Hydrophilic tetrazine-functionalized payloads for the preparation of targeted conjugates

Abstract

The present invention relates to the field of bioconjugation of functional entities (payloads) to targeting agents, in particular biological targeting agents such as antibody drug conjugates (ADCs), in which one or more payload molecules are conjugated to a targeting agent, for example a monoclonal antibody. More specifically, the present invention relates to novel hydrophilic tetrazine molecules and their preparation, which allow more efficient conjugation of payload molecules to targeting agents such as monoclonal antibodies. The present invention also relates to certain tetrazine intermediates useful for the preparation of corresponding functionalized payload molecules. The present invention also relates to the respective conjugates, in particular bioconjugates, and methods for their preparation. The present invention also relates to the medical use of such conjugates of the present invention, to the corresponding pharmaceutical compositions, and to the corresponding diagnostic and analytical kits.

Description

[Technical field]

[0001] The present invention relates to the field of bioconjugation of entities (payloads) to targeting agents, particularly biological targeting agents, such as antibody-drug conjugates (ADCs), in which one or more payload molecules are conjugated to the targeting agent, for example a monoclonal antibody. More specifically, the present invention relates to novel hydrophilic tetrazine molecules and their preparation, which allow for more efficient conjugation of payload molecules to targeting agents, such as monoclonal antibodies. The present invention also relates to certain tetrazine intermediates useful for the preparation of corresponding functionalized payload molecules. The present invention also relates to the respective conjugates, particularly bioconjugates, and methods for their preparation. The present invention also relates to the use of such conjugates of the present invention in medicine, to the corresponding pharmaceutical compositions, and to the corresponding diagnostic and analytical kits. [Background technology]

[0002] ADCs are a rapidly growing class of oncology therapeutics that are attracting significant attention, which is reflected in the increasing number of approved ADC drugs and clinical trials.

[0003] The conjugation required to attach each payload to monoclonal antibodies (mAbs) is often performed by random conjugation. This typically results in heterogeneity with current ADC species, with drug-antibody ratios (DARs) ranging from fully unmodified mAbs to those with a number of unfavorable cytotoxic molecules attached. This can lead to batch-to-batch variability issues and associated side effects such as aggregation, rapid clearance, immunogenicity, and hepatotoxicity.

[0004] A solution to overcome these limitations is the production of homogenous ADC / radioimmunoconjugates (RICs) by site-specific conjugation, which allows for tight control of the DAR and pharmacokinetic properties and the production of uniform batches of the drug to be administered. This also improves the therapeutic index by eliminating the side effects of undesirable ADC species.

[0005] Another important component of any ADC is the linker used to attach the payload.

[0006] In general, there is a distinction between non-cleavable linkers, which release the cargo only after proteasomal degradation, and cleavable linkers, which release the parent drug upon enzymatic, reductive or acid cleavage. Furthermore, the chemistry of the linker also influences the properties of the active metabolites that are actually released. For example, increased hydrophilicity reduces the diffusion rate through membranes, increases intracellular retention and reduces susceptibility to multidrug resistance mechanisms in cancer cells.

[0007] The inventors have demonstrated the genetic encoding of strained cyclooctyne-lysine derivatives for click reactions to proteins (T.P.S., S. Mills, C. Koehler, C. Schultz, E.A. Lemke, Journal of the Deutsche Kammerschutz 2011, 50, 3878-81) and the synthesis and genetic encoding of ncAAs capable of (strain-promoted) inverse electron-demand Diels-Alder cycloadditions (IEDDA) with 1,2,4,5-tetrazines (T.P.S., S. Mills, C. Koehler, J. Zymanski, R. Muller et al., Journal of the Deutsche Kammerschutz 2012, 51, 4166-70; I. Nikic, T.P.S., O. Schreitt, J. Zymanski, J.A.G. Briggs et al., Journal of the Deutsche Kammerschutz 2012, 51, 4166-70). 2014, 53, 2245-9; E. Kozma, I. Nikic, B. R. Varga, I. V. Aramburu, J. H. Kang et al., ChemBioChem 2016, 17, 1518-24; G. E. Hoffman, T. P. S., I. Nikic, I. V. Aramburu, C. Koehler et al., Chem. Eur. J. 2015, 21, 12266-70). An extension of this GCE technology has made it possible to site-specifically introduce such ncAAs into non-glycosylated immunoglobulins produced by insect cells and subsequently modify them via this click chemistry (C. Koehler, P. F. Sauter, M. Wallis-Jin, G. E. Girona, K. Gupta et al., Nat. Methods 2016, 13, 997-1000).

[0008] Mao et al. described in the Journal of the German Chemical Society (2019), 58, 1106 an organocatalytic and scalable synthesis of unsymmetrical 1,2,4,5-tetrazines by a thiol-containing promoter. In particular, a series of unsymmetrically substituted 1,2,4,5-tetrazine derivatives were synthesized by applying 3-mercaptopropionic acid as a catalyst in the reaction of two different nitrile extracts with hydrazine hydrate in ethanolic solution, followed by oxidation with sodium nitrite. One particular compound was a 1,2,4,5-tetrazine derivative substituted at position 1 with a methylphosphonate group and at position 6 with a methyl group. This phosphonate precursor was further derivatized by the Horner-Wadsworth-Emmons reaction to introduce side chains containing different trans-alkene moieties.

[0009] US Patent Application Publication No. US2019247513A1 discloses tetrazine compounds and dienophiles capable of undergoing inverse electron demand Diels-Alder reactions with said tetrazines, as well as their use in bioorthogonal drug activation. In particular, compounds 333 and 14.5-14.7 relate to tetrazine derivatives that present fluorescent payloads, spaced from the tetrazine core by spacers of various chemical natures, such as aryl- or peptidyl- or alkyl-based spacers, as well as polyoxyalkyl-based hydrophilic groups.

[0010] International Publication WO2020 / 256544A1 relates to substituted tetrazines characterized by high click coupling yields: the tartrazine core is linked to a pyridyl-based spacer, which itself is linked via a glutaric group to a payload R87 (such as a cytotoxic drug) and / or a polyoxyalkyl hydrophilic group.

[0011] Mao et al. (Deutsche Kapitel Zeitung (2021), 60, 2393-2397) and International Publication WO 2020 / 239039 A1 disclose tetrazine compounds that include hydrophilic or chemical moieties capable of forming chemical bonds (such as carboxyl, hydroxyl or phosphate-based groups) spaced from the tetrazine core by an alkyl-based spacer. In that regard, Mao et al. also describe compounds in which the hydrophilic or chemical moieties capable of forming chemical bonds are replaced with fluorescent groups.

[0012] Chaignan, B. A. et al. (CHEMICAL ABSTRACTS SERVICE, Columbus, Ohio, USA, Database Accession No. 1983:453711) relates to tetrazines having an alkylsulfone group as the para-substituent.

[0013] International Publication No. WO2014 / 081301A1 discloses tetrazines in which the tetrazine core is linked to various hydrophilic or chemical moieties capable of forming chemical bonds via aryl-based spacers.

[0014] Oller-Salvia et al. (Deutsche Kapitel Zeitung (2018), 572831-2834) in the field of antibody-drug conjugates have disclosed a structure in which trastuzumab is site-selectively conjugated to a tetrazine-modified monomethyl auristatin E (MMAE) via an inverse electron-demand Diels-Alder cycloaddition reaction. The tetrazine handle used for this site-specific conjugation consists of an aryl moiety attached to a tetrazine core.

[0015] Handler et al. (Molecules 2021, 26, 4640) relate to the use of bioorthogonal reactions such as the IEDDA reaction in pre-targeting strategies. A variety of tetrazine surrogates are disclosed as suitable dienes, which are characterized by the presence of a p-substituted tetrazine core presenting alkyl and / or aryl spacers. Figure 2 of the disclosure relates to a classification of said tetrazine surrogates based on their respective reactivities.

[0016] There is a need for further improved 1,2,4,5-tetrazine derivatives that allow for more favorable conjugation of 1,2,4,5-tetrazine functionalized payload molecules to corresponding functionalized target molecules via bioorthogonal reactions. In particular, there is a need for further improved 1,2,4,5-tetrazine derivatives that allow such bioorthogonal reactions to proceed via strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC) in an aqueous, optionally buffered environment without significant steric hindrance. More specifically, there is a need for such tetrazine derivatives whose increased hydrophilicity allows for the conjugation of more hydrophobic payloads and, in the case of pharmacologically active conjugates, provides a better functional profile of such conjugates in vivo. Summary of the Invention

[0017] The above problems have been surprisingly solved by providing 1,2,4,5-tetrazine functionalized payload molecules with small hydrophilic groups, such as phosphonate residues, as tetrazine C-substituents. The present invention allows the easy use of payloads for SPIEDAC-mediated bioorthogonal bioconjugation in aqueous buffers without the need for the addition of potentially disruptive organic solvents and the associated use of sensitive biological agents. It also helps to prevent aggregation and improve the solubility of the formed bioconjugation agents. Due to their relatively small size, the smallest size payloads can be attached with little steric hindrance compared to larger solubilizing units such as PEG, glycosides, etc. For example, bulky PEG linkers are not necessarily required in the conjugates, thus reducing the size of the final payload or the respective targeting conjugates. Payloads of similar size based on the corresponding methyltetrazine groups cannot be conjugated to antibodies under the same conditions. It was also observed that the increased hydrophilicity in the claimed method can provide conjugates, especially ADCs, with improved pharmacokinetics, while at the same time the entire payload is better masked by the antibody due to the reduced size of the hydrophilic groups of the tetrazine. More specifically, the above problems are solved by providing a derivative of tetrazine having a phosphonate group, which further has a chemical moiety that allows coupling with a payload molecule, for example via amide coupling or carbamate formation. The corresponding tetrazine-functionalized payload molecule is represented by general formula I, as described herein below, and the corresponding tetrazine-functionalized intermediate is represented by general formula II, as described herein below. [Brief description of the drawings]

