Anthracycline drug and conjugate thereof

Abstract

The present invention relates to analogs of nemorubicin bearing a series of substituents on the morpholino ring other than 2 "-OMe, as well as PNU-159, 682, which have a beneficial effect on the toxicity of toxins compared to molecules bearing the 2"-OMe group. In addition, it was found that PNU variants having a modified 2 ''-O-alkyl chain exhibit enhanced tolerance in vivo. Consequently, by modifying the 2 ''-O-alkyl group, ADC with elaborately tailored potency and tolerance can be produced, thereby increasing the administration dose of a patient, therefore the present invention relates to compounds according to Structure (1) and conjugates thereof, and pharmaceutical compositions and methods for targeting tumor cells and treating cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine. More particularly, the present invention relates to anthracyclines and antibody-drug conjugates prepared therefrom, particularly antibody-drug conjugates containing analogs of PNU-159,682 as cytotoxic payloads, which are suitable for treating cancer. Background Art

[0002] Antibody-drug conjugates (ADCs) are considered one of the main types of targeted therapies and consist of an antibody with a pharmaceutical agent attached. Antibodies (also called ligands) can be in the form of small proteins (scFv, Fab fragments, DARPins, affibodies, etc.), but are typically monoclonal antibodies (mAbs), which are selected based on their high selectivity and affinity for specific antigens, their long circulation half-life, and almost no immunogenicity. Therefore, mAbs, as protein ligands for carefully selected biological receptors, provide an ideal targeting platform for the selective delivery of drugs. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used to deliver chemically conjugated cytotoxic agents to tumors through binding, internalization, intracellular processing, and ultimately the release of active catabolites. Cytotoxic agents can be small molecule toxins, protein toxins, or other forms, such as oligonucleotides. As a result, tumor cells can be selectively eliminated while sparing normal cells that are not targeted by the antibody. Similarly, chemical conjugation of antibacterial drugs (antibiotics) to antibodies can be used to treat bacterial infections, while conjugates of anti-inflammatory drugs are being studied for the treatment of autoimmune diseases, and, for example, attaching oligonucleotides to antibodies is a potential and promising approach for treating neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to specific cellular locations of choice is a powerful approach for treating a variety of diseases and has many advantages over delivering the same drug systemically.

[0003] ADCs are prepared by conjugating a linker-drug to a protein, a process known as bioconjugation. Many techniques are known for bioconjugation, as summarized in G.T. Hermanson, "Bioconjugate Techniques," Elsevier, 3rd ed. 2013, which is incorporated by reference. Conceptually, the process for preparing ADCs by bioconjugation requires that x reactive moieties F present on the antibody react with complementary reactive moieties Q present on the drug (payload), see Figure 1 .

[0004] Typically, a chemical linker exists between Q and the payload. This linker needs to possess a number of key properties, including the requirement to remain stable in plasma for extended periods after drug administration. A stable linker enables the ADC to localize to the intended site or cell in the body and prevents premature release of the payload into the circulation, which could indiscriminately induce a variety of undesirable biological responses and thus reduce the ADC's therapeutic index. After internalization, the ADC should be processed to effectively release the payload, enabling it to bind to its target. The linker may also contain a spacer element. There are two types of linkers: non-cleavable and cleavable. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload and are completely stable under physiological conditions, regardless of the organ or biological compartment in which the antibody-drug conjugate is located. Therefore, payload release from ADCs with non-cleavable linkers relies on complete (lysosomal) degradation of the antibody after the ADC is internalized into the cell. As a result of this degradation, the payload is released, still carrying the linker, as well as peptide fragments and / or amino acids from the antibody to which the linker was originally attached. Cleavable linkers exploit the inherent properties of cells or cellular compartments to selectively release the payload from the ADC, which typically leaves no trace of the linker after processing. There are three common mechanisms for cleavable linkers: (1) susceptibility to specific enzymes, (2) pH sensitivity, and (3) sensitivity to the redox state of the cell (or its microenvironment). Cleavable linkers can also contain self-destructive units, such as those based on the p-aminobenzyl alcohol group and its derivatives. Linkers can also contain additional elements, often called spacer units or stretcher units, to connect the linker to the reactive group for reaction with the antibody.

[0005] The reactive moiety F may be naturally present in the antibody, for example the reactive moiety may be a lysine or cysteine ​​side chain, which may be available for acylation (lysine side chain) or alkylation (cysteine ​​side chain).

[0006] Acylation of the ε-amino group in the lysine side chain is usually achieved by subjecting the protein to treatment with reagents based on activated esters or activated carbonate derivatives, such as SMCC used to make

[0007] A variety of reagents are known to be used to alkylate thiol groups in cysteine ​​side chains, see Figure 2. Among the strategies for cysteine ​​alkylation, the vast majority are based on the use of maleimide reagents, such as in the manufacture of In addition to standard maleimide derivatives, a series of maleimide variants can also be used for more stable cysteine ​​conjugation, as demonstrated by James Christie et al., J. Contr. Rel. 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol. 2014, 32, 1059-1062, both of which are incorporated by reference. Other methods for cysteine ​​alkylation include, for example, nucleophilic substitution with haloacetamides (typically bromoacetamide or iodoacetamide), see, for example, Alley et al., Bioconj. Chem. 2008, 19, 759-765, incorporated herein by reference, or various methods based on nucleophilic addition to unsaturated bonds, such as reaction with acrylate reagents, see, for example, Bernardim et al., Nat. Commun. 2016, 7, 13128 and Ariyasu et al., Bioconj. Chem. 2017, 28, 897-902, both incorporated herein by reference, with phosphonamidates. e), see, for example, Kasper et al., Angew. Chem. Int. Ed. 2019, 58, 11625-11630, incorporated herein by reference, reactions with allenamide, see, for example, Abbas et al., Angew. Chem. Int. Ed. 2014, 53, 7491-7494, incorporated herein by reference, reactions with cyanoethynyl reagents, see, for example, Kolodych et al., Bioconj. Chem. 2015, 26, 197-200, incorporated herein by reference, reactions with vinyl sulfones, see, for example, Gil de Montes et al., Chem. Sci. 2019, 10, 4515-4522, incorporated herein by reference, or for reactions with vinylpyridine, see, for example, Seki et al., Chem. Sci. 2021, 12, 9060-9068 and https: / / iksuda.com / science / permalink / (Accessed July 26, 2020). Alternative methods for antibody conjugation that do not require reengineering the antibody include reducing the interchain disulfide bridges followed by the addition of a payload attached to a cysteine ​​cross-linking reagent, such as a bissulfone reagent, see, e.g., Balan et al., Bioconj. Chem. 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceutics 2015, 12, 1872-1879, both incorporated by reference, mono- or dibromomaleimide, see, e.g., Smith et al., J. Am. Chem. Soc. 2010, 132, 1960-1965 and Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261–7269, both incorporated by reference, bismaleimide reagents, see, e.g., WO 2014114207, bis(phenylthio)maleimide, see, e.g., Schumacher et al., Org. Biomol. Chem. 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem. 2018, 29, 3516-3521, both incorporated by reference, bisbromopyridazinedione, see, e.g., Robinson et al., RSC Advances 2017, 7, 9073-9077, incorporated by reference, bis(halomethyl)benzene, see, e.g., Ramos-Tomillero et al., Bioconj. Chem. 2018, 29, 1199-1208, incorporated by reference, or other bis(halomethyl)aromatic compounds, see, e.g., WO 2013173391. Typically, ADCs prepared by cysteine ​​cross-linking have a drug antibody loading of approximately 4 (DAR4). Another useful technique for conjugation to cysteine ​​side chains is through disulfide bonds, a bioactivatable linkage that has been used to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci. 2017, 8, 366-370, incorporated by reference).

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

[0009] An example of a non-natural reactive functional group F that can be used for linker-drug bioconjugation is an oxime group (suitable for oxime connection) or an azido group (suitable for click chemistry conjugation). Oxime or azide can be installed in the antibody by the genetic coding of non-natural amino acids, such as p-acetylphenylalanine (suitable for oxime connection) or p-azidomethylphenylalanine or p-azidophenylalanine (suitable for click chemistry conjugation), such as Axup et al. Proc. Nat. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States] 2012, 109, 16101-16106, which is incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem. [Biological Conjugation Chemistry] 2014, 25, 351-361 (which is incorporated by reference) introduced azidomethylphenylalanine (AzPhe) into monoclonal antibodies using a cell-free protein synthesis method for conversion into ADC by metal-free click chemistry. In addition, Nairn et al., Bioconj. Chem. [Bioconjugation Chemistry] 2012, 23, 2087-2097 (incorporated by reference) also demonstrated that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins by auxotrophic bacteria and further converted into protein conjugates by click chemistry. Finally, Nguyen et al., J. Am. Chem. Soc. [American Chemical Society] 2009, 131, 8720-8721 (incorporated by reference) demonstrated the use of pyrrolysyl-tRNA synthetase / tRNA CUAThe genetic coding of aliphatic azides in recombinant proteins and labeling is achieved by click chemistry (by copper-catalyzed alkyne-azide cycloaddition (CuAAC) or strain-promoted alkyne-azide cycloaddition (SPAAC)). In addition, CuAAC and SPAAC, i.e., bioconjugation of linker-drugs to antibodies (and other biomolecules, such as polysaccharides, nucleic acids) can be achieved by a series of other metal-free click chemistries, see, for example, Nguyen and Prescher, Nature Rev. Chem. [Natural Review Chemistry] 2020, 4, 476-489, which are incorporated by reference. For example, oxidation of specific tyrosine in proteins can produce ortho-quinones, which are easily cycloadded with strained olefins (such as TCO) or strained alkynes, see, for example, Bruins et al., Chem. Eur. J. [Chemistry - European Journal] 2017, 24, 4749-4756, which are 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. [Chemical Science] 2020, 11, 9011-9016, which is incorporated by reference. Tetrazine moieties can also be introduced into proteins or polysaccharides by various means, such as genetic encoding or chemical acylation, and can also undergo cycloaddition with cyclic alkenes and alkynes. Figure 4 A list of functional group F and Q pairs for metal-free click chemistry is provided.

[0010] In SPAAC bioconjugation, the linker-drug is functionalized with a cyclic alkyne, and the cycloaddition with the azide-modified antibody is driven 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. Figure 5 Various strained alkynes suitable for metal-free click chemistry are shown.

[0011] An increasingly popular approach in the field of ADCs is based on the enzymatic installation of non-natural functional groups F. For example, Lhospice et al., Mol. Pharmaceut. 2015, 12, 1863-1871 (incorporated by reference) utilized the bacterial enzyme transglutaminase (BTG or TGase) to install azide moieties on antibodies. Cheng et al., Mol. Cancer Therap. 2018, 17, 2665-2675 (incorporated by reference) reported a genetic approach based on C-terminal TGase-mediated azide introduction followed by conversion to ADCs via metal-free click chemistry.

[0012] In WO 2014065661, van Geel et al., Bioconj. Chem. 2015, 26, 2233-2242, Verkade et al., Antibodies 2018, 7, 12 and Wijdeven et al., MAbs 2022, 14, 2078466 (all incorporated by reference) demonstrated that enzymatic remodeling of native antibody glycans at N297 can introduce azide-modified sugars suitable for attachment of cytotoxic payloads using metal-free click chemistry, see Figure 6 Similarly, free thiol groups can be installed on antibodies using enzymatic glycan remodeling protocols (see Figure 7 ), to be conjugated based on any of the above-mentioned cysteine ​​conjugation methods.

[0013] Although ADC has shown clinical and preclinical activity, it is not clear what other factors determine such efficacy in addition to targeting antigen expression on tumor cells. For example, drug-antibody ratio (DAR), ADC binding affinity, the efficacy of payload, receptor expression level, internalization rate, transportation, multidrug resistance (MDR) status and other factors are considered to be involved and affect the results of in vitro ADC treatment. In addition to directly killing antigen-positive tumor cells, ADC also has the ability to kill adjacent antigen-negative tumor cells: the so-called "bystander killing" effect, as originally reported by Sahin et al., Cancer Res. [Cancer Research] 1990, 50, 6944-6948 (incorporated by reference), and for example Li et al., Cancer Res. [Cancer Research] 2016, 76, 2710-2719 (incorporated by reference). In general, neutral cytotoxic payloads will show bystander killing effects, while ionic (charged) payloads will not, because ionic species are not easy to pass through the cell membrane by passive diffusion. Payloads with established bystander effects are, for example, MMAE and DXd. Examples of payloads that do not show bystander killing are MMAF or The active catabolite of lysine (lysine-MCC-DM1).

[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, tubulysin], DNA damaging agents [e.g., calicheamicin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiapine dimers, duocarmycin, anthracyclines], topoisomerase inhibitors [e.g., DXd, exitecan, SN-38], or RNA polymerase II inhibitors [e.g., amanitin]. ADCs that have received market approval include, for example, payloads MMAE, MMAF, DM1, calicheamicin, SN-38, DXd, and PBD dimers, while ADCs based on duocarmycin or DM4 are undergoing various pivotal trials. A wider variety of payloads are still undergoing clinical evaluation or have been clinically tested in the past, such as eribulin, indoline benzodiazepine dimer, PNU-159,682, amanitin, hemicycline, doxorubicin, vinca alkaloids, etc. Finally, various ADCs in the late preclinical stage are conjugated with new payloads, such as KSP inhibitors, MMADs, cryptophycin, etc.

[0015] In addition to gosartumomab In addition, all clinical and marketed ADCs contain cytotoxic drugs that are not suitable as stand-alone drugs. SN-38 is an exception because it uses SN-38 as the cytotoxic payload, which is also the active catabolite of irinotecan (an SN-38 prodrug). Several other payloads currently used in clinical ADCs were initially evaluated as free drugs for chemotherapy, such as calicheamicin, PBD dimer, and eribulin, but were not readily available due to the extremely high potency of the cytotoxic agents (picomolar to low nanomolar IC 50 values), while standard chemotherapy drugs such as paclitaxel and doxorubicin often have low micromolar potency and therefore fail.

[0016] Another cytotoxin that is gaining increasing attention in ADC applications is PNU-159,682 (see Figure 9), an anthracycline derivative that is >1000-fold more potent than doxorubicin. PNU-159,682, one of the oxidative catabolites of nemorubicin (MMDX), was developed as a synthetic analog of doxorubicin without the cardiotoxicity associated with the latter. PNU-159,682 is the bioactivated product of nemorubicin formed in the human liver by CYP3A following oral administration. Interestingly, two other oxidative catabolites, nemorubicin N-oxide and PNU-159,696, exhibit potency similar to that of doxorubicin. Due to its high potency, PNU-159,682 is being actively investigated as a payload for ADCs, as reported by Dal Corso et al., J. Contr. Rel. 2017, 264, 211-218 (incorporated by reference), which reported that a non-internalizing antibody-drug conjugate based on an antibody specific for tenascin C mediated potent therapeutic activity when formulated with PNU-159,682, which was linked to the antibody via maleimide-based cysteine ​​alkylation and a Val-Cit-PABC-based protease-sensitive cleavable linker, and a dimethylethylenediamine (DMEDA) cyclization-cleavage element was linked to the free 14-hydroxyl group of PNU-159,682 via a carbamate group ( Figure 9 The ADC was found to be stable in serum but efficiently cleaved in the subendothelial extracellular matrix by proteases released by dying tumor cells, leading to good tumor regression in various in vivo models. Stefan et al., Mol. Cancer Ther. 2017, 16, 879-892 (incorporated by reference) reported a similar PNU-159,682 ADC based on 14-OH acylation (with Val-Cit-PABC-DMEDA) using sortase-mediated antibody conjugation (SMAC). TM ) attached the linker-drug to the C-terminus of various antibodies (the anti-HER2 antibody trastuzumab and the anti-CD30 antibody brentuximab) (see Figure 10 In this study, the DMEDA-conjugated ADC was compared head-to-head with another PNU-159,682 derivative prepared by oxidation of the hydroxyketone group to a carboxylic acid followed by amidation with a diglycyl-ethylenediamine (EDA) linker ( Figure 9 Characterization of the resulting ADCs showed that they exhibited potency exceeding that of ADCs based on conventional tubulin-targeting payloads, such as those based on the same antibody. and However, the same report also showed that the cytotoxic selectivity of PNU-derived ADCs based on EDA-amide linkers was more selective for target-positive cells than similar ADCs based on DMEDA-carbamate linkers, likely due to the specific release of PNU-159,682 by the latter ADCs. Therefore, the EDA-amide-based technology was selected for further development and is currently being applied in various clinical programs, including NBE-002 and SO-N102, ADCs targeting ROR1 and claudin 18.2, respectively.

[0017] A similar ADC generation method has been disclosed based on the oxidation of the hydroxyketone of PNU-159,682 followed by coupling of the resulting acid, see WO 2016127081. Various derivatives of PNU include amides, hydrazides, and acylhydroxylamine derivatives.

[0018] Beyond the modification and covalent attachment of PNU-159,682 via a hydroxyketone moiety, surprisingly few reports detail the use of methoxy-morpholino groups for antibody attachment. WO 2009099741 demonstrates how PNU-159,682 can be conjugated to antibodies with engineered cysteines via a hydroxyketone moiety and suggests preparing conjugates by attaching at various positions on the morpholino group, including replacing 2″-OMe with a carbamate linker. However, no morpholine-linked structures were achieved.

[0019] Details of the tolerability of NBE-002 in cynomolgus monkeys have been disclosed (AACR 2018, Abstract No. 737), indicating an MTD of approximately 3 mg / kg with a qw 3 x 3 dosing schedule, but it is worth noting that one of the monkeys developed an immune response after the third dose. A Phase 1 study is currently underway (clinical trial NCT04441099), and the MTD in humans remains to be seen. Given the exceptional efficacy of PNU in preclinical models (with a MED as low as 0.033 mg / kg), the MTD in humans is likely to be (significantly) lower than 1 mg / kg. Therefore, after administration (usually intravenous), receptor saturation in the body may not be achieved, resulting in suboptimal tumor uptake and enhanced ADC clearance.

[0020] One way to increase the ADC administration dose of a patient is to reduce the drug loading of the antibody. For example, a DAR1 form with the same payload may be preferred because the MTD may be twice as high as that of a similar DAR2 version. Ruddle et al., ChemMedChem [Chemistry and Medicinal Chemistry] 2019, 14, 1185-1195 recently showed 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. In subsequent publications, White et al., MAbs [monoclonal antibodies] 2019, 11, 500-515, and in WO2019034764 (incorporated by reference), it was shown that DAR1 conjugates can also be prepared from full IgG antibodies using Flexmab technology. It has been shown that Flexmab-derived DAR1 ADC is highly resistant to payload loss in serum and exhibits strong anti-tumor activity in a HER2-positive gastric cancer xenograft model. Furthermore, this ADC exhibited a twice-higher tolerable dose in rats compared to a site-specific DAR2 ADC prepared using a single maleimide-containing PBD dimer.

[0021] While DAR1-format ADCs may offer advantages, to date no DAR1 technology has been reported to improve the therapeutic index relative to DAR2 ADCs. Furthermore, no technology has been reported to generate DAR1 ADCs from whole antibodies without the need to re-engineer the monoclonal antibody, making the generation of DAR2 ADCs inherently easier.

[0022] Another approach to increasing the dosage level of ADCs (particularly PNU-based ADCs) involves generating analogs with reduced potency. For example, Holte et al., Bioorg. Med. Chem. Lett. [Bioorganic & Medicinal Chemistry Express] 2020, 30, 127640 (incorporated by reference), generated a series of PNU analogs with broad cytotoxic activity by oxidatively modifying the hydroxyketone portion of the molecule. Exploring structure-activity relationships led to the development of six linker-drugs for antibody conjugation. Compared to conventional PNU-159,682, these ADCs demonstrated increased MEDs of approximately 1 mg / kg in various preclinical models.

[0023] A final approach to modulating the potency of PNU-159,682 is to modify the morpholino group, particularly the 2″-OMe group. As a single example, WO 2012073217 reports the preparation and in vitro evaluation of a 2″-OEt analog of PNU-159,682, which exhibited 3-8 times higher in vitro potency compared to the OMe variant in two different cell lines (A2780 and MCF7). Summary of the Invention

[0024] The inventors unexpectedly discovered that analogs of nemorubicin and PNU-159,682 bearing a range of substituents other than 2"-OMe on the morpholino ring were significantly less potent in vitro than molecules bearing a 2"-OMe group (i.e., where R 1 =Me). Similar potency reductions were 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), where covalent attachment was ensured by carbamylation or oxidation of the hydroxyketone group followed by coupling with the resulting carboxylic acid. Furthermore, it was discovered that covalent attachment to antibodies could also be achieved by installing chemoselective handles, including but not limited to amino, thiol, or hydroxyl groups, in the 2″-O-alkyl chain, while leaving the hydroxyketone group (as in doxorubicin) or the methylketone group (as in daunorubicin) intact. Furthermore, it was discovered that PNU variants with modified 2″-O-alkyl chains exhibited enhanced tolerability in vivo. Thus, by modifying the 2″-O-alkyl group, ADCs with carefully tailored potency and tolerability can be generated, thereby increasing the dose administered to patients.

[0025] The present invention relates to a novel toxin according to structure (1) and a conjugate thereof according to structure (2). In this connection, the present invention relates to a method for preparing a conjugate according to the present invention. In a further aspect, the present invention relates to a method for targeting tumor cells. In this connection, there is a first medical use of the conjugate according to the present invention and a second medical use for treating cancer. DETAILED DESCRIPTION definition

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

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

[0028] A linker is defined herein as a moiety that connects (covalently links) two or more elements of a compound. A linker may comprise one or more spacer moieties. A spacer moiety is defined herein as a moiety that separates (i.e., provides distance between) two (or more) parts of a linker and covalently links them together. A linker can be, for example, part of a linker-construct, a linker-conjugate, a linker-payload (e.g., a linker-drug), or an antibody-conjugate, as defined below.

[0029] "Hydrophilic group" or "polar linker" is defined herein as any molecular structure containing one or more polar functional groups that impart improved polarity, and therefore improved water solubility, to the molecule to which it is attached. Preferred hydrophilic groups are selected from carboxylic acid groups, alcohol groups, ether groups, polyethylene glycol groups, amino groups, ammonium groups, sulfonic acid groups, phosphoric acid groups, acylsulfonamide groups, or carbamoylsulfonamide groups. In addition to higher solubility, other effects of the hydrophilic group include improving click conjugation efficiency and, once incorporated into the antibody-drug conjugate, reducing aggregation, improving pharmacokinetics, and thereby improving efficacy and in vivo tolerability.

[0030] The term "salt thereof" means a compound formed when an acidic proton (usually the proton of an acid) is replaced by a cation (such as a metal cation or an organic cation, etc.). Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts that are not intended to be administered to a patient. For example, in a salt of a compound, the compound can be protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid serves as the anionic component of the salt. The term "pharmaceutically acceptable" salt means a salt that is acceptable for administration to a patient (such as a mammal) (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from a pharmaceutically acceptable inorganic or organic base, as well as a pharmaceutically acceptable inorganic or organic acid. "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which are derived from various organic and inorganic counterions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium salts, and the like; and when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

[0031] The term "enediyne" or "enediyne antibiotic" or "enediyne-containing cytotoxin" refers to any cytotoxin known in the art that is characterized by the presence of a 3-ene-1,5-diyne structural feature as part of a cyclic molecule, and includes neocarzinostatin (NCS), C-1027, cadacetin (KED), maduratide (MDP), N1999A2, sporolide (SPO), cyanosporaside (CYA and CYN), as well as figilide, calicheamicin (CAL), esperamicin (ESP), danemycin (DYN), namenamicin, shishijimicin, and uncialamycin (UCM).

[0032] As used herein, the term "alkylamino sugar" refers to a tetrahydropyranyl moiety which is linked to an alcohol function via its 2-position to form an acetal function and is further substituted with (at least) one N-alkylamino group at position 3, 4 or 5. "N-alkylamino group" in this context refers to an amino group having one methyl, ethyl or 2-propyl group.

[0033] The term "click probe" refers to a functional part that can undergo a click reaction, that is, two compatible click probes undergo a click reaction with each other so that they are 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, the click probe Q in the compound according to the present invention is capable of reacting with the click probe F on the (modified) protein so that a conjugate is formed when a click reaction occurs, wherein the protein is conjugated to the compound according to the present invention. In this article, F and Q are compatible click probes.

[0034] The term "(hetero)alkyl" refers to both alkyl and heteroalkyl groups. A heteroalkyl group is one in which one or more carbon units in the alkyl chain (e.g., CH2, CH or C) are replaced by heteroatoms (e.g., O, S, S(O), S(O)2 or NR 4 ) substituted alkyl groups. In other words, the alkyl chain is selected from O, S, S(O), S(O)2 and NR 4 The (hetero)alkyl group is an alkyl radical, for example, an alkyl radical, which is a radical which is interrupted by one or more elements of the alkyl radical. Such interruptions are different from substituents because they occur within the chain of the alkyl group, whereas substituents are side chain groups, attached in a monovalent form to, for example, a carbon atom of the alkyl chain. In a preferred embodiment, the (hetero)alkyl group is an alkyl radical, for example, an 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] Likewise, the term "(hetero)aryl" refers to both aryl and heteroaryl groups. A heteroaryl group is one in which one or more carbon units (e.g., CH) in the ring are replaced by heteroatoms (e.g., O, S, N, or NR). 4 ) substituted aryl groups.

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

[0037] A "domain" can be any region of a protein, generally defined by sequence homology, and is generally associated with a specific structural or functional entity. CEACAM family members are known to be composed of Ig-like domains. The term "domain" is used in this document to refer to either a single Ig-like domain, such as an "N-domain," or a group of consecutive domains, such as an "A3-B3 domain."

[0038] A "coding sequence" or a sequence "encoding" an expression product (e.g., 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] The term "gene" means a DNA sequence that encodes or corresponds to a specific amino acid sequence, which contains 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, for example, the conditions for gene expression. Some genes are not structural genes and may be transcribed from DNA into RNA but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term "gene" may refer to a genomic sequence that encodes a protein, i.e., a sequence that includes regulators, promoters, introns, and exon sequences.

[0040] The term "glycoprotein" is used herein in its normal scientific sense and refers to a protein comprising one or more monosaccharide or oligosaccharide chains ("glycans") covalently bound to the protein. 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 the protein (N-glycoproteins), such as asparagine or arginine, or to carbons on the protein (C-glycoproteins), such as tryptophan. Glycoproteins can contain more than one type of glycan, can contain a combination of one or more monosaccharide and one or more oligosaccharide glycans, and can contain a combination of N-linked, O-linked, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and can therefore be 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 normal scientific sense and refers to a monosaccharide or oligosaccharide chain connected to a protein. Therefore, the term "glycan" refers to the carbohydrate portion of a glycoprotein. A glycan is attached to a protein via the C-1 carbon of a sugar, which may not be further substituted (monosaccharide) or may be further substituted (oligosaccharide) at one or more hydroxyl groups. Naturally occurring glycans typically contain 1 to about 10 sugar moieties. However, when a longer sugar chain is attached to a protein, the sugar chain is also considered a glycan herein. The glycans of a glycoprotein may be a monosaccharide. Typically, the monosaccharide glycans of a glycoprotein are composed of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently attached to a protein. A glycan may also be an oligosaccharide. The oligosaccharide chains of a glycoprotein may be linear or branched. In oligosaccharides, the sugar directly attached to a protein is referred to as a core sugar. In oligosaccharides, the sugar that is not directly attached to a protein but is attached to at least two other sugars is referred to as an internal sugar. Among oligosaccharides, the sugar that is not directly attached to the protein but is attached to a single other sugar, that is, the sugar that does not carry an additional sugar substituent at one or more of its other hydroxyl groups is called a terminal sugar. For the avoidance of doubt, there may be multiple terminal sugars in the oligosaccharides of a glycoprotein, but there can only be one core sugar. Glycans can be O-linked glycans, N-linked glycans, or C-linked glycans. In O-linked glycans, the monosaccharide or oligosaccharide glycan is bound to the O atom of a protein amino acid, typically through the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, the monosaccharide or oligosaccharide glycan is bound to the protein through the N atom of a protein amino acid, typically through the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). In C-linked glycans, the monosaccharide or oligosaccharide glycan is bound to the C atom of a protein amino acid, typically to the C atom of tryptophan (Trp).

[0042] The term "antibody" (AB) is used in this article with its normal scientific meaning. Antibodies are proteins that the immune system produces that can recognize and bind specific antigens. Antibodies are an example of glycoproteins. The term "antibody" herein is used in its broadest sense, and particularly includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (such as bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. The term "antibody" herein is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is intended to include whole antibodies, but also includes antibody fragments, such as antibody Fab fragments, F (ab') 2, Fv fragments or Fc fragments, scFv-Fc fragments, miniantibodies, diabodies, or scFv from cleavage antibodies. In addition, the term also includes derivatives of genetically engineered antibodies and antibodies. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art.

