl and heteroalkyl groups. Heteroalkyl groups are groups in which one or more carbon units (e.g., CH2, CH, or C) in the alkyl chain is replaced with O, S, S(O), S(O)2, or NR 4 In other words, the alkyl chain is an alkyl group substituted with a heteroatom such as O, S, S(O), S(O)2, and NR 4The (hetero)alkyl group is interrupted by one or more elements selected from the group consisting of: (i) and (ii). Such interruptions are distinct from substituents, since they are present within the alkyl group chain, whereas substituents are, for example, pendant groups monovalently attached to carbon atoms of the alkyl chain. In preferred embodiments, the (hetero)alkyl group is an alkyl group, such as ethyl (Et), isopropyl (i-Pr), n-propyl (n-Pr), tert-butyl (t-Bu), isobutyl (i-Bu), n-butyl (n-Bu), or n-pentyl. The (hetero)alkyl group can be linear, branched, or cyclic.

[0035] Similarly, the term "(hetero)aryl" refers to aryl and heteroaryl groups. Heteroaryl groups are groups in which one or more carbon units in the ring (e.g., CH) are replaced with O, S, N, or NR 4 and the like.

[0036] An "acylsulfamide moiety" is defined herein as a sulfamide moiety (H2NSO2NH2) that is N-acylated or N-carbamoylated at one end of the molecule and N-alkylated (mono- or bis-) at the other end of the molecule. In the context of the present invention, particularly in the Examples, this group is also referred to as "HS."

[0037] A "domain" is generally defined based on sequence homology and can be any region of a protein that is often associated with a particular structural or functional entity. CEACAM family members are known to be composed of Ig-like domains. The term domain is used herein to refer to either an individual Ig-like domain, e.g., an "N-domain," or a group of consecutive domains, e.g., an "A3-B3 domain."

[0038] A "coding sequence," or a sequence that "encodes" an expression product such as an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme; i.e., the nucleotide sequence encodes the amino acid sequence of that polypeptide, protein, or enzyme. A protein-coding sequence may include a start codon (usually ATG) and a stop codon.

[0039]

[0039] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence, including all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term gene may be intended for a genomic sequence that encodes a protein, i.e., a sequence that includes regulatory elements, promoter, intron, and exon sequences.

[0040] The term "glycoprotein" is used herein in its ordinary scientific sense to refer to a protein containing one or more monosaccharide or oligosaccharide chains ("glycans") covalently attached to the protein. The glycans can be attached to hydroxyl groups on the protein (O-linked glycans), such as those of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline, or to amide functional groups on proteins (N-glycoproteins), such as asparagine or arginine, or to carbons on proteins (C-glycoproteins), such as tryptophan. A glycoprotein may contain two or more glycans, a combination of one or more monosaccharides and one or more oligosaccharide glycans, or a combination of N-linked, O-linked, and C-linked glycans. It is estimated that over 50% of all proteins have some form of glycosylation and are therefore considered glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (candida antarctica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins and antibodies.

[0041] The term "glycan" is used herein in its ordinary scientific sense to refer to a monosaccharide or oligosaccharide chain linked to a protein. Thus, the term glycan refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of one sugar, which may be unsubstituted (monosaccharide) or may be further substituted with one or more of its hydroxyl groups (oligosaccharide). Naturally occurring glycans typically contain one to about ten sugar moieties. However, when a longer sugar chain is linked to a protein, the sugar chain is also considered a glycan herein. The glycan of a glycoprotein may be a monosaccharide. Typically, a monosaccharide glycan of a glycoprotein consists of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man), or fucose (Fuc) covalently attached to the protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In an oligosaccharide, the sugar directly attached to the protein is called the core sugar. In an oligosaccharide, a sugar that is not directly attached to the protein and is attached to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to the protein but is attached to a single other sugar, i.e., a sugar that does not have an additional sugar substituent on one or more of its other hydroxyl groups, is called a terminal sugar. For the avoidance of doubt, an oligosaccharide of a glycoprotein may have multiple terminal sugars but only one core sugar. Glycans can be O-linked glycans, N-linked glycans, or C-linked glycans. In O-linked glycans, monosaccharides or oligosaccharide glycans are attached to an O-atom in an amino acid of the protein, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, monosaccharides or oligosaccharide glycans are attached to the protein via an N atom in an amino acid of the protein, typically via the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In C-linked glycans, a monosaccharide or oligosaccharide glycan is attached to a C atom in an amino acid of a protein, typically the C atom of tryptophan (Trp).

[0042] The term "antibody" (AB) is used herein in its usual scientific sense. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein in its broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" is also intended herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is intended to include not only whole antibodies but also antibody fragments, such as antibody Fab fragments, F(ab')2, Fv fragments, or Fc fragments, scFv-Fc fragments, minibodies, diabodies, or scFvs derived from cleaved antibodies. Furthermore, the term includes recombinant antibodies and antibody derivatives. Antibodies, antibody fragments, and recombinant antibodies can be obtained by methods known in the art.

[0043]

[0043] An antibody may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (K). Light chains contain two domains or regions, a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity for antigens. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties to the antibody chain, such as antibody chain assembly, secretion, transplacental mobility, complement fixation, and Fc receptor (FcR) binding. An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, or allotype (e.g., human G1m1, G1m2, Gm3, non-G1m1 (that is, any allotype other than G1m1), G1m17, G2m23, G3m21, G3m28, G3m1.1, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2, and Km3) of immunoglobulin molecule. Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m17,1, G1m3, G1m3.1, G1m3.2, or G1m3.1.2. More preferably, the allotype is selected from the group consisting of G1m17,1 or G1m3 allotypes. Antibodies can be engineered in the Fc domain to enhance or impair binding to Fc-γ receptors, as summarized by Saunders et al. Front. Immunol. 2019, 10, doi:10.3389 / fimmu.2019.01296 and Ward et al., Mol. Immunol. 2015, 67, 131-141.For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. Elimination of binding to Fc-γ receptors can also be achieved by mutating the N297 amino acid to any amino acid other than asparagine, the T299 amino acid to any amino acid other than threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies, for example, with PNGase or endoglycosidase. Immunoglobulins can be derived from any species, including human, mouse, or rabbit origin. Each chain contains distinct sequence domains.

[0044] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, and the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the full length of the reference sequence.

[0045] The term "CDR" refers to complementarity-determining region: the specificity of an antibody resides in the structural complementarity between the antibody-binding site and an antigenic determinant. An antibody-binding site is composed of residues primarily from hypervariable or complementarity-determining regions (CDRs). Optionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and thus the binding site. Thus, complementarity-determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. Each immunoglobulin light and heavy chain has three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen-binding site contains six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. "CDR"

[0046] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule of a single amino acid sequence, which is directed against a particular antigen, and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be produced recombinantly, i.e., by protein engineering.

[0047]

[0047] The term "chimeric antibody," in its broadest sense, refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal in association with the CH and CL domains of another antibody, in one embodiment a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, or rabbit. A chimeric antibody can also refer to a multispecific antibody having specificity for at least two different antigens.

[0048] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and has been modified, for example by substituting certain amino acids in the framework regions of the VH and VL domains, to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most often human CH and CL domains. A "fragment" of a (traditional) antibody comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0049] [The present invention] In a first aspect, the present invention relates to a conjugate in which a compound represented by structure (1) is conjugated to a cell-binding agent via a linker, wherein structure (1) is as follows: [ka] During the ceremony, -R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C 6~15 Alkyl, C 2~15 Alkenyl, C 2~15 Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2, wherein the optional substituents are halogen, C 1~12 (Hetero)alkyl, (hetero)aryl, C 2~15 Alkenyl, C 2~15 Alkynyl, X 2 R 4, N(R 4 )2, NO2, and the substituent C 1~12 The (hetero)alkyl and (hetero)aryl are optionally C 1~6 (hetero)alkyl, X 2 R 4 and N(R 4 )2; each Sp may be individually further substituted with C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1~12 (hetero)alkylene, and the (hetero)alkylene or (hetero)arylene is selected from the group consisting of halogen, X 2 R 4 , N(R 4 )2, C 1~4 and each R is optionally substituted with one or more substituents selected from alkyl and NO. 4 are individually, H, C 1~4 alkyl or adamantyl, and X 2 is C(O), C(O)O, C(O)NH, O, S, S(O), S(O)2, S(O)NH or S(O)2NH, and R 12 is β-glucuronide acid, PO3 (2-) , OPO3 (2-) , CO2 (-) , SO3 (-) or N(C 1~4 alkyl)3 (+) and; -R 2 is H, S(O)2OH or P(O)2OH, and R 3 is OH, or R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; - Y 5 are CH2-Y, C(O)-Y, C(=N(R 20 ))-Y, C(R 9 )=NY, where R 9 is an OH group or O(CO)C 1~6 C optionally substituted with alkyl 1~4alkyl, and R 20 is NR 4 -C(O)-N(R 4 )2, NR 4 -C(O)-Sp-N(R 4 )2, NR 4 -C(O)-R 12 , N.R. 4 -C(O)-Sp-R 12 where Sp, R 4 and R 12 is as defined above; The compound of structure (1) is linked to a cell binding agent via Y. Also contemplated in this aspect are salts of compounds represented by structure (1), where each ion, if present, is in equilibrium with one or more pharmaceutically acceptable counterions.

[0050] In a second aspect, the present invention provides a novel toxin represented by structure (1): [ka] (In the formula, -R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C 6~15 Alkyl, C 2~15 Alkenyl, C 2~15 Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2, wherein the optional substituents are halogen, C 1~12 (Hetero)alkyl, (hetero)aryl, C 2~15 Alkenyl, C 2~15 Alkynyl, X 2 R 4 , N(R 4 )2, NO2, and the substituent C 1~12The (hetero)alkyl and (hetero)aryl are optionally C 1~6 (hetero)alkyl, X 2 R 4 and N(R 4 )2; each Sp may be individually further substituted with C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1~12 (hetero)alkylene, and the (hetero)alkylene or (hetero)arylene is selected from the group consisting of halogen, X 2 R 4 , N(R 4 )2, C 1~4 and each R is optionally substituted with one or more substituents selected from alkyl and NO. 4 are individually, H, C 1~4 alkyl or adamantyl, and X 2 is C(O), C(O)O, C(O)NH, O, S, S(O), S(O)2, S(O)NH or S(O)2NH, and R 12 is β-glucuronide acid, PO3 (2-) , OPO3 (2-) , CO2 (-) , SO3 (-) or N(C 1~4 alkyl)3 (+) and; -R 2 is H, S(O)2OH or P(O)2OH, and R 3 is OH, or R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; - N % is N or N→O; - Y 5 are CH2-Y, C(O)-Y, C(=N(R 20 ))-Y, C(R 9 )=NY, C(R 9 )=N(R 20 ), where R 9 is an OH group or O(CO)C1~6 C optionally substituted with alkyl 1~4 alkyl, and R 20 is NR 4 -C(O)-N(R 4 )2, NR 4 -C(O)-Sp-N(R 4 )2, NR 4 -C(O)-R 12 , N.R. 4 -C(O)-Sp-R 12 where Sp, R 4 and R 12 is as defined above; - Y is NR 4 -Sp 3 -N(R 4 )2, NR 4 -Sp 3 -X 2 (R 4 ), N(R 4 )2, NR 4 -Sp 3 -X 2 (R 4 ), R 12 , Sp 3 R 12 , N.R. 4 -Sp 3 -X 2 -Sp 3 -R 12 , OH, CH3 or CH2OH, where Sp 3 is a spacer; - Y 5 is C(O)-CHOH, then R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl); Also contemplated in this aspect are salts of compounds represented by structure (1), where each ion, if present, is in equilibrium with one or more pharmaceutically acceptable counterions.

[0051]

[0051] As used herein, the compounds of structure (1) can be in conjugated form (i.e., conjugated to a cell-binding agent) or in free form (i.e., as a small molecule). Unless otherwise specified, everything defined for the conjugated form of structure (1), except for the connection to the cell-binding agent via a linker, applies to the free form of structure (1), and vice versa.

[0052]

[0052] Salts of the antibody conjugate represented by structure (1), preferably pharmaceutically acceptable salts, are also contemplated within the scope of the present invention. The compound represented by structure (1) in conjugated and free form may be in salt form, but the conjugated form of the compound represented by structure (1) is typically not in salt form, while the compound represented by structure (1) in free form may be in salt form or neutral form. When the compound of structure (1) is charged, it is typically balanced with one or more pharmaceutically acceptable counterions.

[0053]

[0053] Below, the compound represented by structure (1) is first defined. The structural features of the compound represented by structure (1) also apply to the conjugate represented by structure (2) and the linker-toxin construct represented by structure (5). Furthermore, the structural features of the cell-binding agent represented by structure (4) also apply to the conjugate represented by structure (2). Those skilled in the art will understand that any structural feature that remains unchanged in the conjugation reaction is equally defined for each of the molecules according to the present invention. In the conjugation reaction, only the reactive moieties F and Q are attached to the linking group Z upon reaction of the linker-toxin construct represented by structure (5) with the antibody represented by structure (3). 1 is converted to

[0054] In a further aspect, the present invention relates to the application of the conjugate represented by structure (2) for targeting tumor cells. In this regard, the present invention relates to a first medical use and a second medical use of the conjugate represented by structure (2).

[0055] As will be appreciated by those skilled in the art, the definitions of chemical moieties and their preferred embodiments apply to all aspects of the present invention.

[0056] [Compound represented by structure (1)] The present invention provides a compound represented by structure (1): [ka] (In the formula, -R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C 6~15 Alkyl, C 2~15 Alkenyl, C 2~15 Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2, wherein the optional substituents are halogen, C 1~12 (Hetero)alkyl, (hetero)aryl, C 2~15 Alkenyl, C 2~15 Alkynyl, X 2 R 4 , N(R 4 )2, NO2, and the substituent C 1~12 The (hetero)alkyl and (hetero)aryl are optionally C 1~6 (hetero)alkyl, X 2 R 4 and N(R 4 )2; each Sp may be individually further substituted with C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1~12 (hetero)alkylene, and the (hetero)alkylene or (hetero)arylene is selected from the group consisting of halogen, X 2 R 4 , N(R4 )2, C 1~4 and each R is optionally substituted with one or more substituents selected from alkyl and NO. 4 are individually, H, C 1~4 alkyl or adamantyl, and X 2 is C(O), C(O)O, C(O)NH, O, S, S(O), S(O)2, S(O)NH or S(O)2NH, and R 12 is β-glucuronide acid, PO3 (2-) , OPO3 (2-) , CO2 (-) , SO3 (-) or N(C 1~4 alkyl)3 (+) and; -R 2 is H, S(O)2OH or P(O)2OH, and R 3 is OH, or R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; - Y 5 are CH2-Y, C(O)-Y, C(=N(R 20 ))-Y, C(R 9 )=NY, C(R 9 )=N(R 20 ), where R 9 is an OH group or O(CO)C 1~6 C optionally substituted with alkyl 1~4 alkyl, and R 20 is NR 4 -C(O)-N(R 4 )2, NR 4 -C(O)-Sp-N(R 4 )2, NR 4 -C(O)-R 12 , N.R. 4 -C(O)-Sp-R 12 where Sp, R 4 and R 12 is as defined above; - Y is NR 4 -Sp 3 -N(R4 )2, NR 4 -Sp 3 -X 2 (R 4 ), N(R 4 )2, R 12 , Sp 3 R 12 , N.R. 4 -Sp 3 -X 2 -Sp 3’ -R 12 , OH, CH3 or CH2OH, where Sp 3 and Sp 3’ is a spacer; - N % is N or N→O).

[0057] The compounds represented by structure (1) may be linked to a cell-binding agent (i.e., a conjugate) or may contain a reactive group (i.e., in free form or a small molecule) that can react with an appropriately functionalized cell-binding agent or with a linker that is conjugated to the cell-binding agent in a subsequent step. In certain preferred embodiments of the compounds represented by structure (1), the linkage to the cell-binding agent or reactive moiety may be at any position on the compound. Preferably, this linkage is at Y or R 1 and most preferably via Y. Reactive groups that can link a compound of structure (1) to a linker or cell binding agent include, for example, N(R 4 )2 or X 2 (R 4 ) group or R 1 X in 2 R 4 Or it may be an N3 group.

[0058]

[0058] Salts thereof, particularly pharmaceutically acceptable salts, are also contemplated in the present invention. Typically, R 2 S(O)2OH or P(O)2OH group or R 12 The analogous group is Na + , K. + , NH4 + or NEt4 +The compound of formula (1) may be present in a salt form containing a pharmaceutically acceptable cation such as ##STR00001## Salts are particularly contemplated when the compound of formula (1) is in a free form that is not conjugated to a cell-binding agent. Conjugates are rarely in a salt form.

[0059]

[0059] R 1 The properties of the methyl group (R 1 We have found that replacing Y with a larger substituent can beneficially affect the toxicity of a toxin. We are the first to tailor this substituent to improve toxicity while conjugating a toxin to a cell-binding agent via Y.

[0060]

[0060] R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C 6~15 Alkyl, C 2~15 Alkenyl, C 2~15 Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2. Preferably, R 1 is optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, n-hexyl, C 7~12 Alkyl, C 3~12 Alkenyl, C 3~12 Alkynyl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-OR 4 , Sp-N3, Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2 is selected.

[0061] In an alternative preferred embodiment, R1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 and Sp-N(R 4 In a further preferred embodiment, R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3, Sp-X 2 -Sp-R 12 and Sp-N(R 4 In another preferred embodiment, R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-X 2 R 4 , Sp-N3, Sp-X 2 -Sp-R 12 and Sp-N(R 4 In another preferred embodiment, R 1 is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 In another preferred embodiment, R 1 is optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 In one embodiment, R 1 is as defined above, but Sp-X 2 -Sp-R 12 No.

[0062]

[0062] R 1 The group may be optionally substituted with one or more substituents, the optional substituents being halogen, C 1~12 (Hetero)alkyl, (hetero)aryl, C 2~15 Alkenyl, C 2~15 Alkynyl, X 2 R 4 , N(R 4 )2 and NO2, preferably halogen, X 2 R 4 and N(R 4 )2. Most preferably, the optional substituents are selected from OH, SH and NH2. When present, the substituents are selected from R 1 In a preferred embodiment, R 1 The carbon atom located immediately adjacent to the O atom to which R is attached does not have a substituent so that it is attached only to carbon and / or hydrogen atoms, which has been found to improve the stability of the compound. 1 The group contains 0 to 2 substituents, more preferably 0 or 1 substituent, and most preferably R 1 The group is unsubstituted.

[0063] Optional Substituent C 1~12 (Hetero)alkyl and (hetero)aryl themselves also include C 1~6 (hetero)alkyl, X 2 R 4 and N(R 4 )2. X may be further substituted with an optional substituent selected from 2 and R 4 A preferred embodiment for C 1~12 These optional substituents apply equally to (hetero)alkyl and (hetero)aryl substituents. In one embodiment, C 1~12 The (hetero)alkyl and (hetero)aryl substituents do not contain any further substituents.

[0064] In this specification, X 2is C(O), C(O)O, C(O)NH, O, S, S(O), S(O)2, S(O)NH or S(O)2NH, preferably X 2 is O, S, S(O) or S(O)2. Preferably, X 2 is not S. Therefore, X 2 is preferably C(O), C(O)O, C(O)NH, O, S(O), S(O), S(O)NH or S(O)NH. In a preferred embodiment, X 2 is O, S(O) or S(O)2. Most preferably, X 2 is O. R 4 is H, C 1~4 alkyl and adamantyl. Preferably, R 4 is H or C 1~4 Each X is an alkyl. 2 and R 4 , as well as each optional substituent, may be individually selected.

[0065]

[0065] R 12 is β-glucuronide acid, PO3 (2-) , OPO3 (2-) , CO2 (-) , SO3 (-) or N(C 1~4 alkyl)3 (+) Preferably, R 12 is β-glucuronide acid, PO3 (2-) or SO3 (-) Most preferably, R 12 is a β-glucuronide acid.

[0066] Sp is an alkyl or aryl spacer. More specifically, Sp is C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (Hetero)alkylene-(hetero)arylene or (hetero)arylene-C 1~12 (hetero)alkylene. The carbon atoms of Sp are selected from halogen, X 2 R 4 , N(R 4 )2, C 1~4X may be substituted with one or more substituents selected from alkyl, and NO2. 2 and R 4 The preferred embodiments for apply equally to these optional substituents of Sp. In preferred embodiments, the optional substituents are F, Cl, Br, OH, OR 4 , SH, NH2, Et, Me, and NO2. In particularly preferred embodiments, the spacer Sp contains 0 to 2 substituents, more preferably 0 or 1 substituent, and most preferably the spacer Sp is unsubstituted. The (hetero)alkylene and (hetero)arylene groups may optionally be interrupted by one or more elements selected from O, S, S(O), S(O)2, or NR4. Each Sp and each optional substituent may be selected individually.

[0067]

[0067] R 1 Preferred options are represented by structures (D1) to (D67) shown below. [ka] [ka]

[0068]

[0068] In this specification, the following applies: n and n' are each an integer in the range of 0 to 10, preferably in the range of 1 to 10, more preferably in the range of 1 to 5. - X 3 OH, NH2, OR 6 , N(R 6 )2, N(+)(R 6 )3, SR 6 , S(O)R 6 , S(O)2R 6 , N3 and SH. - Y 4 , NH, NR 6 , N(+)(R 6 )2, S(O) and S(O)2. - Each R 6is hydrogen, C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C3-C 12 Cycloalkanes, C3-C 12 Cycloalkenyl, C3-C 12 are individually selected from cycloalkynyl, (hetero)aryl, and polyethylene glycol (PEG). As used herein, PEG typically has the structure (CH2CH2O) m R 10 wherein m is 1, 2, or 3; and R 10 is H, CH3 or CH2CH3. -R 7 is H or (CH2) n It is CH3. -R 8 is a (hetero)aryl group. -R 12 is β-glucuronide acid, PO3 2- , OPO3 2- , CO2 - , SO3 - and N(C 1~4 alkyl)3 + is.

[0069]

[0069] R 1 The preferred options (D1) to (D67) also include their halogenated and / or unsaturated forms. Thus, any hydrogen atom directly bonded to a carbon atom may be substituted with a halogen, preferably F or Cl, more preferably F. Most preferably, no hydrogen atom is substituted with a halogen atom. Similarly, any two adjacent saturated carbon atoms may also contain a double or triple bond between them, if possible. In other words, a CH2-CH2 fragment may be substituted with a CH=CH fragment or a C≡C fragment, a CH2-CH fragment may be substituted with a CH=C fragment, and a CH-CH fragment may be substituted with a C=C fragment. Most preferably, no carbon-carbon double or triple bonds are present, except as explicitly shown in the structures of (D1) to (D67). In a preferred embodiment, R 1is selected from (D1) to (D61).

[0070]

[0070] Y 5 typically contains a carbonyl moiety as present in the parent anthracycline compound. Alternatively, the carbonyl group can be replaced with a methylene group, an imine group, or a hydrazone group. Hydrazones are cleaved under the low pH conditions of endosomes and / or lysosomes, but are stable in the blood circulation. The hydrazone moiety can be introduced by reacting the ketone of the parent anthracycline with YC(O)-NH-NH2. Preferably, Y 5 contains a carbonyl moiety or a hydrazone moiety, most preferably a carbonyl moiety. 5 are CH2-Y, C(O)-Y, C(=N(R 20 ))-Y, C(R 9 )=NY, or C(R 9 )=N(R 20 ), and preferably Y 5 is C(O)-Y, C(=N(R 20 ))-Y, C(R 9 )=NY, or C(R 9 )=N(R 20 ), and most preferably Y 5 is C(O)-Y.

[0071]

[0071] Y 5 When R contains an imine or hydrazone moiety, 9 is a substituent on a carbon, and R 20 is a substituent on the nitrogen. 9 is an OH group or O(CO)C 1~6 C optionally substituted with alkyl groups 1~4 alkyl, and R 20 is NR 4 -C(O)-N(R 4 )2, NR 4 -C(O)-Sp-N(R 4 )2, NR 4 -C(O)-R 12 , N.R. 4 -C(O)-Sp-R 12 where Sp, R4 and R 12 are as defined above. In this specification, Sp, R 4 and R 12 is as defined above. Preferably, R 9 is Me, CH2OH or CH2OC(O)C 1~6 alkyl, more preferably R 9 is Me, CH2OH or CH2OC(O)C4H9, most preferably R 9 is Me. R 20 is preferably NR 4 -C(O)-R 12 or NR 4 -C(O)-Sp-R 12 and most preferably R 20 is NR 4 -C(O)-Sp-R 12 Sp, R 4 and R 12 , as well as preferred embodiments thereof, are defined above. 20 In relation to R 4 is hydrogen and C 1~4 alkyl, more preferably H and Me, and most preferably R 4 is preferably Me. 20 In relation to 1~4 Preferably, Sp is alkylene, and most preferably, Sp is CH2. R 20 In relation to R 12 is N(C 1~4 alkyl)3 (+) , more preferably N(Me)3 (+) It is preferable that:

[0072]

[0072]

[0073] In a preferred embodiment, the compound represented by structure (1) can be conjugated to a cell-binding agent via Y. In this embodiment, R 1 Preferably, R does not contain a reactive moiety for conjugation to a cell-binding agent. 1 is selected from (D1) to (D52), wherein X3 is OR 6 , N(R 6 )2, N (+) (R 6 )3, SR 6 , S(O)R 6 , S(O)2R 6 is selected from Y 4 is NR 6 , N (+) (R 6 )2, S(O) and S(O)2, R 6 Each occurrence of C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C3-C 12 Cycloalkanes, C3-C 12 Cycloalkenyl, C3-C 12 are individually selected from cycloalkynyl, (hetero)aryl and PEG, i.e., R 6 is not hydrogen.

[0073]

[0074] In an alternative preferred embodiment, the compound represented by structure (1) is R 1 In this embodiment, R 1 contains a reactive moiety for conjugation to a cell-binding agent. 1 is preferably selected from (D10) to (D15), (D18) to (D26), (D31 to (D37), (D41) to (D44), (D48) and (D53) to (D61), and in the formula, X 3 are OH, NH2, and NHR 6 , N3, SH, and / or Y 4 is NH.

[0074]

[0075] The compound is R 1 When conjugated to a cell-binding agent via 5 is preferably selected from the structures (Y11) to (Y16) shown below: [ka]

[0075]

[0076] In a preferred embodiment, Y 5 is C(=N(R 20 ))-Y or C(R 9 )=N(R 20 ) and R 20 is NR 4 -C(O)-R 12 or NR 4 -C(O)-Sp-R 12 Hydrazone and ionic R 12 The combination of groups allows the ionic cap to prevent the payload from entering the cell when the payload is released prematurely, while the ionic R 12 The group improves the therapeutic window of the conjugates according to the invention because it reduces aggregation while still being bound to the antibody.

[0076]

[0077] In one embodiment, R 1 is not CH2CH2SH or benzyl when Y=CH2OH, more preferably R 1 is not CH2CH2SH or benzyl, regardless of Y. In one embodiment, R 1 is not unsubstituted ethyl, CHCHSH or benzyl when Y=CHOH, more preferably R 1 is not unsubstituted ethyl, CH2CH2SH, or benzyl, regardless of Y.

[0077]

[0078] The compounds according to the invention contain an oxane ring and a morpholine ring, which may be joined together in a "closed" tricyclic structure containing an intermediate oxazolidine ring, or the structure may be "open". 2 and R 3 is a substituent on the oxane ring and the morpholine ring. In one embodiment, R 2 and R 3 are fused together via an ether moiety, thus forming a five-membered oxazolidine ring. In an alternative embodiment, the ring structure is open and R 2 is H, S(O)2OH or P(O)2OH, and R 3is OH. In this specification, S(O)2OH and P(O)2OH may be in salt form. In the open ring form, R 2 is H and R 3 is preferably OH. In the most preferred embodiment, the structure is open and R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring.

[0078]

[0079] R 5 is a substituent on the outer phenyl ring of the tetracyclic moiety. 5 is either H or OCH. In a preferred embodiment, R 5 is OCH3. Preferably, R 2 is H and R 3 is OH, or R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring, and R 5 is OCH3, more preferably R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring, and R 5 is OCH3.

[0079]

[0080] Y is NR 4 -Sp 3 -N(R 4 )2, NR 4 -Sp 3 -X 2 (R 4 ), N(R 4 )2, R 12 , N.R. 4 -Sp 3 -X 2 -Sp 3’ -R 12 , OH, CH or CHOH. In a preferred embodiment, Y is NR 4 -Sp 3 -N(R 4 )2, NR 4 -Sp 3 -X 2 (R 4 ), N(R 4)2, CH3 or CH2OH, more preferably Y is NR 4 -Sp 3 -N(R 4 )2, N(R 4 )2, CH3 or CH2OH. 2 and R 4 is as defined above, including preferred embodiments thereof, and Sp 3 is a spacer. Spacer Sp 3 is preferably C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1~12 alkylene, wherein alkylene or (hetero)arylene is selected from halogen, X 2 R 4 , N(R 4 )2, C 1~4 optionally substituted with one or more substituents selected from alkyl and NO; 1~4 The alkyl substituent is NR 4 A ring structure formed by bonding with a moiety, particularly NR with the bond marked with * 4 The alkylene may optionally be formed with a pyrrolidine moiety, and the X 2 and NR 4 The spacer may be interrupted by one or more heteroatoms selected from the group consisting of: 3 As for C 1~4 Included are alkylenes, which are optionally substituted as defined above, where the substituents are R 4 The substituents may be joined together to form a ring structure. A preferred ring structure is a pyrrolidine ring, particularly Sp 3 -N(R 4 )2 together form a proline amino acid, i.e., Y=NR 4 -CH2-pyrrolidine-N*, where N* is R 4 The linker may contain a substituent group, but is connected to the cell binding agent. 4 is preferably CH3 or H.