[0018] [Figure 1] IEDDA of TCO derivatives site-specifically incorporated into mAbs, including 1,2,4,5-tetrazine. [Diagram 2] Purification of trastuzumab A132TCO*-5. A: Superdex S200 run; B: Coomassie stained SDS-PAGE analyzing fractions 10-20 of the S200 run. [Diagram 3] Purification of trastuzumab A132TCO*-11: A Superdex S200 run, B: Coomassie-stained SDS-PAGE analyzing fractions 10-20 of the S200 run [Figure 4] Cytotoxicity assay. Measurements of trastuzumab-5 (=compound 5), trastuzumab-11 (=compound 11) and trastuzumab WT are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] A. Abbreviation ADC = Antibody Drug Conjugate APC = antibody payload complex aq. = aqueous Bps = base pairs BCN = 2-amino-6-(9-biocyclo[6.1.0]non-4-ynylmethoxycarbonylamino)hexanoic acid BOC = 2-amino-6-(tert-butoxycarbonylamino)hexanoic acid, in the example "BOC" specifically refers to (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoic acid = Boc-L-Lys- OH=N-α-tert-butyloxycarbonyl-L-lysine Conc. = concentrated DAR=drug-antibody ratio DCM = dichloromethane DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DIPEA = N,N-diisopropylethylamine DMF = Dimethylformamide DMSO = dimethyl sulfoxide EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide eq.=equivalent EtOH = ethanol GCE = Genetic Code Extension h=time HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, coupling agent HOBt = hydroxybenzotriazole IEDDA = inverse electron demand Diels-Alder cycloaddition kDa = kilodaltons min=minutes MMAE = monomethylauristatin E ((S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide), an antitumor agent MeOH = methanol ncAA = nonstandard amino acid NES = nuclear export signal NLS = nuclear localization signal O-tRNA = orthogonal tRNA O-RS = Orthogonal RS PBS = phosphate buffered saline PMSF = phenylmethylsulfonyl fluoride PNP chloroformate = 4-nitrophenyl chloroformate POI = Polypeptide of Interest pRS = prokaryotic RS ptRNA = prokaryotic tRNA PylRS = pyrrolysyl-tRNA synthetase PylRS AF = Mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase containing the amino acid substitutions Y306A and Y384F RCF (rcf) = relative centrifugal force RP-HPLC = Reversed Phase High Performance Liquid Chromatography RS = aminoacyl-tRNA synthetase RT = Room temperature / ambient temperature (20~25℃) SCO = 2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid [ka] SDS-PAGE = sodium sulfate polyacrylamide gel electrophoresis SPIEDAC = Strain-Promoted Inverse Electron Demand Diels-Alder Cycloaddition 5-TAMRA = 5-carboxytetramethylrhodamine 5-TAMRA-OSu = 5-carboxytetramethylrhodamine N-succinimidyl ester, fluorophore TCO = transcyclooctene TCO-Lys = N-ε-((trans-cyclooct-4-en-1-yloxy)carbonyl)-L-lysine TCO * -Lys = N-ε-((trans-cyclooct-2-en-1-yloxy)carbonyl)-L-lysine TCO # -Lys = N-ε-((trans-cyclooct-3-en-1-yloxy)carbonyl)-L-lysine TCO-E-Lys = N6-((((R,E)-cyclooct-4-en-1-yl)oxy)carbonyl)-L-lysine [ka] TCO * A-Lys = N6-((((S,E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine [ka] TFA = trifluoroacetic acid THF = tetrahydrofuran TLC = Thin Layer Chromatography tRNA Pyl = a tRNA that can be acylated with pyrrolysine by wild-type or modified PylRS and has an anticodon that is the reverse complement of the selector codon for site-specific incorporation of an ncAA into the POI. UNAA = unnatural amino acid, synonymous with ncAA U6 promoter = the promoter that normally controls the expression of U6RNA (a small nuclear RNA) in mammalian cells UHPLC-MS = Ultra High Performance Liquid Chromatography / Mass Spectrometry

[0020] B. Definition

[0021] B1. General Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0022] The terms "purified," "substantially purified," and "isolated" as used herein refer to the absence of other distinct compounds with which the compounds of the invention are normally associated in their natural state, and thus "purified," "substantially purified," and "isolated" refer to at least 0.5%, 1%, 5%, 10%, or 20% by weight of a given sample, or at least 50% or 75% by weight. In one embodiment, these terms refer to compounds of the invention that constitute at least 95, 96, 97, 98, 99, or 100% by weight of a given sample. As used herein, the terms "purified," "substantially purified," and "isolated" when referring to nucleic acids or proteins also refer to a state of purification or concentration different from that which occurs naturally in a prokaryotic or eukaryotic environment, such as a bacterial or fungal cell, or in a mammalian organism, particularly the human body. Included within the meaning of "isolated" is a degree of purification or enrichment greater than that which occurs naturally, including (1) purification from other associated structures or compounds, or (2) association with structures or compounds not normally associated in the prokaryotic or eukaryotic environment. The nucleic acids or proteins, or classes of nucleic acids or proteins, described herein may be isolated or associated with structures or compounds with which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.

[0023] In the context of this specification and the appended claims, the use of "or" means "and / or" unless specifically stated otherwise.

[0024] Similarly, the terms "containing," "comprises," "having," "including," "including," and "including" are interchangeable and are not intended to be limiting.

[0025] Furthermore, when the term "comprising" is used in describing various embodiments, those of skill in the art will understand that in certain cases, the embodiments can be described using the terms "consisting essentially of" or "consisting of."

[0026] The term "one or more," or the similar term "at least one," refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0027] Where a lower and upper limit of a numerical range is disclosed, each numerical value falling within that range, and any range encompassed by that upper and lower limit, is specifically disclosed. In particular, any range of values ​​disclosed herein should be understood to mean every value and narrower range that is included within that broader range.

[0028] The term "about" indicates a potential variation of ±25%, specifically ±15%, ±10%, and more specifically ±5%, ±2%, or ±1% of the stated value.

[0029] The term "substantially" refers to a range of values ​​such as about 80-100%, for example 85-99.9%, particularly 90-99.9%, more particularly 95-99.9%, or 98-99.9%, especially 99-99.9%.

[0030] The term "mainly" refers to a proportion in the range of 50% or more, for example, a range of 51 to 100%, particularly a range of 75 to 99.9%, and more specifically, 85 to 98.5%, 95 to 99%, and the like.

[0031] Where the present disclosure refers to features, parameters, and ranges thereof of differing preference (including general, not explicitly preferred, features, parameters, and ranges thereof), any combination of two or more of such features, parameters, and ranges thereof, regardless of their respective preferences, unless otherwise stated, is encompassed by the disclosure herein.

[0032] B2.Chemical definition The term halogen in each case denotes a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical.

[0033] "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 6, in particular 1 to 4 or 1, 2 or 3 carbon atoms. Examples include methyl, C1-C4-alkyl residues (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl or tert-butyl); n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl; n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl.

[0034] "Lower alkyl" refers to a straight or branched alkyl group having 1, 2, 3 or 4, especially 1 or 2, carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl or tert-butyl.

[0035] "Alkenyl" refers to a monovalent unsaturated hydrocarbon radical containing a single chemical carbon-carbon double bond having 2, 3, 4, 5, or 6 carbon atoms, such as vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, methallyl (2-methylprop-2-en-1-yl), and the like.

[0036] "Lower alkenyl" refers to a monovalent unsaturated hydrocarbon radical containing a single chemical carbon-carbon double bond having two, three or four carbon atoms, e.g., vinyl, allyl (2-propen-1-yl), 1-propen-1-yl, 2-propen-2-yl, methallyl (2-methylprop-2-en-1-yl), and the like.

[0037] "Alkynyl" and "lower alkynyl" are related to analogs of the alkenyl or lower alkenyl groups described above and refer to monovalent unsaturated hydrocarbon radicals containing a single chemical carbon-carbon triple bond.

[0038] "Alkylene" refers to straight or branched alkylene groups having 1 to 6, especially 1 to 4, carbon atoms. Examples include methylene, ethylene, 1,2-ethylene, 1,3-propylene, isopropylene, 1-4-butylene, 1-5-pentylene, 1-6-hexylene, and their branched analogs.

[0039] "Lower alkylene" refers to a straight or branched alkylene group having from 1 to 4 carbon atoms. Examples include methylene, ethylene, 1,2-ethylene, 1,3-propylene, isopropylene, 1-4-butylene, and their branched analogs.

[0040] "Alkoxy" refers to a radical of the formula RO-, where R is a straight or branched alkyl group as defined herein having 1 to 6, particularly 1 to 4 or 1 to 3, carbon atoms as defined herein. Non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, isobutoxy or tert-butoxy.

[0041] "Alkyleneoxy" refers to a radical of the formula -RO-, where R is a straight-chain or branched alkylene group having 1 to 6, particularly 1 to 4 or 1 to 3, carbon atoms as defined herein.

[0042] "Lower alkyleneoxy" refers to a radical of the formula -RO- where R is a straight or branched lower alkylene group having 1 to 4 or 1 to 3 carbon atoms as defined herein. Examples include methyleneoxy, ethyleneoxy, 1,2-ethyleneoxy, 1,3-propyleneoxy, isopropyleneoxy, and 1-4-butyleneoxy.

[0043] "Polyalkyleneoxy" relates to moieties comprising at least two, e.g. 2-20, 2-15, 2-10 or 2-5 repeat units of covalently bonded, identical or different, especially identical, lower alkyleneoxy groups having at least 2 carbon atoms as defined above, especially polyethyleneoxy and polypropyleneoxy groups having 2-20, 2-15, 2-10 or 2-5 identical repeat units.

[0044] "Alkenoxy" refers to a radical of the formula RO-, where R is a straight-chain or branched alkenyl group having 1 to 6, particularly 1 to 4 or 1 to 3, carbon atoms as defined herein.

[0045] "Alkanoyloxy" refers to a radical of the formula R-(CO)-O-, where R is a straight or branched alkyl group having 1 to 6, particularly 1 to 4 or 1 to 3, carbon atoms as defined herein.

[0046] "Alkylaminocarbonyloxy" refers to a radical of the formula R-NH-(CO)-O-, where R is a straight or branched alkyl group having 1 to 6, particularly 1 to 4 or 1 to 3, carbon atoms, as defined herein.

[0047] "Alkylthio" refers to a radical of the formula RS-, where R is an alkyl radical having one to four, preferably one to three, carbon atoms, as defined herein.

[0048] "Alkylamino" refers to a radical of the formula R-NH-, where R is an alkyl radical having 1 to 6, particularly 1 to 4, carbon atoms as defined herein. Examples include methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, 2-butylamino, isobutylamino, tert-butylamino, and the like.