[0043] Antibodies can be natural or conventional antibodies, in which two heavy chains are interconnected by disulfide bonds and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (kappa). The light chain consists of two domains or regions, namely a variable domain (VL) and a constant domain (CL). The heavy chain consists of four domains, namely a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light and heavy chains (VL and VH) determine the binding recognition and specificity for the antigen. The constant region domains of the light and heavy chains (CL and CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and is composed of the variable portion 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., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass, or allotype (e.g., human G1m1, G1m2, Gm3, non-G1m1 [i.e., 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). 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. The antibody can be engineered in the Fc domain to enhance or nihilate binding to Fc-γ receptors, as summarized by Saunders et al. Front. Immunol. [Immunology Frontier] 2019, 10, doi: 10.3389 / fimmu.2019.01296 and Ward et al. Mol. Immunol. [Molecular Immunology] 2015, 67, 131-141. For example, the combination of Leu234Ala and Leu235Ala (commonly referred to as the LALA mutation) eliminates FcγRIIa binding. Binding to Fc-γ receptors can also be eliminated by mutating the N297 amino acid to any amino acid other than asparagine, by mutating the T299 amino acid to any amino acid other than threonine or serine, or by enzymatic deglycosylation or trimming of fully glycosylated antibodies using, for example, PNGase F or endoglycosidase.Immunoglobulins can be derived from any species, including human, murine, or rabbit. Each chain contains distinct sequence domains.

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

[0045] The term "CDR" refers to complementarity determining region: the specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody combining site is composed of residues primarily from the hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and thus the binding site. Thus, the complementarity determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen-binding site includes 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 that is directed against a single amino acid sequence of a specific antigen and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a single B cell clone or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0047] The term "chimeric antibody" refers to an engineered antibody that, in its broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In an embodiment, a chimeric antibody comprises a VH domain and a VL domain derived from an antibody of a non-human animal, and a CH domain and a CL domain of another antibody (in an embodiment, a human antibody). As a non-human animal, any animal such as a mouse, rat, hamster, rabbit, etc. can be used. A chimeric antibody can also refer to a multispecific antibody that is specific 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 to replace certain amino acids, for example, in the framework regions of the VH and VL domains, to avoid or minimize human immune responses. The constant domains of humanized antibodies are mostly human CH and CL domains. A "fragment" of a (conventional) antibody comprises a portion of a complete antibody, in particular the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific antibodies and multispecific antibodies formed from antibody fragments. A conventional antibody fragment can also be a single domain antibody, such as a heavy chain antibody or VHH. The present invention

[0049] In a first aspect, the present invention relates to a conjugate wherein a compound according to structure (1) is conjugated to a cell binding agent via a linker, wherein structure (1) is as follows: in: -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, heterocyclic, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 ) 2, wherein the optional substituent is selected from 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 wherein the substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups may optionally be further substituted with C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 )2 substituted; wherein each Sp is independently C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1-12(Hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2, wherein each R 4 H, C alone 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 wherein R 12 Is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) ; -R 2 is H, S(O)2OH or P(O)2OH and R 3 is OH, or R 2 and R 3 fused together through the ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; -Y 5 is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY, where R 9 Selected from optionally OH groups or O(CO)C 1-6 Alkyl substituted C 1-4 Alkyl, and R 20 It is NR 4 -C(O)-N(R 4 )2、NR 4 -C(O)-Sp-N(R 4 )2、NR 4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 As defined above; - the compound according to structure (1) is linked via Y to the cell binding agent. Also contemplated in this aspect are salts of compounds according to structure (1) wherein each ion, if present, is balanced with one or more pharmaceutically acceptable counterions.

[0050] In a second aspect, the present invention relates to a novel toxin according to structure (1): in: -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, heterocyclic, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 ) 2, wherein the optional substituent is selected from 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 wherein the substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups may optionally be further substituted with C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 )2 substituted; wherein each Sp is independently C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1-12 (Hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2, wherein each R 4 H, C alone 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 wherein R 12 Is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) ; -R 2 is H, S(O)2OH or P(O)2OH and R 3 is OH, or R 2 and R 3 fused together through the ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; -N ％ is N or N→O; -Y 5 is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY、C(R 9 )=N(R 20 ), where R 9 Selected from optionally OH groups or O(CO)C 1-6 Alkyl substituted C 1-4 Alkyl, and R 20 It is NR 4 -C(O)-N(R 4 )2、NR 4 -C(O)-Sp-N(R 4 )2、NR 4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 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 NR 4 -Sp 3 -X 2 -Sp 3 -R 12 , OH, CH3 or CH2OH, where Sp 3 It is a spacer; - and where Y 5When it is C(O)-CH2OH, R 1 Not unsubstituted ethyl, CH2CH2SH or benzyl; Also contemplated in this aspect are salts of compounds according to structure (1) wherein each ion, if present, is balanced with one or more pharmaceutically acceptable counterions.

[0051] As used herein, the compound of structure (1) may be in conjugated form (i.e., conjugated to a cell-binding agent) or in free form (i.e., as a small molecule). Unless otherwise indicated, all definitions for the conjugated form of structure (1) apply to the free form of structure (1), and vice versa, except for the connection to the cell-binding agent via a linker.

[0052] The present invention also contemplates salts of the antibody-conjugate according to structure (1), preferably pharmaceutically acceptable salts. Although both the conjugated form and the free form of the compound according to structure (1) can be in the form of a salt, the conjugated form of the compound according to structure (1) is generally not in the form of a salt, whereas the free form of the compound according to structure (1) can be in the form of a salt and a neutral form. If the compound having structure (1) is charged, it is generally in equilibrium with one or more pharmaceutically acceptable counterions.

[0053] In the following, the compound according to structure (1) is first defined. The structural features of the compound according to structure (1) also apply to the conjugate according to structure (2) and the linker-toxin construct according to structure (5). Further, the structural features of the cell binding agent according to structure (4) also apply to the conjugate according to structure (2). It is understood by those skilled in the art that any structural feature that remains unchanged in the conjugation reaction is defined identically for each molecule according to the present invention. In the conjugation reaction, when the linker-toxin construct according to structure (5) reacts with the antibody according to structure (3), only the reactive moieties F and Q are converted into the linker group Z 1 .

[0054] In a further aspect, the present invention relates to the use of the conjugate according to structure (2) for targeting tumor cells. In connection therewith, the present invention relates to a first medical use and a second medical use of the conjugate according to structure (2).

[0055] As will be understood by those skilled in the art, the definitions of chemical moieties and their preferred embodiments apply to all aspects of the invention. Compounds according to structure (1)

[0056] The present invention relates to compounds according to structure (1): in: -R 1is 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, heterocyclic, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 ) 2, wherein the optional substituent is selected from 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 wherein the substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups may optionally be further substituted with C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 )2 substituted; wherein each Sp is independently C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1-12 (Hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2, wherein each R 4 H, C alone 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 wherein R 12 Is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) ; -R 2 is H, S(O)2OH or P(O)2OH and R 3 is OH, or R 2 and R 3fused together through the ether moiety to form an oxazolidine ring; -R 5 is H or OCH3; -Y 5 is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY、C(R 9 )=N(R 20 ), where R 9 Selected from optionally OH groups or O(CO)C 1-6 Alkyl substituted C 1-4 Alkyl, and R 20 It is NR 4 -C(O)-N(R 4 )2、NR 4 -C(O)-Sp-N(R 4 )2、NR 4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 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、R 12 、Sp 3 R 12 NR 4 -Sp 3 -X 2 -Sp 3’ -R 12 , OH, CH3 or CH2OH, where Sp 3 and Sp 3’ It is a spacer; -N ％ It is N or N→O.

[0057] The compounds according to structure (1) may be linked to a cell binding agent (i.e., a conjugate) or may contain a reactive group (i.e., a free form or a small molecule) that is capable of reacting with a suitably functionalized cell binding agent or with a linker to be subsequently conjugated to the cell binding agent. For certain preferred embodiments of the compounds according to structure (1), the linkage to the cell binding agent or reactive moiety may be at any position in the compound. Preferably, such linkage is via Y or R1 The reactive group capable of linking the compound according to structure (1) to a linker or cell binding agent may be, for example, N(R 4 )2 or X 2 (R 4 ) group or R 1 X in 2 R 4 or N3 group.

[0058] The present invention also contemplates salts thereof, especially pharmaceutically acceptable salts. 2 S(O)2OH or P(O)2OH group or R 12 Similar groups may exist in salt form, containing pharmaceutically acceptable cations, such as Na + , K + NH4 + or NEt4 + If the compound according to structure (1) is in free form (not conjugated to a cell binding agent), salts are particularly contemplated. Conjugates rarely exist in salt form.

[0059] R 1 The present invention has found that the methyl group (R 1 =Me) with a larger substituent can beneficially affect the toxicity of the toxin. The present inventors have for the first time adjusted this substituent to improve toxicity while conjugating the toxin to a cell binding agent via Y.

[0060] R 1 is selected from 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, heterocyclic, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2. Preferably, R 1 is selected from the group consisting of 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 3-12Alkynyl, Sp-(hetero)aryl, Sp-heterocyclyl, Sp-OR 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 )2.

[0061] In an alternative preferred embodiment, R 1 is selected from 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 )2. In another preferred embodiment, R 1 is selected from 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 )2. In another preferred embodiment, R 1 is selected from 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 )2. In another preferred embodiment, R 1 Selected from 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 )2. In another preferred embodiment, R 1 Selected from 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 )2. In one embodiment, R 1 As defined above but not Sp-X 2 -Sp-R 12.

[0062] R 1 The group may be optionally substituted by one or more substituents, wherein the optional substituents are selected from 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 selected from halogen, X 2 R 4 and N(R 4 )2. Most preferably, the optional substituents are selected from OH, SH and NH2. If present, the substituents may be located at R 1 In a preferred embodiment, with R 1 The carbon atom directly adjacent to the attached O atom does not bear a substituent, so that it is only attached to carbon and / or hydrogen atoms, which has been found to increase the stability of the compound. In a particularly preferred embodiment, R 1 The group contains 0-2 substituents, more preferably 0 or 1 substituent, most preferably R 1 The group is unsubstituted.

[0063] Optional substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups themselves may also be further selected from C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 ) 2 is optionally substituted by a substituent. 2 and R 4 The preferred embodiment is also applicable to C 1-12 These optional substituents of (hetero)alkyl and (hetero)aryl substituents. In one embodiment, C 1-12 (Hetero)alkyl and (hetero)aryl substituents do not contain any further substituents.

[0064] In this article, 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, preferably X 2 is O, S, S(O) or S(O)2. Preferably, X 2 Not S. Therefore, X 2 Preferably, X is selected from C(O), C(O)O, C(O)NH, O, S(O), S(O)2, S(O)NH or S(O)2NH. 2 is O, S(O) or S(O)2. Most preferably, X 2 It is O.R.4 Selected from H, C 1-4 Preferably, R 4 Is H or C 1-4 Alkyl. Each X 2 and R 4 And each optional substituent can be selected individually.

[0065] R 12 Is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) Preferably, R 12 Is β-glucuronide, PO3 (2-) or SO3 (-) Most preferably, R 12 It is β-glucuronide.

[0066] Sp is an alkyl or aryl spacer. More particularly, Sp is selected from C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene or (hetero)arylene-C 1-12 (Hetero)alkylene. The carbon atom of Sp can be selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2. 2 and R 4 The preferred embodiments of the present invention also apply to these optional substituents of Sp. In a preferred embodiment, the optional substituents are selected from F, Cl, Br, OH, OR 4 , SH, NH 2 , Et, Me and NO 2 . In particularly preferred embodiments, the spacer Sp comprises 0-2 substituents, more preferably 0 or 1 substituents, and most preferably the spacer Sp is unsubstituted. (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 NR 4 . Each Sp and each optional substituent may be selected individually.

[0067] R 1 Preferred options are according to structures (D1)-(D67) depicted below.

[0068] In this article, the following applies: n and n' are independently integers in the range of 0-10, preferably in the range of 1-10, more preferably in the range of 1-5. -X 3 Selected from 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 Selected from NH, NR 6 、N (+) (R6)2, S(O) and S(O)2. -Each R 6 Individually selected from hydrogen, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C3-C 12 Cycloalkanes, C3-C 12 Cycloalkenyl, C3-C 12 Cycloalkynyl, (hetero)aryl and polyethylene glycol (PEG). In this context, PEG generally has the structure (CH2CH2O) m R 10 , where m is 1, 2 or 3 and R 10 It is H, CH3 or CH2CH3. -R 7 is H or (CH2) n CH3. -R 8 is a (hetero)aryl group. -R 12 Is β-glucuronide, PO3 2- 、OPO3 2- 、CO2 - 、SO3 - and N(C 1-4 Alkyl)3 + .

[0069] R 1Preferred options (D1)-(D67) also include halogenated and / or unsaturated versions thereof. Thus, any hydrogen atom directly bonded to a carbon atom may be replaced by a halogen, preferably by F or Cl, more preferably by F. Most preferably, no hydrogen atom is replaced by a halogen atom. Likewise, any two adjacent saturated carbon atoms may also contain a double bond or a triple bond (if possible). In other words, the CH2-CH2 fragment may be replaced by a CH=CH fragment or a C≡C fragment, the CH2-CH fragment may be replaced by a CH=C fragment, and the CH-CH fragment may be replaced by a C=C fragment. Most preferably, there are no carbon-carbon double or triple bonds except those explicitly indicated in the structures (D1)-(D67). In a preferred embodiment, R 1 Selected from (D1)-(D61).

[0070] Y 5 Typically, it contains the same carbonyl moiety as that present in the parent anthracycline compound. Alternatively, the carbonyl group may be replaced by a methylene group, an imine group, or a hydrazone group. Hydrazones are cleaved under the low pH conditions of endosomes and / or lysosomes but remain 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 is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY、or C(R 9 )=N(R 20 ), preferably Y 5 is C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY、or C(R 9 )=N(R 20 ), most preferably Y 5 It is C(O)-Y.

[0071] If Y 5 Contains an imine or hydrazone moiety, then R 9 is a substituent on carbon, and R 20 is a substituent on nitrogen. 9 Selected from optionally OH groups or O(CO)C 1-6 Alkyl-substituted C 1-4 Alkyl, and R 20 It is NR 4 -C(O)-N(R 4 )2、NR 4 -C(O)-Sp-N(R 4 )2、NR4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 As defined above. In this article, Sp, R 4 and R 12 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 It's Me. 20 Preferably NR 4 -C(O)-R 12 or NR 4 -C(O)-Sp-R 12 , most preferably R 20 It is NR 4 -C(O)-Sp-R 12 .Sp、R 4 and R 12 And preferred embodiments thereof are as defined above. 20 In the context of 4 Selected from hydrogen and C 1-4 Alkyl, more preferably selected from H and Me, most preferably R 4 It's Me. 20 In the context of 1-4 Alkylene, most preferably Sp is CH2. 20 In the context of 12 It is N(C 1-4 Alkyl)3 (+) , more preferably N(Me)3 (+) .

[0073] In a preferred embodiment, the compound according to structure (1) can be conjugated to a cell binding agent via Y. In this embodiment, it is preferred that R 1 does not contain a reactive moiety for conjugation to a cell binding agent. Therefore, it is preferred that R 1 Selected from (D1)-(D52), wherein X 3 Selected from OR 6 、N(R 6 )2、N (+) (R 6 )3.SR 6 、S(O)R 6 、S(O)2R6 , and Y 4 Selected from NR 6 、N (+) (R6)2, S(O) and S(O)2, where each occurrence of R 6 Selected solely from C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C3-C 12 Cycloalkanes, C3-C 12 Cycloalkenyl, C3-C 12 Cycloalkynyl, (hetero)aryl and PEG, i.e. R 6 Not hydrogen.

[0074] In an alternative preferred embodiment, the compound according to structure (1) can be obtained by R 1 Conjugated to a cell binding agent. In this example, R 1 Contains a reactive moiety for conjugation to a cell binding agent. Therefore, it is preferred that R 1 is selected from (D10)-(D15), (D18)-(D26), (D31-(D37), (D41)-(D44), (D48) and (D53)-(D61), wherein X 3 Selected from OH, NH2, NHR 6 , N3, SH and / or Y4 are NH.

[0075] It is preferred if the compound is 1 Conjugated with a cell binding agent, then Y 5 Selected from structures (Y11)-(Y16) depicted below:

[0076] In a preferred embodiment, Y 5 is C(=N(R 20 ))-Y or C(R 9 )=N(R 20 ), and R 20 It is NR 4 -C(O)-R 12 or NR 4 -C(O)-Sp-R 12 hydrazone and ion R 12 The combination of groups improves the therapeutic window of the conjugates according to the present invention, because the ionic cap can prevent the payload from entering the cell if the payload is released prematurely, while the ionic R 12 The group can reduce aggregation while the payload remains attached to the antibody.

[0077] In one embodiment, if Y=CH2OH, then R 1is not CH2CH2SH or benzyl, more preferably whatever Y is, R 1 is not CH2CH2SH or benzyl. In one embodiment, if Y=CH2OH, then R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl, more preferably whatever Y is, R 1 Not unsubstituted ethyl, CH2CH2SH or benzyl.

[0078] The compounds according to the present invention contain an oxane ring and a morpholine ring. They can be linked together to form a "closed" tricyclic structure containing the middle oxazolidine ring, or the structure can 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 through the ether moiety and thus form 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 3 Herein, S(O)2OH and P(O)2OH may be in salt form. In the case of open form, R 2 is H and R 3 In the most preferred embodiment, the structure is closed and R 2 and R 3 The oxazolidine ring is formed by fusion of the ether moiety.

[0079] R 5 It is a substituent on the outer benzene ring of the tetracyclic ring. 5 is H or OCH3. In a preferred embodiment, R 5 is OCH3. Preferably R 2 is H and R 3 is OH, or R 2 and R 3 The oxazolidine ring is fused together through the ether portion and R 5 is OCH3, more preferably R 2 and R 3 The oxazolidine ring is fused together through the ether portion and R 5 It is OCH3.

[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 NR 4 -Sp 3 -X 2 -Sp 3‘ -R 12 , OH, CH3 or CH2OH. 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. In this article, X 2 and R 4 As defined above, including preferred embodiments thereof, and Sp 3 Is a spacer. Spacer Sp 3 Preferably selected from 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 the (hetero)arylene may be optionally selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2, wherein the C 1-4 The alkyl substituents may optionally be separated by contact with NR 4 Partially connected to form a ring structure, especially with NR with a bond marked with * 4 The pyrrolidine formed by the moiety, and the alkylene group may be optionally selected from X 2 and NR 4 is interrupted by one or more heteroatoms. 3 Including C 1-4 Alkylene, which is optionally substituted as defined above, and wherein the substituents may be 4 The substituents are linked together to form a ring structure. A preferred ring structure is a pyrrolidine ring, particularly one in which Sp 3 -N(R 4 )2 together to form proline amino acid, ie, wherein Y=NR 4 -CH2-pyrrolidine-N*, wherein N* may contain R 4Substituents, connected to the cell binding agent via a linker. In this article, R 4 Preferably it is CH3 or H.

[0081] When the compound according to general structure (1) is in free form or not conjugated to a cell binding agent via Y, particularly preferred options for Y are selected from NR 4 -(CH2)-N(R 4 )2、NR 4 -Sp 3 -X 2 (R 4 )、N(R 4 )2, CH3 or CH2OH.

[0082] The compound according to structure (1), if in free form, may be in the amine form (N ％ =N) or N-oxide form (N ％ =N→O). If the compound according to 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 ％ =N. In a particularly preferred embodiment, the compound is in the form of an amine (ie, N ％ =N) and the structure is closed (ie R 2 and R 3 fused together through the ether moiety to form an oxazolidine ring).

[0083] Particularly preferred compounds according to structure (1) contain R 1 moiety selected from 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. Preferably, R 1 is selected from optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, aryl, Bn, Sp-N3 and Sp-N(R 4 )2. In this article, the optional substituents Sp, X 2 and R 4 As defined above, including preferred embodiments thereof. In the context of this embodiment, it is preferred that R 1 Selected from 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 selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, wherein Sp is C 1-4 Alkylene or C 1-4 More preferably, R 1 is i-Pr, Bn or Sp-N3, wherein Sp is CH2CH2, CH2CH2CH2 or CH2(Ph). In this context, CH2(Ph) may be CH2(2-Ph), CH2(3-Ph) or CH2(4-Ph), preferably CH2(4-Ph). In a particularly preferred embodiment, R 1 It is i-Pr, Bn, CH2CH2N3, CH2CH2CH2N3 or CH2((4-N3)Ph).

[0084] The compound according to structure (1) may contain a hydrophilic portion R 12 It is believed that the hydrophilic portion reduces aggregation of the ADC and increases efficacy and / or improves toxicity profile. 12 Selected from β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) and N(C 1-4 Alkyl)3 (+) , wherein these anions may also be in their protonated form. In a preferred embodiment, the conjugate according to the present invention is linked via Y, and R 1 It is Sp-R 12 or Sp-X 2 -Sp-R 12 More preferably, each Sp is independently C1-C5 alkyl and X 2 is NHC(O). Most preferably, R 1 Selected from:

[0085] In another embodiment, the compound according to structure (1) comprises a hydrophilic portion R 12 , and the conjugate is formed by R 1 In this context, Y is preferably R 12 、Sp 3 R 12 or NR 4 -Sp 3 -X 2 -Sp 3’ -R 12 In this embodiment, it is preferred that R 12 It's SO (3-) or N(C 1-4Alkyl)3 (+) More preferably, Y is selected from NHCH2CH2NHC(O)CH2SO3 (-) 、NHCH2CH2NHC(O)CH2NMe3 (+) 、CH2SO3 (-) and CH2NMe3 (+) , even more preferably, Y is 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.

[0086] The inventors have obtained particularly beneficial results in terms of improved efficacy with compounds according to structure (1). Therefore, in the context of conjugates according to structure (2) as further defined below, it is preferred that the payload D is a compound according to this preferred embodiment. In these conjugates, the compound according to structure (1) can be conjugated to the compound by R 1 or Y connection. If R 1 Selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, wherein Sp is C 1-4 Alkylene or C 1-4 More preferably, R 1 is i-Pr, Bn or Sp-N3, wherein Sp is CH2CH2 or CH2(4-Ph).

[0087] In one embodiment of the compound according to structure (1), when Y is CH2OH and Y 5 When it is C(O)-Y, 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 Not unsubstituted or substituted ethyl, CH2CH2SH or benzyl.

[0088] Preferably, when R 2 and R 3 When fused together through the ether portion to form an oxazolidine ring, R 1 Not alcohols, thiols or amines. Conjugates having the general structure (2)

[0089] In a first aspect, the present invention relates to conjugates wherein a compound according to structure (1) is conjugated to a cell binding agent via a linker. Such conjugates generally have the general structure (2): CB-Z 1 -LZ 2 -D (2) in: -CB is the cell binding agent; -D is the compound according to structure (1); -L is a linker; -Z 1 is a linker group connecting the cell binding agent CB to the linker; and -Z 2 is a linking group that connects the compound D to the linker. Cell binding agent CB

[0090] The conjugates according to the present invention contain a cell binding agent that is capable of targeting cells, for example, by interacting with extracellular receptors on the cell surface. 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, that is capable of such interaction with a specific receptor and is capable of targeting specific cells. Advantageously, these specific cells are tumor cells. In a most preferred embodiment, the cell binding agent (CB) is an antibody (Ab), typically an antibody that is capable of binding to a specific extracellular receptor on the cell surface, such that the antibody is capable of targeting the specific cell.

[0091] Antibodies are known in the art and include IgA, IgD, IgE, IgG, IgM, Fab, VHH, scFv, diabodies, minibodies, affibodies, affylin, affimer, atrimer, fynomer, cysteine ​​knot, DARPin, adnectin / centryin, knottin, anticalin, FN3, Kunitz domain, Obody, bicyclic peptides and tricyclic peptides. Preferably, the antibody is a monoclonal antibody, more preferably selected from the group consisting of: IgA, IgD, IgE, IgG and IgM antibodies. Even more preferably, Ab is an IgG antibody. The IgG antibody can be any IgG isotype. The antibody can be any IgG isotype, such as IgG1, IgG2, IgI3 or IgG4. Preferably, Ab is a full-length antibody, but Ab can also be an Fc fragment.

[0092] The antibody Ab is typically specific for an extracellular receptor on a tumor cell, preferably wherein the extracellular receptor on the 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 , 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, guanylate 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, adhesion molecule-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tenascin.

[0093] The conjugate according to the present invention contains a linker group Z 1, which is formed during the conjugation reaction, in which the cell binding agent, which can be appropriately modified, is combined with a cell binding agent containing LZ 2 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 link between the cell binding agent and the toxin. A portion of the cell binding agent can be a reactive group F or a linker group Z. 1 Linker L attached to the peptide portion of the cell binding agent 6 Preferably, the linking group Z 1 The cell-binding agent CB is linked to 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.

[0094] Therefore, the conjugate according to the present invention is preferably represented by: CB-[(L 6 ) b -{Z 1 -LZ 2 -D} x ] y (3) in: -b is 0 or 1; -L 6 Y-GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ -, wherein G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-; -x is 1 or 2; and -y is 1, 2, 3, or 4. Linker L 6

[0095] If the reactive group F is directly linked to CB, then CB is linked to F or Z 1 Linker L 6 (For conjugates with structure (1)) is absent and b = 0. This is the case, for example, for cysteine ​​conjugation and lysine conjugation. Alternatively, CB can be linked to F or Z using 1 Linker L 6 The reactive group F is introduced onto the antibody, in this case L 6exists and b=1. If there is L 6 , then the reactive group F is typically introduced into the glycan of the antibody. This is the case, for example, when conjugation is performed by artificially introduced reactive groups F, such as using transglutaminase, using sortase, or by enzymatic glycan modification (e.g., glycosyltransferase or α-1,3-mannosyl-glycoprotein-2-β-N-acetylglucosaminyltransferase). For example, a modified sugar residue S(F) can be introduced into the glycan x , thereby extending the glycan with one monosaccharide residue S, which introduces x reactive groups F on the glycan of the antibody. In the most preferred embodiment, the conjugation occurs via the glycan of the antibody and b = 1. The conjugation site is preferably located on the heavy chain of the antibody.

[0096] If present, L 6 Connect CB to F or Z 1 The linker is composed of -GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’ - represents, wherein G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or a sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 Is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. Usually, L 6 At least in part, it is formed by the glycans of the antibody. All recombinant antibodies produced in mammalian host systems contain a conserved N-glycosylation site at or near the asparagine residue at position 297 (Kabat numbering) of the heavy chain, which is modified by a complex type of glycan. It is preferred to use this naturally occurring glycosylation site of the antibody, but other glycosylation sites, including artificially introduced glycosylation sites, can also be used in the linker L. 6 Therefore, in a preferred embodiment, L 6 It is linked to an amino acid located within the range of positions 250-350 of the heavy chain of the antibody, preferably within the range of positions 280-310 of the heavy chain, more preferably within the range of positions 295-300 of the heavy chain, and most preferably at position 297 of the heavy chain.

[0097] L 6 -GlcNAc(Fuc) w -(G) j - is a glycan or a portion thereof. Thus, -GlcNAc(Fuc) w -(G) j- Typically derived from the original antibody, where GlcNAc is the N-acetylglucosamine moiety and Fuc is the fucose moiety. Fuc is typically bound to GlcNAc via an α-1,6-glycosidic bond. Typically, antibodies 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 can be achieved using fucosylated (w=1) and non-fucosylated (w=0) antibody conjugates. The GlcNAc residue may also be referred to as a core-GlcNAc residue and is a monosaccharide directly attached to the peptide portion of the antibody.

[0098] S can be directly linked to the core-GlcNAc(Fuc) w part, i.e. j = 0, means that before S is attached, the rest of the glycan is separated from the core-GlcNAc(Fuc) w Such trimming of glycans is well known in the art and can be achieved by the action of endoglycosidases. Alternatively, core-GlcNAc(Fuc) w There are one or more monosaccharide residues between moiety and S, i.e. j is an integer in the range of 1-10, preferably j=2-5. In a preferred embodiment, (G) j is the oligosaccharide fraction containing j monosaccharide residues G, where j is an integer in the range of 2-5. (G) j Typically linked to GlcNAc(Fuc) via a β-1,4 linkage w In a preferred embodiment, j is 3, 4 or 5. Although any monosaccharide present in the polysaccharide 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, mannose. Antibodies and antibody conjugates with j = 0 do not bind to Fc-γ receptors or have significantly reduced binding, while antibodies and antibody conjugates with j in the range of 4-10 do bind to Fc-γ receptors. Therefore, by selecting a certain value for j, the desired degree of binding to the Fc-γ receptor can be obtained. Therefore, preferably j = 0, 4, 5, 6, 7, 8, 9 or 10, more preferably j = 0, 4 or 5, and most preferably the antibody is trimmed and j = 0.