[0080]

[0081] A particularly preferred option for Y is when the compound of general structure (1) is in free form or conjugated to a cell-binding agent without Y, and is NR 4 -(CH2)-N(R 4 )2, NR 4 -Sp 3 -X 2 (R 4 ), N(R 4 )2, CH3 or CH2OH.

[0081]

[0082] The compound represented by structure (1) may be in the amine form (N % =N) or N-oxide form (N % =N→O). When the compound represented by structure (1) is conjugated to a cell-binding agent, it is always in the amine form. In a preferred embodiment, the compound in free form is in the amine form, and N % In a particularly preferred embodiment, the compound is in the amine form (i.e., N % =N) and the structure is closed (i.e., R 2 and R 3 are fused together via an ether moiety to form an oxazolidine ring).

[0082]

[0083] Particularly preferred compounds of structure (1) are optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 )2 selected from R 1 Preferably, R 1 is arbitrarily substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, aryl, Bn, Sp-N3 and Sp-N(R 4 )2. As used herein, the optional substituents Sp, X 2 and R 4is as defined above, including preferred embodiments thereof. In the context of this embodiment, R 1 is optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, N-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 )2, more preferably i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, where Sp is C 1~4 Alkylene or C 1~4 More preferably, R 1 is i-Pr, Bn or Sp-N3, where Sp is CH2CH2, CH2CH2CH2 or CH2(Ph), where CH2(Ph) can be CH2(2-Ph), CH2(3-Ph) or CH2(4-Ph), preferably it is CH2(4-Ph). In one particularly preferred embodiment, R 1 is i-Pr, Bn, CH2CH2N3, CH2CH2CH2N3 or CH2((4-N3)Ph).

[0083]

[0084] The compound represented by structure (1) has a hydrophilic moiety R 12 The hydrophilic moiety is believed to reduce aggregation of the ADC and also improve efficacy and / or toxicity profiles. 12 is β-glucuronide acid, PO3 (2-) , OPO3 (2-) , CO2 (-) , SO3 (-) and N(C 1~4 alkyl)3 (+) wherein the anions may also be in their protonated form. In a preferred embodiment, the conjugates according to the invention are linked via Y and R 1 is Sp-R 12 or Sp-X 2 -Sp-R 12 More preferably, each Sp is individually C1-C5 alkyl and X 2 is NHC(O). Most preferably, R1 is selected from: [ka]

[0084]

[0085] In another embodiment, the compound represented by structure (1) comprises a hydrophilic moiety R 12 and the conjugate comprises R 1 wherein Y is preferably R 12 , Sp 3 R 12 or NR 4 -Sp 3 -X 2 -Sp 3’ -R 12 In this embodiment So, R 12 SO (3-) or N(C 1~4 alkyl)3 (+) More preferably, Y is NHCH2CH2NHC(O)CH2SO3 (-) , NHCH2CH2NHC(O)CH2NMe3 (+) , CH2SO3 (-) and CH2NMe3 (+) and even more preferably Y is selected from NHCH2CH2NHC(O)CH2SO3 (-) or NHCH2CH2NHC(O)CH2NMe3 (+) and Y 5 is C(O)-Y, or Y is CH2SO3 (-) or CH2NMe (3) and Y 5 contains a hydrazone group.

[0085]

[0086] The present inventors have obtained particularly beneficial results in terms of improved efficacy with compounds represented by structure (1). Therefore, in relation to conjugates represented by structure (2), further defined below, it is preferred that payload D is a compound according to this preferred embodiment. In these conjugates, the compound represented by structure (1) is 1 or may be linked via Y.1 is selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, Sp is C 1~4 Alkylene or C 1~4 More preferably, R 1 is i-Pr, Bn, or Sp-N3, where Sp is CH2CH2 or CH2(4-Ph).

[0086]

[0087] In one embodiment of the compound represented by structure (1), Y is CHOH and Y 5 is C(O)-Y, then R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl. Preferably, when Y is CH2OH, R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl. More preferably, R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl. Preferably, in the context of this embodiment, R 1 is not unsubstituted or substituted ethyl, CH2CH2SH or benzyl.

[0087]

[0088] Preferably, R 2 and R 3 are fused together through an ether moiety to form an oxazolidine ring, R 1 is not an alcohol, thiol or amine.

[0088] [Conjugates of general structure (2)]

[0089] In a first aspect, the present invention relates to a conjugate in which a compound represented by structure (1) is conjugated to a cell-binding agent via a linker. Such a conjugate typically has the general structure (2): CB-Z 1 -LZ 2 -D (2) (In the formula, - CB is a cell binding agent; D is a compound represented by structure (1); - L is a linker; -Z1 is a linking group that connects the cell-binding agent CB to the linker; -Z 2 is a linking group connecting compound D to the linker).

[0089] [Cell binding agent CB]

[0090] The conjugate according to the present invention contains a cell-binding agent capable of targeting cells, for example, by interacting with an extracellular receptor on the surface of the cell. The cell-binding agent is typically a peptide (e.g., an antibody), a small molecule, or an aptamer. Preferably, the cell-binding agent is a peptide, such as a polypeptide, capable of interacting with a specific receptor, which allows it to target specific cells. Advantageously, these specific cells are tumor cells. In the most preferred embodiment, the cell-binding agent (CB) is an antibody (Ab), typically an antibody capable of binding to a specific extracellular receptor on the surface of a cell, so that the antibody can target that specific cell.

[0090]

[0091] Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabodies, minibodies, affibodies, affilins, affimers, atrimers, finomers, cys-knots, DARPins, adnectins / centrins, knottins, anticalins, FN3, Kunitz domains, obodies, bicyclic peptides, and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of IgA, IgD, IgE, IgG, and IgM antibodies. Even more preferably, the Ab is an IgG antibody. The IgG antibody can be of any IgG isotype. The antibody can be any IgG isotype, for example, IgG1, IgG2, IgI3, or IgG4. Preferably, the Ab is a full-length antibody, but the Ab can also be an Fc fragment.

[0091]

[0092] The antibody Ab is typically specific for an extracellular receptor on a tumor cell, and preferably the extracellular receptor on a tumor cell is selected from the group consisting of 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, and CD33. , CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tenascin.

[0092]

[0093] The conjugates according to the invention comprise a linking group Z formed during the conjugation reaction. 1 wherein the cell binding agent, which may be suitably modified, contains LZ 2 -D. In the bioconjugation reaction, the reactive group F on the cell-binding agent reacts with the reactive group Q on the linker-toxin construct, thereby forming a covalent bond between the cell-binding agent and the toxin. A portion of the cell-binding agent is either the reactive group F or the linking group Z 1 a linker L connecting the peptide portion of the cell-binding agent to 6 Preferably, the linking group Z 1 is linked to the cell-binding agent CB via a lysine residue of the CB, a glutamine residue of the CB, a cysteine residue of the CB, a tyrosine residue of the CB, a threonine residue of the CB, or a glycan of the CB.

[0093]

[0094] Thus, the conjugate according to the invention preferably comprises: CB-[(L 6 ) b -{Z 1 -LZ 2 -D} x ] y (3) (In the formula, - b is 0 or 1; -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, where G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is —N(H)C(O)CH—, —N(H)C(O)CF—, or —CH—; - x is 1 or 2; - y is 1, 2, 3 or 4).

[0094] [Linker L 6 ]

[0095] When the reactive group F is directly linked to CB, CB can be replaced by F or Z 1 Linker L 6 (For conjugates of structure (1)) is absent and b=0. This is the case, for example, for cysteine and lysine conjugations. Alternatively, the reactive group F can also convert CB to F or Z. 1 Linker L 6 may be introduced onto the antibody using 6 exists and b=1. L 6 If present, the reactive group F is typically introduced into the glycan of the antibody. This is the case for conjugation via an artificially introduced reactive group F, for example, using transglutaminase, using sortase, or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase). For example, the modified sugar residue S(F) x can be introduced into the glycan, elongating the glycan with one monosaccharide residue S, thereby introducing x reactive groups F onto the antibody glycan. In the most preferred embodiment, conjugation occurs via the antibody glycan, and b=1. The site of conjugation is preferably in the heavy chain of the antibody.

[0095]

[0096] If present, L 6 CB is F or Z 1 -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7is —N(H)C(O)CH—, —N(H)C(O)CF— or —CH—. Typically, L 6 is formed at least in part by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at an asparagine residue at or near position 297 (Kabat numbering) of the heavy chain, which is decorated with complex-type glycans. This naturally occurring glycosylation site of the antibody is preferably used, although other glycosylation sites, including artificially introduced ones, may also be used in the linker L. 6 Thus, in a preferred embodiment, L 6 is linked to an amino acid of the antibody located at a position in the range of 250 to 350 of the heavy chain, preferably at a position in the range of 280 to 310 of the heavy chain, more preferably at a position in the range of 295 to 300 of the heavy chain, and most preferably at position 297 of the heavy chain.

[0096]

[0097] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan or a part thereof. Thus, the -GlcNAc(Fuc) of the glycan w -(G) j - is typically derived from the original antibody, where GlcNAc is an N-acetylglucosamine moiety and Fuc is a fucose moiety. Fuc is typically attached to GlcNAc via an α-1,6-glycosidic bond. Usually, the antibody may be fucosylated (w=1) or non-fucosylated (w=0). In the context of the present invention, the presence of the fucosyl moiety is irrelevant, and similar effects are obtained with fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue, sometimes referred to as the core-GlcNAc residue, is the monosaccharide that is directly attached to the peptide portion of the antibody.

[0097]

[0098] S is core-GlcNAc(Fuc) w The moiety can be directly linked to the core-GlcNAc(Fuc) moiety, i.e., j=0, which means that the remainder of the glycan is core-GlcNAc(Fuc) before S is attached. wSuch glycan trimming is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, the core-GlcNAc(Fuc) w There are one or more monosaccharide residues between the moiety and S, i.e., j is an integer ranging from 1 to 10, preferably j=2 to 5. In a preferred embodiment, (G) j is an oligosaccharide fraction containing j monosaccharide residues G, where j is an integer ranging from 2 to 5. (G) j is typically linked to GlcNAc(Fuc) via a β-1,4-linkage w In a preferred embodiment, j is 3, 4, or 5. While any monosaccharide that may be present in a glycan can be used as G, each G is preferably individually selected from the group consisting of galactose, glucose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and N-acetylneuraminic acid. More preferred options for G are galactose, N-acetylglucosamine, and mannose. Antibodies and antibody conjugates with j = 0 exhibit no or significantly reduced binding to Fc-γ receptors, while antibodies and antibody conjugates with j in the range of 4 to 10 bind to Fc-γ receptors. Therefore, by selecting a specific value for j, the desired degree of binding to Fc-γ receptors can be obtained. Therefore, j = 0, 4, 5, 6, 7, 8, 9, or 10 is preferred, more preferably j = 0, 4, or 5, and most preferably the antibody is trimmed and j = 0.

[0098]

[0099] S is a sugar or sugar derivative. The term "sugar derivative" is used herein to refer to a monosaccharide sugar, i.e., a derivative of a monosaccharide containing a substituent and / or functional group. Suitable examples of S include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugars and sugar acids, such as glucosamine (GlcNH), galactosamine (GalNH), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), also known as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Preferably, S is selected from Glc, Gal, GlcNAc, and GalNAc. In a particularly preferred embodiment, S is GalNAc.

[0099]

[0100] x is a linking group Z bonded to the sugar (derivative) S 1 or an integer indicating the number of reactive groups F. Thus, the antibody preferably contains a moiety S comprising x reactive moieties F. Each of these reactive groups F is reacted with a reactive moiety Q of a linker-toxin construct such that x linking groups Z are formed, and x compounds of general structure (1) are attached to a single occurrence of S. x is 1 or 2, preferably x=1.

[0100]

[0101] Linking group Z 1 Alternatively, the reactive group F may be directly bonded to S or may be bonded to S and Z 1 or a linker L between F 7 may be present. Therefore, L 7 may be present (w'=1 or 2) or absent (w'=0). Typically, each moiety Z is 7 and thus in one embodiment, x with w'=0. Preferably, L 7 is absent and each linking moiety Z is directly bonded to S. If present, L 7may be selected from -N(H)C(O)CH-, -N(H)C(O)CF- or -CH-. In a preferred embodiment, x=1 and w'=0 or 1, most preferably x=1 and w'=0.

[0101]

[0102] y is an integer indicating the number of sugar (derivative) S, each bearing x reactive groups F, or linked to x linking groups Z1, which are linked to CB. y is 1, 2, 3, or 4, preferably y=2 or 4, and most preferably y=1. Thus, the antibody contains y moieties S, each of which contains x reactive groups F. Each of these reactive groups F is reacted with a reactive moiety Q of the linker-toxin construct, thereby forming an x×y linking group Z 1 is formed, and x×y compounds of general structure (1) are attached to a single CB. Each linker-toxin construct can contain multiple payloads, for example, by branching nitrogen atoms N* in L. Preferably, each linker-toxin construct contains one or two occurrences of D, most preferably one occurrence of D. In particularly preferred embodiments, the linker L 1 contains a branched nitrogen atom N* to which the second occurrence of D is attached.

[0102]

[0103] The amount of toxin D (a compound represented by general structure (1)) conjugated to a single antibody is known in the art as the DAR (drug-to-antibody ratio). In the context of the present invention, the DAR is preferably an integer ranging from 1 to 8, more preferably 2 or 4, and most preferably DAR=2. In other words, the DAR is preferably an integer ranging from (x×y) to [(x×y)×2], and most preferably DAR=[(x×y)×2]. For the preferred values where x is 1 and y is 1, the DAR is preferably 2. It will be understood that these are theoretical DAR values; in practice, the DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as a stochastic mixture of antibody-drug conjugates, and DAR values vary between individual conjugates. Depending on the conjugation technique used, the DAR may have a wide distribution (e.g., DAR=0-10) or a narrow distribution (e.g., 3-4). In such mixtures, the DAR often refers to the average DAR of the mixture. This is well known in the art of bioconjugation. However, when conjugation occurs via glycans (i.e., b=1 and L 6 is present), the conjugates according to the invention have a DAR close to the theoretical. For example, if the theoretical DAR is 4, AR values greater than 3.6, or even greater than 3.8, are readily obtained, indicating that most of the antibodies in the reaction mixture are fully reacted and have a DAR of 4.

[0103] [Linking group Z 1 ]

[0104] Z 1is a linking group that covalently links both parts of the conjugate according to the present invention. The term "linking group" as used herein refers to a structural element resulting from the reaction between Q and F, which links one part of a conjugate with another part of the same conjugate. As will be understood by those skilled in the art, the nature of the linking group will depend on the type of reaction that results in the link between the parts of said compound. As an example, when the carboxyl group of RC(O)-OH is reacted with the amino group of HN-R' to form RC(O)-N(H)-R', R is linked to R' via the linking group Z, and Z can be represented by a -C(O)-N(H)- group. The linking group Z 1 is derived from the reaction between Q and F and can therefore take any form.

[0104]

[0105] Because two or more reactive groups F may be present in or introduced into an antibody, the antibody-conjugate according to the present invention may contain two or more payloads D, for example, 1 to 8 payloads D, preferably 1, 2, 3, or 4 payloads D, more preferably 2 or 4 payloads D, per biomolecule. The number of payloads is typically an even number, taking into account the symmetry of the antibody. In other words, if one side of the antibody is functionalized with F, the symmetrical counterpart will also be functionalized. Alternatively, if the naturally occurring thiol group of a cysteine residue of a protein is used as F, the value of m can be any and can vary between individual conjugates.

[0105]

[0106] In the compound represented by structure (1), the linking group Z 1 is optionally linked to L via a linker L 6 D is linked to CB via a number of reactions known in the art for the attachment of reactive group Q to reactive group F. Thus, a wide variety of linking groups Z 1 may be present in the conjugates according to the invention. In one embodiment, the reactive group Q is selected from the options described above, preferably a complementary reactive group F and the resulting linking group Z, as shown in Figures 2, 4 or 5. 1When a linker-conjugate containing Q is conjugated to a biomolecule containing a complementary reactive group F, F and a linking group Z that will be present in the bioconjugate 1 Some examples of suitable combinations are shown in FIG.

[0106]

[0107] For example, when F contains or is a thiol group, the complementary group Q includes an N-maleimidyl group, an alkenyl group, and an allenamide group. For example, when F contains or is an amino group, the complementary group Q includes a ketone group and an activated ester group. For example, when F contains or is a ketone group, the complementary group Q includes an (O-alkyl)hydroxylamino group and a hydrazine group. For example, when F contains or is an alkynyl group, the complementary group Q includes an azide group. For example, when F contains or is an azide group, the complementary group Q includes an alkynyl group. For example, when F contains or is a cyclopropenyl group, trans-cyclooctene group, cycloheptyne, or cyclooctyne group, the complementary group Q includes a tetrazinyl group. In these particular cases, as shown in Figure 4, Z is merely an intermediate structure that releases N2, thereby generating a dihydropyridazine (from reaction with an alkene) or a pyridazine (from reaction with an alkyne).

[0107]

[0108] Further suitable combinations of F and Q and the resulting linking groups Z 1The properties of are known to those skilled in the art and are described, for example, in Hermanson, "Bioconjugate Techniques", Illesvier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), in particular Chapter 3, pages 229-258 (incorporated by reference). A list of complementary reactive groups suitable for bioconjugation processes is disclosed in Table 3.1, pages 230-232, Chapter 3 of GT Hermanson, "Bioconjugate Techniques", Illesvier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), the contents of which are expressly incorporated herein by reference.

[0108]

[0109] In a preferred embodiment, the linking group Z 1 is obtained by cycloaddition or nucleophilic reaction, preferably the cycloaddition is a [4+2] cycloaddition or a 1,3-dipolar cycloaddition, or the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Such cycloaddition or nucleophilic reaction occurs via a reactive group Q linked to D via reactive groups F and L linked to S. Conjugation reactions via cycloaddition or nucleophilic reaction are known to those skilled in the art, and they can select appropriate reaction partners F and Q to obtain the resulting linking group Z. 1 will understand the nature of

[0109]

[0110] In a first preferred embodiment, Z 1is formed by cycloaddition. Preferred cycloadditions are (4 + 2)-cycloadditions (e.g., Diels-Alder reaction) or (3 + 2)-cycloadditions (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably wherein Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art, and those skilled in the art will recognize how to carry them out.

[0110]

[0111] Preferably, Z 1 contains a moiety selected from the group consisting of triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, piperazine, thioether, amide, or imide group. The triazole moiety is 1 In one embodiment, Z 1 is formed from Q containing a (hetero)cycloalkene moiety, i.e., Q containing a (hetero)cycloalkyne moiety. In an alternative embodiment, Z 1 is formed from Q containing a (hetero)cycloalkane moiety, i.e., Q containing a (hetero)cycloalkene moiety. As used herein, aromatic rings such as triazole rings are considered heterocycloalkane rings because they are formed by the reaction of an alkyne moiety with an azide moiety. In a preferred embodiment, Z 1 has the structure (Z1): [ka]

[0111]

[0094] In this specification, [ka] A bond shown as is a single or double bond. Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za) to (Zj) as defined below, where the carbon atom marked with ** is the carbon atom of (Z1) to which ring Z is fused. [ka] corresponding to the two carbon atoms of the bond shown as: -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and are selected from the group consisting of two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31 where S (+) is B (-)is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked via L; - u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4 or 5, where u+u'=0, 1, 2, 3, 4, 5, 6, 7 or 8; - v=an integer in the range 8 to 16; Ring Z is formed by cycloaddition and is preferably selected from (Za) to (Zj).

[0112] In a preferred embodiment, u+u'=0, 4, 5, 6, 7 or 8, more preferably 0, 4 or 5. [ka] When the bond shown as is a double bond, it is preferred that u+u'=4, 5, 6, 7 or 8, more preferably u+u'=4 or 5. [ka] When the bond shown as is a single bond, it is preferred that u+u'=0 or 5. Preferably, the wavy bond marked with * is optionally L 6 The bond is connected to CB via , and the wavy bond labeled with ** is connected to L.

[0113]

[0096] Z 1 It is particularly preferred that the formula (I) contains a (hetero)cycloalkene moiety, i.e. [ka] In a preferred embodiment, the bond shown as Z 1 is selected from structures (Z2) to (Z20) shown below: [ka]

[0114]

[0097] In this specification, the connection to L is shown as a wavy bond. (-) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by a cycloaddition reaction and is preferably triazole, cyclohexene, cyclohexadiene, [2.2.2]-bicyclooctadiene, [2.2.2]-bicyclooctene, isoxazoline, isoxazolidine, pyrazoline, or piperazine. Most preferably, ring Z is a triazole ring. Ring Z may have a structure selected from (Za) to (Zj) shown below, where the carbon atoms marked with ** correspond to two carbon atoms of the (hetero)cycloalkane ring of (Z2) to (Z20) to which ring Z is fused. Since the linking group Z is formed by reaction with a (hetero)cycloalkyne in the context of this embodiment, [ka] The bond shown as is a double bond. [ka]

[0115] In a further preferred embodiment, Z 1 is selected from structures (Z21) to (Z38) shown below: [ka]

[0116]

[0099] In this specification, the connection to L is shown as a wavy bond. Structure (Z29) can be in the endo or exo configuration, preferably in the endo configuration. In structure (Z38), B (-) is an anion, preferably a pharmaceutically acceptable anion. Ring Z is selected from structures (Za) to (Zj) defined above.

[0117] In a preferred embodiment, Z 1 Z preferably comprises a (hetero)cyclooctene or (hetero)cycloheptene moiety represented by the structure (Z8), (Z26), (Z27), (Z28) or (Z37), which is optionally substituted. 1 Each of these preferred options for is further defined herein below.

[0118]

[0101] Therefore, in a preferred embodiment, Z 1 comprises an optionally substituted heterocycloheptene moiety represented by structure (Z37). Preferably, the heterocycloheptene moiety represented by structure (Z37) is unsubstituted.

[0119] In a preferred embodiment, Z 1 comprises a (hetero)cyclooctene moiety represented by structure (Z8), or more preferably (Z29), which is optionally substituted. Preferably, the cyclooctene moiety represented by structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z 1 preferably comprises a (hetero)cyclooctene moiety represented by the structure (Z39) shown below, wherein V is (CH) l wherein l is an integer ranging from 0 to 10, preferably from 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably, l is 0, 1 or 2, and most preferably, l is 0 or 1. In relation to group (Z39), l is most preferably 1. Most preferably, Z 1 is represented by structure (Z42), further defined below.

[0120] In an alternative preferred embodiment, Z 1 comprises a (hetero)cyclooctene moiety represented by the structure (Z26), (Z27) or (Z28), which is optionally substituted. 1preferably comprises a (hetero)cyclooctene moiety represented by the structure (Z40) or (Z41) shown below, wherein Y 1 is O or NR 11 and R 11 are independently hydrogen, straight-chain or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring of (Z40) is optionally O-sulfonylated at one or more positions, while the ring of (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety represented by structure (Z40) or (Z41) is not further substituted. Most preferably, Z is represented by structure (Z43), further defined below.

[0121] In an alternative preferred embodiment, Z 1 contains a heterocycloheptenyl group and is represented by structure (Z37). [ka]

[0122] In a particularly preferred embodiment, Z 1 contains a cyclooctenyl group and is represented by structure (Z42): [ka] During the ceremony, - the bond labeled * is connected to CB, and the wavy bond labeled ** is connected to L; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and are selected from the group consisting of two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is a second occurrence of Z (or Q) or D linked via a spacer moiety; - l is an integer ranging from 0 to 10.

[0123] In a preferred embodiment of the group represented by structure (Z42), R 15 is hydrogen, halogen, -OR 16, a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 15 is hydrogen or C1-C6 alkyl, more preferably R 16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the group represented by structure (Z42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the group represented by structure (Z42), R 19 is H. In preferred embodiments of the group represented by structure (Z42), i is 0 or 1, and more preferably, l is 1.

[0124] In a particularly preferred embodiment, Z 1 contains a (hetero)cyclooctenyl group and is represented by structure (Z43): [ka] During the ceremony, - the bond labeled * is connected to CB, and the wavy bond labeled ** is connected to L; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and are selected from the group consisting of two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y is N or CR 15 is.

[0125] In a preferred embodiment of the group represented by structure (Z43), R 15 is hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 is hydrogen and -S(O)3 (-) In preferred embodiments of the group represented by structure (Z43), Y is N or CH, more preferably Y=N.

[0126] In a particularly preferred embodiment, Z 1 contains a heterocycloheptenyl group and is according to structure (Z37), where ring Z is a triazole. [ka]

[0127] In an alternative preferred embodiment, Z 1 represents a (hetero)cycloalkane moiety, i.e., [ka] The bond shown as is a single bond. The (hetero)cycloalkane group may also be referred to as a heterocycloalkyl group or a cycloalkyl group, preferably a cycloalkyl group, where the (hetero)cycloalkyl group is optionally substituted. Preferably, the (hetero)cycloalkyl group is a (hetero)cyclopropyl group, a (hetero)cyclobutyl group, a norbornyl group, a norbornenyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group (all of which may be optionally substituted). Particularly preferred are a (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group, where the (hetero)cyclopropyl group, a (hetero)cycloheptyl group, or a (hetero)cyclooctyl group is optionally substituted. Preferably, Z 1 includes a cyclopropyl moiety represented by structure (Z44), a heterocyclobutane moiety represented by structure (Z45), a norbornane or norbornene group represented by structure (Z46), a (hetero)cycloheptyl moiety represented by structure (Z47), or a (hetero)cyclooctyl moiety represented by structure (Z48). 3 is C(R 23 )2, NR 23 or O, and each R 23 are individually hydrogen, C1-C6 alkyl, or optionally linked to L via a spacer and labeled [ka] is a single bond or a double bond. In a further preferred embodiment, a cyclopropyl group is represented by structure (Z49). In another preferred embodiment, a (hetero)cycloheptane group is represented by structure (Z50) or (Z51). In another preferred embodiment, a (hetero)cyclooctane group is represented by structure (Z52), (Z53), (Z54), (Z55), or (Z56). [ka]

[0128]

[0111] In this specification, the R group on Si in (Z50) and (Z51) is typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is selected from structures (Zk) to (Zn), and the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring of (Z44) to (Z56) to which ring Z is fused, and the carbon atom marked with * is connected to CB. Linking group Z 1 is formed in the context of this embodiment by reaction with a (hetero)cycloalkene, [ka] The bond shown above as is a single bond. [ka]

[0129] In a second preferred embodiment, Z 1 is formed by a nucleophilic reaction, preferably by nucleophilic substitution or Michael addition, preferably by Michael addition. A preferred Michael reaction is thiol-maleimide ligation, most preferably Q is maleimide and F is a thiol group, the thiol being part of a disulfide bridge. Preferably, the thiol is present in the side chain of a cysteine residue. Such a conjugation reaction with a thiol may also be referred to as thiol alkylation or thiol arylation. In a preferred embodiment, the linking group Z 1 contains a succinimidyl ring or its ring-opened succinamide derivative, which can be formed by hydrolysis of the succinimidyl ring.

[0130]

[0113] Or, Z 1is formed by a nucleophilic reaction at the amino group in the side chain of a lysine residue (F), which can be reacted with an amino-reactive group Q. Such a conjugation reaction with a thiol can also be referred to as amide bond formation or carbamate bond formation. Exemplary amino-reactive groups Q include N-hydroxysuccinimidyl (NHS) ester, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanate, isothiocyanate, and benzoyl halides.

[0131]

[0114] Linking group Z 1 Preferred options for include moieties selected from (Z57) to (Z71) shown herein below. [ka]

[0132]

[0115] In the present specification, the wavy bond marked with * in (Z57) to (Z66) is a wavy bond that is linked to CB and is not labeled to a payload via a linker L. Furthermore, R 29 is C 1~12 Alkyl or 1 to 24 polyethylene glycol units, preferably C 1~4 alkyl or 6 to 14 polyethylene glycol units, most preferably ethyl or 12 polyethylene glycol units; X 1 is O or S, preferably X 1 =O. Or, R 29 is C 1~12 Alkyl, preferably C 1~4 alkyl, most preferably ethyl; X 1 is O or S, preferably X 1 =O. The nitrogen atoms marked with ** in (Z67) to (Z71) correspond to the nitrogen atoms of the side chains of lysine residues of the antibody, and contain an unlabeled wavy bond to the payload via the linker L. The carbon atoms of the phenyl groups in (Z69) and (Z70) are optionally substituted, and preferably optionally fluorinated.