[0049] "Dialkylamino" refers to a radical of the formula RR'N-, where R and R' are each independently an alkyl radical having 1 to 6, particularly 1 to 4, carbon atoms as defined herein. Examples include dimethylamino, diethylamino, N-methyl-N-ethylamino, and the like.

[0050] "Alkenylamino" refers to a radical of the formula R-NH-, where R is an alkenyl radical having 2 to 6, especially 2 to 4, carbon atoms as defined herein. Examples include vinylamino, allylamino (2-propen-1-ylamino), 1-propen-1-ylamino, 2-propen-2-ylamino, methallylamino (2-methylprop-2-en-1-ylamino), and the like.

[0051] "N-alkyl-N-alkenylamino" refers to a radical of the formula RR'N-, where R is an alkyl radical having 1 to 6, especially 1 to 4, carbon atoms as defined herein and R' is an alkenyl radical having 2 to 6, especially 2 to 4, carbon atoms as defined herein. Examples include N-methyl-N-vinylamino, N-methyl-N-allylamino (N-methyl-N-2-propen-1-ylamino), N-methyl-N-1-propen-1-ylamino, N-methyl-N-2-propen-2-ylamino, N-methyl-N-methallylamino (N-methyl-N-2-methylprop-2-en-1-ylamino), and the like.

[0052] "Dialkenylamino" refers to a radical of the formula RR'N-, where R and R' are each independently an alkyl radical having 2 to 6, in particular 2 to 4, carbon atoms as defined herein. Examples include divinylamino, diallylamino (di-(2-propen-1-yl)-amino), N-vinyl-N-allylamino, and the like.

[0053] "Aryl" refers specifically to a monovalent monocyclic or polycyclic aromatic moiety having six to fourteen ring carbon atoms, especially phenyl, fluorenyl, naphthenyl, and phenanthrenyl.

[0054] "Arylene" refers to the divalent analogues of the aryl groups listed above, particularly 1,2-, 1,3- and 1,4-phenylene.

[0055] "Halogen" refers to F, Cl, Br or I.

[0056] Unless otherwise stated, the term "substituents" is selected from halogen, C1-C4-alkyl, CN, CF3, hydroxyl, -O-CF3, C1-C4-alkoxy, C2-C4-alkanoyloxy, C1-C4-alkylaminocarbonyloxy and C1-C4-alkylthio, carboxy and carboxy-C1-C4-alkyl.

[0057] Unless otherwise stated, the term "substituted" means that a radical is substituted with 1, 2 or 3, particularly 1 or 2, substituents.

[0058] A "bond" is formed between two adjacent structural motifs of the compounds of the invention and, unless otherwise specified, is a chemical bond or is selected from an ether, thioether, ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide, or linear or branched polyalkylene oxide bond in any possible orientation.

[0059] An "ether" linkage contains at least one group of the (-O-) type.

[0060] A "thioether" bond contains at least one group of the (-S-) type.

[0061] An "amide" bond contains at least one group of the type -C(=O)N(R)- or -(R)NC(=O)-.

[0062] A "carbamate" linkage contains at least one group of the type -OC(=O)-N(R)- or -N(R)-C(=O)-O-.

[0063] A "dicarbamate" linkage contains at least one group of the type -N(R)-C(=O)O-R'-OC(=O)-N(R)-.

[0064] A "carbonate" bond contains at least one group of the type -C(=O)O-, -OC(=O)-, -OC(=O)O-, or -OC(=O)-O-.

[0065] A "hydrazine" linkage contains at least one group of the type --NH--NH--.

[0066] A "urea" linkage contains at least one group of the type -N(R)-C(=O)-N(R)-.

[0067] The "alkylene oxide" bond has -((CH2) n -O)- or -(O-(CH2) n )-type group, where n is 1, 2, 3 or 4, in particular 1 or 2.

[0068] A "polyalkylene oxide" linkage contains repeating units of the same or different alkylene oxide groups as defined above and can be linear or branched, especially linear, e.g., -((CH2) n -O) m - or -(O-(CH2) n ) m with n=1, 2, 3 or 4, in particular 1 or 2, and m=2-20, 2-15, 2-10 or 2-5.

[0069] In the above chemical formula of the particular bond, the residues R can, independently of one another, represent H or lower alkyl, lower alkenyl or lower alkenyl, in particular methyl, or ethyl, and R' represents a lower alkylene or lower alkenylene group, in particular methylene or ethylene.

[0070] "Cleavable group" includes any group that can be cleaved enzymatically or chemically, particularly under in vivo or in vitro conditions. Enzymatic cleavage can be achieved, for example, by the action of a protease, and chemical cleavage can be achieved, for example, by hydrolytic or reductive cleavage of an S-S bond.

[0071] A "tetrazine" group in the present invention, unless otherwise defined, refers to a residue consisting of a six-membered aromatic ring containing four nitrogen atoms and having the molecular formula -C2N4-, in particular derived from 1,2,4,5-tetrazine or an s-tetrazine isomer, and bonded to an adjacent group via ring carbon positions 3 and 6.

[0072] A "tetrazine reactive group" is a chemical moiety that has the ability to chemically react with a tetrazine group as defined herein, in particular via the so-called "bio-orthogonal" or "click reaction". In particular, such a tetrazine reactive group is selected from dienophiles. More specifically, it is selected from dienophiles that have the ability to react with a tetrazine group in a biological environment. Non-limiting examples include isonitrile groups, norbornene groups, bicyclononyl groups, cyclooctenyl groups, cyclooctynyl groups, cyclopropenyl groups, cyclobutynyl groups, spirohexenyl groups and their stereoisomers, alkenes or allyl groups or dihydroazeto groups.

[0073] "Tetrazine ligation" refers to the reaction of trans-cyclooctene with s-tetrazine in a reverse demand Diels-Alder reaction followed by the removal of nitrogen (N2) via a retro-Diels-Alder reaction. This type of reaction proceeds rapidly and allows the modification of biomolecules at extremely low concentrations.

[0074] The compounds described herein may contain one or more asymmetric elements, such as stereocenters, stereoaxes, for example, asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.These compounds may be, for example, racemic or optically active.All stereoisomers, diastereomers, Z-forms and E-forms are included in purified form and in mixture form.Therefore, when a compound is described by a specific name or a class of compounds is described, it is intended that all these forms are included.

[0075] The compounds described herein may also exist in the form of structural isomers, also called configurational or positional isomers, which are molecules that have the same overall chemical formula but differ only in the order of the atoms or atomic groups.

[0076] Thus, unless otherwise indicated, for each compound, biomolecule, and complex described herein, all such potential stereoisomeric or regioisomeric forms, or mixtures of multiple stereoisomeric and / or regioisomeric forms, are within the scope of the invention.

[0077] The "inverse electron demand Diels-Alder (IEDDA) cycloaddition" is a reaction between an electron-deficient diene and an electron-rich dienophile, and is just one example of the various types of "bioorthogonal reactions". The diene used can be a 1,2,4,5-tetrazine or a 1,2,4-triazine. The dienophiles include a variety of molecules containing strained cyclic alkenes, such as transcyclooctene (TCO, norbornene, cyclopropene, azetine). Of these, the reaction of tetrazine with TCO is the fastest reported to date and is suitable for in vivo applications (Smake et al., Current Opinion in Chemical Biology Volume 60, February 2021, pp. 79-88).

[0078] The term "bioorthogonal" refers to chemical reactions that can occur inside a living system, i.e., in an aqueous environment, without interfering with native biochemical processes. For example, the "tetrazine ligation reaction" is one type of bioorthogonal reaction. Bioorthogonal chemistry typically proceeds in two steps. First, a cellular substrate is modified with a bioorthogonal functional group (also called a chemical reporter), such as one of the "tetrazine reactive groups" identified above. Cellular substrates include, for example, immunoglobulins, such as natural antibodies and recombinant antibodies. The chemical reporter must not dramatically change the structure of the substrate so as not to affect the biological activity of the substrate. In the second step, a probe containing a complementary functional group, such as a tetrazine group as described herein, is introduced to react with and label the substrate.

[0079] The "acid or base addition salts" of the compounds of the present invention are particularly addition salts with physiologically acceptable acids or bases. Physiologically acceptable acid addition salts can be formed by treating the base forms of the compounds of the present invention with appropriate organic or inorganic acids. Compounds of the present invention that contain acidic protons can be converted into non-toxic metal or amine addition salt forms by treating with appropriate organic and inorganic bases. The compounds and salts of the present invention also include hydrates and solvent addition forms, such as hydrates, alcoholates, and the like.

[0080] A "physiologically acceptable" acid or base is, in particular, an acid or base that is tolerated by the system used to incorporate the first and second dienophiles (e.g., a biological system such as a translation system used to prepare a polypeptide containing a transcyclooctenyl or cyclooctynyl group), and is, for example, substantially non-toxic to living cells.

[0081] A "pharmaceutical composition" comprises, in addition to the ADC of the invention, one or more substances selected from the group consisting of a pharma- ceutical acceptable preservative, a pharma- ceutical acceptable colorant, a pharma- ceutical acceptable protective colloid, a pharma- ceutical acceptable pH adjusting agent, and a pharma- ceutical acceptable osmolality adjusting agent. Such substances are described in the art. A more detailed description of the pharmaceutical compositions of the invention is provided below.

[0082] As used herein, the term "effective amount" refers to the amount of a therapy sufficient to reduce or alleviate the severity and / or duration of a disease or one or more symptoms thereof, prevent the progression of a disease, cause regression of a disease, prevent the recurrence, onset, development or progression of one or more symptoms associated with a disease, detect a disease, or enhance or improve the prophylactic or therapeutic effects of another therapy (e.g., a prophylactic or therapeutic agent).

[0083] B.3 Biochemical definition A "polypeptide" is an oligomer of amino acid residues (natural or non-natural, or a combination thereof) of any length, usually but not necessarily linked by covalent peptide bonds. A polypeptide can be obtained from any source, such as a naturally occurring polypeptide, a polypeptide produced by recombinant molecular genetic techniques, a polypeptide from a cell or translation system, or a polypeptide produced by cell-free synthetic means. A polypeptide is characterized by its amino acid sequence, e.g., the primary structure of its constituent amino acid residues. As used herein, the amino acid sequence of a polypeptide is not limited to a full-length sequence, but may be a partial sequence or a complete sequence. Furthermore, it is not intended that a polypeptide be limited by whether or not it has a particular biological activity.