[0099] S is sugar or sugar derivative.Term " sugar derivative " is used to refer to the derivative of monosaccharide (i.e. the monosaccharide comprising substituent and / or functional group) in this article.The suitable example of S comprises glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), amino sugar and sugar acid, such as glucosamine (GlcNH ), galactosamine (GalNH ), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) (also referred to 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 particularly preferred embodiments, S is GalNAc.

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

[0101] Linking group Z 1 Or the reactive group F can be attached directly to S, or between S and Z 1 or F may have a linker L 7 Therefore, L 7 Can be present (w'=1 or 2) or absent (w'=0). Typically, each Z moiety can be linked to a linker L. 7 Connected to S, so in one embodiment, w'=0 for x. Preferably, L 7 Not present and each linker Z is directly attached to S. If present, L 7 It can be selected from -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-. In a preferred embodiment, x=1 and w'=0 or 1, most preferably x=1 and w'=0.

[0102] y is an integer representing the number of sugars (derivatives) S, each sugar (derivative) having x reactive groups F or connected to x linking groups Z 1 , which are linked to CB. y is 1, 2, 3 or 4, preferably y = 2 or 4, most preferably y = 1. Thus, the antibody contains y S moieties, each of which contains x reactive moieties F. Each of these reactive moieties F reacts with a reactive moiety Q of the linker-toxin construct, such that x x y linker groups Z are formed. 1And x x y compounds according to general structure (1) are attached to a single CB. Each linker-toxin construct can contain multiple payloads, for example by means of a branching nitrogen atom N* in L. It is preferred that each linker-toxin construct contains 1 or 2 occurrences of D, most preferably 1 occurrence of D. In particularly preferred embodiments, linker L 1 Contains a branching nitrogen atom N*, to which the second occurrence of D is attached.

[0103] The amount of toxin D (compound according to general structure (1)) attached to a single antibody is referred to in the art as DAR (drug-to-antibody ratio). In the context of the present invention, it is preferred that DAR is an integer in the range of 1-8, more preferably 2 or 4, and most preferably DAR=2. In other words, DAR is preferably an integer in the range of (x×y) to [(x×y)×2], most preferably DAR=[(x×y)×2]. When the preferred value of x is 1 and the preferred value of y is 1, DAR is preferably 2. It should be understood that these are theoretical DAR values, and in practice, DAR may deviate slightly from this value due to incomplete conjugation. Typically, conjugates are obtained as random mixtures of antibody-drug conjugates, where the DAR values ​​vary between individual conjugates and, depending on the conjugation technique used, DAR may have a broad distribution (e.g., DAR=0-10) or a narrow distribution (e.g., DAR=3-4). In the case of such mixtures, DAR generally refers to the average DAR of the mixture. This is well known in the art of bioconjugation. However, if conjugation occurs via the glycan (ie b = 1 and L 6 If the conjugate is present, the conjugate according to the present invention has a DAR close to the theoretical DAR. For example, when the theoretical DAR is 4, it is easy to obtain a DAR value higher than 3.6 or even higher than 3.8, indicating that most of the antibodies in the reaction mixture have fully reacted and the DAR is 4. Linking group Z 1

[0104] Z 1 is a linking group that covalently links the two parts of the conjugate according to the present invention. The term "linking group" herein refers to the structural element resulting from the reaction between Q and F, which is used to link one part of the conjugate to another part of the same conjugate. It will be understood by those skilled in the art that the nature of the linking group depends on the type of reaction that results in the connection between the parts of the compound. For example, when the carboxyl group of RC(O)-OH reacts with the amino group of H2N-R' to form RC(O)-N(H)-R', R is linked to R' via the linking group Z, and Z can be represented by the group -C(O)-N(H)-. Since the linking group Z 1 Derived from the reaction between Q and F, so it can take any form.

[0105] Since more than one reactive moiety F may be present or introduced into an antibody, the antibody-conjugate according to the present invention may contain more than one payload D / biomolecule, for example 1-8 payloads D, preferably 1, 2, 3 or 4 payloads D, more preferably 2 or 4 payloads D. In view of the symmetry of the antibody, the number of payloads is usually an even number. In other words, when one side of the antibody is functionalized with F, the other symmetrical side will also be functionalized. Alternatively, if a thiol group naturally occurring in a protein cysteine ​​residue is used as F, the value of m can be arbitrary and can vary between individual conjugates.

[0106] In the compound according to structure (1), the linking group Z 1 D is linked to CB via a linker L, optionally via L 6 A variety of reactions are known in the art for attaching reactive groups Q to reactive groups F. Thus, a variety of linking groups Z 1 In one embodiment, the reactive group Q is selected from the above options, preferably as depicted in Figures 2, 4 or 5, and the complementary reactive group F and the linker group Z obtained therefrom 1 It is known to those skilled in the art. Figure 4 shows that when a linker-conjugate comprising Q is conjugated to a biomolecule comprising a complementary reactive group F, F and Q as well as the linker group Z will be present in the bioconjugate. 1 Several examples of suitable combinations are given below.

[0107] For example, when F contains or is a thiol group, the complementary group Q includes an N-maleimide group, an alkenyl group and an allenamide group. For example, when F contains or is an amino group, the complementary group Q includes a keto group and an activated ester group. For example, when F contains or is a keto group, the complementary group Q includes an (O-alkyl)hydroxyamino group and a hydrazine group. For example, when F contains or is an alkynyl group, the complementary group Q includes an azido group. For example, when F contains or is an azido group, the complementary group Q includes an alkynyl group. For example, when F contains or is a cyclopropenyl group, a trans-cyclooctenyl group, a cycloheptyne or a cyclooctyne group, the complementary group Q includes a tetrazinyl group. In these special cases, Z is only an intermediate structure and will expel N2, thereby generating a dihydropyridazine (generated by reaction with olefins) or a pyridazine (generated by reaction with alkynes), such as Figure 4 shown.

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

[0109] In a preferred embodiment, the linking group Z 1 Obtained by cycloaddition or nucleophilic reaction, preferably wherein 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 the reactive group F connected to S and the reactive group Q connected to D via L. Conjugation reactions via cycloaddition or nucleophilic reaction are known to those skilled in the art, and those skilled in the art are able to select appropriate reaction partners F and Q and will understand the resulting linker Z 1 nature.

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

[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 imine groups. The triazole moiety is particularly preferably present in Z 1 In one embodiment, Z 1comprises a (hetero)cycloalkene moiety, i.e. is formed from a Q comprising a (hetero)cycloalkyne moiety. In an alternative embodiment, Z 1 Contains a (hetero)cycloalkane moiety, i.e., is formed by Q containing a (hetero)cycloalkene moiety. In this article, an aromatic ring (such as a triazole ring) is considered to be a heterocycloalkane ring because it is formed by the reaction of an alkyne moiety and an azide moiety. In a preferred embodiment, Z 1 With structure (Z1):

[0094] In this article, it is described as The bond is either a single bond or a double bond. In addition: - Ring Z is obtained by cycloaddition, preferably ring Z is selected from (Za)-(Zj) defined below, wherein the carbon atom marked with ** corresponds to the carbon atom in (Z1) fused to ring Z is depicted as The bond between the two carbon atoms; -R 15 are independently selected from the group consisting of 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, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 It is C(R 31 )2.O.S.S (+) R 31 、S(O)R 31 、S(O)=NR 31 or NR 31 , where S (+) By B (-) Balanced cationic sulfur atoms, where B (-) is an anion, and each R31 R alone 15 or a connection with D (via L); -u is 0, 1, 2, 3, 4, or 5; - u' is 0, 1, 2, 3, 4 or 5, wherein u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; -v=an integer in the range of 8-16; - Ring Z is formed by cycloaddition and is preferably selected from (Za)-(Zj).

[0095] In preferred embodiments, u+u'=0, 4, 5, 6, 7 or 8, more preferably 0, 4 or 5. If depicted as If the bond is a double bond, it is preferred that u+u'=4, 5, 6, 7 or 8, more preferably u+u'=4 or 5. If the bond is a single bond, then preferably u+u'=0 or 5. Preferably, the wavy bond marked with * is connected to CB, optionally through L 6 , while the tilde key marked with ** is connected to L.

[0096] Especially preferred is Z 1 Contains a (hetero)cyclic olefin moiety, i.e. depicted as is a double bond. In a preferred embodiment, Z 1 Selected from structures (Z2)-(Z20) depicted below:

[0097] In this article, the connection to L is depicted as a wavy bond. (-) Is an anion, preferably a pharmaceutically acceptable anion. Ring Z is formed by a cycloaddition reaction, and is preferably a 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 can have the structure selected from (Za)-(Zj) described below, in which the carbon atom marked with ** corresponds to two carbon atoms of the (hetero)cycloalkane ring fused to ring Z in (Z2)-(Z20). Since in the context of the present embodiment, the linking group Z is formed by reacting with a (hetero)cycloalkyne, the above description is The bond is a double bond.

[0098] In another preferred embodiment, Z 1 Selected from structures (Z21)-(Z38) depicted below:

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

[0100] In a preferred embodiment, Z 1 Contains a (hetero)cyclooctene moiety or a (hetero)cycloheptene moiety, preferably according to structure (Z8), (Z26), (Z27), (Z28) or (Z37), which are optionally substituted. 1 Each of these preferred options is further defined below.

[0101] Therefore, in a preferred embodiment, Z 1 comprising a heterocycloheptene moiety according to structure (Z37), which is optionally substituted. Preferably, the heterocycloheptene moiety according to structure (Z37) is unsubstituted.

[0102] In a preferred embodiment, Z 1 comprising a (hetero)cyclooctene moiety according to structure (Z8), more preferably according to (Z29), which is optionally substituted. Preferably, the cyclooctene moiety according to structure (Z8) or (Z29) is unsubstituted. In the context of this embodiment, Z 1 Preferably comprising a (hetero)cyclooctene moiety according to structure (Z39) as shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Z39), l is most preferably 1. Most preferably, Z 1 is according to Structure (Z42), further defined below.

[0103] In an alternative preferred embodiment, Z 1 comprises a (hetero)cyclooctene moiety according to structure (Z26), (Z27) or (Z28), which is optionally substituted. In the context of this embodiment, Z 1 Preferably comprises a (hetero)cyclooctene moiety according to structure (Z40) or (Z41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 Independently selected from the group consisting of: hydrogen, straight or branched C1-C 12 Alkyl group or C4-C 12(Hetero)aryl groups. The aromatic ring in (Z40) is optionally O-sulfonylated at one or more positions, while the ring in (Z41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctene moiety according to structure (Z40) or (Z41) is not further substituted. Most preferably, Z is according to structure (Z43), further defined below.

[0104] In an alternative preferred embodiment, Z 1 Contains a heterocycloheptenyl group and is according to structure (Z37).

[0105] In a particularly preferred embodiment, Z 1 Contains a cyclooctenyl group and is according to structure (Z42): In this article: - The bond marked with * is connected to CB, while the wavy bond marked with ** is connected to L; -R 15 are independently selected from the group consisting of 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, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 independently selected from the group consisting of 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; -R19 Selected from the group consisting of: 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, these alkyl groups are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Z (or Q) or D connected by a spacer moiety; and -l is an integer in the range of 0 to 10.

[0106] In preferred embodiments of groups according to structure (Z42), R 15 are independently selected from the group consisting of hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In preferred embodiments of groups according to structure (Z42), R 18 independently selected from the group consisting of: hydrogen, a C1-C6 alkyl group, most preferably two R 18 are all H. In preferred embodiments of groups according to structure (Z42), R 19 is H. In preferred embodiments of the group according to structure (Z42), l is 0 or 1, more preferably l is 1.

[0107] In a particularly preferred embodiment, Z 1 Contains a (hetero)cyclooctenyl group and is according to structure (Z43): In this article: - The bond marked with * is connected to CB, while the wavy bond marked with ** is connected to L; -R 15 are independently selected from the group consisting of 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-C24 (hetero)arylalkyl groups, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 .

[0108] In preferred embodiments of groups according to structure (Z43), R 15 are independently selected from the group consisting of hydrogen, halogen, -OR 16 、-S(O)3 (-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from the group consisting of: hydrogen and -S(O)3 (-) In preferred embodiments of groups according to structure (Z43), Y is N or CH, more preferably Y═N.

[0109] In a particularly preferred embodiment, Z 1 Contains a heterocycloheptenyl group and is according to structure (Z37) wherein Ring Z is a triazole.

[0110] In an alternative preferred embodiment, Z 1 Contains a (hetero)cycloalkane moiety, i.e. depicted as The bond of (hetero)cycloalkane group is a single bond. (Hetero)cycloalkane group may also be referred to as heterocycloalkyl group or cycloalkyl group, preferably cycloalkyl group, wherein 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 norbornyl group, a (hetero)cycloheptyl group, a (hetero)cyclooctyl group, all of which may be optionally substituted. Particularly preferred are (hetero)cyclopropyl group, (hetero)cycloheptyl group or (hetero)cyclooctyl group, wherein the (hetero)cyclopropyl group, the (hetero)cycloheptyl group or the (hetero)cyclooctyl group is optionally substituted. Preferably, Z 1Contains a cyclopropyl moiety according to structure (Z44), a heterocyclobutane moiety according to structure (Z45), a norbornene or norbornene group according to structure (Z46), a (hetero)cycloheptyl moiety according to structure (Z47), or a (hetero)cyclooctyl moiety according to structure (Z48). In this context, Y 3 Selected from C(R 23 )2、NR 23 or O, where each R 23 is independently hydrogen, C1-C6 alkyl or linked to L (optionally via a spacer) and is marked with The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropyl group is according to structure (Z49). In another preferred embodiment, the (hetero)cycloheptane group is according to structure (Z50) or (Z51). In another preferred embodiment, the (hetero)cyclooctane group is according to structure (Z52), (Z53), (Z54), (Z55) or (Z56).

[0111] In this context, the R groups on Si in (Z50) and (Z51) are typically alkyl or aryl, preferably C1-C6 alkyl. Ring Z is selected from structures (Zk)-(Zn), wherein the carbon atoms marked with ** correspond to the two carbon atoms of the (hetero)cycloalkane ring fused to ring Z in (Z44)-(Z56), and the carbon marked with * is connected to CB. Since in the context of this embodiment, the linking group Z 1 is formed by reaction with a (hetero)cyclic olefin, and is therefore depicted above as The bond is a single bond.

[0112] In the second preferred embodiment, Z 1 The thiol is formed by a nucleophilic reaction, preferably by nucleophilic substitution or Michael addition, preferably by Michael addition. The preferred Michael reaction is a thiol-maleimide linkage, most preferably wherein Q is a maleimide and F is a thiol group, wherein the thiol may be part of a disulfide bridge. Preferably, the thiol is present in the side chain of a cysteine ​​residue. Such conjugation reactions with thiols may also be referred to as thiol alkylation or thiol arylation. In a preferred embodiment, the linker group Z 1 Contains a succinimidyl ring or a ring-opened succinic acid amide derivative thereof, which can be formed by hydrolysis of the succinimidyl ring.

[0113] Alternatively, Z 1It is formed by a nucleophilic reaction at the amino group (F) in the side chain of a lysine residue, which reacts with an amino reactive group Q. Such conjugation reactions with thiols may also be referred to as amide bond formation or carbamate bond formation. Typical amino reactive groups Q include N-hydroxysuccinimidyl (NHS) esters, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanates, isothiocyanates, and benzoyl halides.

[0114] Linking group Z 1 Preferred options include a moiety selected from (Z57)-(Z71) depicted below:

[0115] In this paper, the wavy bonds marked with * in (Z57)-(Z66) are connected to CB, while the unlabeled wavy bonds are connected to the payload through the linker L. 29 It is C 1-12 Alkyl or 1-24 polyethylene glycol units, preferably C 1-4 alkyl or 6-14 polyethylene glycol units, most preferably ethyl or 12 polyethylene glycol units, and X 1 is O or S, preferably X 1 =O. Alternatively, R 29 It is C 1-12 Alkyl, preferably C 1-4 alkyl, most preferably ethyl, and X 1 is O or S, preferably X 1 =0. The nitrogen atoms marked with ** in (Z67)-(Z71) correspond to the nitrogen atoms of the side chains of the antibody lysine residues, while the unlabeled wavy bonds are connected to the payload via the linker L. The carbon atoms of the phenyl groups of (Z69) and (Z70) are optionally substituted, preferably optionally fluorinated.

[0116] In a preferred embodiment, the linking group Z 1 Comprising a moiety selected from (Z1)-(Z71). Linker L

[0117] Linker L is connected to the linker group Z 2 Combine the payload D with the linker Z 1 (in the conjugates according to the invention) or to link the payload D to the reactive group Q (in the 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 a cleavable linker comprising a peptide spacer and optionally a p-aminobenzyloxycarbonyl (PABC) moiety or a derivative thereof.

[0118] In a preferred embodiment, the structure of the linker L is -(L 1 ) n -(L 2 ) o -(L 3 ) p -, where (L 3 ) p Through the linker Z 2 Connected with the payload D, and (L 1 ) n With Z 1 Or Q connection. In this article, L 1 , L 2 and L 3 is a linker or linking unit, and each of n, o, and p is independently 0 or 1, wherein n+o+p is at least 1. In a preferred embodiment, at least the linker L is present. 1 and L 2 (ie n = 1; o = 1; p = 0 or 1), more preferably there is a linker L 1 , L 2 and L 3 (i.e. n=1; o=1; p=1).

[0119] Therefore, it is preferred that in the conjugate according to the present invention, LZ 2 Has the following structure: *-NR 4 -Sp 3 -NR 4 -(L 3 ) p -(L 2 ) o -(L 1 ) n -** in: - the bond marked with * is connected to the compound according to structure (1); -Bonds marked with ** and linker group Z 1 connect; -Sp 3 It 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 the (hetero)arylene may be optionally selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 Alkyl and one or more substituents of NO2, wherein the C 1-4 The alkyl substituents may optionally be separated by contact with NR4 Partially connected to form a ring structure, especially with NR with a bond marked with * 4 The pyrrolidine formed by the moiety, and the alkylene group may be optionally selected from X 2 and NR 4 is interrupted by one or more heteroatoms; -R 4 and X 2 As defined in claim 1; -L 1 , L 2 and L 3 Each of them is Z 1 A linker connected to D; - n, o and p are each independently 0 or 1, provided that n+o+p=1, 2 or 3.

[0120] Linkers, especially linker L 1 It may contain one or more branch points for attaching multiple payloads to a single linker. In a preferred embodiment, the linker of the conjugate according to the present invention contains a branching moiety. In the context of the present invention, a "branching moiety" refers to a moiety embedded in the linker connecting three moieties. In other words, the branching moiety contains at least three bonds to other moieties, typically one bond to Z. 1 or Q, one bond to the payload D, one bond to the second payload D. If present, the branching moiety is preferably embedded in the linker L 1 Among them, Sp 3 Part of or as NR 13 In the context of the present invention, any moiety containing at least three bonds to other moieties is suitable as a branching moiety. In a preferred embodiment, the branching 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 branching moiety is a nitrogen atom. Linker L 1

[0121] Linker L 1 Either absent (n=0) or present (n=1). Preferably, the linker L 1 exists and n=1. L 1 It can be selected, for example, from the group consisting of: linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylidene group, C3-C 200 Cycloalkylene groups, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene group, C9-C 200 These alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may optionally be substituted, and optionally the groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, preferably selected from the group consisting of: O, S(O) y’ and NR 21 , wherein y' is 0, 1 or 2, preferably y'=2, and R 21 independently selected from the group consisting of 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 group.

[0122] In a preferred embodiment, the linker L 1 Contains polar groups. Such polar groups can be selected from (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 (wherein z' is the number of carbon atoms in the alkane, preferably z'=1-10), -(O) a -C(O)-NH-S(O)2-NR 13 -(as further defined below, see structure (23)), -C(S(O)3 (-) )-、-C(C(O)2 (-) )-、-S(O)2-、-P(O)2 (-) -, -O(CH2CH2O) t -、-NR 30 (CH2CH2NR 30 ) t -, and the following two structures:

[0123] For the polar groups defined above, which end is connected to Z 1 Which end is connected to (L 2 ) o It doesn't matter.

[0124] The polar group may also contain amino acids, preferably selected from Arg, Glu, Asp, Ser and Thr. 13 Structure (23) is further defined below. t is an integer in the range of 0-15, preferably 1-10, more preferably 2-5, and most preferably t=2 or 4. Each R 30 H, C alone 1-12 Alkyl, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl. Linker L 1 There may be more than one such polar group, for example at least two polar groups. Polar groups may also be present in the linker L. 1 In a branch of a branch that branches off from a branching moiety defined elsewhere. Preferably, nitrogen or carbon atoms are used as branching moieties. Particularly preferred is the presence of -O(CH2CH2O) in the branch t -Polar group.

[0125] In a preferred embodiment, the linker L 1 is or comprises a sulfonamide group, preferably a sulfonamide group according to structure (23):

[0126] The wavy line indicates the connection to the rest of the compound, usually to Q and L. 2 , L 3 or D, preferably with Q and L 2 Preferably, (O) a The C(O) part is connected to Q, and NR 13 Part with L 2 , L 3 or D connection, preferably with L 2 connect.

[0127] In structure (23), a=0 or 1, preferably a=1, and R 13 Selected from the group consisting of: hydrogen, 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, these 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 24The (hetero)arylalkyl group is optionally substituted and is optionally selected from O, S and NR 14 is interrupted by one or more heteroatoms, where R 14 are independently selected from the group consisting of: hydrogen and C1-C4 alkyl groups. Alternatively, R 13 is optionally via a spacer, preferably via Sp as defined below 2 D connected to N, in one embodiment, D is connected via -(B) e -(A) f -(B) g -C(O)- is connected to N. Alternatively, R 13 Optionally, it is connected to the rest of the linker via a spacer to form a ring structure. For example, R 13 The linker may be connected via a CH2CH2 spacer moiety to form a piperazinyl ring, wherein the connection to D is through the second nitrogen of the piperazinyl ring.

[0128] In a preferred embodiment, R 13 is hydrogen, C1-C 20 Alkyl groups, preferably C1-C 16 Alkyl groups, more preferably C1-C 10 An alkyl group, or optionally connected to another occurrence of D or to another place in the linker through a spacer. In this context, an alkyl group is optionally substituted and optionally selected from O, S and NR 14 , preferably one or more heteroatoms interrupted by O, wherein R 14 In another preferred embodiment, R 13 It is C1-C 20 Alkyl groups, more preferably C1-C 16 Alkyl groups, even more preferably C1-C 10 An alkyl group, wherein the alkyl group is optionally interrupted by one or more O atoms, and wherein the alkyl group is optionally substituted by an -OH group, preferably a terminal -OH group. In this embodiment, further preferred is R 13 is a (poly)ethylene glycol chain containing a terminal -OH group. In another preferred embodiment, R 13is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl, or optionally linked to an additional occurrence of D or to elsewhere in the linker via a spacer, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and isopropyl, or optionally linked to an additional occurrence of D or to elsewhere in the linker via a spacer, and even more preferably selected from the group consisting of hydrogen, methyl and ethyl, or optionally linked to an additional occurrence of D or to elsewhere in the linker via a spacer. Even more preferably still, R 13 is hydrogen or is optionally linked through a spacer moiety to another occurrence of D or to elsewhere in the linker, and most preferably R 13 It's hydrogen.

[0129] In a preferred embodiment, L 1 According to the structure (24):

[0130] In this paper, a and R 13 As defined above, 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 Independently selected from the group consisting of: linear or branched C1-C 200 Alkylene group, C2-C 200 Alkenylene group, C2-C 200 Alkynylidene group, C3-C 200 Cycloalkylene groups, C5-C 200 Cycloalkenylene group, C8-C 200 Cycloalkynylene group, C7-C 200 Alkyl arylene group, C7-C 200 Aryl alkylene group, C8-C 200 Arylalkenylene groups and C9-C 200 Arylalkynylene groups, these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are optionally substituted and are optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, where R 16 independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24Alkynyl groups and C3-C 24 Cycloalkyl groups, these alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted. When these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are interrupted by one or more heteroatoms as defined above, it is preferred that the groups are interrupted by one or more O atoms and / or by one or more SS groups.

[0131] More preferably, the spacer portion Sp 1 and Sp 2 (if present) independently selected from the group consisting of: linear or branched C1-C 100 Alkylene group, C2-C 100 Alkenylene group, C2-C 100 Alkynylidene group, C3-C 100 Cycloalkylene groups, C5-C 100 Cycloalkenylene group, C8-C 100 Cycloalkynylene group, C7-C 100 Alkyl arylene group, C7-C 100 Aryl alkylene group, C8-C 100 Arylalkenylene groups and C9-C 100 Arylalkynylene groups, these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are optionally substituted and are optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, where R 16 independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 Cycloalkyl groups, these alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0132] Even more preferably, the spacer portion Sp 1 and Sp 2 (if present) independently selected from the group consisting of: linear or branched C1-C 50 Alkylene group, C2-C 50 Alkenylene group, C2-C 50 Alkynylidene group, C3-C 50 Cycloalkylene groups, C5-C 50 Cycloalkenylene group, C8-C50 Cycloalkynylene group, C7-C 50 Alkyl arylene group, C7-C 50 Aryl alkylene group, C8-C 50 Arylalkenylene groups and C9-C 50 Arylalkynylene groups, these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are optionally substituted and are optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, where R 16 independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 Cycloalkyl groups, these alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0133] Still even more preferably, the spacer portion Sp 1 and Sp 2 (if present) independently selected from the group consisting of: linear or branched C1-C 20 Alkylene group, C2-C 20 Alkenylene group, C2-C 20 Alkynylidene group, C3-C 20 Cycloalkylene groups, C5-C 20 Cycloalkenylene group, C8-C 20 Cycloalkynylene group, C7-C 20 Alkyl arylene group, C7-C 20 Aryl alkylene group, C8-C 20 Arylalkenylene groups and C9-C 20 Arylalkynylene groups, these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are optionally substituted and are optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, where R 16 independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 Cycloalkyl groups, these alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0134] In these preferred embodiments, it is further preferred that the alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups are unsubstituted and optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, preferably O, wherein R 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0135] Most preferably, the spacer portion Sp 1 and Sp 2 (if present) independently selected from the group consisting of: linear or branched C1-C 20 Alkylene groups, which are optionally substituted and optionally selected from O, S and NR 16 The group is interrupted by one or more heteroatoms, where R 16 independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Alkenyl groups, C2-C 24 Alkynyl groups and C3-C 24 Cycloalkyl groups, these alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted. In this embodiment, it is further preferred that these alkylene groups are unsubstituted and are optionally selected from O, S and NR 16 The group of is interrupted by one or more heteroatoms, preferably O and / or SS, wherein R 16 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, preferably hydrogen or methyl.

[0136] Therefore, the preferred spacer portion Sp 1 and Sp 2 Including -(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 yet 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, yet even more preferably 1, 2, 3 or 4.

[0137] Alternatively, the preferred linker L 1 Can be -(W) k -(A) d -(B) e -(A) f -(C(O)) g - means, where: - d = 0 or 1, preferably d = 1; - e = an integer in the range of 0-10, preferably e = 0, 1, 2, 3, 4, 5 or 6, preferably an integer in the range of 1-10, 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 1-5; and preferably wherein 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 sulfonamide group according to structure (23): -B is a -CH2-CH2-O- or -O-CH2-CH2- moiety, or (B) e is -(CH2-CH2-O) e1 -CH2-CH2- or -(CH2-CH2-O) e1 -CH2- moiety, wherein e1 is defined the same as e; -W is -OC(O)-, -C(O)O-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)(CH2) m C(O)-, -C(O)(CH2) m C(O)NH- or -(4-Ph)CH2NHC(O)(CH2) m C(O)NH-, preferably wherein W is -OC(O)NH-, -C(O)(CH2) m C(O)NH- or -C(O)NH-, and wherein m is an integer in the range of 0-10, preferably m=0, 1, 2, 3, 4, 5 or 6, most preferably m=2 or 3; - preferably wherein L 1 Pass (W) k Connected to Q and through (C(O)) g , preferably connected to L via C(O) 2 , L 3 or D, preferably L 2 .