[0133] In a preferred embodiment, the linking group Z 1 contains a moiety selected from (Z1) to (Z71).

[0134] [Linker L] The linker L connects the payload D to the linking group Z 2 via the linking group Z 1 (in a conjugate according to the invention), or the payload D is linked to the reactive group Q (in a linker-toxin construct). Linkers are known in the art and may be cleavable or non-cleavable. The linker L preferably contains a self-immolative group or cleavable linker comprising a peptide spacer and optionally a para-aminobenzyloxycarbonyl (PABC) moiety or derivative thereof.

[0135] In a preferred embodiment, the structure -(L 1 ) n -(L 2 ) o -(L 3 ) p -(In the formula, (L 3 ) p is the linking group Z 2 Connected to payload D via (L 1 ) n is Z 1 or Q. As used herein, L 1 , L 2 and L 3 is a linker or linking unit, and each of n, o, and p is individually 0 or 1, and n+o+p is at least 1. In a preferred embodiment, at least the linker L 1 and L 2 is present (i.e., n=1; o=1; p=0 or 1), and more preferably, a linker L 1 , L 2 and L 3 (i.e., n=1; o=1; p=1).

[0136]

[0119] Thus, in the conjugate according to the present invention, LZ 2has the following structure: *-NR 4 -Sp 3 -NR 4 -(L 3 ) p -(L 2 ) o -(L 1 ) n- ** (In the formula, - The bond marked with * is connected to the compound represented by structure (1); - Bonds marked with ** are linking groups Z 1 Concatenated to; -Sp 3 is C 1~12 (Hetero)alkylene, (hetero)arylene, C 1~12 (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1~12 alkylene, wherein the alkylene or (hetero)arylene is selected from the group consisting of halogen, X 2 R 4 , N(R 4 )2, C 1~4 optionally substituted with one or more substituents selected from alkyl and NO; 1~4 The alkyl substituent is NR 4 A ring structure formed by bonding with a moiety, particularly NR with the bond marked with * 4 The alkylene may optionally be formed with a pyrrolidine moiety, and the X 2 and NR 4 may be interrupted by one or more heteroatoms selected from: -R 4 and X 2 is as defined in claim 1; -L 1 , L 2 and L 3 are each individually, Z 1 is a linker that connects together D; - n, o and p are each independently 0 or 1, with the proviso that n+o+p=1, 2 or 3.

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

[0138] [Linker L 1 ] Linker L 1 is either absent (n=0) or present (n=1). Preferably, the linker L 1 where n=1. L 1 is, for example, a linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylene group, C3-C 200 Cycloalkylene group, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkylarylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, and the heteroatoms are preferably O, S(O), y’ and NR 21 wherein y' is 0, 1 or 2, preferably y'=2; 21 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups.

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

[0140]

[0123] With respect to the polar groups defined herein above, which terminus is Z 1 Which end is linked to (L 2 ) o It doesn't matter whether it's connected to

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

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

[0143]

[0126] The wavy lines indicate the linkages, typically Q and L, to the remainder of the compound. 2 , L 3 or D, preferably Q and L 2Preferably, ((O) a The C(O) moiety is linked to Q and NR 13 Part L 2 , L 3 or D, preferably L 2 is linked to.

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

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

[0146] In a preferred embodiment, L 1is represented by structure (24): [ka]

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

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

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

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

[0151] In these preferred embodiments, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are unsubstituted and are substituted with O, S, and NR 16 , optionally interrupted by one or more heteroatoms selected from the group of O, 16 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

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

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

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

[0155]

[0138] In relation to this embodiment, the wavy line in structure (23) represents (W) k , (B) e and (C(O)) g A represents a linkage to an adjacent group such as structure (23) where a=1 and R 13 =H or C1~C 20 is an alkyl group, more preferably R 13 =H or methyl, most preferably R 13 =H).

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

[0157]

[0140] In this specification, when d and / or f=1, a=1 and R 13 Preferably, =H. Most preferably, the linker is structure (A).

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

[0159] Preferred linkers L containing branched nitrogen atoms 1 is the structure - (W) k -(A) d -(B) e -(A) f -(C(O)) g -N*[-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, wherein (i) k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is represented by structure (23), a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2. (j) k = d = g = e' = g' = 1; f = d' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is represented by structure (23), a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2.

[0160] [Linker L 2 ] Linker L 2 is a peptide spacer. Linker L 2 is either absent (o=0) or present (o=1). Preferably, the linker L 2 is present and o=1. Peptide spacer L 2 and a cleavable linker L 3 are well known in the art. However, in the conjugates according to the invention, the same moiety is used in conjunction with the payload D, in particular R 1 or Y, so L 3 For example, the presence of the moiety CH2-Ph-NH-L 2 (Wherein CH2-Ph-NH is R 1 If the compound contains 3 The presence of the additional para-aminobenzyl moiety in is not required for the linker to be self-immolative. Thus, in one preferred embodiment, L 3 does not exist, and L 2 is preferably R 1 or is directly bonded to D via Y.

[0161] The peptide spacer can also be (NH-CR 17 -CO) n where R 17represents an amino acid side chain known in the art. As used herein, amino acids may be natural or synthetic amino acids. Examples of preferred synthetic amino acids are citrulline and cysteic acid. Preferably, all amino acids are in the L-configuration. n is an integer ranging from 1 to 5, preferably from 2 to 4. Thus, the peptide spacer contains 1 to 5 amino acids. Preferably, the peptide is a dipeptide (n=2), tripeptide (n=3), or tetrapeptide (n=4), and most preferably, the peptide spacer is a dipeptide. Although any peptide spacer can be used, preferably the peptide spacer is Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Glu-Val-Ala, Asp-Val-Ala, iGlu-Val-Ala, Glu-Val-Cit, Asp-Val-Cit, iGlu-Val-Cit, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, Phe-Phe, Gly, Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly, Leu-Gly, Tyr-Gly,

[0049] The amino acid sequence is selected from Ala-Gly, Pro-Gly, Phe-Gly, Phe-Gly, Ser-Gly, Gly-Phe-Gly, Gly-Gly-Phe-Gly, Gly-Phe-Gly-Gly, Phe-Gly-Gly-Gly, Gly-Gly-Gly-Phe, Phe-Phe-Gly-Gly, Gly-Gly-Phe-Phe, Gly-Gly-Gly-Phe-Gly and Lys, more preferably Val-Cit, Val-Ala, Glu-Val-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. As used herein, AcLys is ε-N-acetyl lysine and iGlu is isoglutamate. 2 In one embodiment, L 2=Val-Ala.

[0162]

[0145] R 17 represents an amino acid side chain selected from the side chains of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, acetyllysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, tyrosine, and citrulline. Preferred amino acid side chains are those of Val, Cit, Ala, Lys, Arg, AcLys, Phe, Leu, Ile, Trp, Glu, Asp, and Asn, and more preferably those derived from the side chains of Val, Cit, Ala, Glu, and Lys. In other words, R 17 is preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred embodiments of R are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu) and CH(CH3)2(Val). Most preferably, R 17 is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0163] In a particularly preferred embodiment, the peptide spacer is represented by the general structure (25): [ka]

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

[0165] [Linker L 3 ] Linker L 3 is a self-cleavable spacer, also called a self-immolative spacer. 3 is either absent (p=0) or present (p=1). Preferably, the linker L 3 However, there exists a payload of L 2 Z is NH linked to the C(O) terminus of the peptide spacer 2 is connected to the linker via R 1 or when Y (depending on the location of the point of attachment to the compound of structure (1)) contains an aromatic ring as defined herein below for ring A, L 3 is absent and p=0. 2 , Z 2 and Y or R 1 The properties of L 3 This ensures that the linker L is self-cleavable even in the absence of

[0166] Preferably, L 3 is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative represented by structure (26): [ka]

[0167]

[0150] In this specification, the wavy line represents Q or Z. 1 , L 1 or L 2 , and Z 2 Typically, the PABC derivatives are linked to Q, Z via NH. 1 , L 1 or L 2 Preferably, L 2 and Z through OC(O) 2 is linked to.

[0168]

[0151] Ring A is a 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Preferred 5-membered rings are oxazole, thiazole and furan. Preferred 6-membered rings are phenyl and pyridyl. Ring A may contain halogen, X 2 R 4 , N(R 4 )2, C 1~4 As used herein, X may be substituted with a substituent selected from alkyl and NO. 2 and R 4 is as defined above, including preferred embodiments thereof. In preferred embodiments, the optional substituents are F, Cl, Br, OH, OR 4 , SH, NH2, Et, Me, and NO2. In particularly preferred embodiments, ring A contains 0 to 2 substituents, more preferably 0 or 1 substituent, and most preferably ring A is unsubstituted. In preferred embodiments, ring A is 1,4-phenyl, 1,2-phenyl, 2,5-pyridyl, or 3,6-pyridyl. Most preferably, A is 1,4-phenyl.

[0169]

[0152] R 21 H, R 26 , C(O)OH and C(O)R 26 where R 26 is C1~C 24 (Hetero)alkyl groups, C3-C 10(Hetero)cycloalkyl groups, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero)aryl groups and C3-C 10 (hetero)arylalkyl groups, which are O, S and NR 28 and optionally substituted and optionally interrupted by one or more heteroatoms selected from 28 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. 26 is C3~C 10 (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H. The polyalkylene glycol is most preferably polyethylene glycol, preferably —(CHCHO) s H, where s is an integer ranging from 1 to 10, preferably from 1 to 5, and most preferably s=1, 2, 3 or 4. More preferably, R 21 is H or C(O)R 26 where R 26 = 4-methyl-piperazine or morpholine. Most preferably, R 21 is H.

[0170] [Linking group Z 2 and Payload D] D, also referred to in the art as "payload," represents a compound that is or will be linked to CB. D is a compound represented by structure (1) and its preferred embodiments as defined above and below.

[0171] A conjugate according to the present invention may comprise two or more payloads D. When two or more payloads are present, the payloads D may be the same or different, and are typically the same. In the context of the present invention, at least one payload should be a compound represented by structure (1). In a preferred embodiment, the conjugate contains two or four occurrences of D, most preferably two occurrences of D. The second occurrence of D may be within a linker L, which may contain a branching moiety, typically a branching nitrogen atom, linked to the second occurrence of D. Preferably, both occurrences of D are linked to the branching moiety via the same linker. Similarly, a conjugate according to the present invention may comprise a linking group Z 1 Each may contain more than one payload.

[0172] The payload comprises a linking group Z formed by reaction of a compound represented by structure (1) with a linker unit. 2 Thus, the reactive moiety of the compound represented by structure (1) is reacted with the reactive moiety of the linker. 2 The nature of the linking group Z can take any form, depending on the nature of the reactive moiety and the type of reaction performed to link the linker to the payload. 2 is selected from the group consisting of an amide moiety, an ester moiety, a carbamate moiety, a carbonate moiety or a (hetero)aryl moiety, more preferably an amide moiety or a carbamate moiety. When the payload is linked via Y, the linking group Z 2 is most preferably an amide moiety. 1 When the linking group is Z 2 is most preferably a carbamate moiety.

[0173] Conveniently, the reactive moiety of the compound represented by structure (1) is R 1 In particular, R 1 =Sp-N(R 4 )2, or in Y, in particular, Y=N(R 4 )2 or NR 4 -Sp3 -N(R 4 )2 is an amine moiety. 4 The group is replaced by a linkage to L. Thus, the remaining NR 4 The residue is Z, which is formed when the amine group is reacted with the linker. 2 and therefore typically has the structure -(O) a’ -C(O)-NR 4 - (wherein a'=0 or 1), or form an amide or carbamate moiety represented by R 1 contains an azide moiety, a cycloaddition reaction can be carried out to form the linking group Z 2 In this embodiment, Z 2 contains a (hetero)aryl moiety and is not related to the above linking group Z as far as the cycloaddition reaction with the azide moiety is concerned. 1 As defined for

[0174] In the context of the present invention, the link between the compound of structure (1) and the linker L is preferably R 1 Or through Y.

[0175]

[0158] Thus, in a preferred embodiment, the compound represented by structure (1) is linked to the linker L via Y. The present inventors have discovered that by conjugating the compound represented by structure (1) via Y, the substituent R 1 It has been found that the compound represented by structure (1) can be used to modulate or improve the efficacy of the toxin and therefore the efficacy of the entire conjugate. Therefore, it is preferred that the compound represented by structure (1) is linked to the conjugate according to the present invention via Y. Linkage via Y is achieved by linking R 1 optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl and Sp-X 2 R 4 In a particularly preferred embodiment, the link to the compound of structure (1) is through Y and R1 is selected from (D1) to (D52) defined above, more preferably optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 It is selected from alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, preferably from i-Pr, n-Pr, t-Bu, i-Bu, n-Bu and Bn, more preferably from i-Pr and Bn.

[0176] In an alternative embodiment, the compound represented by structure (1) is R 1 is connected to the linker L via R 1 Such a linkage via R is particularly preferred when the compound represented by structure (1) is according to one of the preferred embodiments specified above. 1 The connection via R 1 is particularly preferred when it contains a reactive moiety suitable for linking to a linker L, such as an N, NH, or OH moiety. In a particularly preferred embodiment, the link to the compound represented by structure (1) is 1 It is due to R 1 is selected from (D10) to (D15), (D18) to (D26), (D31 to (D37), (D41) to (D44), (D48) and (D53) to (D61), as defined above, more preferably R 1 Sp-X 2 R 4 , Sp-N3 or Sp-N(R 4 )2, most preferably Sp-N3 or Sp-N(R 4 )2.

[0177] In a particularly preferred embodiment, the conjugate according to the invention comprises optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 )2 1 Preferably, the payload D contains R1 is optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, aryl, Bn, Sp-N3 and Sp-N(R 4 )2. As used herein, the optional substituents Sp, X 2 and R 4 is as defined above, including preferred embodiments thereof. In the context of this embodiment, R 1 is optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, N-pentyl, C 6~12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, Sp-OR 4 , Sp-N3 and Sp-N(R 4 )2, more preferably i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, where Sp is C 1~4 Alkylene or C 1~4 More preferably, R 1 is i-Pr, Bn or Sp-N3, where Sp is CH2CH2, CH2CH2CH2 or CH2(Ph), where CH2(Ph) can be CH2(2-Ph), CH2(3-Ph) or CH2(4-Ph), preferably it is CH2(4-Ph). In one particularly preferred embodiment, R 1 is i-Pr, Bn, CH2CH2N3, CH2CH2CH2N3 or CH2((4-N3)Ph).

[0178] The present inventors have obtained particularly beneficial results in terms of improved efficacy with these compounds of structure (1). In these conjugates, the compound of structure (1) is 1 or may be linked via Y. 1 is selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, Sp is C 1~4 Alkylene or C 1~4 More preferably, R 1 is i-Pr, Bn, or Sp-N3, where Sp is CH2CH2 or CH2(4-Ph).

[0179] Preferred Linkers

[0162] According to a preferred embodiment, LZ 2 The linking moiety of the conjugate according to the present invention, represented by [ka]

[0163] In the formula, - Bonds marked with ** are linked to the linking group Z 1 Concatenated to; - Each R 13 are hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, wherein C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups include O, S and NR 14 and optionally substituted and optionally interrupted by one or more heteroatoms selected from 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is D linked to N via a spacer moiety, and R 13 = hydrogen or D linked to N via a spacer moiety, preferably the spacer moiety is defined above -(B) e -(A) f -(B) g -C(O)-; -L 2 is as defined above, preferably L 2 is a dipeptide, tripeptide or tetrapeptide; - o is 0 or 1, preferably o is 1; -L 3 is as defined above; - p is 0 or 1; z1 is an integer ranging from 1 to 4; z2 is 0 or 1, preferably z2=1; z3 is 0 or 1, preferably z3=0; z4 is 1 or 2, preferably z4=1.

[0180]

[0164] According to a particularly preferred embodiment, LZ 2 The linking moiety of the conjugate according to the present invention represented by has a structure selected from (L4) to (L7): [ka]

[0165] In the formula, - Bonds marked with * are: (a) For (L3) and (L4), they are linked to the C(O) group of the moiety Y of the compound represented by structure (1); (b) For (L6) and (L7), OR directly bonded to the morpholine ring of the compound represented by structure (1) 1 linked to the moiety O; - Bonds marked with ** are linking groups Z 1 Concatenated to; -R 13 is hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, wherein C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups include O, S and NR 14 and optionally substituted and optionally interrupted by one or more heteroatoms selected from 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is D linked to N via a spacer moiety, and R 13 = hydrogen or D linked to N via a spacer moiety, preferably the spacer moiety is defined above -(B) e -(A) f -(B) g -C(O)-; -L 2 is as defined above, preferably L 2 is a dipeptide, tripeptide or tetrapeptide; - o is 0 or 1, preferably o is 1; - ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring, preferably A is 1,4-phenyl or 1,3-phenyl, most preferably A is 1,4-phenyl; z1 is an integer ranging from 1 to 4; z2 is 0 or 1, preferably z2=1.

[0181] A particularly preferred combination is A=1,4-phenyl, R 13 = D linked to N via hydrogen or a spacer moiety, o = 1, L 2 is a dipeptide, containing z2=1 and z1=2.

[0182]

[0167] According to a particularly preferred embodiment, LZ 2 The linking moiety of the conjugate according to the present invention, represented by [ka]

[0168] In the formula, - Bonds marked with * are: (a) For (L8) and (L9), linked to the C(O) group of moiety Y of the compound represented by structure (1), (b) For (L10) and (L11), OR directly bonded to the morpholine ring of the compound represented by structure (1) 1 linked to the moiety O; - Bonds marked with ** are linked to the linking group Z 1 Concatenated to; -L 2 is as defined above, preferably L 2 is a dipeptide, tripeptide or tetrapeptide; - o is 0 or 1, preferably o is 1; - ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring, preferably A is 1,4-phenyl or 1,3-phenyl, most preferably A is 1,4-phenyl; z1 is an integer ranging from 1 to 4; z2 is 0 or 1, preferably z2=1.

[0183]

[0169] A particularly preferred combination is A=1,4-phenyl, o=1, L 2 is a dipeptide and contains z2=1 and z1=2.

[0184]

[0170] According to a particularly preferred embodiment, LZ 2 The linking moiety of the conjugate according to the present invention, represented by [ka]

[0171] In the formula, - Bonds marked with * are: (a) for (L12) and (L13), linked to the C(O) group of the moiety Y of the compound represented by structure (1); (b) For (L14) and (L15), OR directly bonded to the morpholine ring of the compound represented by structure (1) 1 linked to the moiety O; - Bonds marked with ** are linked to the linking group Z 1 Concatenated to; - Each R 17 are individually amino acid side chains, preferably i-Pr, CH or CHCHCHNHC(O)NH; - ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring, preferably A is 1,4-phenyl or 1,3-phenyl, most preferably A is 1,4-phenyl; z1 is an integer ranging from 1 to 4; z2 is 0 or 1, preferably z2=1.

[0185] A particularly preferred combination is A=1,4-phenyl, R 17 = i-Pr, CH3 or CH2CH2CH2NHC(O)NH2, including z2=1 and z1=2.

[0186] In another preferred embodiment, the linker is a cleavable linker represented by structures (L16)-(L20): [ka]

[0187] [Preferred conjugates]

[0174] Preferred antibody conjugates according to the first aspect are selected from the group consisting of compounds (I) to (II), more preferably (II). More preferred conjugates are selected from (III) to (V). Even more preferred conjugates are selected from (X) to (XVII). In a particularly preferred embodiment, the conjugate is selected from (Xb) and (XIb). The structures of these conjugates are defined herein below.

[0188] Conjugate (I) has the following structure: CB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 ) p -D} x ] y (I) During the ceremony, - C.B., L. 6 , Z, D, x and y are as defined above; -L 1 is defined above - (A) d -(B) e -(A) f -(C(O)) g - is a linker represented by -L 2 is a peptide spacer as defined above, preferably Val-Cit or Val-Ala; -L 3 is a PABC derivative represented by structure (26); p=0 or 1.

[0189] In relation to the antibody-conjugate (I), L 1 For d=1 (A represented by structure (23), a=1 and R 13 =H), e=2, f=0 and g=1. 2 = Val-Cit or Val-Ala, more preferably Val-Cit. In relation to antibody conjugate (I), p=1, then R 21 It is preferred that =H.

[0190] The antibody-conjugate (II) has the following structure: CB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-D} x ] y (II) During the ceremony, - C.B., L. 6 , Z, D, x and y are as defined above; -L 1 is -(A)-(B) as defined above e a linker represented by -(C(O))-; -L 2 is a peptide spacer as defined above, preferably Val-Cit or Val-Ala; -L 3 is a PABC derivative represented by structure (26), where R 21 =H.

[0191] In relation to antibody-conjugate (II), L 1 , e=2, for A represented by structure (23), a=1 and R 13 In relation to the antibody-conjugate (II), L is preferably ═H. 2 = Val-Cit or Val-Ala, more preferably Val-Cit.

[0192] According to a preferred embodiment, the conjugate according to the present invention has a structure selected from (III) to (V): [ka]

[0180] In the formula, - Bonds marked with ** are linked to the linking group Z 1 Concatenated to; - Each R 13 are hydrogen, C1 to C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, wherein C1-C 24 Alkyl groups, C3-C24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups include O, S and NR 14 and optionally substituted and optionally interrupted by one or more heteroatoms selected from 14 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 13 is D linked to N via a spacer moiety, and R 13 = hydrogen or D linked to N via a spacer moiety, preferably the spacer moiety is defined above -(B) e -(A) f -(B) g -C(O)-; -L 2 is as defined above, preferably L 2 is a dipeptide, tripeptide or tetrapeptide; - o is 0 or 1, preferably o is 1; -L 3 is as defined above; - p is 0 or 1; z1 is an integer ranging from 1 to 4; z2 is 0 or 1, preferably z2=1; z3 is 0 or 1, preferably z3=0; z4 is 1 or 2, preferably z4=1.

[0193]

[0181] The conjugate (X) has the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0194]

[0182] L 2 may be absent, and preferably, L 2 is present and o=1. For preferred conjugates (Xa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (Xb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. The conjugate (X) preferably has the structure (Xb).

[0195] Conjugate (XI) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0196]

[0184] L 2 may be absent, and preferably, L 2 is present and o=1. For the preferred conjugate (XIa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XIb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XI) preferably has the structure (XIb).

[0197] Conjugate (XII) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0198]

[0186] L 2 may be absent, and preferably, L 2 is present and o=1. For the preferred conjugate (XIIa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XIIb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XII) preferably has the structure (XIIb).

[0199] Conjugate (XIII) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0200]

[0188] L 2 may be absent, and preferably, L 2 is present and o=1. For the preferred conjugate (XIIIa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XIIIb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XIII) preferably has the structure (XIIIb).

[0201] Conjugate (XIV) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0202]

[0190] L 2 may be absent, and preferably, L 2 is present and o=1. For the preferred conjugate (XIVa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XIVb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. With respect to conjugate (XIV), structure (XIVb) is most preferred.

[0203] Conjugate (XV) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0204]

[0192] L 2 may be absent, and preferably, L 2 is present and o=1. For preferred conjugates (XVa), L 2 is represented by the structure (25), and R 17For the preferred conjugate (XVb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. With respect to conjugate (XV), structure (XVb) is most preferred.

[0205] Conjugate (XVI) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L 2 , o and D are as defined above.

[0206]

[0194] L 2 may be absent, and preferably, L 2 is present and o=1. For the preferred conjugate (XVIa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XVIb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. With respect to conjugate (XVI), structure (XVIb) is most preferred.

[0207] Conjugate (XVII) has a linker-payload portion represented by the following structure: [ka] During the ceremony, - Wavy line is Z 1 Shows the connection to; -L2 , o and D are as defined above.

[0208]

[0196] L 2 may be absent, and preferably, L 2 is present and o=1. For preferred conjugates (XVIIa), L 2 is represented by the structure (25), and R 17 For the preferred conjugate (XVIIb), L 2 is represented by the structure (25), and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. With respect to conjugate (XVII), structure (XVIIb) is most preferred.

[0209]

[0197] These preferred conjugates (I) to (V), (X) to (XVII) are further preferably conjugated via a glycan, i.e., b=1, and more preferably conjugated via a trimmed glycan, i.e., j=0. It is further preferred herein that S=GalNAc and w'=0. It is further preferred herein that the linking group Z 1 is formed by azide-alkyne cycloaddition, and preferably the linking group Z 1 =(Z39)(In the formula, ring Z 1 = (Za) and V = CH2). It is more preferred herein that x = 1. It is more preferred herein that y = 2, and more preferably x = 1 and y = 2.

[0210]

[0198] In a most preferred embodiment, the conjugate according to the present invention is represented by structure (Xb) or (XIb) as defined above, where b=1, e=0, S=GalNAc, w'=0, linking group Z1=(Z39), where ring Z=(Za) and V=CH2, x=1 and y=2.

[0211] [Compounds with the general structure (4)] In a further aspect, the present invention relates to linker-toxin constructs. The linker-toxin constructs comprise a compound represented by structure (1) linked to a reactive moiety Q via a linker L, specifically an appropriately functionalized cell-binding agent CB-[(L 6 ) b -{F} x ] y (5) can be used in the preparation of conjugates according to the invention by reaction with (6). In a bioconjugation reaction, the reactive moiety Q of the linker-toxin construct reacts with a reactive group F on a cell-binding agent to form a linking group Z 1 Generate.

[0212]

[0200] The linker-toxin constructs according to the present invention have the general structure (4): QLZ 2 -D (4) During the ceremony, Q is a reactive moiety; - L is a linker; -Z 2 is a linking group connecting L to D; - D is a compound represented by structure (1).

[0213] The linker-drug construct contains the linker L and payload D of the final conjugate. Compounds of general formula (4) can be prepared by one of ordinary skill in the art using standard organic synthesis techniques, as illustrated in the Examples. Linker L and payload D are defined above in relation to the conjugate represented by structure (2).

[0214]

[0202] Also, the linking group Z 2 The linkage between the linker L and the payload D via the compound represented by structure (4) is the same as defined for the conjugate represented by structure (2). Thus, in a preferred embodiment, the compound represented by structure (1) is 1The conjugation is via R or via Y. 1 When the linkage occurs via R 1 =Sp-N(R 4 )2, it occurs through the nitrogen atom. When conjugation occurs through Y, Y=N(R 4 )2 or NR 4 -Sp 3 -N(R 4 )2, the linkage preferably occurs via the nitrogen atom. 4 The remaining NR groups 4 The residue is Z, which is formed when the amine group is attached to the linker. 2 is replaced by a connection to L so that it is part of R. 1 When Y is substituted with Y, it is preferred that Y=CH3 or CH2OH.

[0215] [Reaction part Q]

[0203] The compound represented by general structure (4) comprises a reactive moiety Q. In the context of the present invention, the term "reactive moiety" can refer to a chemical moiety that comprises a reactive group, but can also refer to the reactive group itself. For example, a cyclooctynyl group is a reactive group, i.e., a reactive group that contains a C-C triple bond. Similarly, an N-maleimidyl group is a reactive group that contains a C-C double bond as the reactive group. However, a reactive group, such as an azide-reactive group, a thiol-reactive group, or an alkynyl-reactive group, can be referred to as a reactive moiety herein.

[0216] Q is S(F) xQ functions as a chemical handle for linking to F. In other words, Q is reactive to and complementary to F. As used herein, a reactive group is said to be "complementary" to a reactive group if it selectively reacts with said reactive group, optionally in the presence of another functional group. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Thus, compounds represented by general structure (4) are advantageously used in conjugation reactions in which a chemical reaction between Q and F occurs, thereby forming a conjugate comprising a covalent bond between a payload D and an antibody. This is described in more detail below in connection with a process for synthesizing a conjugate according to the invention.

[0217] The exact nature of Q and F depends on the type of conjugation reaction used. Those skilled in the art will be able to select an appropriate combination of Q and F. Preferably, Q, and therefore F, are reactive in a cycloaddition or nucleophilic reaction. Thus, Q preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety; more preferably, Q is a click probe, a thiol-reactive moiety, or an amine-reactive moiety; most preferably, Q is a click probe. The click probe is reactive in a cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an alkene or alkyne moiety; more preferably, the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or (hetero)cycloalkyne. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, allenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, vinyl sulfones, vinylpyridine moieties, or methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters and other activated esters, p-nitrophenyl carbonate and other activated carbonates, isocyanates, isothiocyanates, haloacetamides, and benzoyl halides. In a preferred embodiment, Q is selected from an alkene moiety, an alkyne moiety, a thiol-reactive moiety, or an amine-reactive moiety, more preferably an alkene moiety or an alkyne moiety, and even more preferably an alkyne moiety. Herein, the alkene is preferably a (hetero)cycloalkene, and the alkyne is preferably a terminal alkyne or (hetero)cycloalkyne. Most preferably, Q is a cyclic (hetero)alkyne moiety. Each of these moieties is further defined herein below.