[0084] As used herein, the term "protein" is synonymous with polypeptide. The term "peptide" refers to small polypeptides, for example, but not limited to, those ranging from 2 to 25 amino acids in length.

[0085] In certain embodiments, proteins incorporating at least one ncAA in their amino acid sequence are utilized to form a "targeting agent". The primary purpose of such a targeting agent is the formation of a covalent or non-covalent bond with a specific "target". The secondary purpose of the targeting agent is the targeted transport of a "payload molecule" to said target. To achieve said secondary purpose, said targeting agent must be associated (reversibly or irreversibly) with at least one "payload molecule". For this purpose, said targeting agent is functionalized with said at least one ncAA. The functionalized targeting agent with said at least one ncAA can be linked to said at least one payload molecule by bioconjugation via said ncAA residue. The ncAA is reactive with the payload molecule, which bears a corresponding moiety, in this case a specific tetrazine moiety that reacts with the ncAA residue of the targeting agent. The bioconjugate thus obtained allows the transfer of the payload molecule to the desired target.

[0086] As used herein, the term "incorporating an unnatural amino acid," e.g., when incorporated into a target polypeptide, refers to the direct addition of the unnatural amino acid to a growing polypeptide chain during the primary assembly of the target polypeptide, e.g., via translation or chemical synthesis.

[0087] Unnatural amino acids ("UNAA") can be directly incorporated into a target polypeptide using any of a number of methods known in the art. In many embodiments, an orthogonal translation system is used as a route to directly incorporate the unnatural amino acid, although other methods of direct incorporation (e.g., in vitro translation systems, solid phase synthesis, etc.) can alternatively be used. It will be understood that in typical embodiments herein, the unnatural amino acid is preferably incorporated into the target polypeptide, i.e., incorporated during construction of the polypeptide, and not added by post-translational chemical derivatization.

[0088] In certain embodiments described herein, unnatural amino acids can be site-specifically incorporated into target polypeptides with high efficiency and fidelity using "orthogonal tRNA / aminoacyl-tRNA synthetase pairs."

[0089] The term "translation system" refers to the components necessary to incorporate amino acids into a growing polypeptide chain (protein). Components of a translation system include, for example, ribosomes, tRNAs, synthetases, mRNAs, etc. A translation system can be an in vivo translation system or an in vitro translation system.

[0090] "In vitro translation system" may be a cell-free translation system. A cell-free translation system is a system that synthesizes a desired protein by obtaining the protein factors required for mRNA translation, for example in the form of a cell extract, and then reconstituting this reaction in vitro. Such cell-free systems and their use in protein synthesis are known in the art. Examples include E. coli extract, wheat germ extract, or rabbit reticulocyte lysate (Spirin and Lord Waltz, Cell-Free Protein Synthesis, Willie VCH Verlag, Weinheim, Germany, 2008).

[0091] An aminoacyl-tRNA synthetase (RS) is an enzyme capable of acylating a tRNA with an amino acid or amino acid analog. Conveniently, the RS used in the methods of the invention is capable of acylating a tRNA with an unnatural amino acid.

[0092] The methods of the present invention advantageously utilize a "tRNA / aminoacyl-tRNA synthetase (tRNA / RS) pair." Preferably, the tRNA / RS pair used in the processes of the present invention is orthogonal to the translation system.

[0093] The term "orthogonal" as used herein refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl-tRNA synthetase (O-RS)) that is used with reduced efficiency by a translation system (e.g., a cell) of interest. Orthogonal refers to an orthogonal tRNA or orthogonal aminoacyl-tRNA synthetase that is unable to function with an endogenous aminoacyl-tRNA synthetase or endogenous tRNA of the translation system of interest or with reduced efficiency (e.g., less than 20%, less than 10%, less than 5%, or less than 1%). For example, an orthogonal tRNA in a translation system of interest is acylated by any endogenous aminoacyl-tRNA synthetase of the translation system of interest, but with reduced or no efficiency compared to when an endogenous tRNA is acylated by an endogenous aminoacyl-tRNA synthetase. In another example, the orthogonal aminoacyl-tRNA synthetase acylates any endogenous tRNA in a translation system of interest, but with reduced or no efficiency compared to acylation of the endogenous tRNA by the endogenous aminoacyl-tRNA synthetase.

[0094] Orthogonal tRNA / RS pairs used in the methods of the invention preferably have the following properties: the O-tRNA is preferentially acylated by the O-RS with an unnatural amino acid of the invention. Furthermore, the orthogonal pair functions in a translation system of interest, e.g., the translation system uses the unnatural amino acid acylated O-tRNA to incorporate an unnatural amino acid of the invention into a polypeptide chain. Incorporation occurs in a site-specific manner, e.g., the O-tRNA recognizes a selector codon, e.g., an amber stop codon, in an mRNA encoding a polypeptide.

[0095] The term "preferentially acylates" means that the O-RS acylates the O-tRNA with the unnatural amino acid with, e.g., about 50% efficiency, about 70% efficiency, about 75% efficiency, about 85% efficiency, about 90% efficiency, about 95% efficiency, or about 99% or greater efficiency, as compared to the endogenous tRNA or amino acid of the translation system of interest. The unnatural amino acid is then incorporated into the growing polypeptide chain with high fidelity, e.g., greater than about 75% efficiency for a given selector codon, greater than about 80% efficiency for a given selector codon, greater than about 90% efficiency for a given selector codon, greater than about 95% efficiency for a given selector codon, or greater than about 99% efficiency for a given selector codon.

[0096] The term "selector codon" refers to a codon that is recognized by the O-tRNA in the translation process and not by endogenous tRNAs. The O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates the amino acid, e.g., an unnatural amino acid, at this site in the polypeptide. Selector codons include, for example, nonsense codons, e.g., stop codons, e.g., amber, ochre, and opal codons, four or more base codons, codons derived from natural or unnatural base pairs, and the like. In a given system, the selector codon may also include one of the natural three-base codons (i.e., a natural triplet), in which case the endogenous system does not use the natural triplet, e.g., a system lacking a tRNA that recognizes the natural triplet, or a system in which the natural triplet is a rare codon.

[0097] An "anti-codon" has the reverse complementary sequence of the corresponding codon.

[0098] An O-tRNA / O-RS pair is composed of an O-tRNA, such as a suppressor tRNA, and an O-RS.

[0099] A "suppressor tRNA" is a tRNA that alters the reading of messenger RNA (mRNA) in a particular translation system. A suppressor tRNA can, for example, read a stop codon, a four-base codon, or a rare codon.

[0100] The O-tRNA is not acylated by endogenous synthetases and is capable of decoding a selector codon as described herein.

[0101] The O-RS recognizes, for example, an O-tRNA that has an extended anticodon loop and preferentially acylates the O-tRNA with an unnatural amino acid.

[0102] The tRNA and RS used in the process of the present invention are naturally occurring or derived by mutation of naturally occurring tRNA and / or RS from various organisms.In various embodiments, tRNA and RS are from at least one organism.In another embodiment, tRNA is derived from naturally occurring tRNA or mutated naturally occurring tRNA from a first organism, and RS is derived from naturally occurring RS or mutated naturally occurring RS from a second organism.

[0103] A suitable tRNA / RS pair can be selected from a library of mutant tRNAs and RSs, for example based on the results of a library screening. Alternatively, a suitable tRNA / RS pair can be a heterologous tRNA / synthetase pair imported into the translation system from a source species. The cells used as the translation system are preferably cells different from the source species.

[0104] For example, suitable orthogonal O-tRNAs may be used in the preparation of tRNAs for the purposes of Methanococcus jannasii, Methanobacterium thermoautotrophicum, Halobacterium (such as Haloferax volcanii and Halobacterium species NRC-I), Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyram pernicus, Methanococcus maripaludis, Methanopyrus candlerii, Methanosarcina spp., and the like. Orthogonal O-RSs can be derived from archaea such as M. zei (Mm), Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus (Ss), Sulfolobus kodai, Thermoplasma acidophilum, and Thermoplasma volcanium, and eubacteria such as Escherichia coli, Thermus thermophilus, Bacillus subtilis, and Bacillus stearothermophilus, while orthogonal O-RSs can be derived from e.g., Methanococcus jannasci. The organisms may be derived from organisms or combinations of organisms such as archaea, such as Methanobacterium thermoautotrophicum, halobacteria, such as Haloferax volcanii and Halobacterium species NRC-J, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, Methanococcus maripaludis, Methanopylus kandleri, Methanosarcina mazei, Methanosarcina bakeri, Methanosarcina hafniense, Pyrobaculum aerophilum, Pyrococcus abyssi, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium, and the like, or eubacteria, such as Escherichia coli, Thermus thermophilus, Bacillus subtilis, and Bacillus stearothermophilus. In one embodiment, eukaryotic sources, such as plants, algae, protists, fungi, yeast, animals, such as mammals, insects, arthropods, etc., can also be used as a source of the O-tRNA and O-RS.

[0105] Methods for evolving tRNA / RS pairs are described, for example, in International Publication Nos. WO 02 / 085923 and WO 02 / 06075.

[0106] Preferably, the RS is a pyrrolysyl-tRNA synthetase (pylRS) capable of acylation of tRNA with an unnatural amino acid of the invention. The pyrrolysyl-tRNA synthetase used in the methods of the invention is a wild-type or engineered pylRS. Examples of wild-type pylRS include, but are not limited to, pylRS from archaea and eubacteria, such as Methanosarcina mazei, Methanosarcina barkeri, Methanococcoides bartoniae, Methanosarcina acetivorans, Methanosarcina thermophila, and Desulfitobacterium hafniense.

[0107] Pyrrolysyl-tRNA synthetase (PylRS) is an aminoacyl-tRNA synthetase (RS). RS is an enzyme capable of acylation of tRNA with an amino acid or an amino acid analogue. Conveniently, the PylRS of the present invention is enzymatically active, i.e., capable of acylation of tRNA with a specific amino acid or an amino acid analogue, preferably UNAA or a salt thereof (tRNA Pyl ) can be acylated.

[0108] As used herein, the term "archaeal pyrrolysyl-tRNA synthetase" (abbreviated as "archaeal PylRS") refers to a PylRS in which at least a portion of the PylRS amino acid sequence, or the entire PylRS amino acid sequence, has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of a native PylRS from an archaea, or to the amino acid sequence of an enzymatically active fragment of such a native PylRS.