[0138] In the context of this example, the wavy line in structure (23) indicates that the adjacent groups (such as (W) k (B) e and (C(O)) g ) is connected. It is preferred that A is according to structure (23) wherein a=1 and R 13 =H or C1-C 20 Alkyl groups, more preferably R 13 =H or methyl, most preferably R 13 =H.

[0139] Preferred linker L 1 With structure-(W) k -(A) d -(B) e -(A) f -(C(O)) g -,in: (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.

[0140] In this context, it is preferred that when d and / or f=1, then a=1 and R 13 =H. Most preferably, the linker is structure (a).

[0141] In a preferred embodiment, the linker L 1 Contains a branched nitrogen atom located between Q or Z and (L 2 ) o and contains another part D as a substituent, which is preferably connected to the branch nitrogen atom through a linker. An example of a branch nitrogen atom is the nitrogen atom NR in structure (23) 13 , where R 13 The second occurrence of D is connected via a spacer moiety. Alternatively, the branching nitrogen atom may be located according to the structure -(W) k -(A) d -(B) e -(A) f -(C(O)) g -L 1 In one embodiment, L 1 By-(W) k -(A) d -(B) e -(A) f -(C(O)) g -N*[-(A) d -(B) e -(A) f -(C(O)) g -]2 represents, wherein A, B, W, d, e, f, g and k are as defined above and are selected individually at each occurrence, and N* is a branching nitrogen atom, two -(A) d-(B) e -(A) f -(C(O)) g - connected to it. In this article, two (C(O)) g Part of them are related to -(L 2 ) o -(L 3 ) p -D connection, where L 2 , L 3 , o, p and D are as defined above and are each selected independently. In a preferred embodiment, L 2 , L 3 , o, p and D for each of (C(O)) g Both parts of the connection are identical.

[0142] Preferred linkers L containing branching nitrogen atoms 1 With structure-(W) k -(A) d -(B) e -(A) f -(C(O)) g -N*[-(A') d’ -(B') e’ -(A') f’ -(C(O)) g’ -]2, where: (i) k = d = g = e' = 1; f = d' = g' = 0; W = -C(O)-; B = B' = -CH2-CH2-O-; A is according to structure (23), where 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 according to structure (23), where a = 0 and R 13 =H; e=1, 2, 3 or 4, preferably e=2. Linker L 2

[0143] Linker L 2 Is a peptide spacer. Linker L 2 Either absent (o=0) or present (o=1). Preferably, the linker L 2 Present and o=1. Peptide spacer L 2 and cleavable linker L 3 However, in the conjugate according to the present invention, L 3The presence of is not essential, since the same unit (motive) may exist in connection with the payload D, especially in R 1 Or within Y. For example, if the conjugate comprises the unit CH2-Ph-NH-L 2 , where CH2-Ph-NH is composed of R 1 Formed, then the linker does not need L when it self-destructs 3 In addition, there is a p-aminobenzyl moiety. Therefore, in a preferred embodiment, L 3 does not exist and L 2 Preferably through R 1 Or Y and D are directly connected.

[0144] The peptide spacer can also be defined as (NH-CR 17 -CO) n , where R 17Represents an amino acid side chain as known in the art. In this article, the amino acid can be a natural amino acid or a synthetic amino acid. Examples of preferred synthetic amino acids are citrulline and cysteic acid. Preferably, the amino acids are all in the L configuration. n is an integer in the range of 1-5, preferably in the range of 2-4. Therefore, the peptide spacer contains 1-5 amino acids. Preferably, the peptide is a dipeptide (n=2), a tripeptide (n=3) or a tetrapeptide (n=4), most preferably, the peptide spacer is a dipeptide. While any peptide spacer can be used, preferably, the peptide spacer is selected from the group consisting of 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, 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-Ph e. 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, most preferably Val-Cit or Val-Ala. In this article, AcLys is ε-N-acetyl lysine and iGlu is isoglutamate. In one embodiment, L 2 =Val-Cit. In one embodiment, L 2 =Val-Ala.

[0145] R 17R represents an amino acid side chain, preferably selected from the group consisting 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, more preferably those of Val, Cit, Ala, Glu and Lys. In other words, R 17 Preferably selected from CH3(Ala), CH2CH(CH3)2(Leu), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2CH2NHC(O)CH3(AcLys), CH2CH2CH2NHC(=NH)NH2(Arg), CH2Ph(Phe), CH(CH3)2(Val), CH(CH3)CH2CH3(Ile), CH2C(O)NH2(Asn), CH2CH2C(O)OH(Glu), CH2C(O)OH(Asp) and CH2(1H-indol-3-yl)(Trp). 17 Particularly preferred examples are CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), CH2CH2C(O)OH(Glu) and CH(CH3)2(Val). Most preferably, R 17 It is CH3(Ala), CH2CH2CH2NHC(O)NH2(Cit), CH2CH2CH2CH2NH2(Lys), or CH(CH3)2(Val).

[0146] In particularly preferred embodiments, the peptide spacer can be represented by the general structure (25):

[0147] In this paper, R 17 As defined above, preferably R 17 Is CH3(Ala) or CH2CH2CH2NHC(O)NH2(Cit). The wavy line indicates the same as (L 1 ) n and (L 3 ) p connection, preferably, according to L of structure (25) 2 Connected to (L 1 ) nand connected to (L 3 ) p . Linker L 3

[0148] Linker L 3 It is a self-cleavable spacer, also known as a self-destructive spacer. 3 Either absent (p=0) or present (p=1). Preferably, the linker L 3 exists and p = 1. However, if the payload passes Z 2 (is NH) is connected to the linker, and Z 2 Connect to L 2 The C(O) terminus of the peptide spacer, and wherein R 1 or Y (depending on the position of the point of attachment to the compound having structure (1)) contains an aromatic ring as defined below for Ring A, then preferably L 3 does not exist and p = 0. 2 , Z 2 and Y or R 1 The properties ensure that even without L 3 In the presence of , linker L can also self-cleave.

[0149] Preferably, L 3 is a p-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative according to structure (26):

[0150] In this article, a wavy line indicates a 1 , L 1 or L 2 and Z 2 Typically, PABC derivatives are connected to Q, Z via NH 1 , L 1 or L 2 , preferably to L 2 and connected to Z via OC(O) 2 .

[0151] Ring A is a 5-membered or 6-membered aromatic or heteroaromatic ring, preferably a 6-membered aromatic or heteroaromatic ring. Suitable 5-membered rings are oxazole, thiazole and furan. Suitable 6-membered rings are phenyl and pyridyl. Ring A can be selected from halogen, X 2 R 4 、N(R 4 )2、C 1-4 In this article, X 2 and R 4As defined above, including preferred embodiments thereof. In preferred embodiments, the optional substituents are selected from F, Cl, Br, OH, OR 4 , SH, NH2, Et, Me, and NO2. In particularly preferred embodiments, Ring A contains 0-2 substituents, more preferably 0 or 1 substituents, 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.

[0152] R 21 Selected from H, R 26 , C(O)OH and C(O)R 26 , where R 26 It is C1-C 24 (hetero)alkyl groups, C3-C 10 (Hetero)cycloalkyl group, C2-C 10 (Hetero)aryl groups, C3-C 10 Alkyl (hetero) aryl groups and C3-C 10 (Hetero)arylalkyl groups, which are optionally substituted and optionally selected from O, S and NR 28 is interrupted by one or more heteroatoms, where R 28 are independently selected from the group consisting of: hydrogen and C1-C4 alkyl groups. 26 It is C3-C 10 (Hetero)cycloalkyl or polyalkylene glycol. 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 -(CH2CH2O) s H, wherein s is an integer in the range of 1-10, preferably 1-5, 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 It’s H. Linking group Z 2 and payload D

[0153] D, also known in the art as "payload", represents a compound that is or will be linked to CB. D is a compound according to structure (1), and preferred embodiments thereof as defined above and below.

[0154] The conjugates according to the present invention may contain more than one payload D. When more than one payload is present, the payloads D may be the same or different, but typically they are the same. In the context of the present invention, at least one payload shall be a compound according to structure (1). In preferred embodiments, the conjugate contains 2 or 4 occurrences of D, most preferably 2 occurrences of D. The second occurrence of D may be present within a linker L, which may contain a branching moiety, typically a branching nitrogen atom, connected to the second occurrence of D. Preferably, both occurrences of D are connected to the branching moiety via the same linker. Likewise, the conjugates according to the present invention may contain more than one payload / linking group Z 1 .

[0155] Payload is linked to the linker Z 2 is linked to a linker L, the linker group being formed by reacting a compound according to structure (1) with a linker unit. Thus, the reactive portion of the compound according to structure (1) reacts with the reactive portion of the linker. Thus, Z 2 The nature of Z depends on the nature of the reactive moiety and the type of reaction performed to connect the linker to the payload and may take any form. 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. If the payload is attached via Y, the linker group Z 2 Most preferably, it is an amide moiety. 1 Connect, then the connecting group Z 2 Most preferred are carbamate moieties.

[0156] Conveniently, the reactive moiety of the compound according to structure (1) is R 1 The amine part in the 1 =Sp-N(R 4 )2, or the amine portion in Y, especially when Y=N(R 4 )2 or NR 4 -Sp 3 -N(R 4 )2. In this article, an R 4 The group is replaced by a link to L. Thus, the remaining NR 4 The residue is Z formed when the amine group reacts with the linker 2 part, usually thus formed according to the structure -(O) a’ -C(O)-NR 4 -amide or carbamate moiety, wherein a' = 0 or 1. Alternatively, if R 1 Contains an azide moiety, a cycloaddition reaction can be performed to generate the linker group Z2 In this embodiment, Z 2 contains a (hetero)aryl moiety and is defined as above for the linker group Z insofar as it is involved in the cycloaddition reaction with the azide moiety 1 As stated.

[0157] In the context of the present invention, the connection between the compound according to structure (1) and the linker L is preferably via R 1 Or through Y.

[0158] Therefore, in a preferred embodiment, the compound according to structure (1) is linked to the linker L via Y. The inventors have found that by conjugating the compound according to structure (1) via Y, the substituent R 1 It can be used to modulate or improve the efficacy of the toxin and thus the efficacy of the entire conjugate. Therefore, it is preferred that the compound according to structure (1) is linked to the conjugate according to the present invention via Y. If R 1 is selected from 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 , then the connection via Y is particularly preferred. In a particularly preferred embodiment, the connection to the compound according to structure (1) is via Y, and R 1 is selected from (D1) to (D52) as defined above, more preferably selected from optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, n-pentyl, C 6-12 Alkyl, (hetero)aryl, Bn, Sp-(hetero)aryl, preferably selected from i-Pr, n-Pr, t-Bu, i-Bu, n-Bu and Bn, more preferably selected from i-Pr and Bn.

[0159] In an alternative embodiment, the compound according to structure (1) is 1 is linked to a linker L. If the compound according to structure (1) complies with one of the above preferred embodiments, then via R 1 Such a connection is particularly preferred. If R 1 Contains a reactive portion suitable for linking to the linker L (such as N3, NH2 or OH moiety), then 1 In a particularly preferred embodiment, the connection to the compound according to structure (1) is via R 1 , and R 1is selected from (D10)-(D15), (D18)-(D26), (D31-(D37), (D41)-(D44), (D48) and (D53)-(D61) as defined above, more preferably R 1 It's Sp-X 2 R 4 , Sp-N3 or Sp-N(R 4 )2, most preferably Sp-N3 or Sp-N(R 4 )2.

[0160] In a particularly preferred embodiment, the conjugate according to the present invention comprises a payload D containing R 1 moiety selected from 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. Preferably, R 1 is selected from optionally substituted i-Pr, n-Pr, t-Bu, i-Bu, n-Bu, aryl, Bn, Sp-N3 and Sp-N(R 4 )2. In this article, the optional substituents Sp, X 2 and R 4 As defined above, including preferred embodiments thereof. In the context of this embodiment, it is preferred that R 1 Selected from 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 selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, wherein Sp is C 1-4 Alkylene or C 1-4 More preferably, R 1 is i-Pr, Bn or Sp-N3, wherein Sp is CH2CH2, CH2CH2CH2 or CH2(Ph). In this context, CH2(Ph) may be CH2(2-Ph), CH2(3-Ph) or CH2(4-Ph), preferably CH2(4-Ph). In a particularly preferred embodiment, R 1 It is i-Pr, Bn, CH2CH2N3, CH2CH2CH2N3 or CH2((4-N3)Ph).

[0161] The inventors have obtained particularly beneficial results in terms of improved efficacy using compounds according to structure (1). In these conjugates, the compounds according to structure (1) can be conjugated to the molecule by R 1 or Y connection. If R 1 Selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, wherein Sp is C 1-4 Alkylene or C 1-4 More preferably, R 1 is i-Pr, Bn or Sp-N3, wherein Sp is CH2CH2 or CH2(4-Ph). Preferred linkers

[0162] According to a preferred embodiment, the linking portion of the conjugate according to the present invention (composed of LZ 2 (L1)-(L3):

[0163] In this article: -Bonds marked with ** and linker group Z 1 connect; -Each R 13 Individually selected from the group consisting of: hydrogen, 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, wherein these 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 The (hetero)arylalkyl group is optionally substituted and is optionally selected from O, S and NR 14 is interrupted by one or more heteroatoms, where R 14 independently selected from the group consisting of hydrogen and a C1-C4 alkyl group, or R 13 D, R are connected to N through the spacer part 13 = hydrogen or D linked to N via a spacer, preferably wherein the spacer is as defined above - (B) e -(A) f -(B) g -C(O)-; -L 2 As defined above, preferably L 2is a dipeptide, tripeptide, or tetrapeptide; - o is 0 or 1, preferably o is 1; -L 3 As defined above; -p is 0 or 1; -z1 is an integer in the range of 1-4; - z2 is 0 or 1, preferably z2=1; - z3 is 0 or 1, preferably z3=0; - z4 is 1 or 2, preferably z4=1.

[0164] According to a particularly preferred embodiment, the linking portion of the conjugate according to the present invention (composed of LZ 2 (L4)-(L7):

[0165] In this article: -Keys marked with * are connected to: (a) for (L3) and (L4), attached to the C(O) group of the Y moiety in the compound according to structure (1), and (b) For (L6) and (L7), with OR directly attached to the morpholine ring of the compound according to structure (1) 1 Some of the O atoms are connected; -Bonds marked with ** and linker group Z 1 connect; -R 13 Selected from the group consisting of: hydrogen, 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, wherein these 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 The (hetero)arylalkyl group is optionally substituted and is optionally selected from O, S and NR 14 is interrupted by one or more heteroatoms, where R 14 independently selected from the group consisting of hydrogen and a C1-C4 alkyl group, or R 13 D, R are connected to N through the spacer part 13= hydrogen or D linked to N via a spacer, preferably wherein the spacer is as defined above - (B) e -(A) f -(B) g -C(O)-; -L 2 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-membered 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 in the range of 1-4; - z2 is 0 or 1, preferably z2=1.

[0166] Particularly preferred combinations include A = 1,4-phenyl, R 13 =Hydrogen or D connected to N through a spacer, o=1, L 2 is a dipeptide, z2=1 and z1=2.

[0167] According to a particularly preferred embodiment, the linking portion of the conjugate according to the present invention (composed of LZ 2 (L8)-(L11):

[0168] In this article: -Keys marked with * are connected to: (a) for (L8) and (L9), attached to the C(O) group of the Y moiety in the compound according to structure (1), and (b) For (L10) and (L11), the OR directly attached to the morpholine ring of the compound according to structure (1) 1 Some of the O atoms are connected; -Bonds marked with ** and linker group Z 1 connect; -L 2 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-membered 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 in the range of 1-4; - z2 is 0 or 1, preferably z2=1.

[0169] Particularly preferred combinations include A = 1,4-phenyl, o = 1, L 2 is a dipeptide, z2=1 and z1=2.

[0170] According to a particularly preferred embodiment, the linking portion of the conjugate according to the present invention (composed of LZ 2 (L12)-(L15):

[0171] In this article: -Keys marked with * are connected to: (a) for (L12) and (L13), attached to the C(O) group of the Y moiety in the compound according to structure (1), and (b) For (L14) and (L15), the OR directly attached to the morpholine ring of the compound according to structure (1) 1 Some of the O atoms are connected; -Bonds marked with ** and linker group Z 1 connect; -Each R 17 is solely an amino acid side chain, preferably i-Pr, CH3 or CH2CH2CH2NHC(O)NH2; - Ring A is an optionally substituted 5-membered 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 in the range of 1-4; - z2 is 0 or 1, preferably z2=1.

[0172] Particularly preferred combinations include A = 1,4-phenyl, R 17 =i-Pr, CH3 or CH2CH2CH2NHC(O)NH2, z2=1 and z1=2.

[0173] In another preferred embodiment, the linker is a non-cleavable linker according to structures (L16)-(L20): Preferred conjugates

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

[0175] Conjugate (I) has the following structure: CB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 ) p -D} x ] y (I) in: -CB, L 6 , Z, D, x and y are as defined above; -L 1 is defined above by -(A) d -(B) e -(A) f -(C(O)) g -represented linker; -L 2 is a peptide spacer as defined above, preferably Val-Cit or Val-Ala; -L 3 is a PABC derivative according to structure (26); -p=0 or 1.

[0176] In the context of antibody-conjugate (I), it is preferred that for L 1 , d = 1 (according to A of structure (23), preferably a = 1 and R 13 =H), e=2, f=0 and g=1. In the context of antibody-conjugate (I), preferred is L 2 =Val-Cit or Val-Ala, more preferably Val-Cit. In the context of the antibody-conjugate (I), it is preferred that p=1, then R 21 =H.

[0177] Antibody-conjugate (II) has the following structure: CB-[(L 6 )-{Z-(L 1 )-(L 2 )-(L 3 )-D} x ] y (II) in: -CB, L 6 , Z, D, x and y are as defined above; -L 1 is defined as above consisting of -(A)-(B) e -(C(O))- represented linker; -L 2 is a peptide spacer as defined above, preferably Val-Cit or Val-Ala; -L 3 is a PABC derivative according to structure (26), wherein R 21 =H.

[0178] In the context of antibody-conjugate (II), it is preferred that for L 1 , e=2, according to the structure (23), preferably a=1 and R 13 =H. In the context of antibody-conjugate (II), preferred is L 2 =Val-Cit or Val-Ala, more preferably Val-Cit.

[0179] According to a preferred embodiment, the conjugate according to the present invention has a structure selected from (III)-(V):

[0180] In this article: -Bonds marked with ** and linker group Z 1 connect; -Each R 13 Individually selected from the group consisting of: hydrogen, 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, wherein these 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 The (hetero)arylalkyl group is optionally substituted and is optionally selected from O, S and NR 14 is interrupted by one or more heteroatoms, where R 14 independently selected from the group consisting of hydrogen and a C1-C4 alkyl group, or R 13D, R are connected to N through the spacer part 13 = hydrogen or D linked to N via a spacer, preferably wherein the spacer is as defined above - (B) e -(A) f -(B) g -C(O)-; -L 2 As defined above, preferably L 2 is a dipeptide, tripeptide, or tetrapeptide; - o is 0 or 1, preferably o is 1; -L 3 As defined above; -p is 0 or 1; -z1 is an integer in the range of 1-4; - z2 is 0 or 1, preferably z2=1; - z3 is 0 or 1, preferably z3=0; - z4 is 1 or 2, preferably z4=1.

[0181] Conjugate (X) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0182] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (Xa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (Xb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (X) preferably has structure (Xb).

[0183] Conjugate (XI) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0184] L 2It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XIa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XIb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XI) preferably has structure (XIb).

[0185] Conjugate (XII) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0186] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XIIa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XIIb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XII) preferably has structure (XIIb).

[0187] Conjugate (XIII) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0188] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XIIIa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XIIIb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Conjugate (XIII) preferably has structure (XIIIb).

[0189] Conjugate (XIV) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0190] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XIVa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XIVb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. In the context of conjugate (XIV), structure (XIVb) is most preferred.

[0191] Conjugate (XV) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0192] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XVa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XVb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. In the context of conjugate (XV), structure (XVb) is most preferred.

[0193] Conjugate (XVI) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0194] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XVIa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XVIb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. In the context of conjugate (XVI), structure (XVIb) is most preferred.

[0195] Conjugate (XVII) has a linker-payload moiety according to the following structure: in: - The wavy line indicates the Z 1 connection; -L 2 , o and D are as defined above.

[0196] L 2 It may be present or absent, preferably L 2 exists and o=1. For the preferred conjugate (XVIIa), L 2 According to structure (25) and R 17 is CH3. For the preferred conjugate (XVIIb), L 2 According to structure (25) and R 17 is CH2CH2CH2NHC(O)NH2. Preferably, R 17 =CH2CH2CH2NHC(O)NH2. In the context of conjugate (XVII), structure (XVIIb) is most preferred.

[0197] It is further preferred that these preferred conjugates (I)-(V), (X)-(XVII) are conjugated via a glycan, i.e. b=1, more preferably a trimmed glycan, i.e. j=0. In this context, it is further preferred that S=GalNAc and w'=0. In this context, it is further preferred that the linker group Z 1 The linker group Z is preferably formed by an azide-alkyne cycloaddition. 1 =(Z39), wherein ring Z 1=(Za) and V=CH2. In this context, further preference is given to x=1. In this context, further preference is given to y=2, more preferably x=1 and y=2.

[0198] In the most preferred embodiment, the conjugate according to the present invention is as defined above according to structure (Xb) or (XIb), wherein b=1, e=0, S=GalNAc, w'=0, the linker Z 1 =(Z39), wherein ring Z=(Za) and V=CH2, x=1 and y=2. Compounds according to general structure (4)

[0199] In a further aspect, the present invention relates to a linker-toxin construct. The linker-toxin construct comprises a compound according to structure (1) linked to a reactive moiety Q via a linker L and can be used to prepare a conjugate according to the present invention, in particular by reacting with a suitably functionalized cell binding agent CB-[(L 6 ) b -{F} x ] y (5) performing a reaction, as further defined below. In a bioconjugation reaction, the reactive portion Q of the linker-toxin construct reacts with the reactive portion F on the cell binding agent to generate the linker group Z 1 .

[0200] Linker-toxin constructs according to the present invention have the general structure (4): QZ 2 -D (4) in: -Q is the reactive part; -L is a linker; -Z 2 It is a linking group connecting L to D; -D is a compound according to structure (1).

[0201] The linker-drug construct contains the linker L and payload D of the final conjugate. Compounds according to formula (4) can be prepared by one skilled in the art using standard organic synthesis techniques and as illustrated in the Examples. Linker L and payload D are as defined above in the context of the conjugate according to structure (2).

[0202] In addition, the compound according to structure (4) is connected between the linker L and the payload D via a linker group Z. 2 The linkages are the same as defined for the conjugates according to structure (2). Thus, in a preferred embodiment, the compound according to structure (1) is connected via R 1 Or conjugated to a cell binding agent via Y. If conjugated via R1 occurs, then when R 1 =Sp-N(R 4 )2, the attachment preferably occurs via the nitrogen atom. If the conjugation occurs via Y, then when Y=N(R 4 )2 or NR 4 -Sp 3 -N(R 4 )2, the connection preferably occurs through the nitrogen atom. In this article, one R 4 The group is replaced by a linker to L such that the remaining NR 4 The residue is the Z formed when the amine group is connected to the linker 2 If the connection is through R 1 , then preferably Y=CH3 or CH2OH. Reaction part Q

[0203] Compounds according to general structure (4) comprise a reactive moiety Q. In the context of the present invention, the term "reactive moiety" may refer to a chemical moiety comprising a reactive group, or to the reactive group itself. For example, a cyclooctynyl group is a reactive group comprising a reactive group, i.e., a C-C triple bond. Similarly, an N-maleimido group is a reactive group comprising a C-C double bond as a reactive group. However, a reactive group (e.g., an azide reactive group, a thiol reactive group, or an alkynyl reactive group) may also be referred to herein as a reactive moiety.

[0203] Q as S(F) x A chemical handle for connection. In other words, Q is reactive to F and complementary to F. As used herein, a reactive group is said to be "complementary" to another reactive group when it selectively reacts with the other reactive group (optionally in the presence of other functional groups). Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below. Thus, compounds according to general structure (4) are conveniently used in conjugation reactions, wherein a chemical reaction occurs between Q and F to form a conjugate comprising a covalent connection between the payload D and the antibody. This will be explained in more detail below in the context of the method for synthesizing the conjugate according to the present invention.

[0204] The exact nature of Q and F depends on the type of conjugation reaction employed. One skilled in the art will be able to select an appropriate combination of Q and F. Preferably, Q, and therefore also F, is reactive in a cycloaddition reaction or a 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. Click probes are reactive in cycloadditions (click reactions) and are preferably selected from azides, tetrazines, triazines, nitrrone, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxythiophenes, sydnone, alkene moieties and alkyne moieties. Preferably, the click probe comprises or is an alkene moiety or an alkyne moiety, more preferably wherein the alkene is a (hetero)cycloalkene and / or the alkyne is a terminal alkyne or a (hetero)cycloalkyne. Typical thiol reaction part is selected from maleimide moiety, haloacetamide moiety, propadiene amide moiety, phosphoramidite (phosphonamidite) moiety, cyanoacetylene moiety, vinyl sulfone, vinyl pyridine moiety or methylsulfonylphenyl oxadiazole moiety.Most preferably, thiol reaction part comprises or is maleimide moiety.Typical amine reaction part is selected from N-hydroxysuccinimidyl ester and other activated esters, p-nitrophenyl carbonate and other activated carbonates, isocyanates, isothiocyanates, haloacetamide and benzoyl halide.In a preferred embodiment, Q is selected from alkene moiety, alkyne moiety, thiol reaction part or amine reaction part, more preferably alkene moiety or alkyne moiety, even more preferably alkyne moiety.Herein, alkene is preferably (hetero) cycloalkene, and alkyne is preferably terminal alkyne or (hetero) cycloalkyne.Most preferably, Q is cyclic (hetero) alkyne moiety.Each in these parts is further defined hereinafter.

[0205] Therefore, in particularly preferred embodiments, Q comprises a cyclic (hetero) alkyne moiety. Alkynyl group may also be referred to as a (hetero) cycloalkynyl group, i.e. a heterocycloalkynyl group or a cycloalkynyl group, wherein 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 this article, (hetero) cycloalkynes 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, wherein the (hetero) cyclooctynyl group is optionally substituted.

[0206] In particularly preferred embodiments, Q comprises a (hetero)cycloalkynyl or (hetero)cycloalkenyl group and is according to structure (Q1): In this article: -Depicted as The bond is a double bond or a triple bond; -R 15 are independently selected from the group consisting of 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, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 It is C(R 31 )2.O.S.S (+) R 31 、S(O)R 31 、S(O)=NR 31 or NR 31 , where S (+) By B (-) Balanced cationic sulfur atoms, where B (-) is an anion, and each R 31 R alone 15 or a connection with D (via L); -u is 0, 1, 2, 3, 4, or 5; - u' is 0, 1, 2, 3, 4 or 5, wherein u + u' = 0, 1, 2, 3, 4, 5, 6, 7 or 8; -v=an integer in the range of 0-16.

[0207] Typically, v = (u + u') x 2 (when the connection to L (depicted as a wavy bond) is through Y 2 when the bond to L (depicted as a wavy bond) is through a carbon atom in u and u'.

[0208] In preferred embodiments of structure (Q1), the reactive group Q comprises a (hetero)cycloalkynyl group and is according to structure (Q1a): In this article, -R 15 and Y 2 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 in the range of 8-16.

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

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

[0211] In preferred embodiments, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q2)-(Q20) depicted below.

[0212] Herein, the connection to L (depicted as a wavy bond) can be any available carbon or nitrogen atom of Q. The nitrogen atoms of (Q10), (Q13), (Q14), and (Q15) can be connected to L, or can contain a hydrogen atom or be optionally functionalized. (-) is an anion, which is preferably selected from (-) OTf、Cl (-) Br (-) or I (-) , most preferably B (-) yes (-) OTf. In the conjugation reaction, B (-) does not need to be a pharmaceutically acceptable anion, since B (-) In any case it will be exchanged with anions present in the reaction mixture. If (Q19) is used as Q, the negatively charged counterion is preferably pharmaceutically acceptable when isolating the conjugate according to the invention, so that the conjugate can be readily used as a drug.

[0213] In another preferred embodiment, Q is a (hetero)cycloalkynyl group selected from the group consisting of (Q21)-(Q38) depicted below.

[0214] In structure (Q38), B (-) is an anion, which is preferably selected from (-)OTf、Cl (-) Br (-) or I (-) , most preferably B (-) yes (-) OTf.

[0215] In preferred embodiments, Q comprises a (hetero)cyclooctyne moiety or a (hetero)cycloheptyne moiety, preferably according to structure (Q8), (Q26), (Q27), (Q28) or (Q37), which are optionally substituted. Each of these preferred options for Q is further defined below.