[0218]

[0205] Therefore, in a particularly preferred embodiment, Q comprises a cyclic (hetero)alkyne moiety. The alkynyl group may also be referred to as a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, where the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptynyl group, a (hetero)cyclooctynyl group, a (hetero)cyclononynyl group, or a (hetero)cyclodecynyl group. In the present specification, the (hetero)cycloalkyne may be optionally substituted. Preferably, the (hetero)cycloalkynyl group is an optionally substituted (hetero)cycloheptynyl group or an optionally substituted (hetero)cyclooctynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, where the (hetero)cyclooctynyl group is optionally substituted.

[0219]

[0206] In particularly preferred embodiments, Q comprises a (hetero)cycloalkynyl group or a (hetero)cycloalkenyl group and is represented by structure (Q1): [ka] During the ceremony, - Bonds shown as [ka] is a double or triple bond; -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and are selected from the group consisting of two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y 2 is C(R 31 )2, O, S, S (+) R 31 , S(O)R 31 , S(O)=NR 31 or NR 31 where S (+) is B (-) is a cationic sulfur atom offset by B (-) is an anion, and each R 31 are individually, R 15 or a linkage to D linked via L; - u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4 or 5, where u+u'=0, 1, 2, 3, 4, 5, 6, 7 or 8; - v=an integer in the range 0 to 16.

[0220] Typically, v = (u + u') x 2 (where the connection to L indicated by the wavy bond is Y 2 through the carbon atoms of u and u') or [(u+u')×2]-1 (when the connection to L represented by the wavy bond is through one of the carbon atoms of u and u').

[0221] In a preferred embodiment of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and is represented by structure (Q1a): [ka] During the ceremony, -R 15 and Y 2 is as defined above, - u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4 or 5, where u+u'=4, 5, 6, 7 or 8; - v=an integer between 8 and 16.

[0222]

[0209] In a preferred embodiment, u+u'=4, 5 or 6, more preferably u+u'=5.

[0223]

[0210] In preferred embodiments, v=8, 9 or 10, more preferably v=9 or 10, and most preferably v=10.

[0224]

[0211] In a preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2) to (Q20) shown herein below. [ka]

[0225]

[0212] Herein, the link to L, represented by a wavy bond, can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14) and (Q15) may have a link to L, or may contain a hydrogen atom, or may be optionally functionalized. B (-) is preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably, B (-) teeth, (-) OTf. In the conjugation reaction, B (-)will be exchanged with anions present in the reaction mixture anyway, so B (-) does not need to be a pharmaceutically acceptable anion. When (Q19) is used for Q, the negatively charged counterion is preferably pharmaceutically acceptable upon isolation of the conjugate according to the invention so that the conjugate can be readily used as a pharmaceutical.

[0226]

[0213] In a further preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21) to (Q38) shown herein below. [ka]

[0227] In the structure (Q38), B (-) is preferably (-) OTf, Cl (-) , Br (-) or I (-) and most preferably, B (-) teeth, (-) It is OTf.

[0228]

[0215] In preferred embodiments, Q comprises an optionally substituted (hetero)cyclooctyne or (hetero)cycloheptyne moiety, preferably represented by the structures (Q8), (Q26), (Q27), (Q28), or (Q37). Each of these preferred options for Q is further defined herein below.

[0229]

[0216] Thus, in a preferred embodiment, Q comprises a heterocycloheptyne moiety represented by structure (Q37), also known as TMTHSI, which is optionally substituted. Preferably, the heterocycloheptyne moiety represented by structure (Q37) is unsubstituted.

[0230] In an alternative preferred embodiment, Q comprises a cyclooctyne moiety represented by structure (Q8), more preferably represented by (Q29), also referred to as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety represented by structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety represented by structure (Q39) shown below, wherein V is (CH2) l wherein l is an integer ranging from 0 to 10, preferably from 0 to 6. More preferably, l is 0, 1, 2, 3, or 4, more preferably, l is 0, 1, or 2, and most preferably, l is 0 or 1. In relation to group (Q39), l is most preferably 1. Most preferably, Q has structure (Q42), further defined below.

[0231] In an alternative preferred embodiment, Q comprises an optionally substituted (hetero)cyclooctyne moiety represented by structure (Q26), (Q27) or (Q28), also referred to as a DIBO, DIBAC, DBCO or ADIBO group. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety represented by structure (Q40) or (Q41) shown below, wherein Y 1 is O or NR 11 and R 11 are independently hydrogen, straight-chain or branched C1-C 12 Alkyl group or C4-C 12 (hetero)aryl groups. The aromatic ring of (Q40) is optionally O-sulfonylated at one or more positions, while the ring of (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety represented by structure (Q40) or (Q41) is not further substituted. Most preferably, Q is represented by structure (Q43), further defined below.

[0232] In an alternative preferred embodiment, Q comprises a heterocycloheptynyl group and is represented by structure (Q37). [ka]

[0233] In a particularly preferred embodiment, Q comprises a cyclooctynyl group and is represented by structure (Q42): [ka] During the ceremony, -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl groups are optionally substituted and are selected from the group consisting of two substituents R 15 may be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, R 16 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; -R 18 are independently hydrogen, halogen, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24(hetero)arylalkyl groups; -R 19 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups, wherein the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted, or R 19 is a second occurrence of Q or D linked via a spacer moiety; - l is an integer ranging from 0 to 10.

[0234] In a preferred embodiment of the reactive group represented by structure (Q42), R 15 are independently hydrogen, halogen, -OR 16 , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 15 is hydrogen or C1-C6 alkyl, and more preferably, R 16 are independently selected from the group consisting of hydrogen and C1-C6 alkyl, and most preferably, all R 15 is H. In a preferred embodiment of the reactive group represented by structure (Q42), R 18 are independently selected from the group consisting of hydrogen, C1-C6 alkyl groups, and most preferably both R 18 is H. In a preferred embodiment of the reactive group represented by structure (Q42), R 19 is H. In preferred embodiments of the reactive group represented by structure (Q42), l is 0 or 1, and more preferably, l is 1.

[0235] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and is represented by structure (Q43): [ka] During the ceremony, -R 15 are independently hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1~C 24 Alkyl groups, C5-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 and (hetero)arylalkyl groups, wherein the alkyl group, the (hetero)aryl group and the (hetero)arylalkyl group are optionally substituted and include two substituents R 15 are joined together to form an optionally substituted cyclized cycloalkyl or an optionally substituted cyclized (hetero)arene substituent R 16 are hydrogen, halogens, C1-C 24 Alkyl groups, C6-C 24 (Hetero)aryl groups, C7-C 24 Alkyl (hetero)aryl groups and C7-C 24 (hetero)arylalkyl groups; - Y is N or CR 15 is.

[0236] In a preferred embodiment of the reactive group represented by structure (Q43), R 15 are independently hydrogen, halogen, -OR 16 , -S(O)3 (-) , a C1-C6 alkyl group, and a C5-C6 (hetero)aryl group; R 16 is hydrogen or C1-C6 alkyl, and more preferably, R 15 are independently hydrogen and -S(O)3 (-) In preferred embodiments of the reactive group represented by structure (Q43), Y is N or CH, more preferably Y=N.

[0237]

[0224] In an alternative preferred embodiment, Q comprises a cyclic alkene moiety. The alkenyl group Q may also be referred to as a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, where the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a norbornene group, a norbornadiene group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecenyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, and trans-(hetero)cyclooctenyl groups, where the (hetero)cyclopropenyl, trans-(hetero)cycloheptenyl, and trans-(hetero)cyclooctenyl groups are optionally substituted. Preferably, Q comprises a cyclopropenyl moiety represented by structure (Q44), a heterocyclobutene moiety represented by structure (Q45), a norbornene or norbornadiene group represented by structure (Q46), a trans-(hetero)cycloheptenyl moiety represented by structure (Q47), or a trans-(hetero)cyclooctenyl moiety represented by structure (Q48). As used herein, Y 3 is C(R 23 )2, NR 23 or O, and each R 23 are individually hydrogen, C1-C6 alkyl, or optionally linked to L via a spacer and labeled [ka] is a single or double bond. In a further preferred embodiment, the cyclopropenyl group is represented by structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is represented by structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctene group is represented by structure (Q52), (Q53), (Q54), (Q55), or (Q56). [ka]

[0238]

[0225] In this specification, the R group on Si in (Q50) and (Q51) is typically alkyl or aryl, preferably C1 to C6 alkyl.

[0239] In an alternative preferred embodiment, Q is a thiol-reactive probe. In this embodiment, Q is a reactive group compatible with cysteine conjugation. Such probes are known in the art and may be selected from the group consisting of a maleimide moiety, a haloacetamide moiety, an allenamide moiety, a phosphonamidite moiety, a cyanoethynyl moiety, a vinyl sulfone, a vinylpyridine moiety, or a methylsulfonylphenyloxadiazole moiety. Most preferably, Q comprises a maleimide moiety. The reagent may be mono-alkylated or may be a cross-linker for reaction with two cysteine side chains.

[0240]

[0227] In a further preferred embodiment, probe Q is selected from the group consisting of (Q57) to (Q71) shown below. [ka] During the ceremony, - X 6 is H, halogen, PhS, MeS, preferably halogen, for example Cl, Br, I; - X 7is a halogen, such as PhS, MeS, preferably Cl, Br, I; -R 24 is H or C 1~12 Alkyl, preferably H or C 1~6 is alkyl; -R 25 is H, C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkal or C 1~12 aralkyl, preferably H or para-methylphenyl; wherein the aromatic rings of (Q61) and (Q63) may optionally be heteroaromatic rings such as phenyl or pyridine rings.

[0241]

[0228] In a preferred embodiment of the thiol-reactive probe (Q57), probe Q is selected from the group consisting of (Q72) to (Q74) shown herein below. [ka] During the ceremony, -R 27 is C 1~12 Alkyl, C 1~12 Aryl, C 1~12 Alkal or C 1~12 It is aralkyl; t is an integer ranging from 0 to 15, preferably from 1 to 10.

[0242] In an alternative preferred embodiment, Q is an amine-reactive probe. In this embodiment, Q is a reactive group compatible with lysine conjugation. Such probes are known in the art and may be selected from the group consisting of N-hydroxysuccinimidyl groups, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanate groups, isothiocyanate groups, and benzoyl halide groups. Most preferably, Q comprises or is an N-hydroxysuccinimidyl ester, p-nitrophenyl carbonate moiety, or pentafluorophenyl carbonate moiety.

[0243]

[0230] In a further preferred embodiment, probe Q is selected from the group consisting of (Q75) to (Q80) shown below. [ka] In this specification, X 2 is a halogen, preferably F.

[0244]

[0231] In a preferred embodiment, Q is selected from the group consisting of (Q1) to (Q80).

[0245] [Cell-binding agent represented by the general structure (5)]

[0232] The cell-binding agent used in the bioconjugation reaction with the linker-toxin construct has the general structure (5): CB-[(L 6 ) b -{F} x ] y (5) During the ceremony, - CB is a cell binding agent; - b is 0 or 1; -L 6 is -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’-, where G is a monosaccharide, j is an integer ranging from 0 to 10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine, Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is —N(H)C(O)CH—, —N(H)C(O)CF—, or —CH—; - F is a reactive moiety; - x is 1 or 2; y is 1, 2, 3 or 4.

[0246]

[0233] Cell-binding agents of general structure (5) may also be referred to as "(modified) cell binders", preferably "(modified) antibodies", because they contain a reactive group F, where the reactive group F is naturally occurring or the cell-binding agent is modified to incorporate the reactive group F. (Modified) cell-binding agents or antibodies of general structure (5) may be prepared by one skilled in the art using standard organic and / or enzymatic synthetic techniques, as illustrated in the Examples. The cell-binding agent CB, the linker L 6 , b, x and y are defined above in relation to the conjugate represented by structure (2).

[0247] [Reaction part F]

[0234] F is reactive towards Q in a conjugation reaction as defined below; preferably, the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. As will be understood by those skilled in the art, the options for F are the same as the options for Q, except that F and Q are reactive with each other. Thus, F preferably comprises a click probe, a thiol, a thiol-reactive moiety, an amine, or an amine-reactive moiety; more preferably, F is a click probe, a thiol, or an amine; most preferably, F is a click probe. The click probe is reactive to cycloaddition (click reaction) and is preferably selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, sydnones, alkene moieties, and alkyne moieties. Preferably, the click probe comprises or is an azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imines, diazo compounds, ortho-quinones, dioxothiophenes, or sydnones; most preferably, it is an azide. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, allenamide moieties, phosphonamidite moieties, cyanoethynyl moieties, ortho-quinone moieties, vinyl sulfone, vinylpyridine moieties, and methylsulfonylphenyloxadiazole moieties. Most preferably, the thiol-reactive moiety comprises or is a maleimide moiety. Typical amine-reactive moieties are selected from N-hydroxysuccinimidyl esters, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanates, isothiocyanates, and benzoyl halides. In a preferred embodiment, F is a click probe or thiol, more preferably F is an azide or thiol, and most preferably F is an azide.

[0248] Two or more reactive groups F may be present in the antibody. The reactive group F in the antibody may be naturally occurring or may be placed in the antibody by specific techniques, such as (bio)chemical or recombinant genetic techniques. The reactive group placed in the antibody is prepared by chemical synthesis, for example, azide or terminal alkyne. Methods for preparing modified antibodies are known in the art, for example, from WO 2014 / 065661, WO 2016 / 170186 and WO 2016 / 053107, which are incorporated herein by reference. From the same documents, the conjugation reaction between modified antibodies and linker-toxin-constructs is known to those skilled in the art.

[0249]

[0236] Preferably, F is a click probe reactive to (hetero)cycloalkenes and / or (hetero)cycloalkynes, typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and sydnones. Preferred structures for the reactive group are structures (F1) to (F10) shown herein below. [ka]

[0250]

[0237] Here, the wavy bond represents a link to a payload. For (F3), (F4), (F8) and (F9), the payload can be linked to any one of the wavy bonds. Then, the other wavy bond can be linked to a hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl(hetero)aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 sulfonyl groups, each of which (excluding hydrogen) may optionally be linked to an R group selected from O, S, and NR 32and optionally interrupted by one or more heteroatoms selected from, where R 32 are independently hydrogen and The reactive group F is selected from the group consisting of C1-C4 alkyl groups. Those skilled in the art will understand which R groups can be applied to each of the groups F. For example, the R group linked to the nitrogen atom of (F3) can be selected from alkyl and aryl, and the R group linked to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl, and sulfonyl. Preferably, the reactive group F is selected from azide or tetrazine. Most preferably, the reactive group F is azide.

[0251] In a second preferred embodiment, F is a thiol or a precursor thereof. The thiol or precursor thereof F is used in a conjugation reaction to link the linker-toxin-construct to a (modified) cell-binding agent. F is reactive to a thiol-reactive probe Q in thiol ligation. The thiol is preferably the thiol of the side chain of a cysteine amino acid naturally occurring in the antibody AB, in which case the linker L 6 is absent (b=0), but it can also be optionally linked by a linker L 6 Thiol precursors relevant to bioconjugation are known in the art and include disulfides that may be naturally occurring disulfide bridges present in antibodies, or synthetically introduced disulfides that are reduced as known in the art. Preferably, F is a thiol group of a cysteine side chain.

[0252] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or precursor thereof F is used in a conjugation reaction to link the linker-toxin-construct to a (modified) antibody. F is reactive to an amine-reactive probe Q in a nucleophilic substitution. The amine is typically a primary amine, preferably the amine of the side chain of a lysine amino acid naturally occurring in the antibody AB, in which case the linker L 6is absent (b=0), but it can also optionally be linked by a linker L 6 Preferably, F is a primary amine group of a lysine side chain.

[0253] [Method for synthesizing conjugates represented by general structure (2)] In a further aspect, the present invention relates to a method for the preparation of a conjugate according to the invention, the method comprising the step of reacting Q of a toxin-linker-construct according to the invention with a reactive group F. Linker-toxin-constructs represented by general structure (4), and preferred embodiments thereof, are described in more detail above. The method is carried out under conditions such that Q reacts with F to covalently attach the cell-binding agent CB (5) to the payload D. In the method according to the invention, Q reacts with F to form a covalent bond between the cell-binding agent and the compound according to the invention. Complementary reactive groups Q and reactive groups F are known to those skilled in the art and are described in more detail below.

[0254]

[0241] Any conjugation technique known in the art can be used to prepare the conjugates of the present invention. Suitable conjugation techniques include thiol ligation, lysine ligation, and cycloaddition (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cycloaddition, and strain-promoted quinone-alkyne cycloaddition). In other words, the conjugation technique is selected from amide bond formation, carbamate bond formation, thiol alkylation, thiol arylation, and cycloaddition reactions. Preferred conjugation techniques used in connection with the present invention include nucleophilic reactions and cycloadditions, and preferably, the cycloaddition is a [4+2] cycloaddition or a [3+2] cycloaddition, and the nucleophilic reaction is a Michael addition or a nucleophilic substitution. Suitable conjugation techniques are disclosed, for example, in Hermanson, "Bioconjugate Techniques", Elsevier, 3rd Ed. 2013 (ISBN: 978-0-12-382239-0), WO 2014 / 065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, PCT / EP2021 / 050594, PCT / EP2021 / 050598 and NL 2026947.

[0255]

[0242] Therefore, in a preferred embodiment of the conjugation process according to the present invention, the conjugation is achieved via a nucleophilic reaction, such as a nucleophilic substitution or a Michael reaction. A preferred nucleophilic reaction is the acylation of a primary amino group with an activated ester. A preferred Michael reaction is the maleimide-thiol reaction, which is widely used in bioconjugation.

[0256]

[0243] Thus, in a preferred embodiment of the conjugation process according to the present invention, the conjugation is achieved via cycloaddition. Preferred cycloadditions are (4 + 2)-cycloadditions (e.g., Diels-Alder reaction) or (3 + 2)-cycloadditions (e.g., 1,3-dipolar cycloaddition). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is an inverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, most preferably wherein Q is or contains an alkyne group and F is an azide group. Cycloadditions such as Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art, and those skilled in the art will recognize how to carry them out.

[0257]

[0244] The method according to this embodiment preferably relates to a Click reaction, more preferably a 1,3-dipolar cycloaddition, and most preferably an alkyne / azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azide group. Click reactions, such as 1,3-dipolar cycloadditions, are known in the art and the skilled artisan will be aware of how to carry them out.

[0258]

[0245] Thus, a method for preparing a conjugate according to the present invention comprises reacting a modified cell-binding agent of structure (5) with a linker-toxin construct represented by structure (4) to obtain a conjugate of structure (2).

[0259] In a preferred embodiment, the method for preparing an antibody conjugate according to the present invention comprises: (i) an antibody comprising y core N-acetylglucosamine (GlcNAc) moieties, where y=1, 2, 3, or 4, is synthesized by the method of formula S(F) x -P(in the formula, S(F) xis a sugar derivative containing x reactive groups F capable of reacting with reactive group Q, x is 1 or 2, and P is a nucleoside mono- or diphosphate, in the presence of a catalyst, wherein the catalyst is x 3. Transferring the moiety to the core-GlcNAc moiety to give the product of formula (26): AB-[GlcNAc(Fuc) w -SCIENCE FICTION} x ] y (26) (In the ceremony - AB is an antibody; - Fuc is fucose; - w is 0 or 1) and (ii) The modified antibody is reacted with a compound having the structure (4): QLZ 2 -D (4) (In the formula, Q is a reactive moiety; - L is a linker; -Z 2 is a linking group; - D is a compound represented by the general structure (1) by reacting with a compound represented by the formula: obtaining an antibody-conjugate represented by structure (2).

[0260] [Process (i)] In step (i), an antibody containing one, two, three, or four core N-acetylglucosamine moieties is reacted with a compound of formula S(F) in the presence of a catalyst. x -P(in the formula, S(F) x is a sugar derivative containing x reactive groups F capable of reacting with reactive group Q, where x is 1 or 2, P is a nucleoside mono- or diphosphate, and the catalyst is S(F) xThe antibody is then contacted with a compound (which can transfer a core-GlcNAc moiety to a core-GlcNAc moiety). As used herein, the antibody is typically an antibody that has been trimmed to a core-GlcNAc residue, as further described below. Step (i) yields a modified antibody represented by formula (26).

[0261]

[0248] Starting materials, i.e., antibodies containing a core-GlcNAc substituent, are known in the art and can be prepared by methods known to those skilled in the art. In one embodiment, the method according to the present invention further comprises deglycosylating the antibody glycan containing the core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody containing a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH and / or combinations thereof, the selection depending on the nature of the glycan. EndoSH is described in PCT / EP2017 / 052792, see Examples 1-3 and SEQ ID NO: 1, which is incorporated herein by reference.

[0262] The structural features S and x are defined above for the conjugates according to the invention and apply equally to this embodiment. x The formula S(F) xCompounds of formula S(F)-P are known in the art. For example, Wang et al., Chem. Eur. J. 2010, 16, 13343-13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462, and WO 2009 / 102820 (all incorporated herein by reference) describe a number of compounds S(F) x -P and their synthesis. In a preferred embodiment, S(F) x The nucleoside monophosphate or diphosphate P in -P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP), more preferably P is selected from the group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP), most preferably P = UDP. Preferably, S(F) x -P is selected from the group consisting of GalNAz-UDP, F2-GalNAz-UDP (N-(azidodifluoro)acetyl-galactosamine), 6-AzGal-UDP, 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP), 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, GlcNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2-(but-3-ynoic acid amido)-2-deoxy-galactose-UDP. Most preferably, S(F) x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0263]

[0250] S(F) x Suitable catalysts capable of transferring a moiety to a core-GlcNAc moiety are known in the art. Suitable catalysts are those that catalyze the transfer of a particular sugar derivative nucleotide S(F) to a core-GlcNAc moiety in that particular process. x-P is a substrate. More specifically, the catalyst catalyzes the formation of a β(1,4)-glycosidic bond. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosaminyltransferases, more preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases (GalNAcTs) and β(1,4)-galactosyltransferases (GalTs), and most preferably from the group of β(1,4)-N-acetylgalactosaminyltransferases having a mutant catalytic domain. Suitable catalysts and mutants thereof are disclosed in WO 2014 / 065661, WO 2016 / 022027, and WO 2016 / 170186 (all of which are incorporated herein by reference). In one embodiment, the catalyst is a wild-type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosaminyltransferase, preferably an N-acetylgalactosaminyltransferase. The mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts can recognize internal sugars and sugar derivatives as acceptors. Thus, the sugar derivative S(F) x is linked to the core-GlcNAc substituent in step (i), regardless of whether said GlcNAc is fucosylated or not.

[0264] Step (i) is preferably carried out in a suitable buffer solution, such as, for example, phosphate, buffered saline (e.g., phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris, and glycine. Suitable buffers are known in the art. Preferably, the buffer solution is phosphate-buffered saline (PBS) or Tris buffer. Step (i) is preferably carried out at a temperature in the range of about 4°C to about 50°C, more preferably in the range of about 10°C to about 45°C, even more preferably in the range of about 20°C to about 40°C, and most preferably in the range of about 30 to about 37°C. Step (i) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 85, more preferably in the range of about 6 to about 8. Most preferably, step (i) is carried out at a pH in the range of about 7 to about 8.

[0265] [Step (ii)] In step (ii), the modified antibody is reacted with a compound of general structure (4) comprising a reactive group Q capable of reacting with a reactive group F and a payload D to form a linking group Z resulting from the reaction of Q with F. 1 A conjugate according to the present invention is obtained, which contains: Such a reaction occurs under conditions such that reactive group Q reacts with reactive group F of the antibody, covalently linking the antibody to the compound represented by general structure (4). Step (ii) may also be referred to as a conjugation reaction.

[0266] In a preferred embodiment, in step (ii), the azide on the azide-modified antibody is reacted with an alkynyl group, preferably a terminal alkynyl group, or a (hetero)cycloalkynyl group, of a compound represented by general structure (4) via a cycloaddition reaction. This cycloaddition reaction between an azide-containing molecule and a molecule containing a terminal alkynyl group or a (hetero)cycloalkynyl group is a type of reaction known in the art as "click chemistry." In the case of a linker-toxin construct containing a terminal alkynyl group, the cycloaddition reaction must be carried out in the presence of a suitable catalyst, preferably a Cu(I) catalyst. However, in a preferred embodiment, the linker-toxin construct contains a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required, and the reaction can also occur spontaneously via a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions known in the art as "metal-free click chemistry."

[0267] [Application]

[0254] The toxins according to the present invention having structure (1) are particularly suitable for preparing conjugates, such as the conjugates according to the present invention, which are then particularly suitable for the treatment of cancer. Compounds represented by structure (1) are further suitable for killing cells. In that respect, the present invention also relates to the use of compounds represented by structure (1) for killing cells, as well as to methods for killing cells, comprising contacting a cell with a compound represented by structure (1). The uses and methods are typically ex vivo or in vitro.

[0268] The conjugates of the present invention are particularly suitable for the treatment of cancer. In this regard, the present invention further relates to a method for the treatment of cancer, comprising administering a conjugate according to the present invention to a subject in need thereof. The subject in need thereof is typically a cancer patient. The use of conjugates, such as antibody-drug conjugates, is well known in the field of cancer treatment, and the conjugates according to the present invention are particularly suitable in this regard. The described method is typically suitable for the treatment of cancer. In the method according to this embodiment, the antibody-conjugate is typically administered in a therapeutically effective dose. This embodiment of the present invention can also be expressed as a conjugate according to the present invention for use in the treatment of cancer. In other words, this embodiment relates to the use of a conjugate according to the present invention for the preparation of a medicament or pharmaceutical composition for use in the treatment of cancer. In this context, the treatment of cancer is assumed to include treating, imaging, diagnosing, preventing tumor growth, inhibiting tumors, and reducing tumors.

[0269] This aspect of the present invention can also be described as a method for targeting tumor cells expressing a specific extracellular receptor, comprising contacting a conjugate according to the present invention with cells that may possibly express the extracellular receptor, and wherein the antibody specifically targets the extracellular receptor. Thus, the method according to this aspect is suitable for determining whether cells express a desired extracellular receptor. These tumor cells may be present in a subject, in which case the method comprises administering a conjugate according to the present invention to a subject in need thereof. Alternatively, the method is performed ex vivo or in vitro. In a preferred embodiment, the cells that may possibly express the extracellular receptor are cells that express the extracellular receptor. Targeting tumor cells preferably includes one or more of treating, imaging, diagnosing, preventing growth, inhibiting, and reducing tumor cells.

[0270] In the context of diagnosis, it is generally unclear whether the contacted cells actually express the specific extracellular receptor being investigated.For example, in the diagnosis of HER2-positive breast cancer, a conjugate containing an antibody targeting HER2, such as trastuzumab, can be contacted with the cells.If the tumor cells actually express HER2, the conjugate will target the cells, but if the tumor cells do not express HER2, the conjugate will not target the cells.Similarly, in the treatment of cancer cells that specifically express an extracellular receptor, those skilled in the art will understand that a cell binding agent, such as an antibody that targets that specific extracellular receptor, should be used.

[0271] In the method of the present invention, the extracellular receptor may be 5T4, ADAM-9, AMHRII, ASCT2, ASLG659, ASPHD1, av-integrin, Axl, B7-H3, B7-H4, BAFF-R, BCMA, BMPR1B, brevican, c-KIT, c-Met, C4.4a, CA-IX, cadherin-6, CanAg, CD123, CD13, CD133, CD138 / syndecan-1, CD166, CD19, CD20, CD203c, CD205, CD21, CD22, CD228, CD25, CD30, CD324, CD33, CD37, CD38, CD45, CD46, CD48a, CD56, CD70, CD71, CD72, CD74, CD79a, CD79b, CEACAM5, claudin-18.2, claudin-6, CLEC12A, CLL-1, Cripto, CRIPTO, CS1, CXCR5, DLK-1, DLL3, DPEP3, E16, EGFR, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FAP, FcRH1, FcRH2, FcRH5, FGFR2, fibronectin, FLT3, folate receptor alpha, Gal-3BP, GD3, GDNF-Ra1, GEDA, GFRA1, Globo H, gpNMB, GPR172A, GPR19, GPR54, guanylyl cyclase C, HER2, HER3, HLA-DOB, IGF-1R, IL13R, IL20Rα, Lewis Y, LGR5, LIV-1, LRRC15, LY64, Ly6E, Ly6G6D, LY6K, MDP, MFI2, MICA / B, MOSPD2, MPF, MSG783, MUC1, MUC16, NaPi2b, NCA, nectin-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCAx hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema Preferably, the extracellular receptor is selected from the group consisting of 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), and tenascin. Likewise, preferably, the tumor cells express an extracellular receptor selected from the same group.One skilled in the art can match a desired extracellular receptor with a suitable cell binding agent that can target that extracellular receptor.

[0272]

[0259] In this respect, the present invention also relates to a pharmaceutical composition comprising a conjugate according to the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition typically contains the conjugate according to the present invention in a pharmaceutically effective dose.