[0109] The PylRS of the present invention may comprise a mutant archaeal PylRS, or an enzymatically active fragment thereof.

[0110] Generally, a "mutant archaeal PylRS" or "mutant archaeal PylRS" differs from the corresponding wild-type PylRS by including the addition, substitution, and / or deletion of one or more amino acid residues. Preferably, these are modifications that improve PylRS stability, alter PylRS substrate specificity, and / or enhance PylRS enzymatic activity. Particularly preferred "mutant archaeal PylRS" or "mutant archaeal PylRS" are described in more detail below.

[0111] The term "nuclear export signal" (abbreviated "NES") refers to an amino acid sequence capable of directing the export of a polypeptide containing it (such as the NES-containing PylRS of the present invention) from the nucleus of a eukaryotic cell. This export is believed to be primarily mediated by Crm1 (chromosomal region maintenance 1, also known as karyopherin exportin 1). NESs are known in the art. For example, the database ValidNES (http: / / validness.ym.edu.tw / ) provides sequence information for experimentally validated NES-containing proteins. Furthermore, NES databases such as NESbase1.0 (see www.cbs.dtu.dk / databased / NESbase-1.0 / ; La Cour et al., NuclAcidsRes 31(1), 2003), NetNES (see www.cbs.dtu.dk / services / NetNES / ; La Cour et al., La Cour et al., ProteinEngDesSel 17(6):527-536, 2004), NESpredictor (NetNES, http: / / www.cbs.dtu.dk / ; Hu et al., NuclAcidsRes 41:D338-D343, 2013; La Cour et al., ProteinEngDesSel 17(6):527-536, 04)) and NESsential (a web interface in combination with ValidNES) are publicly available. Hydrophobic leucine-rich NESs are the most common and best characterized group of NESs to date. Hydrophobic leucine-rich NESs are nonconserved motifs with three or four hydrophobic residues. Many of these NESs are composed of the conserved amino acid sequence pattern LxxLxL (SEQ ID NO:111) or LxxxLxL (SEQ ID NO:112), where each L is independently selected from leucine, isoleucine, valine, phenylalanine, and methionine amino acid residues, and each x is independently selected from any amino acid (see La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004).

[0112] The term "nuclear localization signal" (abbreviated "NLS", also referred to in the art as "nuclear localization sequence") refers to an amino acid sequence that can direct the transport of a polypeptide that contains it (e.g., wild-type archaeal PylRS) to the nucleus of a eukaryotic cell. This transport is thought to be mediated by the binding of the NLS-containing polypeptide to importins (also known as karyopherins) to form a complex that passes through nuclear pores. NLSs are known in the art. There are many publicly available NLS databases and NLS prediction tools, including NLSdb (see Nair et al., NuclAcidsRes 31(1), 2003), cNLSMapper (see www.nls-mapper.aib.keio.ac.jp; Kosugi et al., ProcNatlAcadSciUSA. 106(25):10171-10176, 2009; Kosugi et al., JBiolChem 284(1):478-485, 2009), SeqNLS (see Lin et al., PLoSOne 8(10):e76864, 2013), and NucPred (see www.sbc.su.se / ~maccallr / nucpred / ; Brammaire et al., Bioinformatics 23(9):1159-60, 2007).

[0113] The mutant archaeal PylRS of the invention as defined above can be further modified by removing any NLS present in said native PylRS from which the mutant is derived and / or by introducing at least one NES, which can be identified using known NLS detection tools, such as, for example, cNLSMapper.

[0114] Removal of the NLS and / or introduction of an NES from an archaeal PylRS or a mutant thereof can alter the localization of the thus modified polypeptide when expressed in a eukaryotic cell, and in particular can avoid or reduce accumulation of the polypeptide in the nucleus of the eukaryotic cell. Thus, the localization of the PylRS mutant of the invention expressed in a eukaryotic cell can be altered compared to a PylRS or PylRS mutant, which differs from the PylRS mutant of the invention in that it (still) contains an NLS and lacks an NES.

[0115] When an archaeal PylRS of the invention contains an NES but (still) contains an NLS, the NES is preferably selected such that the strength of the NES exceeds that of the NLS, preventing accumulation of the PylRS in the nucleus of a eukaryotic cell.

[0116] When the NLS is removed from the wild-type or mutant PylRS and / or the NES is introduced into the wild-type or mutant PylRS to obtain the PylRS of the present invention, the PylRS enzyme activity is not lost. Preferably, the PylRS enzyme activity is maintained at essentially the same level, i.e., the PylRS of the present invention has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the enzyme activity of the corresponding wild-type or mutant PylRS.

[0117] The NES is appropriately positioned within the PylRS or mutant PylRS of the present invention so that the NES is functional. For example, the NES can be bound to the C-terminus (e.g., C-terminus of the last amino acid residue) or N-terminus (e.g., between amino acid residue 1, the N-terminal methionine, and amino acid residue 2) of the wild-type or mutant archaeal PylRS.

[0118] The disclosure of International Publication No. WO2018 / 06948, which discloses mutant PylRS modified by the incorporation of an NES and / or deletion of an NLS sequence, is expressly incorporated by reference herein.

[0119] C. Specific Embodiments The tetrazine compounds of the present invention having the general formula (I) described below are used for the preparation of conjugates, in particular bioconjugates. For this purpose, the compounds of formula (I) react via their functional tetrazine moiety with a suitable tetrazine reactive second functional group of a conjugation partner, such as, for example, a biological molecule, in particular a target molecule, for example, an antibody molecule.

[0120] The tetrazine reactive functional group can be, for example, a cyclooctynyl or transcyclooctenyl group.

[0121] The invention provides methods for preparing such polypeptides in vivo or in vitro. In particular, the tetrazine reactive functional group can be translationally incorporated into a polypeptide encoded by a polynucleotide that includes one or more selector codons.

[0122] The present invention relates to the following main aspects and specific embodiments thereof:

[0123] 1. First Aspect of the Invention A first aspect of the present invention relates to payload molecules functionalized with specific tetrazine groups, which are suitable for conjugation with a second molecule, in particular a biomolecule, having a functional corresponding group that reacts with the tetrazine group of the functionalized payload molecule.

[0124] According to a first embodiment of the present invention, there is provided a tetrazine-functionalized compound of general formula I: [ka] Where: m is 0 or 1. n represents an integer selected from 1 and 2. o is 0 or an integer selected from 1 or 2; A represents a cleavable moiety. Sp 1 and Sp2 represent, independently of each other, a spacer moiety. X represents the self-immolative portion. Y represents the payload residue (or cargo). Z represents a hydrophilic group.

[0125] According to a particular embodiment, m is 0. According to another particular embodiment, m is 1. According to another particular embodiment, n is 1. According to another particular embodiment, n is 2.

[0126] According to another particular embodiment, o is 0. According to another particular embodiment, o is 1. According to another particular embodiment, o is 2.

[0127] According to another particular embodiment, Sp 1 and Sp 2 is different. According to another particular embodiment, Sp 1 and Sp 2 are identical.

[0128] m, n, o, and Sp 1 and Sp 2 Specific examples of parameter combinations are as follows:

[0129] [Table 1]

[0130] According to a second embodiment of the invention, the residue Z in general formula I of the compound of the first embodiment is selected from one of the following hydrophilic groups:

[0131] a) phosphorus- and / or sulfur-containing hydrophilic moieties Z, in particular (R 1 O)2P(O)-, (R 1a O)2P(O)-O-, (R 2 O) 3P-O-, R 3S(O)2-, (R 4 O)S(O)2O-, or (R 4 aO)S(O)2- Where: residue R 1 ~R 4 , R 1a and R 4a are the same or different and independently of one another represent H or lower alkyl, in particular methyl or ethyl, or a salt form of said phosphorus and / or sulfur containing hydrophilic moiety, such as a physiologically acceptable salt.

[0132] b) linear or branched mono- or polyalkylene oxide moieties Z, in particular linear moieties -((CH2) x -O) y -R 5 , -(O-(CH2) x ) y -H and -(O-(CH2) x ) y -OR 6 and branched chain analogs thereof; Where: residue R 5 and R 6 represent, independently of one another, H or lower alkyl, in particular H, methyl or ethyl, x's each independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, The y's independently represent an integer from 1 to 20, in particular from 1 to 15, 1 to 10 or 1 to 4, for example 1, 2, 3 or 4.

[0133] Regarding group a According to a particular embodiment, Z is (R 1 O)2P(O)-, (R 1a O)2P(O)-O- or (R 2 O)3P-O-, more specifically (R 1 O)P(O)-, Where: residue R 1 , R 2 , and R 1aare independently the same or different and represent H, methyl, or ethyl.

[0134] Regarding group b According to another particular embodiment, Z is a linear mono- or polyalkylene oxide moiety. According to yet another particular embodiment, Z is a linear moiety -((CH2) x -O) y -R 5 , -(O-(CH2) x ) y -H, and -(O-(CH2) x ) y -OR 6 wherein the residue R 5 and R 6 are each independently H, methyl or ethyl, According to yet another particular embodiment, the x's represent, independently of one another, an integer selected from 1 or 2.

[0135] Parameters Z, m, n, o and Sp 1 and Sp 2 Specific examples of parameter combinations are as follows:

[0136] [Table 2]

[0137] Here, R 1 is H or lower alkyl; or

[0138] [Table 3]

[0139] or

[0140] [Table 4]

[0141] or

[0142] [Table 5]

[0143] Z, m, n, o, and Sp 1 and Sp 2 Further specific examples of parameter combinations are as follows:

[0144] [Table 6]

[0145] where residue R 6 represents H or lower alkyl, in particular H, methyl or ethyl; x represents an integer selected from 1 or 2; y represents an integer of 1 to 20, in particular 1 to 15, 1 to 10 or 1 to 4, for example 1, 2, 3 or 4.