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

[0217] In alternative preferred embodiments, Q comprises a cyclooctyne moiety according to structure (Q8), more preferably according to (Q29), also known as a bicyclo[6.1.0]non-4-yn-9-yl] group (BCN group), which is optionally substituted. Preferably, the cyclooctyne moiety according to structure (Q8) or (Q29) is unsubstituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q39) as shown below, wherein V is (CH2) l and l is an integer in the range of 0 to 10, preferably in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2, and most preferably l is 0 or 1. In the context of group (Q39), l is most preferably 1. Most preferably, Q is according to structure (Q42), as further defined below.

[0218] In alternative preferred embodiments, Q comprises a (hetero)cyclooctyne moiety according to structure (Q26), (Q27) or (Q28), also known as a DIBO, DIBAC, DBCO or ADIBO group, which is optionally substituted. In the context of this embodiment, Q is preferably a (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) as shown below, wherein Y 1 Is O or NR 11 , where R 11 Independently selected from the group consisting of: hydrogen, straight or branched C1-C 12 Alkyl group or C4-C 12(Hetero)aryl groups. The aromatic ring in (Q40) is optionally O-sulfonylated at one or more positions, while the ring in (Q41) may be halogenated at one or more positions. Preferably, the (hetero)cyclooctyne moiety according to structure (Q40) or (Q41) is not further substituted. Most preferably, Q is according to structure (Q43), further defined below.

[0219] In an alternative preferred embodiment, Q comprises a heterocycloheptenyl group and is according to structure (Q37).

[0220] In particularly preferred embodiments, Q comprises a cyclooctynyl group and is according to structure (Q42): In this article: -R 15 are independently selected from the group consisting of 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, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15 can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 independently selected from the group consisting of 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 Selected from the group consisting of: 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, these alkyl groups are optionally interrupted by one or more heteroatoms selected from the group consisting of: O, N and S, wherein the alkyl groups, (hetero)aryl groups, alkyl(hetero)aryl groups and (hetero)arylalkyl groups are independently optionally substituted, or R 19 is the second occurrence of Q or D connected by a spacer portion; and -l is an integer in the range of 0 to 10.

[0221] In a preferred embodiment of the reactive group according to Structure (Q42), R 15 are independently selected from the group consisting of hydrogen, halogen, -OR 16 , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 Independently selected from the group consisting of hydrogen and C1-C6 alkyl, most preferably all R 15 are all H. In a preferred embodiment of the reactive group according to structure (Q42), R 18 independently selected from the group consisting of: hydrogen, a C1-C6 alkyl group, most preferably two R 18 are all H. In a preferred embodiment of the reactive group according to structure (Q42), R 19 is H. In preferred embodiments of the reactive group according to Structure (Q42), l is 0 or 1, more preferably l is 1.

[0222] In particularly preferred embodiments, Q comprises a (hetero)cyclooctynyl group and is according to structure (Q43): In this article: -R 15 are independently selected from the group consisting of 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, and wherein these alkyl groups, (hetero)aryl groups, alkyl (hetero)aryl groups and (hetero)arylalkyl groups are optionally substituted, wherein the two substituents R 15can be linked together to form an optionally substituted cyclic cycloalkyl or an optionally substituted cyclic (hetero)arene substituent, and wherein R 16 independently selected from the group consisting of 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 .

[0223] In a preferred embodiment of the reactive group according to Structure (Q43), R 15 are independently selected from the group consisting of hydrogen, halogen, -OR 16 、-S(O)3 (-) , C1-C6 alkyl groups, C5-C6 (hetero)aryl groups, wherein R 16 is hydrogen or C1-C6 alkyl, more preferably R 15 independently selected from the group consisting of: hydrogen and -S(O)3 (-) In preferred embodiments of the reactive group according to Structure (Q43), Y is N or CH, more preferably Y═N.

[0224] In alternative preferred embodiments, Q comprises cyclic olefin moiety.Alkenyl group Q also can be referred to as (hetero) cycloalkenyl group, i.e. heterocycloalkenyl group or cycloalkenyl group, preferably cycloalkenyl group, wherein this (hetero) cycloalkenyl group is optionally substituted.Preferably, (hetero) cycloalkenyl group is (hetero) cyclopropenyl group, (hetero) cyclobutenyl group, norbornene group, norbornadiene group, trans-(hetero) cycloheptenyl group, trans-(hetero) cyclooctenyl group, trans-(hetero) cyclononenyl group or trans-(hetero) cyclodecenyl group, and they all can be optionally substituted.Especially preferred is (hetero) cyclopropenyl group, trans-(hetero) cycloheptenyl group or trans-(hetero) cyclooctenyl group, wherein this (hetero) cyclopropenyl group, this trans-(hetero) cycloheptenyl group or this trans-(hetero) cyclooctenyl group are optionally substituted. Preferably, Q comprises a cyclopropenyl moiety according to structure (Q44), a heterocyclobutene moiety according to structure (Q45), a norbornene or norbornadiene group according to structure (Q46), a trans-(hetero)cycloheptenyl moiety according to structure (Q47), or a trans-(hetero)cyclooctenyl moiety according to structure (Q48). In this context, Y 3 Selected from C(R 23 )2、NR 23 or O, where each R 23 is independently hydrogen, C1-C6 alkyl or linked to L (optionally via a spacer) and is marked with The bond is a single bond or a double bond. In another preferred embodiment, the cyclopropenyl group is according to structure (Q49). In another preferred embodiment, the trans-(hetero)cycloheptene group is according to structure (Q50) or (Q51). In another preferred embodiment, the trans-(hetero)cyclooctenene group is according to structure (Q52), (Q53), (Q54), (Q55) or (Q56).

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

[0226] 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 can be selected from the group consisting of: a maleimide moiety, a haloacetamide moiety, a malonamide moiety, a phosphoramidite moiety, a cyanoethynyl moiety, a vinyl sulfone, a vinyl pyridine moiety, or a methylsulfonylphenyl oxadiazole moiety. Most preferably, Q comprises a maleimide moiety. The reagent can be a monoalkylated type, or it can be a cross-linker that reacts with two cysteine ​​side chains.

[0227] In another preferred embodiment, probe Q is selected from the group consisting of (Q57)-(Q71) depicted below. in: -X 6 is H, halogen, PhS, MeS, preferably halogen (such as Cl, Br, I); -X 7 is halogen, PhS, MeS, preferably halogen (such as Cl, Br, I); -R 24 Is H or C 1-12 Alkyl, preferably H or C 1-6 alkyl; -R 25 It is H, C 1-12 Alkyl, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl, preferably H or p-methylphenyl; - wherein the aromatic rings of (Q61) and (Q63) may optionally be heteroaromatic rings, such as phenyl or pyridine rings.

[0228] In a preferred embodiment of the thiol-reactive probe (Q57), the probe Q is selected from the group consisting of (Q72)-(Q74) depicted below. in: -R 27 It is C 1-12 Alkyl, C 1-12 Aryl, C 1-12 Alkaryl or C 1-12 Aralkyl; -t is an integer in the range of 0-15, preferably 1-10.

[0229] In alternative preferred embodiments, Q is an amine reactive probe. In this embodiment, Q is a reactive group that is conjugated to lysine. Such probes are known in the art and can be selected from the group consisting of: N-hydroxysuccinimide groups, p-nitrophenyl carbonate, pentafluorophenyl carbonate, isocyanate groups, isothiocyanate groups, and benzoyl halide groups. Most preferably, Q comprises or is N-hydroxysuccinimide ester, p-nitrophenyl carbonate moiety, or pentafluorophenyl carbonate moiety.

[0230] In another preferred embodiment, probe Q is selected from the group consisting of (Q75)-(Q80) depicted below. In this article, X 2 is halogen, preferably F.

[0231] In a preferred embodiment, Q is selected from the group consisting of: (Q1)-(Q80). Cell binding agents according to general structure (5)

[0232] Cell binding agents used in bioconjugation reactions with linker-toxin constructs have the general structure (5): CB-[(L 6 ) b -{F} x ] y (5) in: -CB is a cell-binding agent; -b is 0 or 1; -L 6 Y-GlcNAc(Fuc) w -(G) j -S-(L 7 ) w’-, wherein G is a monosaccharide, j is an integer in the range of 0-10, S is a sugar or sugar derivative, GlcNAc is N-acetylglucosamine and Fuc is fucose, w is 0 or 1, w' is 0, 1 or 2, and L 7 is -N(H)C(O)CH2-, -N(H)C(O)CF2- or -CH2-; -F is the reactive part; -x is 1 or 2; and -y is 1, 2, 3, or 4.

[0233] The cell binding agent having the general structure (5) may also be referred to as a "(modified) cell binding agent", preferably a "(modified) antibody", because it contains a reactive group F, wherein the reactive group F is naturally occurring or the cell binding agent is modified to contain the reactive group F. The (modified) cell binding agent or antibody according to the general formula (5) can be prepared by a person skilled in the art using standard organic and / or enzymatic synthesis techniques and as illustrated in the Examples. Cell binding agent CB, linker L 6 , b, x and y are as defined above in the context of the conjugate according to structure (2). Reaction Part F

[0234] F is reactive toward Q in a conjugation reaction defined below, preferably wherein the conjugation reaction is a cycloaddition reaction or a nucleophilic reaction. It will be understood by those skilled in the art that the options for F are the same as those for Q, provided that F and Q are reactive toward 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 in a cycloaddition (click reaction) and is preferably selected from azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazonium compound, o-quinone, dioxythiophene, sidone, alkene moiety, and alkyne moiety. Preferably, the click probe comprises or is azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazonium compound, o-quinone, dioxythiophene, or sidone, most preferably azide. Typical thiol-reactive moieties are selected from maleimide moieties, haloacetamide moieties, malonamide moieties, phosphoramidite moieties, cyanoethynyl moieties, o-quinone moieties, vinyl sulfone, vinyl pyridine 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, p-nitrophenyl carbonates, pentafluorophenyl carbonates, isocyanates, isothiocyanates, and benzoyl halides. In preferred embodiments, F is a click probe or a thiol, more preferably F is an azide or a thiol, and most preferably F is an azide.

[0235] More than one reactive group F may be present in an antibody. The reactive group F in an antibody may be naturally occurring or may be placed in the antibody by a specific technique (e.g., (bio)chemical or genetic techniques). The reactive group placed in the antibody is prepared by chemical synthesis, such as an azide or a 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 literature, the conjugation reaction between the modified antibody and the linker-toxin-construct is known to those skilled in the art.

[0236] Preferably, F is a click probe reactive toward (hetero)cycloalkenes and / or (hetero)cycloalkynes, and is typically selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, o-quinones, dioxythiophenes, and sidrone. Preferred structures of the reactive group are structures (F1)-(F10) depicted below.

[0237] In this article, the wavy bond represents the connection to the payload. For (F3), (F4), (F8) and (F9), the payload can be connected to any of the wavy bonds. The other wavy bonds can then be connected to R groups selected from the following: hydrogen, C1-C 24 Alkyl groups, C2-C 24 Acyl group, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero) aryl groups, C3-C 24 (Hetero)arylalkyl groups and C1-C 24 Sulfonyl groups, each of which (except hydrogen) may be optionally substituted and is optionally selected from O, S and NR 32 is interrupted by one or more heteroatoms, where R 32 Independently selected from the group consisting of: hydrogen and C1-C4 alkyl groups. Those skilled in the art understand which R groups are applicable to each group F. For example, the R group attached to the nitrogen atom in (F3) can be selected from alkyl and aryl groups, while the R group attached to the carbon atom in (F3) can be selected from hydrogen, alkyl, aryl, acyl and sulfonyl groups. Preferably, the reactive moiety F is selected from azide or tetrazine. Most preferably, the reactive moiety F is an azide.

[0238] In a second preferred embodiment, F is a thiol or a precursor thereof. The thiol or its precursor F is used in a conjugation reaction to link the linker-toxin-construct to the (modified) cell-binding agent. In the thiol linker, F is reactive toward the thiol-reactive probe Q. The thiol is preferably a thiol of the cysteine ​​amino acid side chain, which is naturally present in antibodies AB, in which case the linker L 6 is not present (b=0), however it can also be introduced synthetically, optionally via a linker L 6 Thiol precursors in the context of bioconjugation are known in the art and include disulfides, which may be naturally occurring disulfide bridges present in antibodies or synthetically introduced disulfides, which are reduced as known in the art. Preferably, F is a thiol group of a cysteine ​​side chain.

[0239] In a third preferred embodiment, F is an amine or a precursor thereof, preferably an amine. The amine or its precursor F is used in a conjugation reaction to link the linker-toxin-construct to the (modified) antibody. In a nucleophilic substitution, F is reactive toward the amine-reactive probe Q. The amine is typically a primary amine, preferably an amine of the side chain of a lysine amino acid, which is naturally present in the antibody AB, in which case the linker L 6 is not present (b=0), however it can also be introduced synthetically, optionally via a linker L 6 Preferably, F is a primary amine group of a lysine side chain. Synthesis of conjugates according to general structure (2)

[0240] In a further aspect, the present invention relates to a method for the preparation of a conjugate according to the invention, comprising the step of reacting Q of a toxin-linker-construct according to the invention with a reactive group F. The linker-toxin-construct according to the general structure (4) and preferred embodiments thereof are described in more detail above. The method takes place under conditions in which Q reacts with F to covalently link 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 link 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.

[0241] Any conjugation technique known in the art can be used to prepare the conjugate according to the present invention. Suitable conjugation techniques include thiol connection, lysine connection, cycloaddition (e.g., copper-catalyzed click reaction, strain-promoted azide-alkyne cycloaddition, strain-promoted quinone-alkyne cycloaddition). In other words, conjugation techniques are selected from amide bond formation, carbamate bond formation, thiol alkylation, thiol arylation and cycloaddition reaction. Preferred conjugation techniques used in the context of the present invention include nucleophilic reaction and cycloaddition, preferably wherein the cycloaddition is [4+2] cycloaddition or [3+2] cycloaddition, and the nucleophilic reaction is Michael addition or nucleophilic substitution. Suitable conjugation techniques are disclosed, for example, in GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 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.

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

[0243] Therefore, in a preferred embodiment of the conjugation method according to the present invention, conjugation is accomplished by cycloaddition. Preferred cycloadditions are (4+2)-cycloadditions (e.g., Diels-Alder reactions) or (3+2)-cycloadditions (e.g., 1,3-dipolar cycloadditions). Preferably, the conjugation reaction is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. Preferred Diels-Alder reactions are inverse electron demand Diels-Alder cycloadditions. In another preferred embodiment, 1,3-dipolar cycloadditions are used, more preferably alkyne-azide cycloadditions, and most preferably wherein Q is or comprises an alkyne group and F is an azido group. Cycloadditions (e.g., Diels-Alder reactions and 1,3-dipolar cycloadditions) are known in the art, and those skilled in the art will know how to perform them.

[0244] The method according to this aspect preferably involves a click reaction, more preferably a 1,3-dipolar cycloaddition, most preferably an alkyne / azide cycloaddition. Most preferably, Q is or comprises an alkyne group and F is an azido group. Click reactions (such as 1,3-dipolar cycloadditions) are known in the art, and those skilled in the art know how to perform them.

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

[0246] In a preferred embodiment, the method for preparing the 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) and a polypeptide having the formula S(F) x -P compound in the presence of a catalyst, wherein S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q (x is 1 or 2), and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) x The modified antibody is transferred to the core-GlcNAc portion to obtain a modified antibody according to formula (26): AB-[GlcNAc(Fuc) w -S{F} x ] y (26) in -AB is antibody; -Fuc is fucose; -w is 0 or 1; and (ii) reacting the modified antibody with a compound according to structure (4): QZ 2 -D (4) in: -Q is the reactive part; -L is a linker; -Z 2 is a linking group; -D is a compound according to general structure (1); to obtain the antibody-conjugate according to structure (2). Step (i)

[0247] In step (i), an antibody comprising 1, 2, 3 or 4 core N-acetylglucosamine moieties is combined with an antibody having the formula S(F) x -P compound in the presence of a catalyst, wherein S(F) x is a sugar derivative comprising x reactive groups F capable of reacting with reactive group Q (x is 1 or 2), and P is a nucleoside monophosphate or diphosphate, and wherein the catalyst is capable of converting S(F) xThe antibody is typically trimmed to core-GlcNAc residues, as further described below. Step (i) provides a modified antibody according to formula (26).

[0248] The starting material, i.e., an antibody comprising a core-GlcNAc substituent, is 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 deglycosylation of the antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, a suitable endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of: EndoS, EndoA, EndoE, EfEndo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and / or a combination thereof, the selection of which depends on the nature of the glycan. EndoSH is described in PCT / EP2017 / 052792, see Examples 1-3 and SEQ.ID No: 1, which is incorporated herein by reference.

[0249] The structural features S and x are as defined above for the conjugates according to the invention, which also apply to the present invention. x -P compound, wherein nucleoside monophosphate or nucleoside diphosphate P and sugar derivative S (F) x For example, Wang et al., Chem. Eur. J. [Chemistry - European Journal] 2010, 16, 13343-13345; Piller et al., ACS Chem. Biol. [ACS Chemical Biology] 2012, 7, 753; Piller et al., Bioorg. Med. Chem. Lett. [Biological Organic and Medicinal Chemistry Express] 2005, 15, 5459-5462 and WO 2009 / 102820 (all of which are incorporated herein by reference) disclose many compounds S (F) x -P and its 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)acetylgalactosamine), 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 amide)-2-deoxy-galactose-UDP. Most preferably, S(F) x -P is GalNAz-UDP or 6-AzGalNAc-UDP.

[0250] Able to convert S(F) x Suitable catalysts for transferring the moiety to the core-GlcNAc moiety are known in the art. Suitable catalysts are those that allow the specific sugar derivative nucleotide S(F) in this specific method to x -P is a catalyst for the catalysis of substrates. More particularly, the catalyst catalyzes the formation of β (1,4) -glycosidic bonds. Preferably, the catalyst is selected from the group of galactosyltransferases and N-acetylgalactosamine transferases, more preferably selected from the group of β (1,4) -N-acetylgalactosamine transferases (GalNAcT) and β (1,4) -galactosamine transferases (GalT), most preferably selected from the group of β (1,4) -N-acetylgalactosamine transferases with mutant catalytic domains. Suitable catalysts and mutants thereof are disclosed in WO 2014 / 065661, WO 2016 / 022027 and WO 2016 / 170186, which are incorporated herein by reference. In one embodiment, the catalyst is wild-type galactosyltransferase or N-acetylgalactosamine transferase, preferably N-acetylgalactosamine transferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or N-acetylgalactosamine transferase, preferably a mutant N-acetylgalactosamine transferase. The mutant enzymes described in WO 2016 / 022027 and WO 2016 / 170186 are particularly preferred. These galactosyltransferase (mutant) enzyme catalysts are able to recognize internal sugars and sugar derivatives as acceptors. Therefore, the sugar derivative S (F) x In step (i) attachment is made to a core-GlcNAc substituent, whether or not said GlcNAc is fucosylated.

[0251] 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 a phosphate-buffered saline (PBS) or a 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°C 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 8.5, 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. Step (ii)

[0252] In step (ii), the modified antibody is reacted with a compound according to general structure (4) comprising a reactive group Q capable of reacting with a reactive group F and a payload D to obtain a conjugate according to the invention comprising a linker group Z resulting from the reaction between Q and F. 1 Such a reaction occurs under conditions whereby reactive group Q reacts with reactive group F of the antibody to covalently link the antibody to the compound according to general structure (4). Step (ii) may also be referred to as a conjugation reaction.

[0253] In a preferred embodiment, in step (ii), the azide on the azide-modified antibody reacts with an alkynyl group, preferably a terminal alkynyl group, or a (hetero)cycloalkynyl group of a compound according to general structure (4) by a cycloaddition reaction. This cycloaddition reaction of a molecule comprising an azide and a molecule comprising a terminal alkynyl group or a (hetero)cycloalkynyl group is one of the reactions referred to in the art as "click chemistry". In the case of a linker-toxin construct comprising a terminal alkynyl group, the cycloaddition reaction needs to be carried out in the presence of a suitable catalyst (preferably a Cu(I) catalyst). However, in a preferred embodiment, the linker-toxin construct comprises a (hetero)cycloalkynyl group, more preferably a strained (hetero)cycloalkynyl group. When the (hetero)cycloalkynyl group is a strained (hetero)cycloalkynyl group, the presence of a catalyst is not required, and the reaction can even occur spontaneously by a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC). This is one of the reactions referred to in the art as "metal-free click chemistry". application

[0254] The toxins according to the invention having structure (1) are particularly suitable for preparing conjugates (such as the conjugates according to the invention), which in turn are particularly suitable for treating cancer. In addition, the compounds according to structure (1) are also suitable for killing cells. In view of this, the present invention also relates to the use of the compounds according to structure (1) for killing cells, and a method for killing cells, which method comprises contacting cells with a compound according to structure (1). The use and method are generally ex vivo or in vitro.

[0255] The conjugates of the present invention are particularly suitable for treating cancer. In view of this, the present invention further relates to a method for treating cancer, which method comprises administering a conjugate according to the present invention to a subject in need. The subject in need 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 for this aspect. The described methods are generally suitable for treating cancer. In the method according to this aspect, the antibody-conjugate is generally administered in a therapeutically effective dose. This aspect of the present invention can also be expressed as the conjugate according to the present invention is used for use in treating cancer. In other words, this aspect relates to the use of a conjugate according to the present invention in the preparation of a medicament or pharmaceutical composition for use in treating cancer. In this context, cancer treatment is considered to cover treating tumors, imaging tumors, diagnosing tumors, preventing tumor proliferation, preventing the spread of tumors (containing tumors) and reducing tumors.

[0256] This aspect of the invention can also be expressed as a method for targeting tumor cells that express specific extracellular receptors, the method comprising contacting a conjugate according to the invention with cells that may express extracellular receptors, and wherein the antibody specifically targets the extracellular receptor. Therefore, the method according to this aspect is suitable for determining whether a cell expresses the desired extracellular receptor. These tumor cells may be present in a subject, in which case the method comprises administering a conjugate according to the invention to a subject in need. Alternatively, the method occurs ex vivo or in vitro. In a preferred embodiment, the cell that may express the extracellular receptor is a cell that expresses the extracellular receptor. Targeting tumor cells preferably includes treating tumor cells, imaging tumor cells, diagnosing tumor cells, preventing tumor cell proliferation, preventing the spread of tumor cells (containing tumor cells) and reducing one or more of tumor cells.

[0257] In the context of diagnosis, it is often unknown whether the cells being contacted actually express the particular extracellular receptor being studied. 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 do express HER2, the conjugate will target these cells, while if the tumor cells do not express HER2, the conjugate will not target these cells. Similarly, when treating cancer cells that specifically express an extracellular receptor, one skilled in the art will understand that a cell-binding agent (such as an antibody) that targets that particular extracellular receptor will be used.

[0258] In the method of the present invention, it is preferred that the extracellular receptor 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, 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, guanylate 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, adhesion molecule-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tenascin. Similarly, it is preferred that the tumor cells express extracellular receptors selected from the same group. Those skilled in the art will be able to match the desired extracellular receptor with a suitable cell-binding agent capable of targeting the extracellular receptor.

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

[0260] The present inventors have unexpectedly discovered that the conjugates according to the present invention are superior in safety and / or efficacy to conventional conjugates containing toxins derived from anthracyclines, resulting in an improved therapeutic index for the antibody-conjugates according to the present invention relative to conventional anthracycline-containing conjugates. In view of the reduced toxicity of the compounds according to 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, which in turn has additional benefits in treatment. Conventional conjugates of anthracyclines with cell-binding agents such as antibodies need to be administered at very low doses, making it not uncommon to administer excessively high doses. This can lead to non-specific cell death, resulting in adverse side effects of cancer treatment. In addition, the administration of anthracycline-antibody conjugates at conventional low doses can negatively affect biodistribution, resulting in reduced tumor targeting efficiency. Therefore, the present inventors have discovered anthracycline-based toxins, i.e., compounds according to structure (1), which have reduced toxicity, resulting in an improved therapeutic index, particularly safety or tolerability, for their conjugates. Compared to conventional conjugates, the improvement in therapeutic efficacy of the conjugates according to the present invention can be manifested as a reduction in tumor size and / or a prolongation of the regression period. Compared to the administration of conventional conjugates, the improvement in tolerability of the conjugates according to the present invention can be manifested as a reduction in toxicity signs. The reduction in signs can also be referred to as a reduction in symptoms or side effects of cancer treatment and can involve one or more clinical signs (such as weight loss, decreased activity, decreased food intake) and / or one or more toxicity parameters (such as improvements in blood chemistry, hematology and / or histopathology).

[0261] In another aspect, the present invention relates to a method for regulating, ameliorating or reducing the toxicity of anthracycline-based toxins, the method comprising introducing a substituent R as defined above 1 This aspect of the invention can also be expressed as the substituent R 1 Use for regulating, improving or reducing the toxicity of anthracycline-based toxins, wherein the substituent R 1 As defined above. In this context, conjugation to the compound according to structure (1) is typically performed via Y as defined above. BRIEF DESCRIPTION OF THE DRAWINGS

[0262] Figure 1 A general scheme for preparing antibody-drug conjugates by reacting a monoclonal antibody (mostly a symmetric dimer) containing x functional groups F is shown. xIncubated with an excess of the linker-drug construct (Q-spacer-linker-payload), the conjugate is obtained by the reaction of F with Q to form the linker group Z.

[0263] Figure 2 depicts a range of reagents suitable for reacting with cysteine ​​side chains. The reagents can be of the monoalkylating type (A) or can be cross-linking reagents that react with two cysteine ​​side chains (B).

[0264] Figure 3 A general method for the non-genetic conversion of monoclonal antibodies (mAbs) into antibodies containing probes (F) for click conjugation is shown. The click probes can be located at different positions on the antibody, depending on the technology used. For example, an antibody can be converted to contain two click probes (left structure), four click probes (bottom structure), or eight probes (right structure) for click conjugation.

[0265] Figure 4 A representative (but not comprehensive) set of functional groups (F) that can be introduced into antibodies by engineering, chemical modification, or enzymatic means to generate linker groups Z after metal-free click reaction with complementary reactive groups Q. Functional groups F can be artificially introduced (engineered) at any position of choice on the antibody. Some functional groups F (e.g., nitrile oxides, quinones) can react with strained alkenes in addition to strained alkynes, such as those depicted by triazines or tetrazines (bottom row). Pyridine or pyridazine linkers are products of rearrangement of tetraazabicyclo[2.2.2]octane linkers, which are formed by the reaction of triazines or tetrazines, respectively, with alkynes (but not alkenes) and the loss of N2. Figure 4 The depicted linking group Z is a preferred linking group for use in the present invention.

[0266] Figure 5 Cyclic alkynes suitable for metal-free click chemistry are shown, along with preferred examples of the reactive moiety Q. This list is not comprehensive, as alkynes can be further activated by, for example, fluorination, substitution of the aromatic ring, or introduction of heteroatoms into the aromatic ring.

[0267] Figure 6Depicted is a specific example of the specific conjugation of the payload site based on the polysaccharide remodeling of full-length IgG and subsequent azide-cyclooctyne click chemistry. First, all different glycoforms are trimmed by endoglycosidase-mediated pruning, followed by glycosyltransferase-mediated transfer of the azide sugar to the core GlcNAc released by the endoglycosidase, and IgG is enzymatically remodeled. In the next step, the IgG remodeled by the azide group is placed in (subjected to) an immune cell-engaging polypeptide, which has been modified with a single cyclooctyne to perform metal-free click chemistry (SPAAC), thereby producing a bispecific antibody in the form of a 2:2 molecule. The figure also depicts that the cyclooctyne-polypeptide construct will have a specific spacer between the cyclooctyne and the polypeptide, which makes it possible to adjust the IgG-polypeptide distance or to impart other characteristics to the resulting bispecific antibody.

[0268] Figure 7 Depicted is a specific example of full-length IgG glycan remodeling and subsequent site-specific conjugation of payloads using thiol alkylation chemistry. IgG is enzymatically remodeled by first endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of thiol-modified (and disulfide-protected) sugar derivatives to the core GlcNAc released by the endoglycosidase. In the next step, the remodeled IgG is reduced (converting disulfides to thiols), possibly followed by oxidation, and then reacted with a payload modified with an appropriate thiol-reactive reagent.

[0269] Figure 8 The structures of daunorubicin, doxorubicin, nemorubicin (MMDX), PNU-159,696, and PNU-159,682 are depicted.