[0273] The present inventors have unexpectedly found that conjugates according to the present invention are superior to conventional conjugates having anthracycline-derived toxins in terms of safety and / or efficacy, such that the therapeutic index of the antibody-conjugates according to the present invention is increased compared to conventional anthracycline-containing conjugates. Considering the reduced toxicity of the compound represented by structure (1), the safety of the conjugates according to the present invention is particularly improved. Therefore, higher doses of the conjugates can be administered to subjects in need thereof, which in turn has further benefits in treatment. Conventional conjugates of anthracyclines and cell-binding agents, such as antibodies, must be administered at very low doses, and as a result, administration of excessively high doses is not uncommon. This can lead to specific cell death and, consequently, undesirable side effects of cancer treatment. Furthermore, administration of these conventional low doses of anthracycline-antibody conjugates adversely affects biodistribution, resulting in less efficient tumor targeting. Thus, the inventors have discovered that anthracycline toxins, i.e., compounds represented by structure (1), have reduced toxicity, resulting in improved therapeutic index, particularly safety or tolerability. Improved therapeutic efficacy of the conjugates according to the invention may take the form of reduced tumor size and / or prolonged regression when compared to conventional conjugates. Increased tolerability of the conjugates according to the invention may take the form of reduced signs of toxicity when compared to administration of conventional conjugates. Reduced signs may also be referred to as reduced symptoms or side effects of cancer treatment, and may involve one or more clinical signs, such as reduced weight, reduced mobility, reduced food intake, and / or one or more toxicity parameters, such as improved blood chemistry, hematology, and / or histopathology.

[0274] In a further aspect, the present invention provides a method of modulating, ameliorating, or reducing the toxicity of an anthracycline toxin, comprising administering to a patient a compound comprising a substituent R as defined above. 1 This aspect of the invention also relates to methods of incorporating substituents R to modulate, ameliorate, or reduce the toxicity of anthracycline toxins. 1 (In the formula, substituent R1 is as defined above). As used herein, conjugation to compounds represented by structure (1) is typically via Y, as defined above. [Brief explanation of the drawings]

[0275] [Figure 1] A general scheme for the preparation of antibody-drug conjugates by reaction of a monoclonal antibody (most often a symmetric dimer) containing x functional groups F is shown. By incubating antibody-(F)x with an excess of linker-drug construct (Q-spacer-linker-payload), a conjugate is obtained by reaction of F with Q to form a linking group Z. [Figure 2A] Various reagents suitable for reaction with cysteine side chains are shown. The reagents may be of the mono-alkylating type (A). [Figure 2B] Various reagents suitable for reaction with cysteine side chains are shown. The reagent may be a cross-linker (B) for reaction with two cysteine side chains. [Figure 3] Figure 1 shows a general process for the non-genetic conversion of a monoclonal antibody (mAb) into an antibody containing probes for click conjugation (F). The click probes can be located at various positions in the antibody depending on the technique used. For example, an antibody can be converted into an antibody containing two click probes (structure on the left), four click probes (structure on the bottom), or eight probes (structure on the right) for click conjugation. [Figure 4]Figure 4 shows a representative (but not comprehensive) set of functional groups (F) that can be introduced into antibodies by genetic engineering, chemical modification, or enzymatic means, which, upon metal-free click reaction with a complementary reactive group Q, yield a linking group Z. The functional group F can be artificially introduced (engineered) into antibodies at any optimal position. Some functional groups F (e.g., nitrile oxides, quinones) can also react with strained alkenes in addition to strained alkynes, as shown for triazines or tetrazines (bottom row) as examples. The pyridine or pyridazine linking group is the product of rearrangement of the tetrazabicyclo[2.2.2]octane linking group, with loss of N, formed upon reaction of triazines or tetrazines with alkynes (but not alkenes), respectively. The linking group Z shown in Figure 4 is a preferred linking group for use in the present invention. [Figure 5] Preferred embodiments are given for cyclic alkynes suitable for metal-free click chemistry, and reactive moieties Q. This list is not exhaustive, for example, alkynes can be further activated by fluorination, substitution of the aromatic ring, or introduction of heteroatoms in the aromatic ring. [Figure 6] We demonstrate the glycan remodeling of a full-length IgG followed by site-specific conjugation of a payload based on azido-cyclooctyne click chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all distinct glycoforms, followed by glycosyltransferase-mediated transfer of the azido sugar onto the core GlcNAc, which is released by the endoglycosidase. In the next step, the azido-remodeled IgG is subjected to a single cyclooctyne-modified immune cell-engaging polypeptide for metal-free click chemistry (SPAAC), resulting in a bispecific antibody in a 2:2 molecular format. We also demonstrate that the cyclooctyne-polypeptide construct has a specific spacer between the cyclooctyne and the polypeptide, which allows for tuning of the IgG polypeptide distance or imparts other properties to the resulting bispecific antibody. [Figure 7]We demonstrate a specific example of glycan remodeling of full-length IgG followed by site-specific conjugation of a payload based on thiol alkylation chemistry. The IgG is first enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of a thiol-modified (and disulfide-protected) sugar derivative onto the core GlcNAc released by the endoglycosidase. In the next step, the remodeled IgG is subjected to reduction (to convert the disulfide to a thiol), optionally followed by oxidation, and then reaction with a payload modified with a suitable thiol-reactive reagent. [Figure 8] The structures of daunorubicin, doxorubicin, nemorubicin (MMDX), PNU-159,696, and PNU-159,682 are shown. [Figure 9] Two linker-modified PNU-159,682 derivatives are shown, one based on carbamoylation of the hydroxyketone group with N,N'-dimethylethylenediamine (DMEDA) and a maleimide-containing linker for antibody conjugation to cysteine, and the other based on oxidation-amide coupling of the hydroxyketone with ethylenediamine (EDA) and a glycine-glycine-containing linker for antibody conjugation under the action of sortase. [Figure 10] 1 shows an ADC obtained by sortase-mediated conjugation of glycine-glycine-EDA modified oxidized PNU-159,682. [Figure 11]Figure 1 shows linker-drug structures based on PNU-159,682 analogs according to the present invention that can be applied to conjugation to antibodies via the reactive moiety Z to generate the corresponding ADCs. Class 1 consists of PNU-analogs modified on the morpholino ring with a substituent different from the methyl group present in PNU-159,682, and the original hydroxyacetone moiety is oxidized to a carboxylic acid to allow activation / conjugation of the linker. Class 2 consists of PNU-analogs in which the original (hydroxy)acetone moiety of doxorubicin / daunorubicin is retained and modified with a linker at the original methyl group present on the morpholino group of PNU-159,682. In both cases, the linker is further modified with a reactive group Z, which can be any functional group that allows conjugation to an antibody, such as a maleimide, activated carbonyl, halide, cycloalkyne, azide, etc. [Figure 12] A synthetic scheme for generating PNU-159,682 analogs 6b-6f with modifications at the morpholino ring based on initial TBS protection of the hydroxyacetone functionality of doxorubicin is shown. [Figure 13] We demonstrate how N-alkylation of the amino sugar of doxorubicin can be achieved for various constructs 8b-8f without prior O-silylation of doxorubicin. This route is also applicable to daunorubicin. [Figure 14A] 1 shows the structure of compound 9a, which is based on the Val-Cit dipeptide and DMEDA linker. [Figure 14B] 1 shows the final steps in the preparation of compounds 9c, 9d, 9f and 9g using Val-Ala dipeptide and EDA linker. [Figure 15] 1 shows the structures of compounds 36 and 39 with Val-Ala dipeptide and conjugation via the anthracycline morpholino group. [Figure 16] 1 shows the structures of compounds 47 and 53, which are based on an EDA linker and Gly-Gly-Phe-Gly or Gly-Gly-Gly peptides, respectively. [Figure 17]Figure 1 shows the in vitro cytotoxicity of trast-9g, trast-9d, trast-9c and trast-36 in four cell lines with variable HER2 expression levels. The TO line indicates the number of viable cells at the start of the assay. [Figure 18A] FIG. 1 shows the time-dependent mean body weight of CD-1 mice administered a single bolus of vehicle (PBS), ADC trastuzumab-9d (20 mg / kg), ADC trastuzumab-36 (20 mg / kg) or reference ADC trastuzumab-9g (5 mg / kg). [Figure 18B] Figure 1 shows the time-dependent body weight of CD-1 mice administered a single bolus of vehicle (PBS), ADC trastuzumab-47 (15 mg / kg), ADC trastuzumab-9c (40 mg / kg) or reference ADC trastuzumab-9g (5 mg / kg). [Figure 19A] Figure 1 shows tumor volume over time in NOD / SCID mice engrafted with the JIMT-1 tumor cell line followed by treatment with low (0.3 mg / kg) or high (1 mg / kg) doses of the reference ADC trastuzumab-9g. [Figure 19B] Figure 1 shows tumor volume over time in NOD / SCID mice engrafted with the JIMT-1 tumor cell line and subsequently treated with low (3 mg / kg) or high (5 mg / kg) doses of the ADC trastuzumab-9d or low (0.6 mg / kg) or high (2 mg / kg) doses of the ADC trastuzumab-36. [Figure 19C] 1 shows tumor volume over time in NOD / SCID mice engrafted with the JIMT-1 tumor cell line and subsequently treated with low dose (0.6 mg / kg) or high dose (2 mg / kg) of the ADC trastuzumab-47 or high dose (2 mg / kg) of the ADC trastuzumab-9c. [Figure 20] Figure 1 shows the in vitro cytotoxicity of compounds 6a, 6b, 6c, 6d, and 6e in four cell lines with varying HER2 expression levels. The TO line indicates the number of viable cells at the start of the assay. [Figure 21]Figure 1 shows the in vitro cytotoxicity of trast-9g, trast-63a, trast-63b, and trast-75 in three cell lines with variable HER2 expression levels. The TO line indicates the number of viable cells at the start of the assay.

[0276] [Example]

[0287] The invention is illustrated by the following examples.

[0277] General procedure for analytical RP-UPLC

[0288] Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL in PBS pH 7.4) was added to 40 μL of 12.5 mM DTT, 100 mM Tris-HCl pH 8.0 and incubated at 37°C for 15 min. The reaction was quenched by adding 50 μL of 49% acetonitrile, 49% water, and 2% formic acid. RP-UPLC analysis was performed on an H-class Acquity UPLC system (Waters). Samples (5 μL) were injected at 0.4 mL / min onto a BioResolve™ RP mAb polyphenyl column (450 Å, 2.7 μm, 2.1 × 150 mm, Waters) at a column temperature of 70°C. A linear gradient was applied, running from 30 to 55% acetonitrile in 0.1% TFA and water over 9 min.

[0278] General Procedure for Analytical SEC

[0289] SE-HPLC analysis was performed on an Agilent 1100 series (Hewlett-Packard) using an Xbridge BEH200A column (3.5 μm, 7.8 × 300 mm, PN 186007640, Waters). Samples were diluted to 1 mg / mL in PBS and run at 0.86 mL / min isocratic flow in 0.1 M sodium phosphate buffer, pH 6.9 (NaHPO4 / Na2PO4) containing 10% isopropanol for 16 min.

[0279] General Procedure for Mass Spectrometric Analysis of Monoclonal Antibodies and ADCs

[0290] Prior to mass spectrometry analysis, IgG was treated with IdeS (Fabricator™) for analysis of the Fc / 2 fragment. A solution of 20 μg of (modified) IgG was incubated with 0.5 μL of IdeS (50 U / μL) in phosphate-buffered saline (PBS) pH 6.6 in a total volume of 10 μL for 1 h at 37 °C. After dilution to 40 μL, the sample was analyzed on a JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) coupled to an HPLC system (Agilent 1100 Series, Hewlett-Packard). The HPLC system was equipped with a MassPREP™ online desalting cartridge (Waters P / N 186002785).

[0280] General Procedure for LC-MS Analysis of Monoclonal Antibodies and ADCs

[0291] For analysis of Fc / 2 fragments, IgG was treated with IdeS (Fabricator™). A solution of 10 μg of (modified) IgG was incubated with 0.5 μL of IdeS (50 U / μL) in phosphate-buffered saline (PBS) pH 7.4 in a total volume of 10 μL for 1 hour at 37°C, followed by dilution to 100 μL using MQ. For analysis of reduced samples, IgG was treated with DTT. A solution of 10 μg of (modified) IgG was incubated with DTT (10 mM) in a final volume of 50 μL of PBS pH 7.4 for 15 minutes at 37°C, followed by addition of 50 μL of quench buffer (49% MQ, 49% acetonitrile, 2% FA). The samples were analyzed on a Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry system (ESI-QTOF) coupled with a UPLC system (Aquity series, Waters). The UPLC system was equipped with a bioZen™ 3.6 μm Intact XB-C8, LC column 50 × 2.1 mm (catalog number 00B-4766-AN).

[0281] [Synthesis of bis-iodo compound 7b] [ka] Example a1. Synthesis of 2,3,4-tri-O-acetyl-β-D-arabinopyranosyl bromide (10)

[0292] Tetraacetylarabinose (24.6 g) was dissolved in a solution of HBr in AcOH (33% HBr, 127 mL). AcO (12 mL) was added and the mixture was stirred at room temperature overnight. DCM (200 mL) was added and the mixture was poured onto ice (300 mL). The two phases were separated and the aqueous layer was extracted with DCM (2 x 250 mL). The combined organic layers were washed with saturated aqueous NaHCO (400 mL) and dried over NaSO. The mixture was concentrated and recrystallized using EtO / heptane to give compound 10 (17.66 g, 67.4%). 1 The H-NMR data were identical to those reported by Grugel et al. Synthesis, 2010, 19, 3248-3258.

[0282] Example a2. Synthesis of 1-(2-azidoethyl)-α-D-arabinopyranoside (11)

[0293] Arabinosyl bromide 10 (2.47 g, 6.45 mmol) was dissolved in dry DCM (0.2 M). Molecular sieves and 2-bromoethanol (5 equiv.) were added, and the mixture was cooled to 0 °C. After the addition of AgCO (1 equiv.), the reaction mixture was warmed to room temperature and stirred for 3 h. The mixture was filtered through Celite, the Celite pad was washed with EtO, and the solvent was evaporated. The crude product was purified using flash column chromatography (0 → 25% EtOAc in heptane). The product-containing fractions were concentrated and dissolved in DMF (0.2 M), followed by the addition of NaN (4 equiv.). The mixture was stirred at 80 °C for 1 h and concentrated. After that, it was redissolved in MeOH (0.1 M) and NaOMe (5.4 M in MeOH, 0.1 equiv.) was added. The reaction was stirred overnight, concentrated, and purified using flash column chromatography (0→10% MeOH in EtOAc) to give product 11 (530 mg, 37.5% over three steps). 1 H-NMR(500MHz,CDCl3)δ(ppm)4.28(d,J=7.1Hz,1H),4.10-3.99(m,2H),3.97(s,1H),3.95-3.90 (m,1H),3.80-3.72(m,2H),3.69(m,2H),3.60-3.53(m,2H),3.45(ddd,J=13.3,5.8,3.6Hz,2H). 13 C-NMR(126MHz, CDCl3)δ(ppm)103.28,72.97,71.42,68.22,68.20,65.91,50.84.

[0283] Example a3. Synthesis of 1,5-dihydroxy-2(S)-(2-azidoethoxy)-3-oxa-pentane (12)

[0294] Arabinoside 11 (530 mg, 2.42 mmol) was dissolved in HO (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed complete consumption of the starting material. The mixture was cooled to 0 °C, and NaBH was added portionwise. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of the diol. EtOAc (10 mL) was added, and the organic layer was separated. The aqueous layer was extracted five times with EtOAc (10 mL). The combined organic layers were dried over NaSO and concentrated to give product 12 (346 mg, 74.8%). 1 H-NMR(400MHz,CDCl3)δ(ppm)4.72(t,J=5.3Hz,1H),3.94-3.83(m,2H),3.82-3.76(m,2H),3.77-3.65(m,4H),3.52-3.38(m,2H). 13 C-NMR(101MHz,CDCl3)δ(ppm)102.88,68.39,66.11,62.34,61.80,50.92.

[0284] Example a4. Synthesis of 1,5-di(p-toluenesulfonyl)oxy-2(S)-(2-azidoethoxy)-3-oxa-pentane (13)

[0295] Diol 12 (346 mg, 1.81 mmol) was dissolved in dry pyridine (0.1 M) and cooled to 0 °C, followed by the addition of p-TsCl (2.5 equiv.). The mixture was stirred overnight, concentrated, and dissolved in EtOAc (20 mL). The solution was washed with 0.1 M HCl (10 mL) and brine (10 mL). The mixture was dried over NaSO, concentrated, and purified using flash column chromatography (0 → 50% EtOAc in heptane) to give product 13 (368 mg, 40.7%). 1H-NMR(400MHz,CDCl3)δ(ppm)7.86-7.77(m,4H),7.47 -7.33(m,5H),4.74(t,J=5.4Hz,1H),4.15(ddd,J=5.4,4.0,1.1Hz,2H),3.97(dd,J=5.4,0.8Hz ,2H),3.85-3.69(m,3H),3.66-3.58(m,1H),3.36(dt,J=5.8,3.9Hz,2H),2.48(d,J=1.5Hz,6H). 13 C-NMR(101MHz,CDCl3)δ(ppm)145.26,145.08,132.82,132.50,130.00,129. 94,128.01,127.96,99.40,68.77,67.86,65.71,64.27,50.67,21.69,21.67.

[0285] Example a5. Synthesis of 1,5-diiodo-2(S)-(2-azidoethoxy)-3-oxa-pentane (7b)

[0296] Bis-tosylate 13 (491 mg, 0.98 mmol) was dissolved in 2-butanone (0.05 M). NaI (7 equiv.) was added, and the mixture was stirred at 90 °C for 24 h. The mixture was concentrated and dissolved in EtOAc (20 mL). The organic layer was washed with HO (20 mL) and brine (20 mL), dried over NaSO, and concentrated. The crude product was purified using flash column chromatography (0 → 5% EtOAc in heptane) to give compound 7b (260 mg, 64.4%). 1 H-NMR(400MHz,CDCl3)δ(ppm)4.80(t,J=5.6Hz,1H),3.98-3.80(m,3H),3.73(dd d,J=10.4,5.8,4.5Hz,1H),3.46(ddd,J=5.9,4.2,1.8Hz,2H),3.37-3.26(m,4H). 13 C-NMR(101MHz,CDCl3)δ(ppm)101.83,66.95,65.05,50.73,3.77,2.33.

[0286] [ka] Example a6. Synthesis of 1-isopropyl-α-D-arabinopyranoside (15)

[0297] Arabinosyl bromide 10 (3.51 g, 10.3 mmol) was dissolved in dry EtO (0.25 M). iPrOH (15 equiv.) was added, followed by AgO (1 equiv.). The mixture was stirred in the dark for 3 h. The reaction was filtered over Celite, the Celite pad was washed with EtO, and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by the addition of NaOMe (5.4 M in MeOH, 0.1 equiv.) and stirred overnight at room temperature. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to give product 15 (1.38 g, 69.4% over two steps). 1 H-NMR(400MHz,D2O)δ(ppm)5.70-5.66(m,1H),5.38(hept,J=6.2Hz,1H),5.26(dd,J=12.4 ,2.9Hz,1H),5.22(dt,J=2.9,1.5Hz,1H),5.00-4.85(m,3H),2.62(dd,J=13.0,6.1Hz,6H). 13 C-NMR(101MHz,D2O)δ(ppm)103.16,74.35,72.50,72.48,69.72,66.84,23.81,22.07.

[0287] Example a7. Synthesis of 1,5-dihydroxy-2(S)-isopropyloxy-3-oxa-pentane (16)

[0298] Arabinoside 15 (585 mg, 3.04 mmol) was dissolved in HO (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed complete consumption of the starting material. The mixture was cooled to 0 °C, and NaBH was added portionwise. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of the diol. EtOAc (10 mL) was added, and the organic layer was separated. The aqueous layer was extracted five times with EtOAc (10 mL). The combined organic layers were dried over NaSO and concentrated to give product 16 (324 mg, 64.8%).1 H-NMR(500MHz,CDCl3)δ(ppm)4.72(dd,J=6.1,4.7Hz,1H),3.93(dq,J=12.3,6.1Hz,1H ),3.87-3.75(m,3H),3.71-3.53(m,3H),1.26(d,J=5.7Hz,2H),1.20(d,J=6.1Hz,3H). 13 C-NMR(126MHz,CDCl3)δ(ppm)100.99,70.29,67.64,63.21,62.02,23.09,22.32.

[0288] Example a8. Synthesis of 1,5-diiodo-2(R)-isopropyloxy-3-oxa-pentane (7c)

[0299] Diol 16 (244 mg, 1.49 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 equiv.), PPh3 (3 equiv.), and then I2 (3 equiv.) were added. The mixture was stirred overnight at room temperature in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% aqueous sodium thiosulfate solution (20 mL), brine (20 mL), and dried over Na2SO4. The mixture was concentrated and purified using column chromatography (0 → 5% EtOAc in heptane) to give product 7c (220 mg, 38.5%). 1 H-NMR(400MHz,CDCl3)δ(ppm)4.76(t,J=5.5Hz,1H),3.94(hept,J=6.2Hz,1H),3. 87-3.71(m,0H),3.33-3.12(m,4H),1.24(d,J=6.2Hz,3H),1.20(d,J=6.1Hz,3H). 13 C-NMR(101MHz,CDCl3)δ(ppm)100.29,70.13,65.65,23.16,22.06,5.74,2.61.

[0289] [ka] Example a9. Synthesis of 1-benzyl-α-D-arabinopyranoside (18)

[0300] Arabinosyl bromide 10 (2.06 g, 6.09 mmol) was dissolved in dry EtO (0.25 M). BnOH (15 equiv.) was added, followed by AgO (1 equiv.). The mixture was stirred in the dark for 3 h. The reaction was filtered over Celite, the Celite pad was washed with EtO, and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by the addition of NaOMe (5.4 M in MeOH, 0.1 equiv.) and stirring at room temperature overnight. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to give product 18 (840 mg, 57.4% over two steps). 1 H-NMR(400MHz,D2O)δ(ppm)7.53-7.37(m,5H),4.92(dd,J=11.6,1.4Hz,1H),4.75(d d,J=11.6,1.2Hz,1H),4.44(d,J=7.5Hz,1H),4.02-3.91(m,2H),3.73-3.55(m,3H). 13 C-NMR(101MHz,D2O)δ(ppm)136.63,128.72,128.64,128.45,102.18,102.13,72.35,71.42,70.72,70.69,68.31,68.24,66.28.

[0290] Example a10. Synthesis of 1,5-dihydroxy-2(S)-benzyloxy-3-oxa-pentane (19)

[0301] Arabinoside 18 (259 mg, 1.08 mmol) was dissolved in HO (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed complete consumption of the starting material. The mixture was cooled to 0 °C, and NaBH was added portionwise. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of the diol. EtOAc (10 mL) was added, and the organic layer was separated. The aqueous layer was extracted five times with EtOAc (10 mL). The combined organic layers were dried over NaSO and concentrated to give product 19 (198 mg, 86.7%). 1H-NMR(400MHz,CDCl3)δ(ppm)7.43-7.17(m,5H),4.75-4.66(m,2H),4.57(d,J=11. 7Hz,1H),3.83(ddd,J=10.6,5.3,3.3Hz,1H),3.77-3.71(m,2H),3.68-3.55(m,2H). 13 C-NMR(101MHz,CDCl3)δ(ppm)137.49,128.55,127.97,127.85,102.29,69.51,68.31,62.44,61.68.

[0291] Example a11. Synthesis of 1,5-diiodo-2(S)-benzyloxy-3-oxa-pentane (7d)

[0302] Diol 19 (579 mg, 2.73 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 equiv.), PPh3 (3 equiv.), and then I2 (3 equiv.) were added. The mixture was stirred overnight at room temperature in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% aqueous sodium thiosulfate solution (20 mL), brine (20 mL), and dried over Na2SO4. The mixture was concentrated and purified using column chromatography (0 → 5% EtOAc in heptane) to give product 7d (723 mg, 61.3%). 1 H-NMR(400MHz,CDCl3)δ(ppm)7.45-7.31(m,5H),4.83(t,J=5.6Hz,1H),4.76(d ,J=11.7Hz,1H),4.66(d,J=11.7Hz,1H),3.94-3.75(m,2H),3.37-3.24(m,4H). 13 C-NMR(101MHz,CDCl3)δ(ppm)137.16,128.57,128.05,127.99,101.12,68.66,66.72,4.52,2.50.

[0292] [ka] [Example a12. Synthesis of Compound 21]

[0303] An ice-cold solution of sodium nitrite (842.3 mg, 12.21 mmol) in water (20 mL) was prepared and transferred to a dropping funnel. This mixture was added to a cold solution of 4-aminobenzyl alcohol (1 g, 8.12 mmol) in HCl (5 M, 5 mL) over 30 min. The reaction mixture changed from bright yellow to pale yellow and finally to off-white. After 30 min, sodium azide (2.1 g, 32 mmol) was added in five portions, and the mixture was allowed to stir. After 1 h, the ice bath was removed, and a solid was observed. After 1.5 h, saturated aqueous NaHCO3 (25 mL) was added, followed by EtOAc (25 mL). The reaction was transferred to a separatory funnel, and the organic layer was separated from the aqueous layer. The organic layer was washed with saturated aqueous NaHCO3 (20 mL), brine (25 mL), and dried over Na2SO4. The drying agent was removed by filtration through a glass filter, and the yellow filtrate was concentrated. The crude yellow oil was purified by flash column chromatography on silica gel (5% to 80% EtOAc in heptane, column preconditioned with 5% EtOAc in heptane) to give the product 21 in 89% (1.08 g, 7.24 mmol). 1 H-NMR(400MHz,CDCl3)δ(ppm)7.36(d,J =8.6Hz,2H),7.08-6.97(m,2H),4.67(d,J=4.3Hz,2H),1.68(t,J=5.2Hz,1H).

[0293] [Example a13. Synthesis of Compound 23]

[0304] To a solution of arabinosyl bromide 10 (1.34 mg, 3.95 mmol) and compound 21 (872 mg, 5.85 mmol) in diethyl ether (anhydrous, 20 mL), silver(I) oxide (916 mg, 3.95 mmol) was added, and the reaction was stirred at room temperature in the dark. After stirring for 10 days, the reaction mixture was filtered through prewetted Celite, washed with diethyl ether, and concentrated. The crude oil was dissolved in MeOH (15 mL), and sodium methoxide (134.4 mg, 2.48 mmol) was added. After stirring at room temperature for 3.5 h, the reaction mixture was neutralized with a few drops of 1 M aqueous HCl and concentrated. Excess 21 was removed by precipitating the desired compound in diethyl ether and filtering it on a glass filter covered with filter paper. Compound 23 was obtained as an off-white solid in 65% yield (714.4 mg, 2.54 mmol). 1 H-NMR(400MHz,MeOD)δ(ppm)7.47(d,J=8.6Hz,2H),7.10-7.04(m,2H),4.86(d,J=11.9Hz, 1H),4.63(d,J=11.9Hz,1H),4.31(d,J=6.9Hz,1H),3.95-3.79(m,3H),3.67-3.49(m,4H).

[0294] [Example a14. Synthesis of Compound 24]

[0305] Compound 23 (714.4 mg, 2.54 mmol, 1.0 equiv) was dissolved in MeOH (3 mL) and water (5 mL) and cooled to 0 °C (in the dark). A solution of sodium acetate (270.9 mg, 3.3 mmol, 1.3 equiv) in water (3 mL) was added in one portion, followed by the portionwise addition of sodium periodate (1.35 g, 6.35 mmol, 2.5 equiv). The reaction mixture was stirred on ice for 15 minutes, after which the ice bath was removed and stirring continued at room temperature for 4.5 hours. After stirring for 4.5 hours, the reaction mixture was cooled again to -10 °C, and sodium borohydride (288.3 mg, 7.62 mmol, 3.0 equiv) was added portionwise. After stirring for 1 hour, EtOAc (50 mL) was added, and the reaction mixture was transferred to a separatory funnel. The organic layer was separated from the aqueous layer, and the aqueous layer was extracted with EtOAc (5 × 50 mL). The organic layers were combined, dried over Na2SO4, filtered through a glass filter with pre-wetted Celite, and concentrated to give compound 24 in 87% yield (637.2 mg, 2.2 mmol). 1 H-NMR(400MHz,MeOD)δ(ppm)7.31(d,J=8.5Hz,2H),6.98-6.91(m,2H),4.63-4.48(m,3H),3.70-3.65(m,1H),3.60-3.56(m,2H),3.53-3.48(m,3H).