[0146] Z, x, y, R 6 , m, n, o, and Sp 1 and Sp 2 Specific examples of parameter combinations are as follows:

[0147] [Table 7]

[0148] or

[0149] [Table 8]

[0150] or

[0151] [Table 9]

[0152] or

[0153] [Table 10]

[0154] According to a third embodiment of the present invention, the spacer Sp of the compound of formula I according to any of the previous embodiments is 1 is absent or, more specifically, is selected from:

[0155] a) monocyclic or polycyclic, optionally mono- or polysubstituted aromatic moieties having 6 to 14 ring carbon atoms, in particular 1,4-phenylene, where said one or more optional substituents are selected independently from one another from -Hal, -CHal3, -OH, -SH, -NR'2, -NO2, -CN, -C(=O)R'', -C(=O)OR''', alkyl, alkenyl, alkynyl and alkoxy, Where: R', R'', and R''', independently of one another, are selected from H and C1-C4-alkyl (moiety M1).

[0156] b) a heterocyclic residue of general formula X, [ka] Where: One, two or three of the ring moieties X1 to X4 represent N and the others represent >CH (moiety M2).

[0157] c) Linear or branched lower alkylene, especially -(CH2) n1 -, where n1 is an integer from 1 to 4, and more particularly methylene (moiety M3).

[0158] d) a combination of at least two identical or, more specifically, different moieties selected from M1, M2, and M3.

[0159] Regarding part M1 According to a particular embodiment, the moiety M1 is a monocyclic unsubstituted aromatic moiety having 6 carbon atoms, in particular 1,4-phenylene, or [ka]

[0160] Regarding part M2 According to a particular embodiment, the moiety M2 represents a heterocyclic residue of general formula X, in which One or two of the ring moieties X1 to X4 represent N and the other represents >CH.

[0161] Regarding part M3 According to a particular embodiment, the moiety M3 is a straight chain lower alkylene, in particular -(CH2) n1 -, where n1 is an integer of 1 or 2, and more specifically is methylene.

[0162] According to a fourth embodiment of the present invention, the spacer Sp of compound (I) of any of the previous embodiments is 2 is selected from the following:

[0163] a) monocyclic or polycyclic, optionally mono- or polysubstituted aromatic moieties having 6 to 14 ring carbon atoms, in particular 1,2-phenylene, 1,3-phenylene or 1,4-phenylene, wherein said one or more optional substituents are selected independently of one another from -Hal, -CHal3, -OH, -SH, -NR'2, NO2, -CN, -C(=O)R'', -C(=O)OR''', alkyl, alkenyl, alkynyl and alkoxy, where R', R'' and R''' are independently selected from H and C1-C4 alkyl (moiety M1).

[0164] b) a heterocyclic residue of general formula X, [ka] Here, one, two or three of the ring moieties X1 to X4 represent N, and the other one represents >CH (moiety M2).

[0165] c) linear or branched lower alkylene, especially -(CH2) n1 - and where n1 is an integer from 1 to 4, more specifically methylene (moiety M3).

[0166] d) Linear or branched polyalkylene oxide moieties, in particular linear moieties -((CH2) x1 -O) y1 - or -(O-(CH2) x1 ) y1 - and branched analogs thereof, Where: x1 independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2, and y1 independently represent an integer from 1 to 20, in particular from 1 to 4 (moiety M4).

[0167] e) a heteroatom-containing moiety is -N(R'''')-, -(CH2) x2 -N(R'''')-, -N(R'''')-(CH2) x3 -C(O)O-, -N(R'''')-(CH2) x3 -C(O)-, -N(R'''')-(CH2) x4 -N(R'''')-, -N(R'''')-C(O)-(CH2) x4 -N(R'''')-, -(CH2) x4 -C(O)-, and -(CH2) x4 -C(O)-, Where: R'''' are each independently selected from H and C1-C4 alkyl; x2 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2; x3 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2; and x4 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2 (moiety M5).

[0168] or f) a combination of at least two identical or more specifically different moieties selected from M1, M2, M3 and M4, or a combination of at least two identical or more specifically different moieties selected from M1, M2, M3, M4 and M5.

[0169] Regarding part M1 According to a particular embodiment, the moiety M1 is a monocyclic unsubstituted aromatic moiety having 6 carbon atoms, in particular 1,2-phenylene, 1,3-phenylene or 1,4-phenylene, more particularly 1,4-phenylene, or [ka]

[0170] Regarding part M2 According to a particular embodiment, the moiety M2 represents a heterocyclic residue of general formula X, in which One or two of the ring moieties X1 to X4 represent N, and the other represents >CH.

[0171] Regarding part M3 According to a particular embodiment, the moiety M3 is a straight chain lower alkylene, in particular -(CH2) n1 -, where n1 is an integer of 1 or 2, and more specifically is methylene.

[0172] Regarding part M4 According to a particular embodiment, moiety 4 represents a linear polyalkylene oxide moiety, the linear moiety -((CH2) x1 -O) y1 - or -(O-(CH2) x1 )y1 - selected from Where: x1 each independently represent an integer selected from 1 or 2; and y1 independently represent an integer from 2 to 20, in particular from 2 to 4.

[0173] Regarding part M5 According to certain embodiments, moiety 5 is -N(R'''')-, -N(R'''')-(CH2) x3 -C(O)- or -(CH2) x2 -N(R'''')-, Where: R"" are independently selected from H and C1-C4-alkyl, more particularly H; x2 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2.

[0174] According to a fifth embodiment of the invention, said group A of compound (I) of any of the previous embodiments is an enzymatically or chemically cleavable linker group and is selected from:

[0175] a) Peptidyl groups, in particular di-, tri- or tetrapeptidyl groups.

[0176] b) Formula - (CR 7 R 8 ) n2 -SS-(CR 7 R 8 ) n2 -X5- or X 5’ -(CR 7 R 8 ) n2 -SS-(CR 7 R 8 ) n2 -X5- disulfide group. where n2 represents an integer from 1 to 4, residue R 7 and R 8 are selected independently of one another from H or lower alkyl, in particular methyl, or two residues R 7 and R8 together with the carbon atom to which they are attached form a cyclic C4 to C8 alkyl group, and the moiety X5 is selected from -C(O)- and -O-; Part X 5’ is selected from -C(O)- and -(O)C-(CH)-NH-.

[0177] c) The hydrazone group is >C=NN(R 9 )- and -N(R 9 )-N=C< Where: R 9 is H or lower alkyl, and

[0178] d) β-glucuronidase-sensitive cleavable linker groups (glucuronide linker groups), in particular those having a β-glucuronic acid derived trigger residue.

[0179] According to a particular embodiment, the cleavable linker is a peptidyl group according to feature a). According to another particular embodiment, the cleavable linker is a glucuronide linker group according to feature d).

[0180] According to a sixth embodiment of the present invention, said self-immolative group X of compound (I) of any of the previous embodiments is selected from:

[0181] a) A group represented by the formula p-aminobenzyl alcohol (PAB) -NH-p-phenylene-CH2-O- or -O-CH2-p-phenylene-NH- or -NH-p-phenylene-CH2-N + (R 20 )2-

[0182] b) -OC(O)-O-

[0183] c)-OC(O)-NR 10 -(CR 12 R 13 ) z -NR11 -C(O)-O- or -X 1 -C(O)-NR 10 -(CR 12 R 13 ) z -NR 11 -C(O)-X 2 - in which z represents an integer selected from 1 to 6, in particular from 1 to 4, R 20 represent, independently of each other, H or a lower alkyl group, R 10 and R 11 represent, independently of each other, H or a lower alkyl group, R 12 and R 13 denote, independently of one another, H, methyl or ethyl, in particular H or methyl, in particular H, and X 1 and X 2 are independently O, S or NR 10 Represents.

[0184] d) Methylene alkoxycarbamate (MAC) type bond -OC(O)-NR 13 -C(R 14 R 15 )-(O)- -OC(O)-NR 13 -C(R 14 R 15 )-(S)- -OC(O)-NR 13 -C(R 14 R 15 )-(NR 16 )-or -OC(O)-NR 13 -C(R 14 R 15 )-(NR 16 -C(O)O)- where R 13 , R 14 , R 15 , and R 16 are each independently H or lower alkyl, in particular C1-C4 alkyl.

[0185] According to a particular embodiment, said self-immolative group X is a PAB-derived group according to feature a).

[0186] According to a seventh embodiment of the invention, the payload moiety Y of the compound of any of the previous embodiments is selected from biologically active compounds, in particular labelling agents such as dyes, radiolabels, proteolytic agents, photosensitisers and chelators.

[0187] According to an eighth embodiment of the invention, the compound of any of the previous embodiments corresponds to a compound of general formula Ia: [ka] wherein the bonds α, β, γ and δ are selected independently from one another from a chemical bond or an ether, thioether, ester, amide, carbamate, carbonyl (especially keto), dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide bond.

[0188] The polyalkylene oxide bond is a linear portion -((CH2) x1 -O) y1 - or -(O-(CH2) x1 ) y1 - selected from where x1 each independently represents an integer selected from 1 or 2; y1 independently represent an integer from 2 to 20, in particular from 2 to 4.

[0189] According to certain embodiments, the bonds α, β, γ and δ are each chemical bonds. According to another particular embodiment, the bonds α, β, γ and δ are chemical bonds. According to another particular embodiment, the bond β is a chemical bond. According to another particular embodiment, the bond γ is a chemical bond. According to another particular embodiment, bonds α and β are each chemical bonds. According to another particular embodiment, the bonds α, β, and γ are each chemical bonds.

[0190] According to another particular embodiment, the bonds α, β, γ are each a chemical bond and δ is an ether, thioether, ester, amide, carbamate, carbonyl (especially keto), dicarbamate, carbonate, hydrazine, urea, alkylene oxide, or a linear or branched polyalkylene oxide bond.

[0191] According to another particular embodiment, the bonds α, β, γ are each a chemical bond and δ is an ester, amide, carbamate, dicarbamate, carbonate, alkylene oxide, or linear or branched polyalkylene oxide bond.

[0192] The polyalkylene oxide bond is a linear portion -((CH2) x1 -O) y1 - or -(O-(CH2) x1 ) y1 - selected from where x1 each independently represents an integer selected from 1 or 2; Each y1 independently represents an integer from 2 to 4.

[0193] According to a ninth embodiment of the present invention, the spacer Sp of the compound of any of the previous embodiments 1 is selected from one of the following combinations of moieties: -M1-M3-, -M2-M3-, -M3-M1- or M3-M2- Where: The bonds between said moieties M1, M2, M3 are independently selected from a chemical bond, an ether, a thioether, an ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine or a urea, and an alkylene oxide or a linear or branched polyalkylene oxide bond.