[0270] Figure 9 Two linker-modified PNU-159,682 derivatives are depicted, one based on carbamylation of the hydroxyketone group with a linker containing N,N'-dimethylethylenediamine (DMEDA) and maleimide for antibody conjugation to cysteine, and the other based on oxidative amide coupling of the hydroxyketone with a linker containing ethylenediamine (EDA) and glycine-glycine for antibody conjugation in the presence of sortase.

[0271] Figure 10 Shown are ADCs obtained by sortase-mediated conjugation of Gly-Gly-EDA-modified oxidized PNU-159,682.

[0272] Figure 11Shown are the structures of linker-drugs based on PNU-159,682 analogs according to the present invention, which can be conjugated to antibodies via a reactive moiety, Z, to generate the corresponding ADCs. The first category consists of PNU analogs modified with substituents on the morpholino ring that differ from the methyl group present in PNU-159,682, and the original hydroxyacetone moiety is oxidized to a carboxylic acid, enabling activation / attachment of the linker. The second category consists of PNU analogs that retain the original (hydroxy)acetone moiety of doxorubicin / daunorubicin and are modified with a linker in place of the original methyl group present on the morpholino group of PNU-159,682. In either case, the linker is further modified with a reactive group, Z, which can be any functional group capable of attaching to an antibody, such as a maleimide, an activated carbonyl, a halide, a cycloalkyne, an azide, and the like.

[0273] Figure 12 Shown is a synthetic scheme based on initial TBS protection of the hydroxyacetone functionality of doxorubicin to generate PNU-159,682 analogs 6b-6f modified at the morpholino ring.

[0274] Figure 13 It is shown how N-alkylation of the amino sugar of doxorubicin can be achieved for various constructs 8b-8f without prior O-silylation of the doxorubicin. This route is also applicable to daunorubicin.

[0275] FIG14A shows the structure of compound 9a based on the Val-Cit dipeptide and a DMEDA linker.

[0276] Figure 14B shows the final step in the preparation of compounds 9c, 9d, 9f, and 9g using the Val-Ala dipeptide and the EDA linker.

[0277] Figure 15 Shown are the structures of compounds 36 and 39 with a Val-Ala dipeptide conjugated via an anthracycline morpholino group.

[0278] Figure 16 The structures of compounds 47 and 53 based on the EDA linker and Gly-Gly-Phe-Gly or Gly-Gly-Gly peptides are shown, respectively.

[0279] Figure 17 Figure 3 shows the in vitro cytotoxicity of trast-9g, trast-9d, trast-9c, and trast-36 against four cell lines with different HER2 expression levels. The T0 line indicates the number of viable cells at the start of the assay.

[0280] Figure 18AShown are the time-dependent mean body weights of CD-1 mice that received a single bolus injection of vehicle (PBS), ADC trastuzumab-9d (20 mg / kg), ADC trastuzumab-36 (20 mg / kg), or the reference ADC trastuzumab-9g (5 mg / kg).

[0281] Figure 18B Shown are the time-dependent body weights of CD-1 mice administered a single bolus of vehicle (PBS), ADC trastuzumab-47 (15 mg / kg), ADC trastuzumab-9c (40 mg / kg), or the reference ADC trastuzumab-9g (5 mg / kg).

[0282] Figure 19A Shown are tumor volumes over time in NOD / SCID mice transplanted with the JIMT-1 tumor cell line and subsequently treated with low (0.3 mg / kg) or high (1 mg / kg) doses of the reference ADC trastuzumab-9g.

[0283] Figure 19B Shown are tumor volumes over time in NOD / SCID mice transplanted 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.

[0284] Figure 19C Shown are tumor volumes over time in NOD / SCID mice transplanted with the JIMT-1 tumor cell line and subsequently treated with low (0.6 mg / kg) or high (2 mg / kg) doses of the ADC trastuzumab-47 or with a high (2 mg / kg) dose of the ADC trastuzumab-9c.

[0285] Figure 20 The in vitro cytotoxicity of compounds 6a, 6b, 6c, 6d, and 6e against four cell lines with different HER2 expression levels is shown. The T0 line indicates the number of viable cells at the start of the assay.

[0286] Figure 21 The in vitro cytotoxicity of trast-9g, trast-63a, trast-63b, and trast-75 against three cell lines with different HER2 expression levels is shown. The T0 line indicates the number of viable cells at the start of the assay. Examples

[0287] The present invention is illustrated by the following examples. 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 12.5 mM DTT, 100 mM TrisHCl (pH 8.0) (40 μL) and incubated at 37°C for 15 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, 2% formic acid (50 μL). RP-UPLC analysis was performed on an H-class Acquity UPLC system (Waters). Samples (5 μL) were injected into the BioResolve HPLC system at 0.4 mL / min. TM RP mAb polyphenylene column ( 2.7 μm, 2.1 x 150 mm, Waters Corporation) with a column temperature of 70° C. A linear gradient of 0.1% TFA and acetonitrile in water from 30% to 55% was applied over 9 minutes. Common Procedures for Analytical SEC

[0289] SE-HPLC analysis was performed on an Agilent 1100 series (Hewlett Packard) using an XbridgeBEH200A column (3.5 μM, 7.8×300 mm, PN 186007640, Waters Corporation). The sample was diluted to 1 mg / mL in PBS and measured at 0.86 mL / min isocratic flow (0.1 M sodium phosphate buffer (pH 6.9) (NaHPO / NaPO) containing 10% isopropanol) for 16 minutes. General Procedure for Mass Spectrometry Analysis of Monoclonal Antibodies and ADCs

[0290] Before mass spectrometry analysis, IgG was purified by IdeS (Fabricator TM ) for analysis of Fc / 2 fragments. 20 μg (modified) IgG solution was incubated with 0.5 μL IdeS (50 U / μL) in a total volume of 10 μL phosphate buffered saline (PBS) (pH 6.6) at 37°C for 1 hour. The sample was diluted to 40 μL and then analyzed on a JEOL AccuTOF LC-plus JMS-T100LP system (ESI-TOF) in conjunction with an HPLC system (Agilent 1100 series, Hewlett-Packard). The HPLC system was equipped with MassPREP TM Online desalting cartridge (Waters P / N 186002785). General Procedure for LC-MS Analysis of Monoclonal Antibodies and ADCs

[0291] To analyze Fc / 2 fragments, IgG was isolated using IdeS (FabricatorTM ) treatment. 10 μg (modified) IgG solution was incubated with 0.5 μL IdeS (50 U / μL) in a total volume of 10 μL phosphate buffered saline (PBS) (pH 7.4) at 37 ° C for 1 hour, and then diluted to 100 μL using MQ. In order to analyze the reduced sample, IgG was treated with DTT. 10 μg (modified) IgG solution was incubated with DTT (10 mM) in a final volume of 50 μL PBS (pH 7.4) at 37 ° C for 15 minutes, and then 50 μL quenching buffer (49% MQ, 49% acetonitrile, 2% FA) was added. The sample was analyzed on a Xevo G2-XS QTof quadrupole time-of-flight mass spectrometry system (ESI-QTOF) together with a UPLC system (Aquity series, Waters Corporation). The UPLC system was equipped with bioZen TM 3.6 μm Intact XB-C8, LC column 50 x 2.1 mm (Cat. No.: 00B-4766-AN). Synthesis of diiodine compound 7b 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). Ac O (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 layer was washed with saturated aqueous NaHCO (400 mL) and dried over Na SO. The mixture was concentrated and recrystallized using Et O / heptane to obtain 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. 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 equivalents) were added and the mixture was cooled to 0 ° C. After adding Ag2CO3 (1 equivalent), 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 Et2O and the solvent was evaporated. The crude product was purified using flash column chromatography (0 → 25% EtOAc in heptane). The fraction containing the product was concentrated and dissolved in DMF (0.2 M), followed by the addition of NaN3 (4 equivalents). The mixture was stirred at 80 ° C for 1 h and concentrated, after which it was redissolved in MeOH (0.1 M) and NaOMe (5.4 M in MeOH, 0.1 equivalent) was added. The reaction was stirred overnight, concentrated, and purified using flash column chromatography (0→10% MeOH in EtOAc) to give the 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. 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 H2O (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO4 (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed that the starting material was completely consumed. The mixture was cooled to 0 ° C., and then NaBH4 was added in batches. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of 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 Na2SO4 and concentrated to obtain product 12 (346 mg, 74.8%). 1H-NMR (400MHz, CDCl3) δ (ppm) 4.72 (t, J = 5.3 Hz, 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. Example a4. 1,5-bis(p-toluenesulfonyl)oxy-2(S)-(2-azidoethoxy)-3-oxa-pentane (13) Synthesis

[0295] Diol 12 (346 mg, 1.81 mmol) was dissolved in dry pyridine (0.1 M), cooled to 0° C., and p-TsCl (2.5 equiv) was added. 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 Na2SO4, concentrated, and purified using flash column chromatography (0→50% EtOAc in heptane) to obtain product 13 (368 mg, 40.7%). 1 H-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, 2 H), 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. Example a5. Synthesis of 1,5-diiodo-2(S)-(2-azidoethoxy)-3-oxa-pentane (7b)

[0296] Bis-toluenesulfonate 13 (491 mg, 0.98 mmol) was dissolved in 2-butanone (0.05 M). NaI (7 equivalents) 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 H2O (20 mL) and brine (20 mL), dried over Na2SO4 and concentrated. The crude product was purified using flash column chromatography (0 → 5% EtOAc in heptane) to obtain 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. Example a6. Synthesis of 1-isopropyl-α-d-arabinopyranoside (15)

[0297] Arabinosyl bromide 10 (3.51 g, 10.3 mmol) was dissolved in dry Et2O (0.25 M). iPrOH (15 equiv.) was added, followed by Ag2O (1 equiv.). The mixture was stirred in the dark for 3 h. The reaction was filtered through celite, the celite pad was washed with Et2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by addition of NaOMe (5.4 M in MeOH, 0.1 equiv.) and stirred at room temperature overnight. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to obtain 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. 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 H2O (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO4 (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed that the starting material was completely consumed. The mixture was cooled to 0 ° C., and then NaBH4 was added in batches. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of 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 Na2SO4 and concentrated to obtain 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. 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.), PPh (3 equiv.) were added, followed by I (3 equiv.). 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 (20 mL), brine (20 mL) and dried over Na SO. The mixture was concentrated and purified by column chromatography (0 → 5% EtOAc in heptane) to obtain product 7c (220 mg, 38.5%). 1 H-NMR (400MHz, CDCl3) δ (ppm) 4.76 (t, J = 5.5 Hz, 1H), 3.94 (hept, J = 6.2 Hz, 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. Example a9. Synthesis of 1-benzyl-α-d-arabinopyranoside (18)

[0300] Arabinosyl bromide 10 (2.06 g, 6.09 mmol) was dissolved in dry Et2O (0.25 M). BnOH (15 equiv.) was added, followed by Ag2O (1 equiv.). The mixture was stirred in the dark for 3 h. The reaction was filtered through celite, the celite pad was washed with Et2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by addition of NaOMe (5.4 M in MeOH, 0.1 equiv.) and stirred at room temperature overnight. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to obtain 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. 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 H2O (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO4 (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed that the starting material was completely consumed. The mixture was cooled to 0 ° C., and then NaBH4 was added in batches. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of 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 Na2SO4 and concentrated to obtain 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. 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 equivalents), PPh (3 equivalents) were added, followed by I (3 equivalents). 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 (20 mL), brine (20 mL) and dried over Na SO. The mixture was concentrated and purified by column chromatography (0 → 5% EtOAc in heptane) to obtain 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. Example a12. Synthesis of Compound 21

[0303] Prepare the ice-cold solution of sodium nitrite (842.3mg, 12.21mmol) in water (20mL) and transfer it to the dropping funnel.This mixture was added in the cold solution of 4-aminobenzyl alcohol (1g, 8.12mmol) in HCl (5M, 5mL) in 30min.Reaction mixture becomes faint yellow from bright yellow, finally becomes off-white.After 30min, add sodium azide (2.1g, 32mmol) and stir the mixture in 5 batches.After one hour, remove ice bath and observe solid.After 1.5h, add saturated NaHCO the aqueous solution (25mL), add EtOAc (25mL) subsequently.Reaction is transferred to separating funnel and organic layer is separated with water layer.With saturated NaHCO for organic layer the aqueous solution (20mL) washing, with salt water (25mL) washing and through Na sO dry.Filter desiccant and concentrate yellow filtrate by glass filter. The crude yellow oil was purified by flash column chromatography on silica gel (5%→80% EtOAc in heptane, pre-conditioned the column with 5% EtOAc in heptane) to give the product 21 (89%) (1.08 g, 7.24 mmol). 1 H-NMR (400MHz, CDCl3) δ (ppm) 7.36 (d, J = 8.6 Hz, 2H), 7.08-6.97 (m, 2H), 4.67 (d, J = 4.3 Hz, 2H), 1.68 (t, J = 5.2 Hz, 1H). Example a13. Synthesis of Compound 23

[0304] To a solution of arabinose bromide 10 (1.34 mg, 3.95 mmol) and compound 21 (872 mg, 5.85 mmol) in diethyl ether (anhydrous, 20 mL), silver oxide (I) (916 mg, 3.95 mmol) is added and the reaction is stirred at room temperature in the dark. After stirring for 10 days, the reaction mixture is filtered through pre-wetted celite and fully washed (washthrough) with diethyl ether and concentrated. The crude oil is dissolved in MeOH (15 mL) and sodium methoxide (134.4 mg, 2.48 mmol) is added. After stirring at room temperature for 3.5 h, the reaction mixture is neutralized and concentrated with a few drops of 1M HCl aqueous solution. Excess 21 is removed by precipitating the desired compound in diethyl ether and filtering through a glass filter covered with filter paper. Compound 23 (yield 65%) (714.4 mg, 2.54 mmol) is obtained as a pale solid. 1H-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). Example a14. Synthesis of Compound 24

[0305] Compound 23 (714.4 mg, 2.54 mmol, 1.0 equivalent) 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 equivalents) in water (3 mL) was immediately added, followed by addition of sodium periodate (1.35 g, 6.35 mmol, 2.5 equivalents) in portions. 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 h. After stirring for 4.5 h, the reaction mixture was cooled to -10 ° C again and sodium borohydride (288.3 mg, 7.62 mmol, 3.0 equivalents) was added in portions. After stirring for one 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 x 50 mL). The organic layers were combined and dried over Na2SO4, filtered through a glass filter with pre-wet celite and concentrated to obtain compound 24 (yield 87%) (637.2 mg, 2.2 mmol). 1 H-NMR (400MHz, MeOD) δ (ppm) 7.31 (d, J = 8.5 Hz, 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). 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 nitrogen flow). Pyridine (537 μ L, 6.64 mmol, 3.0 equivalents), methanesulfonic anhydride (964.2 mg, 5.53 mmol, 2.5 equivalents) and DMAP (27.0 mg, 221.4 mmol, 0.1 equivalent) were added. After stirring for 3.5 h, the reaction mixture was washed with saturated NaHCO aqueous solution (11 mL). The aqueous layer was extracted twice with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The crude orange oil was purified by silica gel flash column chromatography (10% → 80% EtOAc in heptane, the post was pretreated with 10% EtOAc (in heptane)) to give compound 25 (61%) (571.6 mg, 1.4 mmol) as a clear light yellow oil. 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). Example a16. Synthesis of compound 7e

[0307] A solution of compound 25 (571.2 mg, 1.4 mmol, 1.0 equivalent) and sodium iodide (1.42 g, 9.48 mmol, 7 equivalents) in 2-butanone (15 mL) was refluxed in the dark. After reflux 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 silica gel flash column chromatography (0% → 7% EtOAc in heptane) to give compound 7e (47.5%) (313.2 mg, 0.64 mmol) as an opaque light yellow oil. 1 H-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). Example a17. Synthesis of 1-ethyl-α-d-arabinopyranoside (27)

[0308] Arabinosyl bromide 10 (5.31 g, 15.66 mmol) was dissolved in dry Et2O (0.25 M). EtOH (15 equivalents) was added, followed by Ag2O (1 equivalent). The mixture was stirred in the dark for 3 h. The reaction was filtered through celite, the celite pad was washed with Et2O and the ether was removed by rotary evaporation. The crude mixture was dissolved in MeOH (0.1 M), followed by NaOMe (5.4 M in MeOH, 0.1 equivalent) and stirred at room temperature overnight. The product was purified using flash column chromatography (0 → 10% MeOH in EtOAc) to obtain 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. 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 H2O (0.25 M). NaOAc (1.3 equiv.) was added, followed by NaIO4 (2.5 equiv.). After stirring in the dark for 1 h, TLC (10% MeOH in EtOAc) showed that the starting material was completely consumed. The mixture was cooled to 0 ° C., and then NaBH4 was added in batches. After 1 h, TLC (10% MeOH in EtOAc) showed the formation of 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 Na2SO4 and concentrated to obtain 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). 13C-NMR (101MHz, CDCl3) δ (ppm) 102.65, 68.44, 63.66, 62.61, 61.83, 15.29. 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 equivalents), PPh (3 equivalents) were added, followed by I (3 equivalents). 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 Na SO. The mixture was concentrated and purified by column chromatography (0 → 5% EtOAc in heptane) to obtain product 7f (170 mg, 18.1%). 1 H-NMR (400MHz, CDCl3) δ (ppm) 4.72 (t, J = 5.5 Hz, 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). 13 C-NMR (101MHz, CDCl3) δ (ppm) 101.76, 66.90, 62.52, 15.10, 4.78, 2.48. A general approach from doxorubicin to PNU-159,682 analogs

[0311] Figure 12 and Figure 13 The general scheme is depicted in. Similar analogs can be prepared from daunorubicin by omitting the silylation and desilylation steps. 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 a few minutes, TBDMS-Cl (1.90 g, 12.65 mmol) was added. The reaction mixture was stirred at 0 ° C for 5 min, then allowed to warm to room temperature. After stirring at room temperature for 3.5 h, the reaction mixture was purified by silica gel flash column chromatography (the column was pretreated with 1% MeOH / DCM, 1% → 30% MeOH in DCM) to give compound 2 (3.59 g, 5.4 mmol, 100%) in a dark red thick oil. For C 33 H 44 NO 11 Si + (M+H +), LCMS (ESI+) calculated value: 658.27, found value: 658.44. Example a21. Synthesis of compound 3b

[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 at 42° C. and stirred for 25 minutes, after which the heating device was removed and the reaction mixture was left to react at room temperature. After 72 hours at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by silica gel flash column chromatography (0%→3% MeOH in DCM) to give compound 3b (303 mg, 367 μmol, 43.9%) as a dark red oil. For C 39 H 53 N4O 13 Si + (M+H + ), LCMS (ESI+) calculated value: 813.34, found value: 813.51. Example a22. Synthesis of Compound 4b

[0314] A solution of compound 3b (303 mg, 253 μmol, 68 wt%) in anhydrous DCM (31 mL) was cooled to a temperature of -78°C using a dry ice / acetone bath. The mixture was stirred vigorously, followed by the addition of a freshly prepared stock solution of mCPBA in anhydrous DCM (70 mg, 580 mM, 699 μL, 406 μmol). After stirring for 7 minutes, complete conversion was achieved. The RM was quenched with an ice-cold acetone solution (reagent grade, 3.33 mL) and stirred. After 20 min, the cold bath was removed and the RM was allowed to warm to room temperature. The RM was transferred to a separatory funnel and washed twice with saturated NaHCO3 aqueous solution (6 mL). The aqueous layers were combined and extracted once with DCM (8 mL). The combined organic layers were dried over Na2SO4, filtered through filter paper, and concentrated until a volume of 30 mL was obtained. Compound 4b was used in the next step as is without further purification. For C 39 H 53 N4O 14 Si + (M+H + ), LCMS (ESI+) calculated value: 829.33, found value: 829.57. Example a23. Synthesis of Compound 5b

[0315] To a solution of compound 4b (188.7 mg, 227.7 μmol) in DCM (27 mL) was added anhydrous acetonitrile (25 mL). The RM was partially concentrated to remove DCM and obtain the compound in anhydrous acetonitrile (25 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (5 mL). Potassium carbonate (198 mg, 1.43 mmol) was then added, and the RM was cooled to 0 ° C., followed by addition of cyanuric chloride (247.7 mg, 12.1 mL, 111 mM, 1.35 mmol) as a stock solution in anhydrous acetonitrile. After stirring at 0 ° C. for 4 h, the RM was quenched with a solution of 3-aminopropane-1,2 diol in water (489.6 mg, 2.9 mL, 1.85 M, 5.37 mmol). The ice bath was removed after 30 min and then allowed to warm to room temperature. DMF (1.5 mL) was added to the RM and the RM was concentrated until only a DMF / water solution (8 mL) remained, which was purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). The collected fractions were combined and concentrated until 6 mL of acetonitrile remained. They were then dried over Na2SO4, filtered, and the residue was washed thoroughly with anhydrous THF (3 x 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. For C 39 H 51 N4O 13 Si + (M+H + ), LCMS (ESI+) calculated value: 811.32, found value: 811.51. Example a24. Synthesis of compound 6b

[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 RM was cooled to -15 ° C. Then, while the RM was vigorously stirred, TBAF (1M in THF, 1.03 g, 4 mL, 4 mmol) was added in batches (observing that the color changed from red to green and then back to dark red). After stirring for 2.5 h, the RM was quenched with water (15 mL) and the solution was changed from dark red to light red. The RM was stirred on ice for 1 min and then placed at room temperature for another 90 min. The RM was transferred to a separating funnel and extracted with DCM (18 mL). The water layer was extracted with another DCM (2 x 6 mL). The combined organic layer was dried over Na SO , filtered, and further diluted to 55 mL with DCM and purified by flash column chromatography on silica gel (0% → 10% MeOH in DCM). Additional preparative HPLC purification was required (30% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). The collected fractions were combined and partially concentrated to give compound 6b (0.22 mM (according to the HPLC calibration line based on doxorubicin), 45 mL, 9.82 μmol, 4.3% yield over 3 steps) as a solution in 45 mL acetonitrile / water. For C 33 H 37 N4O 13 Si + (M+H + ), LCMS (ESI+) calculated value: 697.24, found value: 697.45. Example a25. Synthesis of compound 3c

[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 placed at room temperature for reaction. After 72 ° C at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by silica gel flash column chromatography (0% → 3% MeOH in DCM) to give compound 3c (241.8 mg, 307.6 μmol, 31.1%) as a dark red oil. For C 40 H 56 NO 13 Si+ (M+H + ), LCMS (ESI+) calculated value: 786.96, found value: 786.64. Example a26. Synthesis of compound 4c

[0318] A solution of 3c (125 mg, 159 μmol) in anhydrous DCM (15 mL) was cooled to a temperature of -78 ° C with a dry ice / acetone cold bath. The mixture was vigorously stirred and then a freshly prepared 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 minutes, complete conversion was achieved. The RM was quenched with an ice-cold acetone solution (reagent grade, 1.46 mL) and stirred. After one hour, the cold bath was removed and the RM was allowed to warm to room temperature. The RM was transferred to a separatory funnel and washed twice with saturated NaHCO3 aqueous solution (12 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered through filter paper, and concentrated until a volume of 12 mL was obtained to give 4c as a red solution. Compound 4c can be used as is without further purification. For C 40 H 56 NO 14 Si + (M+H + ), LCMS (ESI+) calculated value: 802.96, found value: 802.63. Example a27. Synthesis of Compound 5c

[0319] To a solution of 4c (128 mg, 159 μmol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove DCM and obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (20 mL). Potassium carbonate (86 mg, 622 μmol) was then added, and the RM was cooled to -7 ° C, followed by addition of cyanuric chloride (73.6 mg, 3.59 mL, 111 mM, 399 μmol), which was a stock solution in anhydrous acetonitrile. After stirring at -7 ° C for 2 h, the RM was quenched with a solution of 3-aminopropane-1,2 diol in water (179 mg, 854 μL, 2.3 moles, 1.96 mmol). The ice bath was removed after 15 min and then allowed to warm to room temperature. DMF (1 mL) was added to the RM and the RM was concentrated until only a DMF / water solution remained, which was purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30 x 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 thoroughly washed with anhydrous THF (3 x 750 μL). It was concentrated again to a volume of 3 mL and compound 5c was used as is. For C 40 H 54 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 784.94, found value: 786.57. Example a28. Synthesis of compound 6c

[0320] In the compound 5c (121mg, 154μmol) in the mixture of acetonitrile and THF (2.88mL), additional dry THF (2.0mL) is added, and the resulting red solution is cooled to-40°C. Then, while RM is vigorously stirred, TBAF (1M in THF, 484.5mg, 1.85mL, 1.85mmol) is added (observing that color changes from red to green). After stirring for 4h, RM is quenched with water (3.0mL), and the solution changes from green to red. RM is transferred to a separating funnel and extracted with DCM (3x 10mL). The organic layer combined is dried over NaSO, filtered, and passed through silica gel flash column chromatography (0% → 5% MeOH, in DCM), subsequently by preparative HPLC (40% → 95% acetonitrile, in 10mM NHHCO aqueous solution, column: Xbridge prep C18 , 5 μM OBD, 30x100 mm). Compound 6c was obtained as a red solution in DCM (70 mL, 0.086 mM (according to the HPLC calibration line based on doxorubicin), 6.0 μmol, 3.9% yield over 3 steps). 34 H 40 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 670.25, found value: 670.51. Example a29. Synthesis of compound 3d

[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 ° C at room temperature, the reaction mixture was diluted with DCM (12 mL) and purified by silica gel flash column chromatography (0% → 3% MeOH in DCM) to give compound 3d (413.5 mg, 479 μmol, 48.5%) as a dark red oil. For C 44 H 56 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 834.35, found value: 834.50. Example a30. Synthesis of compound 4d

[0322] A solution of compound 3d (125 mg, 159 μmol) in anhydrous DCM (15 mL) was cooled to a temperature of -78°C with a dry ice / acetone bath. The mixture was vigorously stirred and then a freshly prepared 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 RM was quenched with an ice-cold acetone solution (reagent grade, 1.38 mL) and stirred. After 12 min, the cold bath was removed and the RM was allowed to warm to room temperature. The RM was transferred to a separatory funnel and washed twice with a saturated NaHCO3 aqueous solution (12 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered through filter paper, and concentrated until a volume of 12 mL was obtained. Compound 4d was used as is without further purification. For C 44 H 56 NO 14 Si + (M+H + ), LCMS (ESI+) calculated value: 850.35, found value: 850.60. Example a31. Synthesis of compound 5d

[0323] To a solution of compound 4d (127 mg, 134 μmol) in DCM (12 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove DCM and obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (20 mL). Potassium carbonate (73 mg, 530 μmol) was then added, and the RM was cooled to -7 ° C, followed by addition of a solution of cyanuric chloride in anhydrous acetonitrile (62.0 mg, 3.01 mL, 111.8 mM, 336 μmol). After stirring at -7 ° C for 2 h, the RM was quenched with a solution of 3-aminopropane-1,2 diol in water (151 mg, 1.85 mL, 894 mM, 1.65 mmol). The ice bath was removed after 15 min and then allowed to warm to room temperature. DMF (2 mL) was added to the RM and the RM was concentrated until only a DMF / water solution remained and the product was purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18, 5 μM OBD, 30x100 mm). The collected fractions were combined and concentrated until a volume of 4 mL remained. The resulting solution, consisting mainly of acetonitrile, was dried over Na2SO4, filtered, and the drying agent was thoroughly washed with anhydrous THF (3 x 750 μL). The combined organic layer was partially concentrated again to a volume of 4 mL (acetonitrile / THF) and compound 5d was used as is. For C 44 H 54 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 832.34, found value: 832.53. Example a32. Synthesis of Compound 6d

[0324] To the compound 5d (25 mg, 30 μ mol) in the mixture of acetonitrile and THF (4 mL), triethylammonium acetate (97 mg, 96 μ L, 600 μ mol) was added, and the resulting red solution was cooled to -20 ° C. Then, while RM was vigorously stirred, TBAF (1 M in THF, 35 mg, 340 μ L, 340 mmol) was added (observing that the color changes from red to green and then back to red). After stirring for 3 h, RM was quenched with water (2.4 mL). RM was transferred to a separating funnel and extracted with DCM (3 x 5 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated until approximately 10 mL remained (water bath rotary evaporator set at 32°C), and purified by silica gel flash column chromatography (0%→10% MeOH in DCM) to give compound 6d as a red solution in DCM (4 mL, 0.82 mM (according to HPLC calibration line based on doxorubicin), 2.36 mg, 3.29 μmol, 11%). For C 38 H 40 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 718.25, found value: 718.50. Example a33. Synthesis of compound 3e