[0295] [Example a15. Synthesis of Compound 25]

[0306] A solution of 24 (637.2 mg, 2.21 mmol) in dry DCM (8 mL) was cooled to 0 °C (under a stream of nitrogen). Pyridine (537 μL, 6.64 mmol, 3.0 equiv), methanesulfonic anhydride (964.2 mg, 5.53 mmol, 2.5 equiv), and DMAP (27.0 mg, 221.4 mmol, 0.1 equiv) were added. After stirring for 3.5 h, the reaction mixture was washed with saturated aqueous NaHCO (11 mL). The aqueous layer was extracted twice with DCM (10 mL). The combined organic layers were dried over NaSO, filtered, and concentrated. The crude orange oil was purified by flash column chromatography on silica gel (10% to 80% EtOAc in heptane, column preconditioned with 10% EtOAc in heptane) to give compound 25 as a clear, pale yellow oil in 61% (571.6 mg, 1.4 mmol). 1 H-NMR(400MHz,CDCl3)δ(ppm)7.34(d,J=8.5Hz,2H),7.06-7.00(m,2H),4.91(t,J=5.2Hz,1H),4.72(d,J=11.7Hz,1H),4.61(d,J= 11.7Hz,1H),4.37(t,J=4.5Hz,2H),4.24(dd,J=5.2,2.1Hz,2H),3.96-3.88(m,1H),3.86-3.80(m,1H),3.06(s,3H),3.05(s,3H).

[0296] [Example a16. Synthesis of compound 7e]

[0307] A solution of compound 25 (571.2 mg, 1.4 mmol, 1.0 equiv.) and sodium iodide (1.42 g, 9.48 mmol, 7 equiv.) in 2-butanone (15 mL) was refluxed in the dark. After refluxing for 72 h, the reaction mixture was diluted with EtOAc (30 mL) and washed with water (20 mL). The layers were separated, and the organic layer was washed with brine (20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude orange oil was purified by flash column chromatography on silica gel (0% to 7% EtOAc in heptane) to afford compound 7e as an opaque pale yellow oil in 47.5% yield (313.2 mg, 0.64 mmol). 1H-NMR(400MHz,CDCl3)δ(ppm)7.37(d,J=8.6Hz,2H),7.11-6.93(m,2H),4.79(t,J=5.6Hz, 1H),4.70(d,J=11.6Hz,1H),4.59(d,J=11.7Hz,1H),3.90-3.72(m,2H),3.35-3.18(m,4H).

[0297] [ka] Example a17. Synthesis of 1-ethyl-α-D-arabinopyranoside (27)

[0308] Arabinosyl bromide 10 (5.31 g, 15.66 mmol) was dissolved in dry EtO (0.25 M). EtOH (15 equiv.) was added, followed by AgO (1 equiv.). The mixture was stirred in the dark for 3 h. The reaction was filtered over Celite, the Celite pad was washed with EtO, and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by the addition of NaOMe (5.4 M in MeOH, 0.1 equiv.) and stirring at room temperature overnight. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to give product 27 (1.80 g, 64.5% over two steps). 1 H-NMR(400MHz,MeOD)δ(ppm)4.22(d,J=7.1Hz,1H),3.97-3.79(m,3H),3.67-3.50(m,4H),1.26(t,J=7.1Hz,3H). 13 C-NMR(101MHz,MeOD)δ(ppm)104.65,74.33,72.43,69.70,66.91,65.94,15.49.

[0298] Example a18. Synthesis of 1,5-dihydroxy-2(S)-ethoxy-3-oxa-pentane (28)

[0309] Arabinoside 27 (721 mg, 4.05 mmol) was dissolved in HO (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed complete consumption of the starting material. The mixture was cooled to 0 °C, and NaBH was added portionwise. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of the diol. EtOAc (15 mL) was added, and the organic layer was separated. The aqueous layer was extracted five times with EtOAc (15 mL). The combined organic layers were dried over NaSO and concentrated to give product 28 (381 mg, 62.7%). 1 H-NMR(400MHz,CDCl3)δ(ppm)4.62(dd,J=6.1,4.3Hz,1H),3.89-3.81(m,1H),3.79-3.69(m,3H),3.67-3.53(m,4H),1.24-1.19(m,3H). 13 C-NMR(101MHz,CDCl3)δ(ppm)102.65,68.44,63.66,62.61,61.83,15.29.

[0299] Example a19. Synthesis of 1,5-diiodo-2(S)-ethoxy-3-oxa-pentane (7f)

[0310] Diol 28 (381 mg, 2.54 mmol) was dissolved in dry THF (0.15 M). Imidazole (7 equiv.), PPh3 (3 equiv.), and then I2 (3 equiv.) were added. The mixture was stirred overnight at room temperature in the dark. After dilution with EtOAc (20 mL), the organic layer was washed with 10% sodium thiosulfate (20 mL), brine (20 mL), and dried over Na2SO4. The mixture was concentrated and purified using column chromatography (0 → 5% EtOAc in heptane) to give product 7f (170 mg, 18.1%). 1 H-NMR(400MHz,CDCl3)δ(ppm)4.72(t,J=5.5Hz,1H),3.93-3.70(m,3H),3.66-3.56(m,1H),3.32-3.23(m,4H),1.26(t,J=7.1Hz,3H). 13C-NMR(101MHz,CDCl3)δ(ppm)101.76,66.90,62.52,15.10,4.78,2.48.

[0300] [General scheme for pNU-159,682 analogs from doxorubicin]

[0311] The general schemes are shown in Figures 12 and 13. Similar analogs can be prepared from daunorubicin by omitting the silylation and desilylation steps.

[0301] [Example a20. Synthesis of Compound 2]

[0312] A solution of doxorubicin HCl (3.09 g, 5.33 mmol) in anhydrous DMF (35 mL) was cooled to 0° C., and imidazole (1.47 g, 21.6 mmol) was added. After stirring for several minutes, TBDMS-Cl (1.90 g, 12.65 mmol) was added. The reaction mixture was stirred at 0° C. for 5 minutes, after which it was warmed to room temperature. After stirring at room temperature for 3.5 hours, the reaction mixture was purified by flash column chromatography on silica gel (preconditioned column 1% MeOH / DCM, 1% → 30% MeOH in DCM) to give compound 2 as a dark red, thick oil (3.59 g, 5.4 mmol, 100%). 33 H 44 NO 11 Si + (M+H + LCMS (ESI+) calculated for 658.27, found 658.44.

[0302] [Example a21. Synthesis of Compound 3b] [ka]

[0313] To a stock solution of compound 2 (550 mg, 836 μmol) in anhydrous DMF (1 mL) was added (S)-1-(2-azidoethoxy-2-iodo-1-(2-iodoethoxy)ethane 7b (828.5 mg, 2.06 mmol) and DIPEA (437 μL, 2.51 mmol). The reaction mixture was heated to 42° C. and stirred for 25 min, after which the heating was removed and the reaction mixture was allowed to react at room temperature. After 72 h at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography on silica gel (0% to 3% MeOH in DCM) to give compound 3b as a dark red oil (303 mg, 367 μmol, 43.9%). 39 H 53 N4O 13 Si + (M+H + LCMS (ESI+) calculated for 813.34, found 813.51.

[0303] [Example a22. Synthesis of compound 4b] [ka]

[0314] A solution of compound 3b (303 mg, 253 μmol, 68 wt%) in anhydrous DCM (31 mL) was cooled to −78° C. using a dry ice / acetone cooling bath. The mixture was vigorously stirred, and then a freshly made stock solution of mCPBA in anhydrous DCM (70 mg, 580 mM, 699 μL, 406 μmol) was added dropwise. After stirring for 7 minutes, complete conversion was achieved. The reaction mixture was quenched with an ice-cold solution of acetone (reagent grade, 3.33 mL), and the reaction mixture was stirred. After 20 minutes, the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO (6 mL). The aqueous layers were combined and extracted once with DCM (8 mL). The combined organic layers were dried over NaSO, filtered through a filter paper, and concentrated to a volume of 30 mL. Compound 4b was used directly in the next step without further purification. 39 H 53 N4O 14 Si +(M+H + LCMS (ESI+) calculated for 829.33, found 829.57.

[0304] [Example a23. Synthesis of compound 5b] [ka]

[0315] To a solution of compound 4b (188.7 mg, 227.7 μmol) in DCM (27 mL) was added anhydrous acetonitrile (25 mL). The reaction mixture was partially concentrated to remove DCM, yielding the compound in anhydrous acetonitrile (25 mL). After concentrating most of the solvent, the reaction mixture was further diluted with anhydrous acetonitrile (5 mL). Potassium carbonate (198 mg, 1.43 mmol) was then added, and the reaction mixture was cooled to 0 °C. Cyanuric chloride (247.7 mg, 12.1 mL, 111 mM, 1.35 mmol) was then added as a stock solution in anhydrous acetonitrile. After stirring at 0 °C for 4 h, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (489.6 mg, 2.9 mL, 1.85 M, 5.37 mmol) in water. The ice bath was removed after 30 min, after which it was allowed to warm to room temperature. DMF (1.5 mL) was added to the reaction mixture, and the reaction mixture was concentrated until only a DMF / water (8 mL) solution remained, which was then purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting mixture was purified by filtration (5 μM OBD, 30×100 mm). The collected fractions were combined and concentrated until a volume of 6 mL of acetonitrile remained. It was then dried over Na2SO4, filtered, and the residue was washed with anhydrous THF (3×750 μL). The combined organic layer was partially concentrated to a volume of 5 mL (acetonitrile / THF) to give compound 5b as a red solution, which was used without further purification. 39 H 51 N4O 13 Si + (M+H + LCMS (ESI+) calculated for 811.32, found 811.51.

[0305] [Example a24. Synthesis of compound 6b] [ka]

[0316] To a solution of compound 5b (185 mg, 228 μmol) in a mixture of acetonitrile and THF (5 mL), triethylamine acetate (731 μL, 4.56 mmol) was added, and the reaction mixture was cooled to −15° C. Next, TBAF (1 M in THF, 1.03 g, 4 mL, 4 mmol) was added portionwise while the reaction mixture was vigorously stirred (a color change from red to green was observed, then back to dark red). After stirring for 2.5 h, the reaction mixture was quenched with water (15 mL), and the solution changed from dark red to pale red. The reaction mixture was stirred on ice for 1 min and then left at room temperature for an additional 90 min. The reaction mixture was transferred to a separatory funnel and extracted with DCM (18 mL). The aqueous layer was extracted with additional DCM (2 × 6 mL). The combined organic layers were dried over Na2SO4, filtered, further diluted to 55 mL with DCM and purified by flash column chromatography on silica gel (0% → 10% MeOH in DCM). Further preparative HPLC purification (30% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge prep C 18 A 5 μm OBD, 30 × 100 mm column was required. The collected fractions were combined and partially concentrated to give compound 6b (0.22 mM, 45 mL, 9.82 μmol, 4.3% yield over three steps based on a doxorubicin-based calibration line for HPLC) as a solution in 45 mL of acetonitrile / water. 33 H 37 N4O 13 Si + (M+H + LCMS (ESI+) calculated for 697.24, found 697.45.

[0306] [Example a25. Synthesis of Compound 3c] [ka]

[0317] To a stock solution of compound 2 (650 mg, 612 μmol) in anhydrous DMF (1.6 mL) was added (R)-2-(2-iodo-1-(2-iodoethoxy)ethoxy)propane 7c (1.13 g, 2.93 mmol) and DIPEA (516 μL, 2.96 mmol). The reaction mixture was heated to 40° C. and stirred for 10 minutes, after which the heating device was removed and the reaction mixture was allowed to react at room temperature. After 72 hours at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography on silica gel (0% → 3% MeOH in DCM) to give compound 3c (241.8 mg, 307.6 μmol, 31.1%) as a dark red oil. 40 H 56 NO 13 Si + (M+H + LCMS (ESI+) calculated for 786.96, found 786.64.

[0307] [Example a26. Synthesis of compound 4c] [ka]

[0318] A solution of 3c (125 mg, 159 μmol) in anhydrous DCM (15 mL) was cooled to −78 °C in a dry ice / acetone cooling bath. The mixture was stirred vigorously, and then a freshly made stock solution of mCPBA in anhydrous DCM (52.1 mg, 580 mM, 521 μL, 302 μmol) was added dropwise. After stirring at −78 °C for 7 min, complete conversion was achieved. The reaction mixture was quenched with an ice-cold solution of acetone (reagent grade, 1.46 mL), and the reaction mixture was stirred. After 1 h, the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO (12 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over NaSO, filtered through a filter paper, and concentrated to a volume of 12 mL to give 4c as a red solution. Compound 4c was used as is without further purification. 40 H 56 NO 14 Si+ (M+H + LCMS (ESI+) calculated for 802.96, found 802.63.

[0308] [Example a27. Synthesis of Compound 5c] [ka]

[0319] To a solution of 4c (128 mg, 159 μmol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The reaction mixture was partially concentrated to remove DCM, yielding the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the reaction mixture was further diluted with anhydrous acetonitrile (20 mL). Potassium carbonate (86 mg, 622 μmol) was then added, and the reaction mixture was cooled to −7 °C, followed by the addition of cyanuric chloride (73.6 mg, 3.59 mL, 111 mM, 399 μmol) as a stock solution in anhydrous acetonitrile. After stirring at −7 °C for 2 h, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (179 mg, 854 μL, 2.3 mol, 1.96 mmol) in water. The ice bath was removed after 15 min, after which it was allowed to warm to room temperature. DMF (1 mL) was added to the reaction mixture, and the reaction mixture was concentrated until only a DMF / water solution remained, which was then purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The compound was purified by HPLC using a HPLC column (5 μM OBD, 30×100 mm). The collected fractions were combined and concentrated until a volume of 4 mL remained. The resulting solution, consisting mainly of acetonitrile, was then dried over Na2SO4, filtered, and the drying agent was washed with anhydrous THF (3×750 μL). It was concentrated again to a volume of 3 mL, and compound 5c was used directly. 40 H 54 NO 13 Si + (M+H + LCMS (ESI+) calculated for 784.94, found 786.57.

[0309] [Example a28. Synthesis of Compound 6c] [ka]

[0320] To compound 5c (121 mg, 154 μmol) in a mixture of acetonitrile and THF (2.88 mL) was added additional dry THF (2.0 mL), and the resulting red solution was cooled to −40 °C. TBAF (1 M in THF, 484.5 mg, 1.85 mL, 1.85 mmol) was then added while the reaction mixture was vigorously stirred (a color change from red to green was observed). After stirring for 4 h, the reaction mixture was quenched with water (3.0 mL), and the solution changed from green to red. The reaction mixture was transferred to a separatory funnel and extracted with DCM (3 × 10 mL). The combined organic layers were dried over NaSO, filtered, and purified by flash column chromatography on silica gel (0% → 5% MeOH in DCM) followed by preparative HPLC (40% → 95% acetonitrile in 10 mM NHHCO in water, column Xbridge Prep C). 18 The resulting mixture was purified by HPLC (5 μM OBD, 30 × 100 mm). Compound 6c was obtained as a red solution in DCM (70 mL, 0.086 mM, 6.0 μmol, 3.9% yield over three steps based on a calibration line based on doxorubicin for HPLC). 34 H 40 NO 13 + LCMS (ESI+) calculated for (M+H+) 670.25, found 670.51.

[0310] [Example a29. Synthesis of compound 3d] [ka]

[0321] To a stock solution of compound 2 (650 mg, 612 μmol) in anhydrous DMF (1.6 mL) was added (S)-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7d (1.27 g, 2.94 mmol) and DIPEA (516 μL, 2.96 mmol). The reaction mixture was heated to 42° C. and stirred for 10 minutes, after which the heating device was removed and the reaction mixture was allowed to react at room temperature. After 72 minutes at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by flash column chromatography on silica gel (0%→3% MeOH in DCM) to give compound 3d as a dark red oil (413.5 mg, 479 μmol, 48.5%). 44 H 56 NO 13 Si + (M+H + LCMS (ESI+) calculated for 834.35, found 834.50.

[0311] [Example a30. Synthesis of compound 4d] [ka]

[0322] A solution of compound 3d (125 mg, 159 μmol) in anhydrous DCM (15 mL) was cooled to −78° C. using a dry ice / acetone cooling bath. The mixture was vigorously stirred, and then a freshly made stock solution of mCPBA in anhydrous DCM (52.0 mg, 580 mM, 519 μL, 301.5 μmol) was added dropwise. After stirring for 36 minutes, complete conversion was achieved. The reaction mixture was quenched with an ice-cold solution of acetone (reagent grade, 1.38 mL), and the reaction mixture was stirred. After 12 minutes, the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO3 (12 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered through a filter paper, and concentrated to a volume of 12 mL. Compound 4d was used directly without further purification. 44 H 56 NO 14 Si +(M+H + LCMS (ESI+) calculated for 850.35, found 850.60.

[0312] [Example a31. Synthesis of compound 5d] [ka]

[0323] To a solution of compound 4d (127 mg, 134 μmol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The reaction mixture was partially concentrated to remove DCM, yielding the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the reaction mixture was further diluted with anhydrous acetonitrile (20 mL). Potassium carbonate (73 mg, 530 μmol) was then added, and the reaction mixture was cooled to −7° C., followed by the addition of a solution of cyanuric chloride (62.0 mg, 3.01 mL, 111.8 mM, 336 μmol) in anhydrous acetonitrile. After stirring at −7° C. for 2 h, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (151 mg, 1.85 mL, 894 mM, 1.65 mmol) in water. The ice bath was removed after 15 min, after which it was allowed to warm to room temperature. DMF (2 mL) was added to the reaction mixture, and the reaction mixture was concentrated until only a DMF / water solution remained and purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting solution, consisting primarily of acetonitrile, was dried over Na2SO4, filtered, and the drying agent was washed with anhydrous THF (3 x 750 μL). The combined organic layers were again partially concentrated to a volume of 4 mL (acetonitrile / THF), and compound 5d was used directly. 44 H 54 NO 13 Si + (M+H + LCMS (ESI+) calculated for 832.34, found 832.53.

[0313] [Example a32. Synthesis of compound 6d] [ka]

[0324] To compound 5d (25 mg, 30 μmol) in a mixture of acetonitrile and THF (4 mL) was added triethylammonium acetate (97 mg, 96 μL, 600 μmol), and the resulting red solution was cooled to −20° C. Next, TBAF (1 M in THF, 35 mg, 340 μL, 340 mmol) was added while the reaction mixture was vigorously stirred (a color change from red to green and back to red was observed). After stirring for 3 h, the reaction mixture was quenched with water (2.4 mL). The reaction mixture was transferred to a separatory funnel and extracted with DCM (3 × 5 mL). The combined organic layers were dried over Na2SO4, filtered, concentrated (water bath rotary evaporator set at 32 °C) until approximately 10 mL remained, and purified by flash column chromatography on silica gel (0% → 10% MeOH in DCM) to give compound 6d as a red solution in DCM (4 mL, 0.82 mM, 2.36 mg, 3.29 μmol, 11% based on a doxorubicin-based calibration line for HPLC). 38 H 40 NO 13 + LCMS (ESI+) calculated for (M+H+) 718.25, found 718.50.

[0314] [Example a33. Synthesis of compound 3e] [ka]

[0325] To a stock solution of compound 2 (165 mg, 612 μmol) in anhydrous DMF (410 μL) was added (S)-1-azido-4-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7e (294 mg, 603 μmol) and DIPEA (131 μL, 753 μmol). The reaction mixture was vortexed and allowed to react at room temperature. After 120 h at room temperature, the reaction mixture was diluted with DCM (8 mL) and purified by flash column chromatography on silica gel (0% → 3% MeOH in DCM) to give compound 3e as a dark red oil (53.9 mg, 61.6 μmol, 24.6%). 44 H 55 N4O 13 Si + (M+H + LCMS (ESI+) calculated for 875.35, found 875.61.

[0315] [Example a34. Synthesis of compound 4e] [ka]

[0326] A solution of compound 3e (53.9 mg, 61.6 μmol) in anhydrous DCM (10 mL) was cooled to −78 °C using a dry ice / acetone cooling bath. The mixture was vigorously stirred, and then a freshly made stock solution of mCPBA (20.2 mg, 580 mM, 202 μL, 117 μmol) in anhydrous DCM was added dropwise. After stirring at −78 °C for 8 minutes, complete conversion was achieved. The reaction mixture was quenched with an ice-cold solution of acetone (reagent grade, 565 μL), and the reaction mixture was stirred at −78 °C. After 45 minutes, the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO (10 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over NaSO, filtered through a filter paper, and concentrated to a volume of 10 mL. Compound 4e was used as is without further purification. 44 H 55 N4O 14 Si + (M+H +LCMS (ESI+) calculated for 891.35, found 891.52.

[0316] [Example a35. Synthesis of compound 5e] [ka]

[0327] To a solution of compound 4e (54.9 mg, 61.6 μmol) in DCM (10 mL) was added anhydrous acetonitrile (6 mL). The reaction mixture was partially concentrated to remove DCM, yielding the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the reaction mixture was further diluted with anhydrous acetonitrile (10 mL). Potassium carbonate (33.2 mg, 240 μmol) was then added, and the reaction mixture was cooled to −7° C., followed by the addition of a solution of cyanuric chloride in anhydrous acetonitrile (28.4 mg, 1.39 mL, 111 mM, 154 μmol). After stirring at −7° C. for 6 h, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (69 mg, 330 μL, 2.3 M, 758 μmol) in water. The ice bath was removed after 15 min, after which it was allowed to warm to room temperature. DMF (1 mL) was added to the reaction mixture, and the reaction mixture was concentrated until only a DMF / water solution remained and purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting mixture was purified by a cyclohexane-distillation column (5 μM OBD, 30×100 mm). The collected fractions were combined and concentrated until a volume of 4 mL of acetonitrile remained. It was then dried over Na2SO4, filtered, and the residue was washed with anhydrous THF (3×750 μL). The combined organic layer was again partially concentrated to a volume of 4 mL (acetonitrile / THF), and compound 5e was used directly. 44 H 53 N4O 13 Si + (M+H + LCMS (ESI+) calculated for 873.34, found 873.46.

[0317] [Example a36. Synthesis of compound 6e] [ka]

[0328] Compound 5e (27 mg, 31 μmol) in a mixture of acetonitrile and THF (4 mL) was cooled to −40° C. Next, TBAF (1 M in THF, 80 mg, 300 μL, 300 mmol) was added while the reaction mixture was vigorously stirred (a color change from red to green was observed). After stirring for 2.5 h, the reaction mixture was quenched with water (2.5 mL), and the solution changed from green to red. The reaction mixture was transferred to a separatory funnel and extracted with DCM (3 × 7 mL). The combined organic layers were dried over NaSO, filtered, and purified by flash column chromatography on silica gel (0% → 10% MeOH in DCM) to give compound 6e as a red solution in DCM (10 mL, 0.31 mM, 2.3 mg, 3.0 μmol, 9.8% over three steps based on a calibration line based on doxorubicin for HPLC). 38 H 39 N4O 13 + (M+H + LCMS (ESI+) calculated for 759.25, found 759.40.

[0318] [Example a37. Synthesis of Compound 3f] [ka]

[0329] A stock solution of compound 2 (150 mg, 228 μmol) in anhydrous DMF (373 μL) was added to a vial containing 1,5-diiodo-2(S)-ethoxy-3-oxa-pentane 7f (1.27 g, 2.94 mmol), followed by DIPEA (119 μL, 684 μmol). The reaction mixture was heated to 42 °C, rotated for 1 min, and then left at room temperature in the dark. After 96 h at room temperature, the reaction mixture was diluted with DCM (4 mL) and purified by flash column chromatography on silica gel (0% → 3% MeOH in DCM) to give 3f as a dark red oil (43.3 mg, 56.1 μmol, 24.6%). 39 H 54 NO 13Si + (M+H + LCMS (ESI+) calculated for 772.34, found 772.65.

[0319] [Example a38. Synthesis of compound 4f] [ka]

[0330] A solution of compound 3f (43.3 mg, 56.1 μmol) in anhydrous DCM (700 μL) was cooled to −78 °C in a dry ice / acetone bath. The mixture was vigorously stirred, followed by the dropwise addition of a freshly made stock solution of mCPBA in anhydrous DCM (6.19 mg, 462 mM, 77.6 μL, 35.9 μmol). After stirring for 20 min, a second solution of mCPBA in anhydrous DCM (26.5 μL, 462 mM, 12.2 μmol) was added dropwise, and the resulting red solution was stirred for an additional 15 min. Finally, a third batch of mCPBA in anhydrous DCM (0.24 mg, 462 mM, 3.0 μL, 1.4 μmol) was added. The reaction mixture was stirred for an additional 2 min, then quenched with a pre-cooled (−78 °C) solution of acetone (reagent grade, 300 μL), and the reaction mixture was stirred at −78 °C. After 12 minutes, the cooling bath was removed, the reaction mixture was allowed to warm to room temperature, and diluted with additional DCM (3.5 mL). The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO (2 mL). The aqueous layers were combined and extracted twice with DCM (2 mL). The combined organic layers were dried over NaSO and filtered through a membrane filter. The filtrate, a red solution containing compound 4f, was used without further purification. 39 H 54 NO 14 Si + (M+H + LCMS (ESI+) calculated for 788.33, found 788.64.

[0320] [Example a39. Synthesis of compound 5f] [ka]

[0331] To a solution of compound 4f (44.3 μmol) in a mixture (approximately 10 mL) of primarily DCM and minimal acetone, anhydrous acetonitrile (2 mL) was added. The reaction mixture was partially concentrated (to remove DCM) to a volume of approximately 6 mL. Additional anhydrous acetonitrile (4.0 mL) was then added, and the mixture was partially concentrated again to a volume of 4.4 mL. A stir bar was added, and the reaction mixture was analyzed by HPLC-MS to assess the concentration of starting material 4f (showing 44.3 μmol of 4f based on a calibration line based on doxorubicin). Potassium carbonate (31.3 mg, 226 μmol) was then added, and the reaction mixture was cooled to 0 °C, followed by the addition of a solution of cyanuric chloride in anhydrous acetonitrile (20.5 mg, 1.00 mL, 111 mM, 111 μmol). After stirring at 0 °C for 2.5 h, the reaction mixture was treated again with a solution of cyanuric chloride in anhydrous acetonitrile (80 μL, 111 mmol, 8.9 μmol). The reaction mixture was stirred at 0°C for an additional 23 minutes and then quenched with an aqueous solution of 3-aminopropane-1,2-diol (62.9 mg, 345 µL, 2.0 M, 690 µmol). The resulting dark red solution was allowed to warm slowly to room temperature. DMF (3 mL) was added to the reaction mixture, resulting in a red solution with a nearly white precipitate. The solution was decanted, and the residue was washed several times with additional DMF, which was then filtered through a membrane filter and combined with the decanted solution. The solution was partially concentrated under reduced pressure to remove most of the acetonitrile, and then purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C). 18 The resulting solution, consisting primarily of acetonitrile, was dried over NaSO, filtered, and the drying agent was washed with anhydrous THF (three times). The combined organic layers were partially concentrated again to a volume of 1.7 mL (acetonitrile / THF), and compound 5f (9.32 mM, 15.8 mM, 35.7% based on a doxorubicin-based calibration line for HPLC) was used directly. 39 H 52 NO 13 Si + (M+H+ LCMS (ESI+) calculated for 770.32, found 770.65.

[0321] [Example a40. Synthesis of compound 6f] [ka]

[0332] To compound 5f (12.9 mg, 15.8 μmol) in a mixture of acetonitrile and THF (1.7 mL) was added triethylammonium acetate (13.8 mg, 13.7 μL, 85.5 μmol), and the resulting red solution was cooled to −15° C. Next, TBAF (1 M in THF, 22.4 mg, 85.5 μL, 85.5 μmol) was added while stirring the reaction mixture (a color change from red to green and back to red was observed). After stirring for 46 minutes, additional triethylammonium acetate (13.8 mg, 13.7 μL, 85.5 μmol) and TBAF (1 M in THF, 22.4 mg, 85.5 μL, 85.5 μmol) were added. The reaction mixture was stirred for an additional 20 minutes, after which a third batch of TBAF (1 M in THF, 22.4 mg, 85.5 μL, 85.5 μmol) was added, and it was stirred for an additional 30 minutes. Finally, a fourth batch of TBAF (1M in THF, 10.0 mg, 40.0 μL, 40.0 μmol) was added, and the reaction mixture was stirred at −10° C. for an additional 35 minutes before being quenched with water (1.0 mL). The reaction mixture was warmed to room temperature, diluted with DCM (7.5 mL), and transferred to a separatory funnel. The two-phase system was separated, and the aqueous layer was extracted with DCM (2 × 1 mL). The combined organic layers were dried over NaSO, filtered, and subsequently purified by flash column chromatography on silica gel (0% → 6% MeOH in DCM). The pure fractions were combined, partially concentrated to a volume of 4.5 mL, and then diluted with MeOH (7 mL). The solution was then partially concentrated again to a volume of 6 mL and diluted to a volume of 6.7 mL to give compound 6f as a red solution in most MeOH (6.7 mL, 1.55 mM, 10.36 μmol, 65.6% based on a doxorubicin-based calibration line for HPLC). 33 H 38 NO13 + LCMS (ESI+) calculated for (M+H+) 656.23, found 656.59.

[0322] [Example a41. Synthesis of Compound 3a] [ka]

[0333] To a solution of nemorubicin (75 mg, 0.12 mmol) and imidazole (40 mg, 0.58 mmol) in anhydrous DMF (2.0 mL) was added TBDMS-Cl (53 mg, 0.35 mmol, 3 equiv.). The resulting red solution was mixed and allowed to stand at room temperature for 15 minutes. The mixture was then diluted with DCM (22 mL) and purified by flash column chromatography on silica gel (1% → 5% MeOH in DCM) to give compound 3a as a dark red oil (134 mg, quantitative). 38 H 52 NO 13 Si + (M+H + LCMS (ESI+) calculated for 758.32, found 758.59.