[0194] In certain embodiments, the bonds between said moieties M1, M2, M3 are each chemical bonds.

[0195] According to a tenth embodiment of the present invention, the spacer Sp of the compound of any of the previous embodiments 2 is selected from one of the following combinations of moieties: -M1-M3-, -M1-M4-, -M2-M3-, -M2-M5-, -M2-M4-, -M3-M1-, -M3-M2-, -M3-M4-. -M1-M3-M4-, -M1-M4-M3-, -M2-M3-M4-, -M2-M4-M3-, -M3-M2-M4-. -M3-M4-M2- or -M2-M5-M4-, Where: The bonds between said moieties M1, M2, M3, M4 and M5 are independently selected from a chemical bond, an ether, a thioether, an ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine or a urea, and an alkylene oxide or a linear or branched polyalkylene oxide bond.

[0196] In certain embodiments, the bonds between said moieties M1, M2, M3 and M4 are each chemical bonds.

[0197] Sp 1 and Sp 2 Specific examples of combinations of motif M of are shown in the following table (the corresponding spacer moieties can be present in any orientation in the compounds of formula I).

[0198] [Table 11]

[0199] Sp 1 and Sp 2 Specific examples of compounds of formula I having combinations of motifs M of the following are shown in the table below (the corresponding spacer moieties can be present in any orientation in the compounds of formula I):

[0200] [Table 12]

[0201] Here, R 1 is H or lower alkyl, or

[0202] [Table 13]

[0203] or

[0204] [Table 14]

[0205] or

[0206] [Table 15]

[0207] or

[0208] [Table 16]

[0209] Where: residue R 6 represents H or lower alkyl, in particular H, methyl or ethyl; x represents an integer selected from 1 or 2; y is an integer from 1 to 20, in particular from 1 to 15, 1 to 10 or 1 to 4, for example 1, 2, 3 or 4, or

[0210] [Table 17]

[0211] or

[0212] [Table 18]

[0213] [Table 19]

[0214] or

[0215] [Table 20]

[0216] or

[0217] [Table 21]

[0218] 2. Second Aspect of the Invention A second aspect of the invention relates to conjugates, in particular bioconjugates. They are formed by reaction of at least one functionalized payload molecule of general formula (I) of the first aspect of the invention, functionalized with a specific tetrazine group as defined above, said payload molecule being suitable for conjugation with a functionalized targeting agent, in particular a functionalized biomolecule having a functional counterpart group that reacts in a biorthogonal chemical reaction with the tetrazine group of the functionalized payload molecule.

[0219] According to an eleventh embodiment of the present invention, there is provided a conjugate, more particularly a bioconjugate, obtained by reacting a functionalized targeting agent with a tetrazine compound of formula I according to any of the previous embodiments to form a covalent bond between said functionalized targeting agent and said tetrazine compound of formula I.

[0220] According to a twelfth embodiment of the present invention, the functionalized targeting agent of the eleventh embodiment is selected from the corresponding functionalized forms of the following entities: viruses, whole cells, phages, liposomes, biomolecules and low or high molecular weight compounds, antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions, enzymes, proteins, peptides, peptidomimetics, carbohydrates, monosaccharides, polysaccharides, oligonucleotides or polynucleotides, in particular DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins and their derivatives. In a particular embodiment thereof, the targeting agent is selected from antibodies, antibody derivatives, antibody fragments and antibody (fragment) fusions.

[0221] According to a thirteenth embodiment of the present invention, there is provided a conjugate according to any of the eleventh and twelfth embodiments, wherein the functionalized targeting agent comprises, as a functional group, at least one dienophile moiety that reacts with the tetrazine moiety of the compound of formula I.

[0222] According to a fourteenth embodiment of the present invention, there is provided a conjugate of any of embodiments 11 to 13, wherein the functionalized targeting agent comprises at least one polypeptide sequence having at least one unnatural amino acid residue within its amino acid sequence, the unnatural amino acid residue comprising at least one dienophile moiety that reacts with the tetrazine moiety of the compound of formula I.

[0223] According to a fifteenth embodiment of the present invention, there is provided a conjugate according to any of embodiments 11 to 14, wherein the functionalized biomolecule is a polyclonal or monoclonal immunoglobulin molecule, in particular a monoclonal antibody or a fragment thereof.

[0224] According to a sixteenth embodiment of the present invention, there is provided a conjugate of any of the eleventh to fifteenth embodiments, which is formed by biorthogonal bioconjugation of a tetrazine compound of formula I with a functionalized biomolecule having a functional group capable of reacting via a Diels-Alder type cycloaddition reaction, such as, for example, a cyclooctynyl dienophile, a transcyclooctenyl dienophile, a norbornenyl dienophile, a cyclopropenyl dienophile, a cyclobutenyl dienophile, a spirohexenyl dienophile, a BCN dienophile, an azetine dienophile, or an alkene.

[0225] According to a seventeenth embodiment of the present invention, there is provided the conjugate of embodiment 16, wherein the functional group capable of reacting via a Diels-Alder type cycloaddition reaction is selected from: (i) a transcyclooctenyl dienophile group represented by the following formula: [ka] Where: R 1 is hydrogen, halogen, C1-C4-alkyl, (R a O)2P(O)O-C1-C4-alkyl, (R b O)2P(O)-C1-C4-alkyl, CF3, CN, hydroxyl, C1-C4-alkoxy, -O-CF3, C2-C5-alkenoxy, C2-C5-alkanoyloxy, C1-C4-alkylaminocarbonyloxy or C1-C4-alkylthio, C1-C4-alkylamino, di-(C1-C4-alkyl)amino, C2-C5-alkenylamino, C2-C5-alkenyl-C1-C4-alkyl-amino or di-(C2-C5-alkenyl)amino, R a , R b are independently hydrogen or C2-C5-alkanoyloxymethyl, or (ii) a cyclooctynyl dienophile group of the formula: [ka] Where: R 2 is hydrogen, halogen, C1-C4-alkyl, (R c O)2P(O)O-C1-C4-alkyl, (R d O)2P(O)-C1-C4-alkyl, CF3, CN, hydroxyl, C1-C4-alkoxy, -O-CF3, C2-C5-alkenoxy, C2-C5-alkanoyloxy, C1-C4-alkylaminocarbonyloxy or C1-C4-alkylthio, C1-C4-alkylamino, di-(C1-C4-alkyl)amino, C2-C5-alkenylamino, C2-C5-alkenyl-C1-C4-alkyl-amino or di-(C2-C5-alkenyl)amino, R c , R d are independently hydrogen or C2-C5-alkanoyloxymethyl.

[0226] 3. Third aspect of the present invention A third aspect of the invention relates to a method for preparing a bioconjugate.

[0227] According to an eighteenth aspect of the present invention, there is provided a method for preparing a bioconjugate according to any of the 11 to 17 embodiments, comprising reacting in an aqueous, optionally buffered reaction medium, a tetrazine compound as defined in any of the 1 to 10 embodiments with a functionalized biomolecule bearing a functional dienophile group, and carrying out a Diels-Alder type cycloaddition reaction between said molecules.

[0228] 4. Fourth Aspect of the Invention A fourth aspect of the present invention relates to certain tetrazine intermediates useful, for example, in the preparation of tetrazine compounds of formula I.

[0229] According to a nineteenth embodiment of the present invention, there is provided a tetrazine intermediate of general formula II: [ka]

[0230] Where: n3 represents an integer selected from 1 or 2; Sp 1 and Sp 2 is as defined above, the bonds α, β and γ are selected independently from one another from chemical bonds or ether, thioether, ester, amide, carbonyl (in particular keto), carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide or linear or branched polyalkylene oxide bonds, Z is a phosphorus-containing hydrophilic group, particularly (R 1 O)2P(O)-, (R 1a O)2P(O)-O-, and (R 2 O) 3P-O-; Here, R 1 , R 1a , and R 2 are the same or different and independently of one another represent H or lower alkyl, in particular methyl or ethyl, and more particularly H, R represents H or a chemical group capable of forming a chemical bond, or a chemical group capable of forming an ether, a thioether, an ester, an active ester such as a succinimidyl ester or a pentafluorophenyl ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine, a urea, an alkylene oxide, or a linear or branched polyalkylene oxide bond, optionally with the proviso that R does not represent a chemical protecting group, in particular it does not represent a cleavable protecting group, and more particularly it does not represent an N-, O-, or S-protecting group.

[0231] More specifically, R represents an amino group or a carboxyl group. According to a particular embodiment, the bonds α, β, and γ are each chemical bonds. According to another particular embodiment, the bond a is a chemical bond. According to another particular embodiment, the bond β is a chemical bond. According to another particular embodiment, the bond γ is a chemical bond. According to another particular embodiment, bonds α and β are each chemical bonds. According to another particular embodiment, the bonds α, β, and γ are each chemical bonds.

[0232] According to another particular embodiment, Z is (R 1 O)2P(O)-, (R 1a O)2P(O)-O-, and (R 2 O) 3P-O-; Where: R 1 , R 1a and R 2 are the same or different and independently of one another represent H or lower alkyl, in particular methyl or ethyl, and even more particularly H.

[0233] Below are shown particularly preferred structures of compounds of general formula II, in which the residue R 1 are, independently of each other, as defined above. [ka] [ka] [ka]

[0234] The following table shows specific examples of synthetic intermediates of general formula II of the present invention.

[0235] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29]

[0236] 5. Fifth aspect of the present invention A fifth aspect of the present invention relates to a method for preparing certain tetrazine intermediates.

[0237] According to a twentieth embodiment of the present invention, there is provided a method for preparing a tetrazine intermediate of general formula II, comprising the steps of: i. reacting a first cyano compound of general formula III with a second cyano compound of general formula IV in the presence of hydrazine hydrate; [ka] [ka] where Z and Sp 1 is as defined above, the hydroxyl group of the residue Z is provided in the form of an optionally protected ip alkoxy, where R and Sp 2 and n3 is as defined above, ii. subsequently oxidizing the 1,4-dihydro-s-tetrazine compound formed in particular in step 1, using an oxidizing agent selected from, for example, NaNO2, PhI(OAc)2, DDQ, or air oxidation; iii. isolating the resulting tetrazine compound as necessary; and iv. If necessary, deprotect the hydroxyl group of residue Z.