[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 silica gel flash column chromatography (0% → 3% MeOH in DCM) to give compound 3e (53.9 mg, 61.6 μmol, 24.6%) as a dark red oil. For C 44 H 55 N4O 13 Si + (M+H + ), LCMS (ESI+) calculated value: 875.35, found value: 875.61. Example a34. Synthesis of compound 4e

[0326] A solution of compound 3e (53.9 mg, 61.6 μmol) in anhydrous DCM (10 mL) was cooled to a temperature of -78°C with a dry ice / acetone cold bath. The mixture was vigorously stirred and then a freshly prepared stock solution of mCPBA in anhydrous DCM (20.2 mg, 580 mM, 202 μL, 117 μmol) was added dropwise. After stirring at -78°C for 8 minutes, complete conversion was achieved. The RM was quenched with an ice-cold acetone solution (reagent grade, 565 μL) and stirred at -78°C. After 45 min, the cold bath was removed and the RM was allowed to warm to room temperature. The RM was transferred to a separatory funnel and washed twice with saturated NaHCO3 aqueous solution (10 mL). The aqueous layers were combined and extracted once with DCM (10 mL). The combined organic layers were dried over Na2SO4, filtered through filter paper, and concentrated until a volume of 10 mL was obtained. Compound 4e was used as is without further purification. For C 44 H 55 N4O 14 Si + (M+H + ), LCMS (ESI+) calculated value: 891.35, found value: 891.52. Example a35. Synthesis of Compound 5e

[0327] To a solution of compound 4e (54.9 mg, 61.6 μmol) in DCM (10 mL) was added anhydrous acetonitrile (6 mL). The RM was partially concentrated to remove DCM and obtain the compound in anhydrous acetonitrile (3 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (10 mL). Potassium carbonate (33.2 mg, 240 μmol) was then added, and the RM was cooled to -7 ° C, followed by 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 RM was quenched with a solution of 3-aminopropane-1,2 diol in water (69 mg, 330 μL, 2.3 M, 758 μmol). The ice bath was removed after 15 min and then allowed to warm to room temperature. DMF (1 mL) was added to the RM and the RM was concentrated until only a DMF / water solution remained and the product was purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). The collected fractions were combined and concentrated until a volume of 4 mL of acetonitrile remained. They were then dried over Na2SO4, filtered, and the residue was washed thoroughly with anhydrous THF (3 x 750 μL). The combined organic layer was partially concentrated again to a volume of 4 mL (acetonitrile / THF) and compound 5e was used as is. For C 44 H 53 N4O 13 Si + (M+H + ), LCMS (ESI+) calculated value: 873.34, found value: 873.46. Example a36. Synthesis of compound 6e

[0328] Compound 5e (27 mg, 31 μ mol) in a mixture of acetonitrile and THF (4 mL) was cooled to -40 ° C. Then, while RM was vigorously stirred, TBAF (1 M in THF, 80 mg, 300 μ L, 300 mmol) was added (observing that color changes from red to green). After stirring for 2.5 h, RM was quenched with water (2.5 mL), and the solution became red from green. RM was transferred to a separating funnel and extracted with DCM (3 x 7 mL). The combined organic layer was dried over Na SO , filtered, and purified by silica gel flash column chromatography (0% → 10% MeOH in DCM), to provide compound 6e, a red solution (10 mL, 0.31 mM (according to the HPLC calibration line based on doxorubicin), 2.3 mg, 3.0 μ mol, 9.8% in 3 steps) in DCM. For C38 H 39 N4O 13 + (M+H + ), LCMS (ESI+) calculated value: 759.25, found value: 759.40. Example a37. Synthesis of Compound 3f

[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 and swirled for 1 minute, then placed at room temperature in the dark. After 96 h at room temperature, the reaction mixture was diluted with DCM (4 mL) and purified by silica gel flash column chromatography (0% → 3% MeOH in DCM) to give 3f (43.3 mg, 56.1 μmol, 24.6%) as a dark red oil. For C 39 H 54 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 772.34, found value: 772.65. Example a38. Synthesis of Compound 4f

[0330] A solution of compound 3f (43.3 mg, 56.1 μmol) in anhydrous DCM (700 μL) was cooled to a temperature of -78°C with a dry ice / acetone bath. The mixture was stirred vigorously, and then a freshly prepared stock solution of mCPBA in anhydrous DCM (6.19 mg, 462 mM, 77.6 μL, 35.9 μmol) was added dropwise. After stirring for 20 minutes, 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 another 15 minutes. Finally, a third batch of mCPBA (0.24 mg, 462 mM, 3.0 μL, 1.4 μmol) in anhydrous DCM was added. The RM was stirred for another 2 minutes, then quenched with a pre-cooled (-78°C) acetone solution (reagent grade, 300 μL) and stirred at -78°C. After 12 min, the cooling bath was removed and the RM was allowed to warm to room temperature and diluted with additional DCM (3.5 mL). The RM was transferred to a separatory funnel and washed twice with saturated aqueous NaHCO3 (2 mL). The aqueous layers were combined and extracted twice with DCM (2 mL). The combined organic layers were dried over Na2SO4 and filtered through a membrane filter. The filtrate was a red solution containing compound 4f and was used without further purification. 39 H 54 NO 14 Si + (M+H + ), LCMS (ESI+) calculated value: 788.33, found value: 788.64. Example a39. Synthesis of Compound 5f

[0331] To compound 4f (44.3 μmol) in the mixture (about 10mL) mainly consisting of DCM and a small amount of acetone, add anhydrous acetonitrile (2mL). RM is partially concentrated (to remove DCM) to a volume of about 6mL. Next, add other anhydrous acetonitrile (4.0mL), and the mixture is partially concentrated to a volume of 4.4mL again. Add stirring bar, and analyze RM by HPLC-MS to assess the concentration of starting material 4f (according to the calibration line based on doxorubicin, showing that 4f is 44.3 μmol). Next, potassium carbonate (31.3mg, 226 μmol) is added, and RM is cooled to 0 DEG C, followed by addition of a solution (20.5mg, 1.00mL, 111mM, 111 μmol) of cyanuric chloride in anhydrous acetonitrile. After stirring 2.5h at 0 DEG C, RM is treated again with a solution (80 μL, 111 mmoles, 8.9 μmol) of cyanuric chloride in anhydrous acetonitrile. The RM was stirred at 0 ° C for another 23 minutes and then quenched with a solution of 3-aminopropane-1,2 diol in water (62.9 mg, 345 μL, 2.0 M, 690 μmol). The resulting dark red solution was slowly warmed to room temperature. DMF (3 mL) was added to the RM to give a red solution containing mostly white precipitate. The solution was decanted and the residue was washed several times with additional DMF, filtered through a membrane filter, and then combined with the decanted solution. The solution was partially concentrated in vacuo to remove most of the acetonitrile and then analyzed by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5μM OBD, 30x100mm). The collected fractions were combined and concentrated until a volume of 4 mL remained. The resulting solution, mainly composed of acetonitrile, was dried over Na2SO4, filtered, and the drying agent was thoroughly washed with anhydrous THF (3x). The combined organic layer was partially concentrated again to a volume of 1.7 mL (acetonitrile / THF) and compound 5f (9.32 mM (according to the HPLC calibration line based on doxorubicin), 15.8 mM, 35.7%) was used as is. For C 39 H 52 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 770.32, found value: 770.65. Example a40. Synthesis of Compound 6f

[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. Then, while stirring the RM, TBAF (1 M in THF, 22.4 mg, 85.5 μL, 85.5 μmol) was added (observed to change color from red to green and back to red). 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 RM was stirred for an additional 20 minutes before a third batch of TBAF (1 M in THF, 22.4 mg, 85.5 μL, 85.5 μmol) was added and 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 RM was stirred for another 35 minutes at -10 ° C, and the reaction was then quenched with water (1.0 mL). The RM was allowed to warm 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 x 1 mL). The combined organic layers were dried over Na2SO4, filtered, and then purified by flash column chromatography on silica gel (0% → 6% MeOH in DCM). The pure fractions were combined and partially concentrated to a volume of 4.5 mL, which was then diluted with MeOH (7 mL). Next, the solution was partially concentrated again to a volume of 6 mL and diluted until the volume was 6.7 mL to give compound 6f, which was a red solution (6.7 mL, 1.55 mM (according to an HPLC calibration line based on doxorubicin), 10.36 μmol, 65.6%) mainly in MeOH. For C 33 H 38 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 656.23, found value: 656.59. Example a41. Synthesis of compound 3a

[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 equivalents). The resulting red solution was mixed and placed at room temperature for 15 minutes. Next, the mixture was diluted with DCM (22 mL) and purified by silica gel flash column chromatography (1% → 5% MeOH in DCM) to give compound 3a (134 mg, quantitative) in a dark red oil. For C 38 H 52 NO 13 Si + (M+H + ), LCMS (ESI+) calculated value: 758.32, found value: 758.59. Example a42. Synthesis of Compound 4a

[0334] A solution of compound 3a (120 μmol) in anhydrous DCM (15.0 mL) was cooled to a temperature of -78°C with a dry ice / acetone bath. The mixture was stirred vigorously and then a freshly prepared stock solution of mCPBA in anhydrous DCM (33.1 mg, 580 mM, 331 μL, 192 μmol) was added dropwise with vigorous stirring. After stirring for 18 minutes, the mixture was quenched with a pre-cooled (-78°C) acetone solution (reagent grade, 1.1 mL). After 23 min, the cold bath was removed and the RM was allowed to warm to room temperature. The RM was transferred to a separatory funnel and washed twice with saturated NaHCO3 aqueous solution (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 in vacuo to a volume of 12 mL to give compound 4a as a red solution primarily in DCM, which was used without further purification. For C 38 H 52 NO 14 Si + (M+H + ), LCMS (ESI+) calculated value: 774.32, found value: 774.50. Example a43. Synthesis of compound 5a

[0335] To a solution of compound 4a (100.6 μmol) in a mixture (10.1 mL) consisting mainly of DCM and a small amount of acetone, anhydrous acetonitrile (8 mL) was added. The RM was partially concentrated (to remove DCM) to a volume of about 4 mL. Next, potassium carbonate (54.5 mg, 394 μmol) was added, and the RM was cooled to 0 ° C, followed by 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 minutes, the RM was quenched with a solution of 3-aminopropane-1,2 diol in water (1.33 mL, 929 mM, 1.24 mmol). The resulting dark red solution was slowly warmed to room temperature within 20 minutes. DMF (1.33 mL) was added to the RM and the resulting mixture was partially concentrated and then purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). The collected fractions were combined and concentrated until a volume of 4 mL remained. The resulting solution, mainly composed of acetonitrile, was dried over Na2SO4, filtered, and the drying agent was thoroughly washed with anhydrous THF (3x). The combined organic layer was partially concentrated again to a volume of 4.5 mL (acetonitrile / THF) to give compound 5a as a red solution, which was used as is in the next step. For C 38 H 48 NO 13 Si - (MH + ), LCMS (ESI+) calculated value: 754.29, found value: 754.52. Example a44. Synthesis of compound 6a

[0336] To the compound 5a (40mg, 53 μmol) in the mixture of acetonitrile and THF (4.5mL), triethylammonium acetate (170 μL, 1.06mmol) is added, and the gained red solution is cooled to-15 ℃.Then, while stirring RM, TBAF (1M in THF, 65mg, 0.25mL, 0.25mmol) is added (observing that color changes from red to green and then turns back to red). After stirring for 1 minute, other TBAF (1M in THF, 63mg, 0.24mL, 0.24mmol) is added. RM is stirred for another 14 minutes, then the third batch of TBAF (1M in THF, 46mg, 0.17mL, 0.17mmol) is added, and stirred for another 41 minutes. The reaction is quenched with water (2.4mL). RM was allowed to warm to room temperature in 25 minutes, then merged with the 2nd batch of crude compound 6a, which was obtained from compound 5a (10mg, 13.0 μmol) in the same manner as above. After the reaction mixture of two cancellations was merged, DCM (6mL) was added, and the resulting two-phase system was separated. The aqueous layer was extracted twice (4mL, then 2mL), and the organic layer dried (Na2SO4) combined, then purified by silica gel flash column chromatography (0% → 6% MeOH in DCM). It was 8-9mL that the pure fractions were merged and partially concentrated to a volume of 8-9mL, and it was then 10.0mL to be diluted to a volume of 10.0mL with other DCM to provide compound 6a, mainly a red solution (10.0mL, 1.66mM (according to the HPLC calibration line based on doxorubicin), 16.6 μmol, 16.5%, through 2 steps (assuming quantitative conversion in the N-oxide formation process)). For C 32 H 36 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 642.22, found value: 642.46. Example a45. Synthesis of compound 8b

[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 at room temperature in the dark for 3 days, diluted with DCM (1.8 mL) and purified by silica gel flash column chromatography (the column was pretreated with 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8b (10.8 mg, 15.5 μmol, 18.4%) as a red oil. For C 33 H 39 N4O 13 + (M+H + ), LCMS (ESI+) calculated value: 669.25, found value: 699.62. Example a46. Synthesis of Compound 9

[0338] To a solution of 8b (8.1 mg, 12 μmol, 1.0 equivalent) in a 1:1 mixture (150 μL) of MeOH / DCM, PPh3 was added to a 200 mmol solution (177 μL, 31.9 μmol, 2.7 equivalent) and H2O (80 μL). The resulting two-phase system was stirred at room temperature in the dark for 8 hours. Next, the RM was placed in a refrigerator and stored for 8 days, then concentrated in vacuo. The residue was dissolved in DMF and purified by preparative HPLC (5% → 90% acetonitrile in water, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm) to give compound 9 (0.5 mg, 0.7 μmol, 6% yield) as a red residue. The impure fractions from the preparative HPLC purification were combined and purified by a second preparative HPLC (5% → 90% acetonitrile in water, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm) to give additional compound 9 (0.6 mg, 0.9 μmol, 8% yield) as a red residue. 33 H 41 N2O 13 + (M+H + ), LCMS (ESI+) calculated value: 673.26, found value: 673.65. Example a47. Synthesis of compound 8c

[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 at room temperature in the dark for 3 days, diluted with DCM (1.8 mL) and purified by silica gel flash column chromatography (column pretreated with 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8c (12.2 mg, 18.2 μmol, 20.5%) as a red oil. For C 34 H 42 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 672.27, found value: 672.60. Example a48. Synthesis of compound 8d

[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 at room temperature in the dark for 3 days, diluted with DCM (1.8 mL) and purified twice by silica gel flash column chromatography (column pretreated with 2% MeOH / DCM, 0%→13% MeOH in DCM) to give compound 8d (6.4 mg, 8.9 μmol, 10%) as a red oil. For C 38 H 42 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 720.27, found value: 720.64. Example a49. Synthesis of compound 8f

[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 at room temperature in the dark for 4 days, diluted with DCM (1.8 mL) and purified by flash column chromatography on silica gel (the column was pretreated with 2% MeOH / DCM, 2% → 8% MeOH in DCM) to give compound 8f (11.1 mg, 16.9 μmol, 18.7%) as a red oil. For C 33 H 40 NO 13 + (M+H + ), LCMS (ESI+) calculated value: 658.25, found value: 658.70. Example a50. Synthesis of Compound 31

[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 silica gel flash column chromatography (0% → 30% EtOAc in DCM (to remove p-nitrophenol), then 0% → 25% MeOH in DCM) to give the intermediate 30 (32.7 mg, 32 μmol, 43%) as a colorless oil. For C 49 H 62 N7O 13 S + (M+H + ), LCMS (ESI+) calculated value: 988.41, found value: 988.78.

[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 hours, complete conversion was achieved and the reaction mixture was concentrated to afford compound 31 (12.6 mg, 16.5 μmol, 100%) as an oil. For C 34 H 52 N7O 11 S + (M+H+ ), LCMS (ESI+) calculated value: 766.34, found value: 766.65. Example a51. Synthesis of compound 9c

[0344] To compound 6c (3.26mg, 4.87 μ mol) in DCM solution, add MeOH (3mL) and the mixture is concentrated until only MeOH (1.5mL) is left. Add water (200 μ L) and add a solution of sodium periodate in water (60mM, 282 μ L, 16.9 μ mol), and the reaction mixture is stirred in the dark for 41 hours. Once complete conversion is achieved, DMF (800 μ L) is added, and the reaction mixture is concentrated until only 400 μ L DMF (2.19mg, 3.34 μ mol, 68.6%) are left. The intermediate is then added into compound 31 (10.2mg, 13.2 μ mol), followed by DiPEA (2 μ L, 10 μ mol) and HATU (2.1mg, 28 μ L, 200mM, 5.4 μ mol). After stirring at room temperature for 20.5 hours, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). Compound 9c was obtained as a red solution in DMF (250 μL, 3.83 mM (according to the HPLC calibration line based on doxorubicin), 1.34 mg, 0.95 μmol, 28.6%). 67 H 87 N8O 23 S + (M+H + ), LCMS (ESI+) calculated value: 1404.51, found value: 1404.06. Example a52. Synthesis of compound 9d

[0345] To compound 6d (3.4 mg, 4.7 μ mol) in DCM solution, add MeOH (5 mL) and the mixture is concentrated until only MeOH (2.1 mL) is left. Then add a solution of sodium periodate in water (60 mM, 288 μ L, 17.5 μ mol), and the reaction mixture is stirred in the dark for 68 hours. Once complete conversion is achieved, add DMF (600 μ L), and the reaction mixture is concentrated until 490 μ L DMF (1.78 mg, 2.53 μ mol, 53%) are left. The intermediate is then added into compound 31 (6.2 mg, 8.1 μ mol), followed by addition of DiPEA (1.32 μ L, 7.59 μ mol) and HATU (1.15 mg, 15.2 μ L, 200 mM, 3.04 μ mol). After stirring at room temperature for 21.5 hours, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). Compound 9d was obtained as a red solution in DMF (165 μL, 10.14 mM (according to the HPLC calibration line based on doxorubicin), 2.42 mg, 1.67 μmol, 65.9%). 71 H 87 N8O 23 S + (M+H + ), LCMS (ESI+) calculated value: 1452.56, found value: 1452.03. Example a52-2. Synthesis of Compound 9f

[0346] To the solution of compound 6f (6.8mg, 10.4 μmol) in a mixture of MeOH (6.7mL) and water (1.4mL), a solution of sodium periodate in water (62.9mM, 206 μL, 13.0 μmol) is added, and the reaction mixture is stirred at room temperature for 3 hours in the dark. Other sodium periodate (62.9mM, 210 μL, 13.2 μmol) in water is added and the reaction mixture is stirred at room temperature for another 17 hours. Finally, the 3rd batch of sodium periodate (62.9mM, 50 μL, 3.1 μmol) in water is added, and the mixture is stirred at room temperature for 80 minutes, then partially concentrated in vacuo to volume is 5.4mL, and then placed at room temperature for 5 hours. Then DMF (670 μL) is added, and gained red solution is partially concentrated to volume for about 350 μL, obtains white residue and the red solution containing thick intermediate. The mixture is diluted to 666 μ L with other DMF, then 222 μ L (3.45 μ mol) of this solution are used compound 31 stock solution in DMF (110 mmoles, 62.7 μ L, 6.9 μ mol) to process, then with DiPEA (1.79 μ L, 10.4 μ mol) and HATU solution (204mM, 16.9 μ L, 3.45 μ mol) in dry DMF to process. The gained mixture is vortexed and placed at room temperature 31 minutes. Next, add other compound 31 (110 mmoles, 13.9 μ L, 1.53 μ mol) in DMF and HATU (204mM, 33.8 μ L, 6.90 μ mol) in dry DMF. The mixture is vortexed again and placed at room temperature 13 minutes, then put into refrigerator and store 17 hours. Finally, the mixture was taken out of the refrigerator and analyzed by preparative HPLC (40% → 95% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). Compound 9f was obtained as a red solution in DMF (300 μL, 8.2 mM (according to the HPLC calibration line based on doxorubicin), 3.9 mg, 2.47 μmol, 71.6%). 66 H 85 N8O 23 S + (M+H + ), LCMS (ESI+) calculated value: 1389.54, found value: 1390.07. Example a53. Synthesis of Compound 9g (OMe-PNU)

[0347] To compound 6a (2.17mg, 3.38 μmol) in DCM solution, add MeOH (3mL) and the mixture is concentrated until only MeOH (1.35mL) is left.Add water (300 μ L) and add a solution of sodium periodate in water (60mM, 112.4 μ L, 6.8 μ mol), and the reaction mixture is stirred in the dark for 19 hours.Remove stirring bar, and RM is concentrated in vacuo, DMF (200 μ L) is added in residue, obtains white residue and the red solution containing thick intermediate.Then compound 31 (6mg, 7.8 μ mol) is added to this intermediate, then DiPEA (1.8 μ L, 10.5 μ mol) and HATU (2.05mg, 5.4 μ L, 196mM, 5.4 μ mol) are added. After 25 min at room temperature, the reaction mixture was purified by preparative HPLC (40% → 95% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm). Compound 9g was obtained as a red solution in DMF (122 μL, 14.24 mM (according to the HPLC calibration line based on doxorubicin), 2.4 mg, 1.74 μmol, 80.9%). 65 H 83 N8O 23 S + (M+H + ), LCMS (ESI+) calculated value: 1375.53, found value: 1376.01. Example a54. Synthesis of Compound 32

[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 before adding Fmoc-Val-Ala-PAB-OPNP (79.4 mg, 117 μmol). The biphasic mixture was stirred at room temperature for another 16 hours before extraction with DCM (2 x 1 mL) to remove water. The combined organic layers were dried over Na2SO4 and immediately purified by silica gel flash column chromatography (0%→40% EtOAc in DCM (to remove excess Fmoc-Val-Ala-PAB-OPNP), followed by 0%→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%). For C 70 H86 N5O 19 Si + (M+H + ), LCMS (ESI+) calculated value: 1329.54, found value: 1329.00. Example a55. Synthesis of Compound 33

[0349] The solution of compound 32 (123.6 mg, 93 μ mol) in anhydrous DCM (7.7 mL) is concentrated until 2 mL of solution remains. Next, the reaction mixture is cooled to a temperature of -78 ° C with a dry ice / acetone cold bath. The mixture is vigorously stirred, and freshly prepared m-CPBA stock solution (17.66 mg, 580 mM, 176 μ L, 102.3 μ mol) is added dropwise afterwards. After stirring for 17 minutes, complete conversion is reached. RM is quenched with an ice-cold acetone solution (reagent grade, 2.5 mL) and stirred. After one hour, the cold bath is removed, and RM is warmed to room temperature. RM is transferred to a separating funnel and washed twice with saturated NaHCO aqueous solution (10 mL). The water layer is merged and extracted twice with DCM (20 mL). The organic layer merged is dried through Na SO dried, filtered through filter paper, and concentrated until the volume of 19 mL is obtained. 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 value: 1345.54, found value: 1345.14. Example a56. Synthesis of Compounds 34 and 35

[0350] To a solution of compound 33 (108.3 mg, 80.5 μmol) in DCM (19 mL) was added anhydrous acetonitrile (10 mL). The RM was concentrated to remove DCM and obtain the compound in anhydrous acetonitrile (5 mL). After concentrating most of the solvent, the RM was further diluted with anhydrous acetonitrile (45 mL). Potassium carbonate (65 mg, 463 μmol) was then added, and the RM was cooled to -10 ° C, followed by addition of cyanuric chloride (55.7 mg, 2.44 mL, 120 mM, 302.2 μmol), which was a stock solution in anhydrous acetonitrile. After stirring at 0 ° C for 5.5 h, the RM was quenched with a solution of 3-aminopropane-1,2 diol in water (90.3 mg, 1.1 mL, 900 mM, 990 μmol). The ice bath was removed after 15 min and allowed to warm to room temperature. DMF (3 mL) was added to the RM and the RM was concentrated until only a DMF / water solution remained and the product was purified by preparative HPLC (40% → 100% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prepC 18 , 5 μM OBD, 30x100 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 thoroughly washed with anhydrous THF (3x1 mL). Intermediate 34 was obtained as a solution in acetonitrile / THF (14 mL, 8.7 mg, 6.6 μmol, 8.1%). For C 70 H 84 N5O 19 Si + (M+H + ), LCMS (ESI+) calculated value: 1327.52, found value: 1327.07.

[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. Triethylammonium acetate (5.3 mg, 5.3 μmol, 33 μmol) was added thereto, and the resulting red solution was cooled to -15 ° C. Then, while RM was vigorously stirred, TBAF (1M in THF, 17.2 mg, 66 μL, 66 μmol) was added (observing that the color changes from red to green). After stirring for 3 h, RM was quenched with water (1.5 mL), and RM became red again. RM was transferred to a separating funnel and extracted with DCM (20 mL). The combined organic layer was dried over Na2SO4 and directly purified by silica gel flash column chromatography (0% → 10% MeOH, in DCM) to give compound 35 as a red solution (8 mL, 2.3 mg, 1.9 μmol, 29%) in DCM. For C 64 H70 N5O 19 + (M+H + ), LCMS (ESI+) calculated value: 1212.47, found value: 1212.93. Example a57. Synthesis of Compound 36

[0352] The synthesis of BCN-HS-PEG2-OPNP has been described in WO 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 RM was concentrated to remove DCM. A solution of BCN-HS-PEG2-OPNP in DMF (1.2 mg, 25 μl, 93 mM, 2.3 μmol) was then added, followed by triethylamine (2.6 μL, 19 μmol). After 30 hours at room temperature, RM was further diluted with DCM (300 μL) and purified by silica gel flash column chromatography (0% → 15% MeOH in DCM), to give compound 36, a red solution (150 μL, 0.64 mM (according to the HPLC calibration line based on doxorubicin), 0.13 mg, 0.09 μmol, 5%) in DMF. For C 65 H 82 N7O 24 S + (M+H + ), LCMS (ESI+) calculated value: 1377.44, found value: 1377.04. Example a58. Synthesis of Compound 38

[0353] This compound was synthesized according to the literature procedure described in WO 2017137457 A1. Example a59. Synthesis of Compound 39

[0354] To a solution of compound 6e (1.0 mg, 1.3 μmol, 1.00 equivalent) in non-dried THF (90 μL), PPh was added. A solution of PPh in THF (200 mmol, 13 μL, 2.6 μmol, 2.0 equivalent) and H O (13 μL) was added. The resulting red solution was vortexed and then placed at room temperature for 19 hours. The mixture was then transferred to an Eppendorf vial and placed in an Eppendorf shaker, shaken at 37°C and 1400RPM for 6.5 hours, and then shaken at room temperature for another 19 hours at 1400RPM. The reaction mixture is then placed in a refrigerator and stored for 11 days. The mixture is then treated with a solution of compound 38 in DMF (790mM, 2.0 μL, 1.6 μmol, 1.2 equivalents), followed by the addition of HATU (517mM, 1.4 μmol, 1.1 equivalents) in DMF, and finally the addition of a solution of DMAP in DMF (500mM, 1.1 μL, 0.53 μmol, 0.4 equivalents). The resulting mixture is vortexed and placed at room temperature for 3 hours, then placed in a refrigerator and stored for 21 hours. The mixture is taken out from the refrigerator and purified by preparative HPLC (40% → 100% acetonitrile, in 10mM NH4HCO3 aqueous solution, column: Xbridge prep C18, 5 μM OBD, 30x 100mm). Pure fractions were combined and diluted with DMF and partially concentrated to give compound 39 as a red solution in DMF (85 μL, 1.5 mM (according to HPLC calibration line based on doxorubicin), 0.17 mg, 0.13 μmol, 10%). 62 H 77 N6O 22 S + (M+H + ), LCMS (ESI+) calculated value: 1289.48, found value: 1289.99. Example a60. Synthesis of Compounds 42 and 43

[0355] To the vial containing Boc-Gly-Gly-Phe-Gly-OH (500mg, 1.15mmol, 1.00 equivalent) and Fmoc-EDA-H (476mg, 1.69mmol, 1.47 equivalent) was added DMF (1.25mL), followed by Et3N (479 μL, 3.44mmol, 3.00 equivalent), and finally HATU (479mg, 1.26mmol, 1.10 equivalent). The resulting mixture was stirred at room temperature for 2 minutes, then DCM (2mL) was added to generate a clear yellow solution, which was stirred at room temperature for 110 minutes. The reaction mixture was then placed in a refrigerator and stored for 16 hours. The mixture was then taken out from the refrigerator and diluted with DCM (15mL) and directly purified by flash column chromatography (50% → 100% EtOAc, in heptane). The fractions containing the product were concentrated to give compound 42 (357 mg, 509 μmol, 45%) as a white solid. 37 H 45 N6O8 + (M+H + ), LCMS (ESI +) calculated value: 701.33, found value: 701.67. Compound 42 (357 mg, 509 μmol, 1.00 equiv) was suspended in DCM (3.0 mL) and cooled to 0 ° C with an ice bath. Next, TFA (1.37 mL, 17.8 mmol, 35 equiv) was added dropwise to generate a light yellow solution, which was stirred at 0 ° C for 47 minutes. The ice bath was then removed, and the RM was warmed to room temperature and stirred for another 75 minutes. The mixture was concentrated in vacuo and the residue was dissolved in a mixture of DCM (1.5 mL) and toluene (2 mL) and concentrated twice 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%→95% acetonitrile (containing 1% AcOH) in water (containing 1% AcOH, column: Xbridge prep C18, 5 μM OBD, 30×100 mm) to afford compound 43 as an acetate salt. 32 H 37 N6O6 + (M+H + ), LCMS (ESI+) calculated value: 601.28, found value: 601.60. Example a61. Synthesis of Compounds 45 and 46