[0323] [Example a42. Synthesis of Compound 4a] [ka]

[0334] A solution of compound 3a (120 μmol) in anhydrous DCM (15.0 mL) was cooled to −78° C. in a dry ice / acetone cooling bath. The mixture was stirred vigorously, and then a freshly made stock solution of mCPBA (33.1 mg, 580 mM, 331 μL, 192 μmol) in anhydrous DCM was added dropwise with vigorous stirring. After stirring for 18 minutes, the mixture was quenched with a pre-cooled (−78° C.) solution of acetone (reagent grade, 1.1 mL). After 23 minutes, the cooling bath was removed and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO3 (3 mL). The aqueous layers were combined and extracted once with DCM (4 mL). The combined organic layers were dried over Na2SO4 and partially concentrated under reduced pressure to a volume of 12 mL to give compound 4a as a red solution in mostly DCM, which was used without further purification. 38 H 52 NO 14 Si + (M+H + LCMS (ESI+) calculated for 774.32, found 774.50.

[0324] [Example a43. Synthesis of Compound 5a] [ka]

[0335] To a solution of compound 4a (100.6 μmol) in a mixture (10.1 mL) of primarily DCM and minimal acetone, anhydrous acetonitrile (8 mL) was added. The reaction mixture was partially concentrated to remove the DCM to a volume of approximately 4 mL. Potassium carbonate (54.5 mg, 394 μmol) was then added, and the reaction mixture was cooled to 0° C., followed by the addition of a solution of cyanuric chloride in anhydrous acetonitrile (46.4 mg, 2.266 mL, 111 mM, 251.5 μmol). After stirring at 0° C. for 150 min, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (1.33 mL, 929 mM, 1.24 mmol) in water. The resulting dark red solution was allowed to slowly warm to room temperature over 20 min. To the reaction mixture was added DMF (1.33 mL), and the resulting mixture was partially concentrated and then purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting mixture was purified by a 5 μm OBD, 30×100 mm column. The collected fractions were combined and concentrated until a volume of 4 mL remained. The resulting solution, consisting mainly of acetonitrile, was dried over NaSO, filtered, and the drying agent was washed with anhydrous THF (3 times). The combined organic layers were again partially concentrated to a volume of 4.5 mL (acetonitrile / THF) to give compound 5a as a red solution, which was used directly in the next step. 38 H 48 NO 13 Si - (MH + LCMS (ESI+) calculated for 754.29 found 754.52.

[0325] [Example a44. Synthesis of Compound 6a] [ka]

[0336] To compound 5a (40 mg, 53 μmol) in a mixture of acetonitrile and THF (4.5 mL) was added triethylammonium acetate (170 μL, 1.06 mmol), and the resulting red solution was cooled to −15° C. Next, while stirring the reaction mixture, TBAF (1 M in THF, 65 mg, 0.25 mL, 0.25 mmol) was added (a color change from red to green and back to red was observed). After stirring for 1 minute, additional TBAF (1 M in THF, 63 mg, 0.24 mL, 0.24 mmol) was added. After stirring the reaction mixture for an additional 14 minutes, a third batch of TBAF (1 M in THF, 46 mg, 0.17 mL, 0.17 mmol) was added, and it was stirred for an additional 41 minutes. The reaction was quenched with water (2.4 mL). The reaction mixture was allowed to warm to room temperature over 25 min and then combined with a second batch of crude compound 6a, which was obtained in the same manner as above, starting with compound 5a (10 mg, 13.0 μmol). After combining both quenched reaction mixtures, DCM (6 mL) was added and the resulting biphasic system was separated. The aqueous layer was extracted twice (4 mL, then 2 mL), and the combined organic layers were dried (NaSO) and then purified by flash column chromatography on silica gel (0% → 6% MeOH in DCM). Pure fractions were combined, partially concentrated to a volume of 8-9 mL, and then diluted to a volume of 10.0 mL with additional DCM to give compound 6a as a red solution in most of DCM (10.0 mL, 1.66 mM, 16.6 μmol, 16.5% over two steps (assuming quantitative conversion during N-oxide formation) based on a doxorubicin-based calibration line for HPLC). 32 H 36 NO 13 + LCMS (ESI+) calculated for (M+H+) 642.22, found 642.46.

[0326] [Example a45. Synthesis of Compound 8b] [ka]

[0337] A mixture of doxorubicin HCl salt (48.6 mg, 83.8 μmol, 1.00 equiv.) and (S)-1-(2-azidoethoxy)-2-iodo-1-(2-iodoethoxy)ethane 7b (101.0 mg, 245.8 μmol, 2.93 equiv.) was dissolved in anhydrous DMF (175 μL) and DIPEA (58.7 μL, 335 μmol, 4 equiv.) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL), and purified by flash column chromatography on silica gel (preconditioned column 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8b as a red oil (10.8 mg, 15.5 μmol, 18.4%). 33 H 39 N4O 13 + (M+H + LCMS (ESI+) calculated for 669.25, found 699.62.

[0327] [Example a46. Synthesis of Compound 9] [ka]

[0338] To a solution of 8b (8.1 mg, 12 μmol, 1.0 equiv.) in a 1:1 mixture of MeOH / DCM (150 μL) was added a 200 mmol solution of PPh3 in DCM (177 μL, 31.9 μmol, 2.7 equiv.) and HO (80 μL). The resulting biphasic system was stirred in the dark at room temperature for 8 h. The reaction mixture was then stored in a freezer for 8 days and then concentrated under reduced pressure. The residue was taken up in DMF and purified by preparative HPLC (5% → 90% acetonitrile in water, column Xbridge Prep C). 18 The purified compound was purified by a 5 μm OBD column (30×100 mm) to give compound 9 as a red residue (0.5 mg, 0.7 μmol, 6% yield). Impure fractions from the preparative HPLC purification were combined and subjected to a second preparative HPLC purification (5% to 90% acetonitrile in water, column Xbridge Prep C). 18 Purification by column chromatography (5 μm OBD, 30×100 mm) afforded additional compound 9 as a red residue (0.6 mg, 0.9 μmol, 8% yield).33 H 41 N2O 13 + (M+H + LCMS (ESI+) calculated for 673.26, found 673.65.

[0328] [Example a47. Synthesis of Compound 8c] [ka]

[0339] A mixture of doxorubicin HCl salt (51.3 mg, 88.5 μmol, 1.00 equiv.) and (R)-2-(2-iodo-1-(2-iodoethoxy)ethoxy)propane 7c (104 mg, 271 μmol, 3.06 equiv.) was dissolved in anhydrous DMF (175 μL) and DIPEA (61.6 μL, 354 μmol, 4 equiv.) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL), and purified by flash column chromatography on silica gel (preconditioned column 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8c as a red oil (12.2 mg, 18.2 μmol, 20.5%). 34 H 42 NO 13 + LCMS (ESI+) calculated for (M+H+) 672.27, found 672.60.

[0329] [Example a48. Synthesis of Compound 8d] [ka]

[0340] A mixture of doxorubicin HCl salt (49.7 mg, 85.7 μmol, 1.00 equiv.) and (S)-((2-iodo-1-(2-iodoethoxy)ethoxy)methyl)benzene 7d (106.5 mg, 246.5 μmol, 2.88 equiv.) was dissolved in anhydrous DMF (175 μL) and DIPEA (59.7 μL, 343 μmol, 4 equiv.) was added. The red suspension was stirred in the dark at room temperature for 3 days, diluted with DCM (1.8 mL), and purified twice by flash column chromatography on silica gel (preconditioned column 2% MeOH / DCM, 0% → 13% MeOH in DCM) to give compound 8d as a red oil (6.4 mg, 8.9 μmol, 10%). 38 H 42 NO 13 + LCMS (ESI+) calculated for (M+H+) 720.27, found 720.64.

[0330] [Example a49. Synthesis of compound 8f] [ka]

[0341] A mixture of doxorubicin HCl salt (52.3 mg, 90.2 μmol, 1.00 equiv.) and (S)-1-ethoxy-2-iodo-1-(2-iodoethoxy)ethane 7f (102.1 mg, 276.0 μmol, 3.06 equiv.) was dissolved in anhydrous DMF (175 μL) and DIPEA (62.8 μL, 361 μmol, 4 equiv.) was added. The red suspension was stirred in the dark at room temperature for 4 days, diluted with DCM (1.8 mL), and purified by flash column chromatography on silica gel (preconditioned column 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8f as a red oil (11.1 mg, 16.9 μmol, 18.7%). 33 H 40 NO 13 + LCMS (ESI+) calculated for (M+H+) 658.25, found 658.70.

[0331] [Example a50. Synthesis of Compound 31] [ka]

[0342] To a solution of Fmoc-N-ethylene-1,2-diamine.HCl (26 mg, 83 μmol) in anhydrous DMF (200 μL) was added a solution of compound 29 (67 mg, 75 μmol) in anhydrous DCM (800 μL) and triethylamine (32 μL, 23 mg, 230 μmol). After stirring at room temperature for 1 hour, the reaction mixture was purified by flash column chromatography on silica gel (0% → 30% EtOAc in DCM (to remove p-nitrophenol), followed by 0% → 25% MeOH in DCM) to give intermediate 30 as a colorless oil (32.7 mg, 32 μmol, 43%). 49 H 62 N7O 13 S + (M+H + LCMS (ESI+) calculated for 988.41, found 988.78.

[0332]

[0343] To a solution of intermediate 30 (16.3 mg, 16.5 μmol) in DMF (150 μL) was added triethylamine (13.8 μL, 10 mg, 99.0 μmol). After stirring at room temperature for 18 h, complete conversion was obtained and the reaction mixture was concentrated to give compound 31 as an oil (12.6 mg, 16.5 μmol, 100%). 34 H 52 N7O 11 S + (M+H + LCMS (ESI+) calculated for 766.34, found 766.65.

[0333] [ka] [Example a51. Synthesis of Compound 9c]

[0344] To a solution of compound 6c (3.26 mg, 4.87 μmol) in DCM, MeOH (3 mL) was added, and the mixture was concentrated until only MeOH (1.5 mL) remained. Water (200 μL) was added, followed by a solution of sodium periodate in water (60 mM, 282 μL, 16.9 μmol), and the reaction mixture was stirred in the dark for 41 h. Upon complete conversion, DMF (800 μL) was added, and the reaction mixture was concentrated until 400 μL remained in DMF (2.19 mg, 3.34 μmol, 68.6%). This intermediate was then added to compound 31 (10.2 mg, 13.2 μmol), followed by DiPEA (2 μL, 10 μmol) and HATU (2.1 mg, 28 μL, 200 mM, 5.4 μmol). After stirring at room temperature for 20.5 hours, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting solution was purified by HPLC using a 5 μm OBD, 30×100 mm column. Compound 9c was obtained as a red solution in DMF (250 μL, 3.83 mM, 1.34 mg, 0.95 μmol, 28.6% based on a doxorubicin-based calibration line for HPLC). 67 H 87 N8O 23 S + (M+H + LCMS (ESI+) calculated for 1404.51, found 1404.06.

[0334] [Example a52. Synthesis of compound 9d]

[0345] To a solution of compound 6d (3.4 mg, 4.7 μmol) in DCM, MeOH (5 mL) was added, and the mixture was concentrated until only MeOH (2.1 mL) remained. Next, a solution of sodium periodate in water (60 mM, 288 μL, 17.5 μmol) was added, and the reaction mixture was stirred in the dark for 68 h. Once complete conversion was achieved, DMF (600 μL) was added, and the reaction mixture was concentrated until 490 μL remained in DMF (1.78 mg, 2.53 μmol, 53%). This intermediate was then added to compound 31 (6.2 mg, 8.1 μmol), followed by the addition of DiPEA (1.32 μL, 7.59 μmol) and HATU (1.15 mg, 15.2 μL, 200 mM, 3.04 μmol). After stirring at room temperature for 21.5 h, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The mixture was purified by HPLC using a 5 μm OBD, 30×100 mm column. Compound 9d was obtained as a red solution in DMF (165 μL, 10.14 mM, 2.42 mg, 1.67 μmol, 65.9% based on a doxorubicin-based calibration line for HPLC). 71 H 87 N8O 23 S + (M+H + LCMS (ESI+) calculated for 1452.56, found 1452.03.

[0335] [Example a52-2. Synthesis of compound 9f]

[0346] To a solution of compound 6f (6.8 mg, 10.4 μmol) in a mixture of MeOH (6.7 mL) and water (1.4 mL), an aqueous solution of sodium periodate (62.9 mM, 206 μL, 13.0 μmol) was added, and the reaction mixture was stirred at room temperature in the dark for 3 hours. Additional aqueous sodium periodate (62.9 mM, 210 μL, 13.2 μmol) was added, and the reaction mixture was stirred at room temperature for an additional 17 hours. Finally, a third batch of sodium periodate in water (62.9 mM, 50 μL, 3.1 μmol) was added, and the mixture was stirred at room temperature for 80 minutes, then partially concentrated under reduced pressure to a volume of 5.4 mL and then left at room temperature for an additional 5 hours. Next, DMF (670 μL) was added, and the resulting red solution was partially concentrated to a volume of approximately 350 μL, yielding a white residue and a red solution containing the crude intermediate. The mixture was diluted to 666 μL with additional DMF, and then 222 μL (3.45 μmol) of this solution was treated with a stock solution of compound 31 in DMF (110 mmol, 62.7 μL, 6.9 μmol), followed by DiPEA (1.79 μL, 10.4 μmol) and a solution of HATU in dry DMF (204 mM, 16.9 μL, 3.45 μmol). The resulting mixture was vortexed and allowed to stand at room temperature for 31 minutes. Next, additional compound 31 in DMF (110 mmol, 13.9 μL, 1.53 μmol) and HATU in dry DMF (204 mM, 33.8 μL, 6.90 μmol) were added. The mixture was vortexed again, allowed to stand at room temperature for 13 minutes, and then stored in a freezer for 17 hours. Finally, the mixture was removed from the freezer and analyzed by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting solution was purified by HPLC using a 5 μm OBD, 30×100 mm column. Compound 9f was obtained as a red solution in DMF (300 μL, 8.2 mM, 3.9 mg, 2.47 μmol, 71.6% based on a doxorubicin-based calibration line for HPLC). 66 H 85 N8O 23 S + (M+H + LCMS (ESI+) calculated for 1389.54, found 1390.07.

[0336] [Example a53. Synthesis of compound 9g (OMe-PNU)]

[0347] To a solution of compound 6a (2.17 mg, 3.38 μmol) in DCM, MeOH (3 mL) was added, and the mixture was concentrated until only MeOH (1.35 mL) remained. Water (300 μL) was added, followed by a solution of sodium periodate in water (60 mM, 112.4 μL, 6.8 μmol), and the reaction mixture was stirred in the dark for 19 hours. The stir bar was removed, the reaction mixture was concentrated under reduced pressure, and DMF (200 μL) was added to the residue to give a white residue and a red solution containing the crude intermediate. Compound 31 (6 mg, 7.8 μmol) was then added to this intermediate, followed by DiPEA (1.8 μL, 10.5 μmol) and HATU (2.05 mg, 5.4 μL, 196 mM, 5.4 μmol). After 25 min at room temperature, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting solution was purified by HPLC using a 5 μm OBD, 30×100 mm column. Compound 9g was obtained as a red solution in DMF (122 μL, 14.24 mM, 2.4 mg, 1.74 μmol, 80.9% based on a doxorubicin-based calibration line for HPLC). 65 H 83 N8O 23 S + (M+H + LCMS (ESI+) calculated for 1375.53, found 1376.01.

[0337] [Example a54. Synthesis of Compound 32] [ka]

[0348] To a solution of compound 3b (80.4 mg, 98.9 μmol) in MeOH (600 μL) was added a solution of triphenylphosphine in DCM (88.2 mg, 967 μL, 348 mM, 336 μmol) and water (450 μL). The biphasic mixture was stirred at room temperature for 3 hours, after which Fmoc-Val-Ala-PAB-OPNP (79.4 mg, 117 μmol) was added. The biphasic mixture was stirred at room temperature for an additional 16 hours and then extracted with DCM (2 × 1 mL) to remove water. The combined organic layers were dried over Na2SO4 and immediately purified by flash column chromatography on silica gel (0% to 40% EtOAc in DCM (to remove excess Fmoc-Val-Ala-PAB-OPNP), followed by 0% to 15% MeOH in DCM) to give compound 32 as a clear red solution in DCM (7.7 mL, 123.6 mg, 93 μmol, 94.1%). 70 H 86 N5O 19 Si + (M+H + LCMS (ESI+) calculated for 1329.54, found 1329.00.

[0338] [Example a55. Synthesis of Compound 33] [ka]

[0349] A solution of compound 32 (123.6 mg, 93 μmol) in anhydrous DCM (7.7 mL) was concentrated until 2 mL of solution remained. The reaction mixture was then cooled to -78 °C in a dry ice / acetone cooling bath. The mixture was vigorously stirred, and then a freshly made stock solution of m-CPBA in anhydrous DCM (17.66 mg, 580 mM, 176 μL, 102.3 μmol) was added dropwise. After stirring for 17 minutes, complete conversion was obtained. The reaction mixture was quenched with an ice-cold solution of acetone (reagent grade, 2.5 mL), and the reaction mixture was stirred. After 1 hour, the cooling bath was removed, and the reaction mixture was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO3 (10 mL). The aqueous layers were combined and extracted twice with DCM (20 mL). The combined organic layers were dried over Na2SO4, filtered through a filter paper, and concentrated to a volume of 19 mL. Compound 33 (108.3 mg, 80.5 μmol, 86.6%) was used as is without further purification. 70 H 86 N5O 20 Si + (M+H + LCMS (ESI+) calculated for 1345.54, found 1345.14.

[0339] [Example a56. Synthesis of Compounds 34 and 35] [ka]

[0350] To a solution of compound 33 (108.3 mg, 80.5 μmol) in DCM (19 mL) was added anhydrous acetonitrile (10 mL). The reaction mixture was concentrated to remove DCM, yielding the compound in anhydrous acetonitrile (5 mL). After concentrating most of the solvent, the reaction mixture was further diluted with anhydrous acetonitrile (45 mL). Potassium carbonate (65 mg, 463 μmol) was then added, and the reaction mixture was cooled to -10 °C, followed by the addition of cyanuric chloride (55.7 mg, 2.44 mL, 120 mM, 302.2 μmol) as a stock solution in anhydrous acetonitrile. After stirring at 0 °C for 5.5 h, the reaction mixture was quenched with a solution of 3-aminopropane-1,2-diol (90.3 mg, 1.1 mL, 900 mM, 990 μmol) in water. The ice bath was removed after 15 min, and it was allowed to warm to room temperature. DMF (3 mL) was added to the reaction mixture, and the reaction mixture was concentrated until only a DMF / water solution remained and purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C 18 The resulting mixture was purified by HPLC (5 μM OBD, 30×100 mm). The collected fractions were combined and concentrated until a volume of 11 mL of acetonitrile remained. It was then dried over Na2SO4, filtered, and the drying agent was washed with anhydrous THF (3×1 mL). Intermediate 34 was obtained as a solution in acetonitrile / THF (14 mL, 8.7 mg, 6.6 μmol, 8.1%). 70 H 84 N5O 19 Si + (M+H + LCMS (ESI+) calculated for 1327.52, found 1327.07.

[0340]

[0351] A solution of intermediate 34 (8.7 mg, 6.6 μmol) in anhydrous acetonitrile / THF (14 mL) was concentrated until 7 mL of solution remained, and anhydrous THF (1 mL) was added. To this was added triethylammonium acetate (5.3 mg, 5.3 μmol, 33 μmol), and the resulting red solution was cooled to -15 °C. Next, TBAF (1 M in THF, 17.2 mg, 66 μL, 66 μmol) was added while the reaction mixture was vigorously stirred (a color change from red to green was observed). After stirring for 3 h, the reaction mixture was quenched with water (1.5 mL), and the reaction mixture turned red again. The reaction mixture was transferred to a separatory funnel and extracted with DCM (20 mL). The combined organic layers were dried over Na2SO4 and directly purified by flash column chromatography on silica gel (0% to 10% MeOH in DCM) to give compound 35 as a red solution in DCM (8 mL, 2.3 mg, 1.9 μmol, 29%). 64 H 70 N5O 19 + (M+H + LCMS (ESI+) calculated for 1212.47, found 1212.93.

[0341] [Example a57. Synthesis of Compound 36] [ka]

[0352] The synthesis of BCN-HS-PEG2-OPNP is described in International Publication No. 2021144314A1, which is incorporated herein by reference. To a solution of compound 35 (2.3 mg, 1.9 μmol) in DCM (8 mL), DMF (100 μL) was added, and the reaction mixture was concentrated to remove the DCM. Next, a solution of BCN-HS-PEG2-OPNP (1.2 mg, 25 μL, 93 mM, 2.3 μmol) in DMF was added, followed by triethylamine (2.6 μL, 19 μmol). After 30 hours at room temperature, the reaction mixture was further diluted with DCM (300 μL) and purified by flash column chromatography on silica gel (0% → 15% MeOH in DCM) to give compound 36 as a red solution in DMF (150 μL, 0.64 mM, 0.13 mg, 0.09 μmol, 5% based on a doxorubicin-based calibration line for HPLC). 65 H 82 N7O 24 S + (M+H + LCMS (ESI+) calculated for 1377.44, found 1377.04.

[0342] [Example a58. Synthesis of Compound 38] [ka]

[0353] This compound was synthesized according to literature procedures described in WO2017137457A1.

[0343] [Example a59. Synthesis of Compound 39] [ka]

[0354] To a solution of compound 6e (1.0 mg, 1.3 μmol, 1.00 equiv.) in wet THF (90 μL) was added PPh (200 mmol, 13 μL, 2.6 μmol, 2.0 equiv.) in THF and HO (13 μL). The resulting red solution was vortexed and then allowed to stand at room temperature for 19 hours. The mixture was then transferred to an Eppendorf vial and placed in an Eppendorf shaker at 37 °C and 1400 RPM for 6.5 hours, then at room temperature and 1400 RPM for an additional 19 hours. The reaction mixture was then stored in a freezer for 11 days and then treated with a solution of compound 38 (790 mM, 2.0 μL, 1.6 μmol, 1.2 equiv.), followed by the addition of HATU (517 mM, 1.4 μmol, 1.1 equiv.) in DMF, and finally, a solution of DMAP (500 mM, 1.1 μL, 0.53 μmol, 0.4 equiv.) in DMF. The resulting mixture was vortexed, left at room temperature for 3 hours, and then stored in a freezer for 21 hours. The mixture was removed from the freezer and purified by preparative HPLC (40% → 100% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge preparative C18, 5 μm OBD, 30 × 100 mm). Pure fractions were combined, diluted with DMF, and partially concentrated to give compound 39 as a red solution in DMF (85 μL, 1.5 mM, 0.17 mg, 0.13 μmol, 10% based on a doxorubicin-based calibration line for HPLC). 62 H 77 NO 22 S + (M+H + LCMS (ESI+) calculated for 1289.48, found 1289.99.

[0344] [Example a60. Synthesis of compounds 42 and 43] [ka]

[0355] To a vial containing Boc-Gly-Gly-Phe-Gly-OH (500 mg, 1.15 mmol, 1.00 equiv.) and Fmoc-EDA-H (476 mg, 1.69 mmol, 1.47 equiv.), DMF (1.25 mL) was added, followed by EtN (479 μL, 3.44 mmol, 3.00 equiv.) and finally HATU (479 mg, 1.26 mmol, 1.10 equiv.). The resulting mixture was stirred at room temperature for 2 min, after which DCM (2 mL) was added to produce a clear yellow solution, which was stirred at room temperature for 110 min. The reaction mixture was then stored in a freezer for 16 h. The mixture was then removed from the freezer, diluted with DCM (15 mL), and directly purified by flash column chromatography (50 to 100% EtOAc in heptane). The product-containing fractions were concentrated to give compound 42 as a white solid (357 mg, 509 μmol, 45%). 37 H 45 N6O8 + (M+H + LCMS (ESI+) calculated for 42: 701.33, found: 701.67. Compound 42 (357 mg, 509 μmol, 1.00 equiv) was suspended in DCM (3.0 mL) and cooled to 0 °C in an ice bath. TFA (1.37 mL, 17.8 mmol, 35 equiv) was then added dropwise to produce a pale yellow solution, which was stirred at 0 °C for 47 min. The ice bath was then removed, and the reaction mixture was allowed to warm to room temperature and stirred for an additional 75 min. The mixture was concentrated under reduced pressure, and the residue was taken up in a mixture of DCM (1.5 mL) and toluene (2 mL) and concentrated a second time to give compound 43 (TFA salt) as a brittle solid, which was used in the next step without further purification. Alternatively, compound 43 was subjected to preparative HPLC purification (5% to 95% acetonitrile with 1% AcOH in water with 1% AcOH, column Xbridge Prep C18, 5 μM OBD, 30 × 100 mm) to give compound 43 as the acetate salt. 32 H 37 N6O6 + (M+H + LCMS (ESI+) calculated for 601.28, found 601.60.

[0345] [Example a61. Synthesis of Compounds 45 and 46] [ka]

[0356] BCN-OH 44 (40 mg, 1 To a solution of 43 (90% by weight, 240 μmol, 1.00 equiv. by H-qNMR), chlorosulfonyl isocyanate (22 μL, 250 μmol, 1.05 equiv.) was added in one portion to produce a pale yellow solution. The mixture was stirred at −40 °C for 17 min, followed by the addition of EtN (67 μL, 480 μmol, 2.00 equiv.). The resulting solution was stirred at −40 °C for approximately 5 min, followed by the addition of a solution of the acetate salt of compound 43 (80 mg, 120 μmol, 0.51 equiv.) and DIPEA (23 μL) in DMF (250 μL). After 10 min, the TFA salt of compound 43 (115.4 mg, 161.5 μmol, 0.67 equiv.) was added as a suspension in a mixture of DIPEA (46 μL) and EtN (23 μL) in DMF (5 mL). Finally, dimethylacetamide (1 mL) was added, and the reaction mixture was allowed to warm to room temperature over 5 hours. The reaction mixture was then stored in a freezer for 5 days, then removed from the freezer and concentrated under reduced pressure. The residue was purified by preparative HPLC (40% to 90% acetonitrile with 1% AcOH in water, Xbridge preparative C18 column, 5 μm OBD, 30 × 100 mm) to give compound 45 (8.9 mg, 9.5 μmol, 4% yield). 43 H 50 N7O 10 S + (M+H +LCMS (ESI+) calculated for 45: 856.33, found: 856.66. To a solution of compound 45 (8.9 mg, 9.5 μmol) in wet DMF (400 μL) was added EtN (58 μL, 0.42 mmol, 40 equiv). The resulting mixture was mixed and allowed to stand at room temperature for approximately 18 hours, then concentrated under reduced pressure to a volume of approximately 25 μL. This solution was transferred to an Eppendorf vial with additional DMF to give compound 46 as a solution in a final volume of 150 μL of DMF, which was used in the next step without further purification. 28 H 40 N7O8S + (M+H + LCMS (ESI+) calculated for 634.27, found 634.54.

[0346] [Example a62. Synthesis of Compound 47] [ka]

[0357] To a solution of compound 6f (6.8 mg, 10.4 μmol) in a mixture of MeOH (6.7 mL) and water (1.4 mL), a solution of sodium periodate in water (62.9 mM, 206 μL, 13.0 μmol) was added, and the reaction mixture was stirred at room temperature in the dark for 3 hours. Additional aqueous sodium periodate (62.9 mM, 210 μL, 13.2 μmol) was added, and the reaction mixture was stirred at room temperature for an additional 17 hours. Finally, a third batch of sodium periodate in water (62.9 mM, 50 μL, 3.1 μmol) was added, and the mixture was stirred at room temperature for 80 minutes, then partially concentrated under reduced pressure to a volume of 5.4 mL, and then allowed to stand at room temperature for an additional 5 hours. Next, DMF (670 μL) was added, and the resulting red solution was partially concentrated to a volume of approximately 350 μL, yielding a white residue and a red solution containing the crude intermediate. The mixture was diluted to 666 μL with additional DMF, and then 222 μL (3.45 μmol) of this solution was treated with a stock solution of compound 46 in DMF (66.6 mmol, 150 μL, 9.99 μmol), followed by DiPEA (1.80 μL, 10.4 μmol) and a solution of HATU in dry DMF (490 mM, 7.1 μL, 3.5 μmol). The resulting mixture was vortexed and allowed to stand at room temperature for 17 minutes. Next, additional HATU in dry DMF (490 mM, 7.1 μL, 3.5 μmol) was added. The mixture was vortexed again and allowed to stand at room temperature for 23 minutes. A third batch of HATU in dry DMF (490 mM, 7.1 μL, 3.5 μmol) was added. The resulting mixture was vortexed and allowed to stand at room temperature for 14 minutes. Next, additional compound 46 (2.4 mg, 3.8 μmol) was added to the reaction mixture, and 14 minutes later, additional compound 46 (2.4 mg, 3.8 μmol) and a fourth batch of HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF were added. The mixture was stirred for an additional 29 minutes at room temperature, after which additional DiPEA (1.80 μL, 10.4 μmol) was added. The mixture was then passed through a membrane filter and then treated with a fifth and final batch of HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF.The mixture was vortexed and left at room temperature for 80 minutes, then purified by preparative HPLC (40% to 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge preparative C18, 5 μm OBD, 30 × 100 mm). Compound 47 was obtained as a red solution in DMF (196 μL, 3.5 mM, 1.0 mg, 0.684 μmol, 20% based on a doxorubicin-based calibration line for HPLC). 60 H 73 N8O 20 S + (M+H + LCMS (ESI+) calculated for 1257.47, found 1357.86.

[0347] [Example a63. Synthesis of compounds 49 and 50] [ka]

[0358] To a suspension of (tert-butoxycarbonyl)glycylglycylglycine (337.4 mg, 1.17 mmol, 1.00 equiv.) and Fmoc-EDA-H (368.8 mg, 1.306 mmol, 1.12 equiv.) in DMF (1 mL) was added triethylamine (488 μL, 3.50 mmol, 3.00 equiv.). To the resulting suspension was added HATU (494.8 mg, 1.301 mmol, 1.12 equiv.). The resulting yellow mixture was stirred at room temperature for 2 min, followed by the addition of DCM (2 mL), producing a yellow solution that was stirred at room temperature for an additional 90 min. The mixture was then stored in a freezer for 1 day. The mixture was removed from the freezer and then purified by flash column chromatography (0 to 10% MeOH in DCM) to give product 50 (443.5 mg, 801.1 μmol, 68.7%) as a white solid. 28 H 36 N5O7 + (M+H +LCMS (ESI+) calculated for 50: 554.26, found: 554.60. Compound 50 was then dissolved in DCM (2.00 mL), and the resulting mixture was cooled to 0° C. in an ice bath. TFA (400 μL, 5.19 mmol, 7.05 equiv) was then added dropwise to the stirring reaction mixture. Upon completion of the addition, the ice bath was removed, and the resulting solution was stirred at room temperature for 85 minutes. Additional TFA (1.6 mL, 21 mmol, 28 equiv) was added portionwise, and the resulting mixture was stirred at room temperature for an additional 200 minutes. The mixture was then concentrated under reduced pressure, and the residue was then purified by preparative HPLC (5% to 90% acetonitrile in water, column Xbridge Prep C). 18 The mixture was purified by filtration (5 μm OBD, 30×100 mm). Compound 50 was obtained as a white powder (84.9 mg, 186 μmol, 25%). 23 H 28 N5O5 + (M+H + LCMS (ESI+) calculated for 454.21, found 454.54.

[0348] [Example a64. Synthesis of Compounds 51 and 52] [ka]

[0359] BCN-OH 44 (30.5 mg, 1To a solution of 50 (90% by weight by H-qNMR, 183 μmol, 1.00 equiv.) was added chlorosulfonyl isocyanate (16.7 μL, 192 μmol, 1.05 equiv.) in one portion to produce a pale yellow solution. The mixture was stirred at −40° C. for 5 min, followed by the addition of EtN (76.4 μL, 548 μmol, 3.00 equiv.). The resulting solution was stirred at −40° C. for approximately 14 min, followed by the addition of compound 50 (84.9 mg, 187 μmol, 1.02 equiv.) in DMF (450 μL). The reaction mixture was stirred at −40° C. for 10 min and then allowed to warm to room temperature over 2 h. The reaction mixture was then stored in the freezer for 1 day, then removed from the freezer, diluted with DCM, and purified by flash column chromatography (0→13% MeOH in DCM) to give impure intermediate 51 (containing EtN) in two batches (45 μmol total, 25% yield). The two batches were combined and co-evaporated with DMF. The resulting oil was diluted with DCM (10 mL) and washed with saturated aqueous NH4Cl (2 mL, twice). A viscous oil formed during extraction, which was combined with the organic layer and concentrated under reduced pressure to give intermediate 51 as a brown, sticky solid. The residue was taken up in 600 μL of DMF, and Et3N (240 μL, 1.70 mmol, 40 equiv.) was added. The resulting mixture was stirred at room temperature for 1 day and then stored in the freezer for 3 days. The reaction mixture was removed from the freezer and filtered through a membrane filter, which was washed with additional DMF (3 times). The resulting yellow solution was partially concentrated to a volume of approximately 240 μL to give compound 52 as a yellow solution in DMF, which was used in the next step without further purification. 19 H 31 N6O7S + (M+H + LCMS (ESI+) calculated for 487.20, found 487.55.

[0349] [Example a65. Synthesis of Compound 53] [ka]

[0360] To a solution of compound 6f (6.8 mg, 10.4 μmol) in a mixture of MeOH (6.7 mL) and water (1.4 mL), a solution of sodium periodate in water (62.9 mM, 206 μL, 13.0 μmol) was added, and the reaction mixture was stirred at room temperature in the dark for 3 hours. Additional aqueous sodium periodate (62.9 mM, 210 μL, 13.2 μmol) was added, and the reaction mixture was stirred at room temperature for an additional 17 hours. Finally, a third batch of sodium periodate in water (62.9 mM, 50 μL, 3.1 μmol) was added, and the mixture was stirred at room temperature for 80 minutes, then partially concentrated under reduced pressure to a volume of 5.4 mL, and then allowed to stand at room temperature for an additional 5 hours. Next, DMF (670 μL) was added, and the resulting red solution was partially concentrated to a volume of approximately 350 μL, yielding a white residue and a red solution containing the crude intermediate. The mixture was diluted to 666 μL with additional DMF, and then 222 μL (3.45 μmol) of this solution was treated with a stock solution of 52 in DMF (175 mmol, 39.4 μL, 6.9 μmol), followed by DiPEA (1.79 μL, 10.4 μmol) and a solution of HATU (500 mM, 6.90 μL, 3.45 μmol) in dry DMF. The resulting mixture was vortexed and allowed to stand at room temperature for 17 minutes. Next, additional 52 (175 mmol, 157.6 μL, 27.6 μmol) in DMF was added. The mixture was vortexed again and allowed to stand at room temperature for 14 minutes, followed by the addition of additional HATU (500 mM, 13.80 μL, 6.90 μmol) in dry DMF. The mixture was vortexed again and allowed to stand at room temperature for 13 minutes, then stored in the freezer for 2 days. Finally, the mixture was removed from the freezer and purified by preparative HPLC (40% to 95% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge preparative C18, 5 μm OBD, 30 × 100 mm). Compound 53 was obtained as a red solution in DMF (165 μL, 2.5 mM, 0.5 mg, 0.41 μmol, 12% based on a doxorubicin-based calibration line for HPLC). 51 H 64 N7O 19 S + (M+H + LCMS (ESI+) calculated for 1110.40, found 1110.85.

[0350] [Synthesis of Compound 57] [ka] [Example a66. Synthesis of Compound 54]

[0361] This compound was synthesized according to the literature procedure described by Pawar et al. in the International Journal of Pharmaceutics, Volume 436, Issues 1-2, Pages 183-193.

[0351] [Example a67. Synthesis of Compound 55]

[0362] A solution of THF (160 mL) and water (160 mL) was added to a round-bottom flask containing compound 54 (5.241 g, 1 equiv., 6.844 mmol). The resulting red suspension was cooled to 0 °C, and then a cold solution of sodium periodate (1.464 g, 34.22 mL, 200 mmol, 1.00 equiv., 6.844 mmol) in HO was added dropwise over 15 min. The resulting red solution, with some solid at the bottom, was stirred on ice for a total of 10 min. The ice bath was then removed, and the reaction mixture was allowed to warm to room temperature and stirred for 23 h. The reaction mixture was partially concentrated under reduced pressure (to 80 mbar, removing all THF and approximately 50% of the water) to give a red suspension in most of the water. The mixture was treated with DCM (200 mL), and the resulting suspension was rotated at 43 °C for several minutes. The biphasic system was transferred to a separatory funnel. To the remaining residue in the round-bottom flask, an additional 300 ml of DCM was added, and the resulting mixture was rotated again at 43 °C until the remaining solids dissolved. The solution was also added to the separatory funnel, and the resulting two-phase system was shaken and separated. The aqueous layer was extracted twice with additional DCM (2 x 200 ml). The separatory funnel, containing a small amount of dark red residue, was washed with 10% MeOH in DCM (150 mL), which completely solubilized the residue. This organic layer was washed once with the aqueous layer. The resulting organic layer was then combined with the other organic layers. MeOH (40 mL) was added to the combined organic layers, producing a clear solution. The combined organic layers were dried (Na2SO4) and then filtered over a glass filter. This solution was treated with DMF (18 mL) and partially concentrated (to 20 mbar) until mainly DMF remained as the solvent to give intermediate 55 (5.30 g, 6.87 mmol) as a solution in DMF as a dark red solution (18 mL) which was used in the next step without further purification. Quantitative yield was assumed. 41 H 37 NO 13 + [M+H + ] UPLC-MS(ESI+) calculated value 752.23, found value 752.52.

[0352] [Example a68. Synthesis of Compound 56]

[0363] To a round-bottom flask containing compound 55 (4.10 g, 5.46 mmol, 1.00 equiv.) in DMF (15 mL), additional DMF was added until a total volume of approximately 94 mL was reached. Next, allyl (2-aminoethyl)carbamate (2.14 g, 2.72 equiv., 14.9 mmol) was added in dry DMF (9.0 mL), and the reaction mixture was placed in a water bath. Next, HATU (2.18 g, 1.05 equiv., 5.73 mmol) was added, followed within 1 min by DIPEA (2.12 g, 2.85 mL, 3.00 equiv., 16.4 mmol). The resulting dark red solution was stirred at room temperature for approximately 30 min. Additional HATU (455 mg, 1.20 mmol, 0.22 equiv.) in DMF (1.0 mL) was then added, followed after another 55 min by the addition of a third batch of HATU (509 mg, 1.34 mmol, 0.25 equiv.). The reaction mixture was stirred at room temperature for an additional 5 min and then partially concentrated under reduced pressure to a volume of 15 mL. The residue was then diluted with DCM (135 mL) and loaded onto a pre-wetted column. The residue was then purified by flash column chromatography on silica gel (0→20% MeOH / DCM). The product-containing fractions were combined and concentrated to give Intermediate 56 (3.61 g, 4.07 mmol, 74.5%, 99% purity) as a dark red, thick oil. 47 H 48 N3O 14 + UPLC-MS(ESI+) calculated for [M+H+] 878.31, found 878.71.

[0353] [Example a69. Synthesis of Compound 57]

[0364] To a dark red solution of compound 56 (3.61 g, 99 wt%, 1.00 equiv., 4.07 mmol) in a total volume of DMF (17.0 mL) was added triethylamine (2.06 g, 2.84 mL, 5.0 equiv., 20.3 mmol). The reaction mixture turned very dark red and was left stirring at room temperature for 18 h. EtO (78 mL) was then added in one portion (slowly) with rapid stirring. Stirring was stopped, and the ether layer was then decanted, and the remaining dark solid was washed several times with EtO (3 × 100 mL). The solid was concentrated under reduced pressure to give intermediate 57 (4.8 g, 81.9% purity) as a dark red solid, which was used without further purification. 32 H 38 N3O 12 + UPLC-MS(ESI+) calculated for [M+H+] 656.25, found 656.60.

[0354] [Example a70. Synthesis of Compound 58] [ka]

[0365] Intermediate 57 (4.0 g, 90 wt%, 5.5 mmol) was suspended in dry DMF (6.0 mL), followed by the addition of bis-iodosugar 7c (6.3 g, 16 mmol, 3 equiv.) and DiPEA (2.9 mL, 16 mmol, 3 equiv.). The resulting mixture was rotated at 45 °C for 45 min. The remaining chunks were then mostly broken up with a spatula, and the suspension was stirred at 40 °C for 2 days. The reaction mixture was diluted with DCM (100 mL), and the resulting red solution was purified by flash column chromatography on silica gel (0% MeOH / DCM, then 2% → 10% MeOH in DCM) to give compound 58 as a red residue (4.03 μmol, 72%). 39 H 50 N3O 14 + (M+H + LCMS (ESI+) calculated for 784.33, found 784.78.

[0355] [Example a71. Synthesis of Compound 59] [ka]

[0366] A solution of compound 58 (4.21 g, 4.78 mmol, 89 wt%) in a mixture of DCM (130 mL) and MeOH (10 mL) was cooled to -78 °C using a dry ice / acetone bath. The mixture was vigorously stirred, and then a freshly made stock solution of mCPBA in DCM (580 mM, 9.0 mL, 5.22 mmol) was added dropwise over 5-10 min. After stirring for 17 min, a second batch of mCPBA in DCM (580 mM, 10.0 mL, 5.80 mmol) was added over 5 min. The reaction mixture was stirred for an additional 9 min, and finally a third batch of mCPBA in DCM (580 mM, 2.5 mL, 1.50 mmol) was added. The reaction mixture was stirred at -78 °C for an additional 13 min, then quenched with cold (-78 °C) acetone (reagent grade, 43.9 mL). The reaction mixture was stirred for an additional 13 min. After 90 min, the cooling bath was removed and the reaction mixture was allowed to warm to 0 °C over 40 min. The reaction mixture was diluted with DCM (450 mL) and saturated aqueous NaHCO (250 mL) and transferred to a separatory funnel. The resulting two-phase system was separated, and the aqueous layer was extracted twice with DCM (150 mL, 100 mL). The combined organic layer was then washed again with saturated aqueous NaHCO (100 mL). The new aqueous layer was extracted with DCM (40 mL), and the combined organic layer was dried over NaSO, filtered, and then concentrated to give compound 59 (3.30 g, 93% purity, 80% yield, 3.84 mmol), which was used directly in the next step. 39 H 50 N3O 15 + (M+H + LCMS (ESI+) calculated for 800.32, found 800.70.

[0356] [Example a72. Synthesis of Compound 60] [ka]

[0367] To a solution of 59 (181 mg, 60 wt%, 136 μmol, 1.00 equiv) in a mixture of dry DCM (1.0 mL) and anhydrous acetonitrile (4.0 mL) was added potassium carbonate (167.8 mg, 1.21 mmol, 8.94 equiv). The reaction mixture was then cooled to 0 °C in an ice bath, after which a solution of cyanuric chloride in anhydrous acetonitrile (62.6 mg, 2.88 mL, 118 mM, 339 μmol, 2.5 equiv) was added. After stirring at 0 °C for 70 min, additional cyanuric chloride in anhydrous acetonitrile (863 μL, 118 mM, 102 μmol, 0.75 equiv) was added. The reaction mixture was stirred at 0°C for an additional 55 min and then quenched with a solution of 3-aminopropane-1,2-diol (186 mg, 1.02 mL, 2 mol, 2.04 mmol, 15 equiv) in water. The ice bath was removed after 30 min and then diluted with DCM (30 mL) and HO (10 mL). The resulting two-phase system was separated. The aqueous layer was extracted twice more with DCM (10 mL, 2 times). The combined organic layers were dried (NaSO), filtered through a phase separator, and concentrated under reduced pressure. The residue was taken up in DMF and purified by preparative HPLC (50% → 70% acetonitrile in 10 mM NHHCO in water, column Xbridge Prep C). 18 The product was purified by column chromatography (5 μm OBD, 30×100 mm). Fractions containing pure product were combined and concentrated to give pure compound 60 (13.5 mg, 17.3 μmol, 12.7% yield) as a red solid. Fractions containing impure product were further combined to give impure 60 (11.4 mg, 63% purity, 9.20 μmol, 6.7% yield). 39 H 48 N3O 14 + (M+H + LCMS (ESI+) calculated for 782.82, found 782.64.

[0357] [Example a73. Synthesis of Compound 61] [ka]

[0368] To a vial containing compound 60 (6.75 mg, 1 equiv., 8.63 μmol), anhydrous DCM (300 μL) was added. To the resulting red solution, pyrrolidine (1.84 mg, 2.13 μL, 3 equiv., 25.9 μmol) was added, instantly producing a very dark red (almost black) solution. After mixing, Pd(PPh3)4 (1.50 mg, 64.8 μL, 20 mmol, 0.15 equiv., 1.30 μmol) in dry DCM was added. The dark red solution was mixed again and allowed to stand at room temperature for 11 min. An additional 20 mmol of Pd(PPh3)4 (64.8 μL, 0.15 equiv., 1.30 μmol) in dry DCM was added, followed by the third (100 μL, 0.23 equiv., 2.00 μmol) and fourth (64.8 μL, 0.15 equiv., 1.30 μmol) batches 13 and 12 min later, respectively. Finally, following the fourth addition, the reaction mixture was stirred at room temperature for 47 min, then diluted with DMF (300 μL), and then partially concentrated under reduced pressure to 30 mbar to give a dark red solution, which was purified by preparative HPLC (20% → 50% acetonitrile in 10 mM NH4HCO3 in water, column Xbridge Prep C). 18 The product was purified by column chromatography (5 μm OBD, 30×100 mm). Fractions containing the pure product were combined and concentrated to give pure compound 61 (2.4 mg, 3.4 μmol, 40% yield) as a red residue. Fractions containing impure product were further combined to give impure 61 (3.3 mg, 53% purity, 2.5 μmol, 29% yield). 35 H 44 N3O 12 + (M+H + LCMS (ESI+) calculated for 698.29, found 698.56.

[0358] [Example a74. Synthesis of compound BCN-HS-GGFG-OH (62)] [ka]

[0369] A round-bottom flas...

Claims

1. A conjugate in which a compound represented by structure (1) is conjugated to a cell-binding agent via a linker, wherein structure (1) is as follows: 【Chemical 1】 (In the formula, -R １ is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C ６～１５ Alkyl, C ２～１５ Alkenyl, C ２～１５ Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X ２ R ４ , Sp-N ３ , Sp-X ２ -Sp-R １２ or Sp-N(R ４ ) ２ wherein the optional substituents are halogen, C １～１２ (hetero)alkyl, (hetero)aryl, C ２～１５ Alkenyl, C ２～１５ Alkynyl, X ２ R ４ , N(R ４ ) ２ , NO ２ and substituent C is selected from １～１２ The (hetero)alkyl and (hetero)aryl are optionally C １～６ (hetero)alkyl, X ２ R ４ and N(R ４ ) ２ each Sp may be individually further substituted with C １～１２ (Hetero)alkylene, (hetero)arylene, C １～１２ (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C １～１２ (hetero)alkylene, and the (hetero)alkylene or the (hetero)arylene is selected from the group consisting of halogen, X ２ R ４ , N(R ４ ) ２ , C １～４ Alkyl and NO ２ and each R is optionally substituted with one or more substituents selected from ４ are individually H, C １～４ alkyl or adamantyl, and X ２ is C(O), C(O)O, C(O)NH, O, S, S(O), S(O) ２ , S(O)NH or S(O) ２ NH and R １２ is β-glucuronide acid, PO ３ （２－） , O.P.O. ３ （２－） , CO ２ （－） , S.O. ３ （－） or N(C １～４ alkyl) ３ （＋） and -R ２ is H, S(O) ２ OH or P(O) ２ OH and R ３ is OH, or R ２ and R ３ are fused together through an ether moiety to form an oxazolidine ring; -R ５ is H or OCH ３ and -N ％ is N or N→O; - Y ５ is CH ２ -Y,C(O)-Y,C(=N(R ２０ ))-Y,C(R ９ )=N−Y, where R ９ is OH or O(CO)C １～６ C optionally substituted with alkyl groups １～４ alkyl, and R ２０ is NR ４ -C(O)-N(R ４ ) ２ , N.R. ４ -C(O)-Sp-N(R ４ ) ２ , N.R. ４ -C(O)-R １２ , N.R. ４ -C(O)-Sp-R １２ where Sp, R ４ and R １２ is as defined above; - said compound is connected to said linker via Y Conjugates, or salts thereof, wherein each ion, if present, is in equilibrium with one or more pharmaceutically acceptable counterions.

2. Structure (2) CB-Z １ -L-Z ２ -D （２） (In the formula, - CB is a cell binding agent; D is a compound of structure (1); L is a linker; -Z １ is a linking group that connects the cell-binding agent CB to the linker; -Z ２ is a linking group that links said compound D to said linker 2. The conjugate of claim 1, having the formula:

3. The linking group Z １ 3. The conjugate of claim 2, wherein is formed by a conjugation reaction selected from amide bond formation, carbamate bond formation, thiol alkylation, thiol arylation, and cycloaddition reactions.

4. The linking group Z １ is linked to the cell-binding agent CB via a lysine residue of CB, a glutamine residue of CB, a threonine residue of CB, a cysteine residue of CB, a tyrosine residue of CB, or a glycan of CB.

5. The linking group Z ２ is an amide moiety, an ester moiety, a thioether moiety, an ether moiety, a carbamate moiety, a [2.2.2] bicyclic structure, a [2.2.1] bicyclic structure, a disulfide, a carbonate moiety, or a (hetero)aryl moiety.

6. L-Z ２ But the structure: *-NR ４ -Sp ３ -NR ４ -(L ３ ) ｐ -(L ２ ) ｏ -(L １ ) ｎ－ ** (In the formula, - the bond marked with * connects to the C(O) moiety adjacent to Y in the compound represented by structure (1); - The bond marked with ** is the linking group Z １ linked to; - Sp ３ is C １～１２ (Hetero)alkylene, (hetero)arylene, C １～１２ Alkylene-(hetero)arylene, or (hetero)arylene-C １～１２ alkylene, wherein the alkylene or the (hetero)arylene is selected from the group consisting of halogen, X ２ R ４ , N(R ４ ) ２ , C １～４ Alkyl, NO ２ and optionally substituted with one or more substituents selected from １～４ The alkyl substituent is NR ４ The NR moiety is bonded to form a cyclic structure, in particular the NR moiety having the bond marked with *. ４ The alkylene may optionally be formed with a pyrrolidine moiety, and the alkylene may optionally be formed with a X ２ and NR ４ and optionally interrupted by one or more heteroatoms selected from: -R ４ and X ２ is as defined in claim 1; - L １ , L ２ and L ３ are, respectively, Z １ is a linker linking D together; n, o, and p are each independently 0 or 1, with the proviso that n+o+p=1, 2, or 3. The conjugate of any one of claims 2 to 5, having the formula:

7. The conjugate of any one of claims 1 to 6, wherein the cell binding agent is an antibody, a peptide, a small molecule or an aptamer.

8. R １ is Et, i-Pr, t-Bu, Bz, Bn, Sp-N ３ or Sp-NH ２ Preferably, R １ is Et, i-Pr, Bn or Sp-N ３ and Sp is C １～４ Alkylene or C １～４ alkylene-arylene, preferably Sp is CH ２ CH ２ , C.H. ２ CH ２ CH ２ or CH ２ The conjugate of any one of claims 1 to 7, wherein the conjugate is Ph.

9. R ２ and R ３ The conjugate of any one of claims 1 to 8, wherein: are linked together via an ether moiety to form an oxazolidine ring.

10. R １ But CH ２ CH ２ The conjugate of any one of claims 1 to 9, which is not SH, unsubstituted ethyl or benzyl.

11. Compounds represented by structure (1): 【Chemistry 2】 (In the formula, -R １ is optionally substituted Et, i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, cyclopropyl, cyclobutyl, cyclopentyl, C ６～１５ Alkyl, C ２～１５ Alkenyl, C ２～１５ Alkynyl, heterocyclyl, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-X ２ R ４ , Sp-N ３ , Sp-X ２ -Sp-R １２ or Sp-N(R ４ ) ２ wherein the optional substituents are halogen, C １～１２ (hetero)alkyl, (hetero)aryl, C ２～１５ Alkenyl, C ２～１５ Alkynyl, X ２ R ４ , N(R ４ ) ２ , NO ２ and substituent C is selected from １～１２ The (hetero)alkyl and (hetero)aryl are optionally C １～６ (hetero)alkyl, X ２ R ４ and N(R ４ ) ２ each Sp may be individually further substituted with C １～１２ (Hetero)alkylene, (hetero)arylene, C １～１２ (hetero)alkylene-(hetero)arylene, or (hetero)arylene-C １～１２ (hetero)alkylene, and the (hetero)alkylene or the (hetero)arylene is selected from the group consisting of halogen, X ２ R ４ , N(R ４ ) ２ , C １～４ Alkyl and NO ２ and each R is optionally substituted with one or more substituents selected from ４ are individually H, C １～４ alkyl or adamantyl, and X ２ is C(O), C(O)O, C(O)NH, O, S, S(O), S(O) ２ , S(O)NH or S(O) ２ NH and R １２ is β-glucuronide acid, PO ３ （２－） , O.P.O. ３ （２－） , CO ２ （－） , S.O. ３ （－） or N(C １～４ alkyl) ３ （＋） and -R ２ is H, S(O) ２ OH or P(O) ２ OH and R ３ is OH, or R ２ and R ３ are fused together through an ether moiety to form an oxazolidine ring; -R ５ is H or OCH ３ and -N ％ is N or N→O; - Y ５ is CH ２ -Y,C(O)-Y,C(=N(R ２０ ))-Y,C(R ９ ) = N-Y, C(R ９ ) = N(R ２０ ), where R ９ is OH or O(CO)C １～６ C optionally substituted with alkyl groups １～４ alkyl, and R ２０ is NR ４ -C(O)-N(R ４ ) ２ , N.R. ４ -C(O)-Sp-N(R ４ ) ２ , N.R. ４ -C(O)-R １２ , N.R. ４ -C(O)-Sp-R １２ where Sp, R ４ and R １２ is as defined above; - Y is NR ４ -Sp ３ -N(R ４ ) ２ , N.R. ４ -Sp ３ -X ２ (R ４ ), N(R ４ ) ２ , C.H. ３ , R １２ , Sp ３ R １２ , N.R. ４ -Sp ３ -X ２ -Sp ３ -R １２ , OH, or CH ２ OH, where each Sp ３ is a spacer; - Y ５ is C(O)-CH ２ When OH, R １ is unsubstituted ethyl, CH ２ CH ２ not SH or benzyl); or salts thereof, wherein each ion, if present, is in equilibrium with one or more pharmaceutically acceptable counterions.

12. R １ is i-Pr, t-Bu, Bn, Sp-N ３ or Sp-NH ２ Preferably, R １ is i-Pr, Bn or Sp-N ３ and Sp is C １～４ Alkylene or C １～４ alkylene-arylene, preferably Sp is CH ２ CH ２ or CH ２ The compound of claim 11, wherein the compound is (4-Ph).

13. R ２ and R ３ are linked together via an ether moiety to form an oxazolidine ring.

14. Y is CH ２ The compound according to any one of claims 11 to 13, wherein the compound is OH.

15. N ％ The compound according to any one of claims 11 to 14, wherein is N.

16. 16. A conjugate, wherein the compound of claim 15 is conjugated to a cell binding agent via a linker.

17. The compound represented by structure (1) (i) R １ , preferably R １ = Sp-NH ２ or via the nitrogen atom of Sp-SH; or (ii) Y, preferably Y=NH ２ or NH-Sp ３ -NH ２ or NH-Sp ３ Nitrogen atom of -SH 17. The conjugate of claim 16, which is conjugated to the cell-binding agent via

18. L-Z ２ が、(L1)~(L4): 【Chemistry 3】 (In the formula, - Bonds marked with * are: (a) for (L1) and (L2), in the C(O) moiety adjacent to Y of the compound represented by structure (1), and (b) For (L3) and (L4), OR of the compound represented by structure (1) １ linked to an O atom of the moiety; - the bond labeled with ** is linked to the cell-binding agent; -R １３ is hydrogen, C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (hetero)arylalkyl groups, wherein said C １ ~C ２４ Alkyl group, C ３ ~C ２４ Cycloalkyl group, C ２ ~C ２４ (hetero)aryl group, C ３ ~C ２４ Alkyl (hetero)aryl groups and C ３ ~C ２４ (Hetero)arylalkyl groups include O, S and NR １４ and optionally substituted and optionally interrupted by one or more heteroatoms selected from １４ are independently hydrogen and C １ ~C ４ alkyl groups, or R １３ is D optionally linked to N via a spacer moiety, or R １３ is linked elsewhere in the linker, optionally via a spacer moiety, to form a cyclic structure; - L ２ is a dipeptide, tripeptide or tetrapeptide; o is 0 or 1; Ring A is an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring; z1 is an integer ranging from 1 to 4; z2 is 0 or 1 18. The conjugate of claim 16 or 17, having the structure (2) of claim 2, wherein the structure is selected from:

19. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 9 or 15 to 17 and a pharmaceutically acceptable carrier.

20. 20. A method for targeting tumor cells expressing a specific extracellular receptor, the method comprising contacting a conjugate of any one of claims 1 to 9 or 16 to 18 with cells likely to express said extracellular receptor, wherein said antibody specifically targets said extracellular receptor.

21. 20. A method for the treatment of cancer, comprising administering to a subject in need thereof the conjugate of any one of claims 1 to 9 or 16 to 18, wherein the cancer cells specifically express an extracellular receptor.

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

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