[0356] In a dry ice / MeCN bath at -40 °C, BCN-OH 44 (40 mg, 90 wt% (according to 1To a solution of chlorosulfonyl isocyanate (22 μL, 250 μmol, 1.05 equiv) in dry DCM (2.5 mL) was added chlorosulfonyl isocyanate (22 μL, 250 μmol, 1.05 equiv) in a single portion to produce a light yellow solution. The mixture was stirred at -40 ° C for 17 minutes, followed by the addition of Et3N (67 μL, 480 μmol, 2.00 equiv). The resulting solution was stirred at -40 ° C for about 5 minutes, 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), followed by the addition of the TFA salt of compound 43 (115.4 mg, 161.5 μmol, 0.67 equiv) as a suspension of a mixture of DIPEA (46 μL) and Et3N (23 μL) in DMF (5 mL) 10 minutes later. 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 placed in a refrigerator for 5 days, then removed from the refrigerator and concentrated in vacuo. The residue was purified by preparative HPLC (40% → 90% acetonitrile (containing 1% AcOH) in water (containing 1% AcOH), column: Xbridge prep C18, 5 μM OBD, 30x100 mm) to give compound 45 (8.9 mg, 9.5 μmol, 4% yield). For C 43 H 50 N7O 10 S + (M+H + ), LCMS (ESI+) calculated value: 856.33, found value: 856.66. To a solution of compound 45 (8.9 mg, 9.5 μmol) in non-dried DMF (400 μL) was added Et3N (58 μL, 0.42 mmol, 40 equivalents). The resulting mixture was mixed and left at room temperature for about 18 hours, then concentrated in vacuo to a volume of about 25 μL. The solution was transferred to an Eppendorf vial with additional DMF to give compound 46 as a solution in DMF with a final volume of 150 μL, which was used in the next step without further purification. For C 28 H 40 N7O8S + (M+H + ), LCMS (ESI+) calculated value: 634.27, found value: 634.54. Example a62. Synthesis of Compound 47

[0357] To the solution of compound 6f (6.8mg, 10.4 μmol) in a mixture of MeOH (6.7mL) and water (1.4mL), a solution of sodium periodate in water (62.9mM, 206 μL, 13.0 μmol) is added, and the reaction mixture is stirred at room temperature for 3 hours in the dark. Other sodium periodate (62.9mM, 210 μL, 13.2 μmol) in water is added and the reaction mixture is stirred at room temperature for another 17 hours. Finally, the 3rd batch of sodium periodate (62.9mM, 50 μL, 3.1 μmol) in water is added, and the mixture is stirred at room temperature for 80 minutes, then partially concentrated in vacuo to volume is 5.4mL, and then placed at room temperature for 5 hours. Then DMF (670 μL) is added, and gained red solution is partially concentrated to volume for about 350 μL, obtains white residue and the red solution containing thick 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 treatment with a solution of DiPEA (1.80 μL, 10.4 μmol) and HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF. The resulting mixture was vortexed and placed at room temperature for 17 minutes. Next, additional HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF was added. The mixture was vortexed again and placed at room temperature for 23 minutes. The third batch of HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF was added. The resulting mixture was vortexed and placed at room temperature for 14 minutes. Next, additional compound 46 (2.4 mg, 3.8 μmol) was added to the RM, followed by the addition of additional compound 46 (2.4 mg, 3.8 μmol) and the fourth batch of HATU (490 mM, 7.1 μL, 3.5 μmol) in dry DMF 14 minutes later. The mixture was stirred at room temperature for an additional 29 minutes before the addition of additional DiPEA (1.80 μL, 10.4 μmol). The mixture was then passed through a membrane filter and then treated with the 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 before purification by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 aqueous solution, column: Xbridge prepC18, 5 μM OBD, 30x100 mm). Compound 47 was obtained as a red solution in DMF (196 μL, 3.5 mM (according to HPLC calibration line based on doxorubicin), 1.0 mg, 0.684 μmol, 20%).60 H 73 N8O 20 S + (M+H + ), LCMS (ESI+) calculated value: 1257.47, found value: 1357.86. Example a63. Synthesis of Compounds 49 and 50

[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). HATU (494.8 mg, 1.301 mmol, 1.12 equiv) was added to the resulting suspension. The resulting yellow mixture was stirred at room temperature for 2 minutes, followed by addition of DCM (2 mL) to generate a yellow solution, which was stirred at room temperature for another 90 minutes. The mixture was then placed in a refrigerator and stored for 1 day. The mixture was taken out from the refrigerator and then purified by flash column chromatography (0 → 10% MeOH in DCM) to give product 50 (443.5 mg, 801.1 μmol, 68.7%) as a white solid. For C 28 H 36 N5O7 + (M+H + ), LCMS (ESI+) calculated value: 554.26, found value: 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. Next, TFA (400 μL, 5.19 mmol, 7.05 equiv) was added dropwise to the reaction mixture while stirring. After the addition was complete, 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 in batches, and the resulting mixture was stirred at room temperature for another 200 minutes. The mixture was then concentrated in vacuo, and the residue was then purified by preparative HPLC (5% → 90% acetonitrile in water, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm) to obtain compound 50 (84.9 mg, 186 μmol, 25%) as a white powder. 23 H 28 N5O5 + (M+H + ), LCMS (ESI+) calculated value: 454.21, found value: 454.54. Example a64. Synthesis of Compounds 51 and 52

[0359] In a dry ice / MeCN bath at -40 °C, BCN-OH 44 (30.5 mg, 90 wt% (according to 1 To a solution of chlorosulfonyl isocyanate (16.7 μL, 192 μmol, 1.05 equiv) in dry DCM (2.1 mL) was added chlorosulfonyl isocyanate (16.7 μL, 192 μmol, 1.05 equiv) in a single portion to produce a light yellow solution. The mixture was stirred at -40 ° C for 5 minutes, followed by the addition of Et3N (76.4 μL, 548 μmol, 3.00 equiv). The resulting solution was stirred at -40 ° C for approximately 14 minutes, followed by the addition of a solution 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 minutes, and then allowed to warm to room temperature over 2 hours. The reaction mixture is then placed in a refrigerator and stored for 1 day, then taken out from the refrigerator and diluted with DCM and purified by flash column chromatography (0 → 13% MeOH in DCM) to obtain impure intermediate 51 (containing EtN), which is two batches (45 μmol in total, 25% yield). Two batches are merged and co-evaporated with DMF. The gained oil is diluted with DCM (10 mL) and washed twice with saturated aqueous NHCl (2 mL, 2 ×). During extraction, a viscous oil is formed, which is merged with the organic layer and concentrated in vacuo to obtain the intermediate 51 as a brown sticky solid. The residue is dissolved in 600 μL DMF, followed by addition of EtN (240 μL, 1.70 mmol, 40 equivalents). The gained mixture is stirred at room temperature for 1 day, then placed in a refrigerator and stored for 3 days. The reaction mixture is taken out from the refrigerator and filtered through a membrane filter, washed with other DMF (3 ×). 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 value: 487.20, found value: 487.55. Example a65. Synthesis of Compound 53

[0360] To the solution of compound 6f (6.8mg, 10.4 μmol) in a mixture of MeOH (6.7mL) and water (1.4mL), a solution of sodium periodate in water (62.9mM, 206 μL, 13.0 μmol) is added, and the reaction mixture is stirred at room temperature for 3 hours in the dark. Other sodium periodate (62.9mM, 210 μL, 13.2 μmol) in water is added and the reaction mixture is stirred at room temperature for another 17 hours. Finally, the 3rd batch of sodium periodate (62.9mM, 50 μL, 3.1 μmol) in water is added, and the mixture is stirred at room temperature for 80 minutes, then partially concentrated in vacuo to volume is 5.4mL, and then placed at room temperature for 5 hours. Then DMF (670 μL) is added, and gained red solution is partially concentrated to volume for about 350 μL, obtains white residue and the red solution containing thick intermediate. The mixture is diluted to 666 μ L with other DMF, then 222 μ L (3.45 μ mol) of this solution are processed with 52 stock solution (175 mmoles, 39.4 μ L, 6.9 μ mol) in DMF, then with DiPEA (1.79 μ L, 10.4 μ mol) and HATU solution (500 mM, 6.90 μ L, 3.45 μ mol) in dry DMF. The gained mixture is vortexed and placed at room temperature for 17 minutes. Next, add other 52 (175 mmoles, 157.6 μ L, 27.6 μ mol) in DMF. The mixture is vortexed again and placed at room temperature for 14 minutes, then add other HATU (500 mM, 13.80 μ L, 6.90 μ mol) in dry DMF. The mixture is vortexed again and placed at room temperature for 13 minutes, then put into refrigerator and store for 2 days. Finally, the mixture was taken out of the refrigerator and purified by preparative HPLC (40% → 95% acetonitrile in 10 mM NH4HCO3 aqueous solution, column: Xbridge prepC18, 5 μM OBD, 30×100 mm). Compound 53 was obtained as a red solution in DMF (165 μL, 2.5 mM (according to the HPLC calibration line based on doxorubicin), 0.5 mg, 0.41 μmol, 12%). For C 51 H 64 N7O 19 S + (M+H + ), LCMS (ESI+) calculated value: 1110.40, found value: 1110.85. Synthesis of compound 57 Example a66. Synthesis of Compound 54

[0361] This compound was synthesized according to the literature procedure described by Pawar et al., International Journal of Pharmaceutics, Vol. 436, No. 1-2, pp. 183-193. Example a67. Synthesis of Compound 55

[0362] To the round-bottom flask containing compound 54 (5.241g, 1 equivalent, 6.844mmol), a solution of THF (160mL) and water (160mL) was added. The gained red suspension was cooled to 0°C, then a cold solution of sodium periodate in H was added dropwise over 15 minutes (1.464g, 34.22mL, 200 mmoles, 1.00 equivalents, 6.844mmol) in O. The red solution with some solids at the bottom of the gained was stirred on ice for a total of 10 minutes. The ice bath was then removed, and RM was warmed to room temperature and stirred for 23 hours. The RM portion was concentrated in vacuo (removing all THF and approximately 50% water, until 80 mbar) to obtain a red suspension mainly in water. The mixture was processed with DCM (200mL), and the gained suspension was rotated at 43°C for several minutes. The two-phase system was transferred to a separating funnel. To the remaining residue in the round-bottom flask, add another 300ml DCM, and the resulting mixture is rotated at 43 ° C again until the remaining solid dissolves. The solution is also added to a separating funnel, and the obtained two-phase system is vibrated and separated. The water layer is extracted twice with another DCM (2x 200ml). The separating funnel (containing a small amount of dark red residue) is washed with 10% MeOH (in DCM, 150mL) to dissolve the residue completely. The organic layer is washed once with water layer. The resulting organic layer is then merged with other organic layers. MeOH (40mL) is added to the combined organic layer to generate a clear solution. The combined organic layer is dried (Na2SO4), then filtered through a glass filter. The solution is processed with DMF (18mL) and partially concentrated until mainly DMF is left as a solvent (up to 20 mbar), to obtain the intermediate 55 in a dark red solution (18mL), which is a solution (5.30g, 6.87mmol) in DMF, which can be used for the next step without further purification. Quantitative yields were assumed. 41 H 37 NO 13 + [M+H + ], UPLC-MS (ESI+) calculated value: 752.23, found value: 752.52. 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) was added additional DMF until a total of approximately 94 mL was reached. Next, allyl (2-aminoethyl)carbamate (2.14 g, 2.72 equiv, 14.9 mmol) in dry DMF (9.0 ml) was added and the RM was placed in a water bath. Next, HATU (2.18 g, 1.05 equiv, 5.73 mmol) was added, followed by DIPEA (2.12 g, 2.85 mL, 3.00 equiv, 16.4 mmol) over one minute, and the resulting dark red solution was stirred at room temperature for approximately 30 min. Next, additional HATU (455 mg, 1.20 mmol, 0.22 eq) in DMF (1.0 mL) was added, followed by a third batch of HATU (509 mg, 1.34 mmol, 0.25 eq) after another 55 minutes. The RM was stirred at room temperature for an additional 5 minutes and then partially concentrated under vacuum until the volume was 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 fractions containing the product were combined and concentrated to give intermediate 56 (3.61 g, 4.07 mmol, 74.5%, 99% purity) as a dark red thick oil. For C 47 H 48 N3O 14 + [M+H + ], UPLC-MS (ESI+) calculated value: 878.31, found value: 878.71. Example a69. Synthesis of Compound 57

[0364] To a dark red solution of compound 56 (3.61 g, 99% Wt, 1.00 eq., 4.07 mmol) in a total volume of (17.0 mL) DMF was added triethylamine (2.06 g, 2.84 mL, 5.0 eq., 20.3 mmol). The RM turned very dark red and was left to stir at room temperature for 18 hours. Next, Et2O (78 mL) was added (slowly) in one go while stirring rapidly. Stirring was stopped, the ether layer was then decanted, and the remaining dark solid was washed several more times with Et2O (3 x 100 ml). The solid was concentrated in vacuo to give intermediate 57 (4.8 g, 81.9% purity) as a dark red solid which was used without further purification. For C 32 H 38 N3O 12 + [M+H +], UPLC-MS (ESI+) calculated value: 656.25, found value: 656.60. Example a70. Synthesis of Compound 58

[0365] Intermediate 57 (4.0 g, 90 wt %, 5.5 mmol) was suspended in dry DMF (6.0 mL), followed by the addition of diiodine sugar 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 minutes. Next, the remaining block was mostly broken up with a spatula, and the suspension was stirred at 40 ° C for 2 days. The RM was diluted with DCM (100 mL) and the resulting red solution was purified by silica gel flash column chromatography (0% MeOH / DCM, then 2% → 10% MeOH, in DCM) to give compound 58 (4.03 μmol, 72%) as a red residue. For C 39 H 50 N3O 14 + (M+H + ), LCMS (ESI+) calculated value: 784.33, found value: 784.78. Example a71. Synthesis of Compound 59

[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 a temperature of -78°C using a dry ice / acetone bath. The mixture was stirred vigorously before a freshly prepared stock solution of mCPBA in DCM (580 mM, 9.0 mL, 5.22 mmol) was added dropwise over 5-10 minutes. After stirring for 17 minutes, a second batch of mCPBA in DCM (580 mM, 10.0 mL, 5.80 mmol) was added over 5 minutes and the RM was stirred for an additional 9 minutes before a final 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 minutes before being quenched with cold (-78°C) acetone (reagent grade, 43.9 mL) and the RM was stirred. After 90 minutes, the cooling bath was removed and the RM was allowed to warm to 0°C over 40 minutes. RM is diluted with DCM (450mL) and saturated NaHCO3 aqueous solution (250mL) and transferred to a separating funnel. The obtained two-phase system is separated, and the water layer is extracted twice with DCM (150,100mL). The combined organic layer is then washed again with saturated NaHCO3 aqueous solution (100mL). New water layer is extracted with DCM (40mL), and the combined organic layer is dried and filtered through Na2SO4, then concentrated to give compound 59 (3.30g, 93% purity, 80% yield, 3.84mmol), which is used in the next step as it is. For C 39 H 50 N3O 15 + (M+H + ), LCMS (ESI+) calculated value: 800.32, found value: 800.70. Example a72. Synthesis of Compound 60

[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 RM was then cooled to 0 °C with an ice bath before the addition of a solution of cyanuric chloride in anhydrous acetonitrile (62.6 mg, 2.88 mL, 118 mM, 339 μmol, 2.5 equiv). 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 RM was stirred at 0 °C for another 55 min before being quenched with a solution of 3-aminopropane-1,2 diol in water (186 mg, 1.02 mL, 2 moles, 2.04 mmol, 15 equiv). After 30 min, the ice bath was removed and the mixture was diluted with DCM (30 mL) and H2O (10 mL). The resulting biphasic system was separated. The aqueous layer was extracted twice more with DCM (10 mL, 2×). The combined organic layers were dried (Na2SO4), filtered through a phase separator and concentrated in vacuo. The residue was dissolved in DMF and purified by preparative HPLC (50% → 70% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridge prep C 18 , 5 μM OBD, 30x100 mm) was purified. The 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. In addition, the fractions containing impure product were combined to give impure 60 (11.4 mg, 63% pure, 9.20 μmol, 6.7% yield). For C 39 H 48 N3O 14 + (M+H + ), LCMS (ESI+) calculated value: 782.82, found value: 782.64. Example a73. Synthesis of Compound 61

[0368] To the vial containing compound 60 (6.75 mg, 1 equivalent, 8.63 μmol), anhydrous DCM (300 μL) was added and pyrrolidine (1.84 mg, 2.13 μL, 3 equivalents, 25.9 μmol) was added to the resulting red solution to immediately generate a very dark red (almost black) solution. The mixture was mixed and then Pd(PPh 3 ) 4 (1.50 mg, 64.8 μL, 20 mmoles, 0.15 equivalents, 1.30 μmol) in dry DCM was added. The dark red solution was mixed again and placed at room temperature for 11 minutes. An additional 20 mmol of Pd(PPh3)4 (64.8 μL, 0.15 eq., 1.30 μmol) in dry DCM was added, followed by the third (100 μL, 0.23 eq., 2.00 μmol) and fourth (64.8 μL, 0.15 eq., 1.30 μmol) batches after 13 and 12 min, respectively. Finally, after the fourth addition, the RM was stirred at room temperature for 47 min, then diluted with DMF (300 μL) and then partially concentrated in vacuo to 30 mbar to give a deep red solution, which was purified by preparative HPLC (20% → 50% acetonitrile in 10 mM aqueous NH4HCO3, column: Xbridgeprep C 18 , 5 μM OBD, 30x100 mm) was purified. The 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. In addition, the fractions containing the impure product were combined to give impure 61 (3.3 mg, 53% pure, 2.5 μmol, 29% yield). For C 35 H 44 N3O 12 + (M+H + ), LCMS (ESI+) calculated value: 698.29, found value: 698.56. Example a74. Synthesis of compound BCN-HS-GGFG-OH (62)

[0369] The round-bottom flask containing a solution of BCN-OH 44 (150 mg, 92% Wt, 1 equivalent, 919 μmol) in acetonitrile (10 mL) was cooled to 0 ° C with ice. Next, chlorosulfonyl isocyanate (137 mg, 83.9 μ L, 1.05 equivalents, 965 μ mol) was added. RM was stirred for 20 minutes, then treated with triethylamine (279 mg, 384 μ L, 3 equivalents, 2.76 mmol), followed by addition of H-GlyGlyPheGly-OH.TFA (496 mg, 1.2 equivalents, 1.10 mmol) as a solid. Next, water (10 mL) was added and RM was stirred vigorously, followed by addition of other triethylamine (186 mg, 256 μ L, 2 equivalents, 1.84 mmol) to help dissolve. RM was stirred for approximately 3 hours to generate a solution, which was diluted with DCM (20 mL) and water (20 mL) and saline (500 μ L). The obtained two-phase system was shaken and then separated. The aqueous layer was extracted with another DCM (10 mL). The combined aqueous layers were combined and EtOAc (20 mL) was added. 1M aqueous HCl was then added until the pH of the aqueous layer reached about 4. The two layers were separated, and the aqueous layer was extracted twice with EtOAc (2 x 20 ...

Claims

1. A conjugate, wherein a compound according to structure (1) is conjugated to a cell binding agent via a linker, wherein structure (1) is as follows: in: -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, heterocyclic group, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic group, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 ) 2, wherein the optional substituent is selected from 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 wherein the substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups may optionally be further substituted with C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 )2 substituted; wherein each Sp is independently C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1-12 (hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally selected from halogen, X 2 R 4 、N(R 4 2. C 1-4 alkyl and substituted with one or more substituents of NO2, wherein each R 4 H, C alone 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 wherein R 12 It is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) ; -R 2 is H, S(O)2OH or P(O)2OH and R 3 is OH, or R 2 and R 3 The oxazolidine ring is formed by fusion of the ether moiety; -R 5 is H or OCH3; -N ％ is N or N→O; -Y 5 It is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY, where R 9 Selected from optionally OH or O(CO)C 1-6 Alkyl substituted C 1-4 Alkyl, and R 20 YesNR 4 -C(O)-N(R 4 )2. NR 4 -C(O)-Sp-N(R 4 )2. NR 4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 As defined above; - the compound is linked to the linker via Y, or a salt thereof, wherein each ion, if present, is balanced with one or more pharmaceutically acceptable counterions.

2. The conjugate according to claim 1, which has the structure (2) CB-Z 1 -LZ 2 -D (2) in: -CB is the cell binding agent; -D is the compound according to structure (1); -L is a linker; -Z 1 is a linker group that connects the cell binding agent CB to the linker; and -Z 2 is a linking group that connects the compound D to the linker.

3. The conjugate according to claim 2, wherein the linker group Z 1 Formed by a conjugation reaction selected from amide bond formation, carbamate bond formation, thiol alkylation, thiol arylation and cycloaddition reaction.

4. The conjugate according to claim 2 or 3, wherein the linker group Z 1 The cell-binding agent CB is linked to the cell-binding agent CB via a lysine residue of the CB, a glutamine residue of the CB, a threonine residue of the CB, a cysteine ​​residue of the CB, a tyrosine residue of the CB, or a glycan of the CB.

5. The conjugate according to any one of claims 2 to 4, wherein the linking group Z 2 It 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. The conjugate according to any one of claims 2 to 5, wherein LZ 2 With structure: *-NR 4 -Sp 3 -NR 4 -(L 3 ) p -(L 2 ) o -(L 1 ) n -** in: - the bond marked with * is connected to the C(O) moiety adjacent to Y in the compound according to structure (1); -Bonds marked with ** and linking group Z 1 connect; -Sp 3 It 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 may be optionally selected from halogen, X 2 R 4 、N(R 4 2. C 1-4 Alkyl, one or more substituents of NO2, wherein the C 1-4 The alkyl substituent may optionally be connected by 4 Partially connected to form a ring structure, especially with NR with a bond marked with * 4 The pyrrolidine formed by the moiety, and the alkylene group may be optionally selected from X 2 and NR 4 interrupted by one or more heteroatoms; -R 4 and X 2 As defined in claim 1; -L 1 , L 2 and L 3 Each of them is Z 1 A linker connected to D; - n, o and p are each independently 0 or 1, provided that n+o+p=1, 2 or 3.

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

8. The conjugate according to any one of the preceding claims, wherein R 1 is selected from Et, i-Pr, t-Bu, Bz, Bn, Sp-N3 or Sp-NH2, preferably wherein R 1 is Et, i-Pr, Bn or Sp-N3, where Sp is C 1-4 Alkylene or C 1-4 Alkylene-arylene, preferably wherein Sp is CH2CH2, CH2CH2CH2 or CH2Ph.

9. The conjugate according to any one of the preceding claims, wherein R 2 and R 3 The oxazolidine ring is formed by linking together through the ether moiety.

10. The conjugate according to any one of the preceding claims, wherein R 1 Not CH2CH2SH, unsubstituted ethyl or benzyl.

11. A compound according to structure (1): in: -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, heterocyclic group, (hetero)aryl, Sp-(hetero)aryl, Sp-heterocyclic group, Sp-X 2 R 4 、Sp-N3、Sp-X 2 -Sp-R 12 or Sp-N(R 4 ) 2, wherein the optional substituent is selected from 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 wherein the substituent C 1-12 (Hetero)alkyl and (hetero)aryl groups may optionally be further substituted with C 1-6 (Hetero)alkyl, X 2 R 4 and N(R 4 )2 substituted; wherein each Sp is independently C 1-12 (Hetero)alkylene, (hetero)arylene, C 1-12 (Hetero)alkylene-(hetero)arylene, or (hetero)arylene-C 1-12 (hetero)alkylene, wherein the (hetero)alkylene or the (hetero)arylene is optionally selected from halogen, X 2 R 4 、N(R 4 2. C 1-4 alkyl and substituted with one or more substituents of NO2, wherein each R 4 H, C alone 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 wherein R 12 It is β-glucuronide, PO3 (2-) 、OPO3 (2-) 、CO2 (-) 、SO3 (-) or N(C 1-4 Alkyl)3 (+) ; -R 2 is H, S(O)2OH or P(O)2OH and R 3 is OH, or R 2 and R 3 The oxazolidine ring is formed by fusion of the ether moiety; -R 5 is H or OCH3; -N ％ is N or N→O; -Y 5 It is CH2-Y, C(O)-Y, C(=N(R 20 ))-Y、C(R 9 )=NY、C(R 9 )=N(R 20 ), where R 9 Selected from optionally OH or O(CO)C 1-6 Alkyl substituted C 1-4 Alkyl, and R 20 YesNR 4 -C(O)-N(R 4 )2. NR 4 -C(O)-Sp-N(R 4 )2. NR 4 -C(O)-R 12 NR 4 -C(O)-Sp-R 12 , where Sp, R 4 and R 12 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、CH3、R 12 、Sp 3 R 12 NR 4 -Sp 3 -X 2 -Sp 3 -R 12 , OH, or CH2OH, where each Sp 3 It is a spacer; - and where Y 5 When it is C(O)-CH2OH, R 1 is not unsubstituted ethyl, CH2CH2SH or benzyl; or a salt thereof, wherein each ion, if present, is balanced with one or more pharmaceutically acceptable counterions.

12. The compound according to claim 11, wherein R 1 is selected from i-Pr, t-Bu, Bn, Sp-N3 or Sp-NH2, preferably wherein R 1 is i-Pr, Bn or Sp-N3, where Sp is C 1-4 Alkylene or C 1-4 Alkylene-arylene, preferably wherein Sp is CH2CH2 or CH2(4-Ph).

13. The compound according to claim 11 or 12, wherein R 2 and R 3 The oxazolidine ring is formed by linking together through the ether moiety.

14. A compound according to any one of claims 11-13, wherein Y is CH2OH.

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

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

17. The conjugate according to claim 16, wherein the compound according to structure (1) is conjugated to the cell binding agent via: (i)R 1 , preferably through R 1 = a nitrogen atom of Sp-NH2 or Sp-SH; or (ii) Y, preferably through Y = NH2 or NH-Sp 3 -NH2 or NH-Sp 3 -SH nitrogen atom.

18. The conjugate according to claim 16 or 17, which has the structure (2) as defined in claim 2, wherein LZ 2 Having a structure selected from (L1)-(L4): in: -Keys marked with * are connected to: (a) for (L1) and (L2), attached to the C(O) moiety adjacent to Y in the compound according to structure (1), and (b) For (L3) and (L4), the compound according to structure (1) 1 Some of the O atoms are connected; - The bond marked with ** is connected to the cell binding agent; -R 13 Selected from the group consisting of: hydrogen, 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, wherein these 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 The (hetero)arylalkyl group is optionally substituted and is optionally selected from O, S and NR 14 is interrupted by one or more heteroatoms, where R 14 independently selected from the group consisting of hydrogen and a C1-C4 alkyl group, or R 13 is D, or R, optionally linked to N via a spacer moiety 13 optionally linked to elsewhere in the linker via a spacer moiety to form a ring structure; -L 2 is a dipeptide, tripeptide or tetrapeptide; -o is 0 or 1; - Ring A is an optionally substituted 5-membered or 6-membered aromatic or heteroaromatic ring; -z1 is an integer in the range of 1-4; -z2 is 0 or 1.

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

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

21. A method of treating cancer, the method comprising administering to a subject in need thereof a conjugate according to any one of claims 1-9 and 16-18, wherein the cancer cells specifically express an extracellular receptor.

22. The method of claim 19 or 20, wherein the extracellular receptor 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, 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, guanylate 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, adhesion protein-4, Notch3, P-cadherin, P2X5, PD-L1, PMEL17, PRLR, PSCA, PSCA hlg, PSMA, PTK7, RET, RNF43, RON, ROR1, ROR2, Sema 5b, SLITRK6, SSTR2, STEAP1, STEAP2, TAG72, TENB2, TF, TIM-1, TM4SF, TMEFF, TMEM118, TMEM46, transferrin, TROP-2, TrpM4, TWEAKR, receptor tyrosine kinase (RTK), tenascin.

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