[0238] According to a particular embodiment, step i) is carried out in the absence of a catalyst. According to another particular embodiment, step i) is carried out in the presence of a catalyst. According to another particular embodiment of the invention, the catalyst is a metal-containing catalyst, such as, for example, Zn(OTf)2. According to another particular embodiment, the catalyst is a metal-free catalyst. According to another particular embodiment, the metal-free catalyst is an organic catalyst. According to another particular embodiment, the organic catalyst is a sulfur-containing catalyst. According to another particular embodiment, the sulfur-containing catalyst is selected from 3-mercaptopropionic acid, L-cysteine, glutathione, 2-aminoethanethiol, 1,3-propanedithiol, thioglycolic acid and N-acetyl-L-cysteine, in particular 3-mercaptopropionic acid.

[0239] According to another particular embodiment, reaction step i) is carried out with a molar excess of the hydrazine compound relative to the compounds of formulae (III) and (IV), for example a 1- to 20-fold molar excess. According to yet another particular embodiment, reaction step i) is carried out in an alcoholic solvent, in particular ethanol. According to yet another particular embodiment, the compounds of formula (III) and (IV) are used in a molar ratio of 1:10 to 10:1. According to another particular embodiment, reaction step ii) is carried out with a molar excess of the oxidizing agent relative to the compounds of formulae (III) and (IV), for example a 1-20 fold molar excess.

[0240] According to yet another particular embodiment, the oxidizing agent in step ii is NaNO2. According to yet another particular embodiment, the oxidizing agent in step ii is PhI(OAc) 2 . According to yet another particular embodiment, the oxidant in step ii is air.

[0241] 6. Further Aspects of the Invention A further aspect of the present invention relates to the medical use of the complexes of the present invention, and pharmaceutical compositions, diagnostic kits or analytical kits comprising same.

[0242] According to a twenty-first embodiment of the present invention there is provided a conjugate as defined in any of the embodiments 11 to 17 for use in medicine, in particular in diagnosis and / or therapy.

[0243] According to a twenty-second embodiment of the present invention there is provided a pharmaceutical composition comprising at least one conjugate as defined in any of the embodiments 11 to 17 in a pharma- ceutically acceptable carrier.

[0244] According to a twenty-third embodiment of the present invention there is provided a diagnostic or analytical kit comprising at least one tetrazine compound as defined in any of the embodiments 1-10.

[0245] D. Further Embodiments 1. Hydrophilic tetrazine intermediates and their preparation The tetrazine intermediate of general formula II can be prepared in analogy with methods well known in the art.Suitable methods are described in various publications cited herein, all of which are incorporated herein by reference.Some methods are outlined here.

[0246] Metal-catalyzed one-pot synthesis of the tetrazine intermediate of formula II can be carried out as disclosed by Young et al. in Deutsche Kapitel Zeitung (2012), 51, 5222.

[0247] Mao et al. in the Journal of the Deutsche Kapitel Zeitung (2019), 58, 1106 describe an organocatalytic and scalable synthesis of unsymmetrical 1,2,4,5-tetrazines via thiol-containing promoters.

[0248] According to a particular aspect of the present invention, there is provided a process for the preparation of a tetrazine intermediate of general formula II. [ka]

[0249] Generally, the method comprises the following steps. i) reacting a first cyano compound of general formula III with a second cyano compound of general formula IV in the presence of hydrazine hydrate; [ka] [ka] where Z and Sp 1 is as defined above, any hydroxyl group of the residue Z is provided in the form of a protected ip alkoxy, where R and Sp 2 and n3 is as defined above, ii) subsequently oxidizing the product of step i), iii) optionally isolating the resulting tetrazine compound; iv) If necessary, deprotecting any protected hydroxyl groups of residue Z.

[0250] More specifically, step i) is carried out in a one-pot reaction: for this purpose, for example, a solution of the cyano educt of general formulae (III) and (IV) is provided and supplemented with a hydrazine compound, in particular hydrazine hydrate, optionally in the presence of a suitable catalyst.

[0251] Particular catalysts are acidic metal-free organocatalytic mercapto compounds, such as 3-mercaptopropionic acid, L-cysteine, glutathione, 2-aminoethanethiol, 1,3-propanedithiol, thioglycolic acid, and N-acetyl-L-cysteine, in particular 3-mercaptopropionic acid, such as 3-mercaptopropionic acid. Alternative catalysts are metal-containing catalysts, such as Zn(OTf)2.

[0252] The reaction of step i) is carried out under temperature control until completion. The resulting dihydrotetrazine intermediate is then oxidized. For this purpose, conventional oxidizing agents can be applied, in particular sodium nitrite / HCl or nitric acid.

[0253] Optionally, the cyano extract mixture is provided in a suitable solvent. Typical solvents that can be used are selected from polar organic solvents such as THF, and organic alcohols, in particular ethanol.

[0254] Typically, the compounds of formula (III) and (IV) are provided in a molar ratio ranging from 1:10 to 10:1, such as from 1:5 to 5:1. More particularly, the compound of formula III is provided in a 1-10 fold or 1-5 fold molar excess.

[0255] Typically, the hydrazine compound is added in molar excess relative to the cyano extract, for example, a 1-20 fold or 4-15 fold excess relative to compound (III) or (IV).

[0256] Typically, the acid catalyst is used in a catalytic amount, more specifically a molar amount relative to compound (III) or (IV).

[0257] Typically, the acid is applied in at least a molar, more preferably a molar excess relative to the hydrazine compound.

[0258] The reaction temperature is controlled within the range of -10 to +10°C.

[0259] The oxidation in step ii converts the dihydrotetrazine intermediate formed in step i) to the respective tetrazine. Typically, the reaction is carried out in the reaction mixture of step i) in the presence of ambient air or by addition of a suitable oxidizing agent, such as NaNO2, p-benzoquinone, DDQ, or PhI(OAc)2. PhI(OAc)2 as an oxidizing agent is described, for example, by Selvaray, R. et al. in Tetrahedron Lett. 2014;55(34):4795-4797.

[0260] As a non-limiting example, the preparation of two different tetrazine phosphates is shown in the following scheme (the oxidation step is not explicitly shown): [ka] Scheme 1. Synthesis of hydrophilic phosphonate tetrazines.

[0261] The optional step iii to isolate the product of step ii can be carried out by conventional purification methods, in particular chromatographic methods such as flash chromatography or HPLC.

[0262] The method of the present invention also includes step iv, provided that the extract of formula III is applied and the residue Z contains a protected, e.g. esterified, hydroxyl group, and the resulting tetrazine is intended to be used in deprotected form. Methods for protecting esterified hydroxyl groups are well known. Deprotection by treatment with trimethylsilyl bromide in an organic solvent is given as a non-limiting example.

[0263] By applying the above general process, specific derivatives of methylphosphonate tetrazines can be synthesized. These compounds contain functional groups that allow them to react with further chemical moieties, for example via amide coupling or carbamate or ester bond formation. Such chemical moieties are selected from the groups identified above. Sp 2 The spacer moiety (Sp already introduced as part of the above reactant of formula IV) 2 (Optional in addition to the above) A cleavable group X Self-destructing part Y payload residue (cargo) or a combination thereof, e.g. -AXY, -AY, -XY, -AXXY, -XXY -Sp 2 -AXY, -Sp 2 -AY, -Sp 2 -XY, -Sp 2 -AXXY

[0264] 2. Payload molecule Y The payload molecule Y typically used as a component of the tetrazine compound of general formula I can be selected from biologically active compounds, labeling agents, and chelating agents, non-limiting examples of which are given in the following sections.

[0265] 2.1 Bioactive compounds Bioactive compounds include, but are not limited to:

[0266] Biologically active compounds applicable to the present invention include, but are not limited to, small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, peptides, peptoids, amino acids, nucleotides, oligonucleotides or polynucleotides, nucleosides, DNA, RNA, toxins, glycans, immunoglobulins.

[0267] Exemplary classes of biologically active compounds that can be used in the practice of the present invention include, but are not limited to, hormones, cytotoxins, anti-proliferative / anti-tumor agents, antivirals, antibiotics, cytokines, anti-inflammatory agents, antihypertensive agents, chemosensitizers, photosensitizers and radiosensitizers, anti-AIDS agents, antivirals, immunosuppressants, immunostimulants, enzyme inhibitors, anti-Parkinson's agents, neurotoxins, channel blockers, cell proliferation inhibitors and modulators of extracellular matrix interactions including anti-adhesion molecules, inhibitors of DNA, RNA or protein synthesis, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-Alzheimer's agents, and the like.

[0268] In some embodiments, the bioactive compound is a low to medium molecular weight compound (eg, about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0269] Exemplary cytotoxic drugs are those that are used in particular for cancer treatment.Such drugs generally include DNA damage agents, antimetabolites, natural products and their analogs, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylating agents, radiosensitizers, DNA intercalators, DNA cleavage agents, microtubule stabilizers and destabilizers, topoisomerase inhibitors.Examples include, but are not limited to, platinum drugs, anthracyclines, vincas, mitomycin, bleomycin, cytotoxic nucleosides, taxanes, lexitropsins, pteridine drugs, diynenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxol. Particularly useful members of these classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycins, duocarmycins, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs, DXd, tubulysin M, cryptophycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBDs), pyridinobenzodiazepines (PDDs) and indolinobenzodiazepines (IBDs) (see US20210206763A1), methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, DNA minor groove binders, 6-mercaptopurine, cytosine arabinoside, melphalanxins ... Examples of such inhibitors include alan, leurosine, leurosideine, actinomycin, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin), PNU-159682 (see US 10,288,745 B2) and its analogs, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, N8-acetylspermidine, staurosporine, colchicine, camptothecin, esperamicin, enediynes and their analogs, hemiasterin and its analogs.

[0270] Other representative drug classes include angiogenesis inhibitors, cell cycle progression inhibitors, P13K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, and protein degraders (see https: / / pubs.acs.org / doi / 10.1021 / acschembio.0c00285).

[0271] Examples of auristatins include dolastatin 10, monomethylauristatin E (MMAE), auristatin F, monomethylauristatin F (MMAF), auristatin F hydroxypropylamide (AFHPA), auristatin F phenylenediamine (AFP), monomethylauristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP, and auristatin AQ. Suitable auristatins are described in U.S. Patent Application Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248, International Patent Application Publication Nos. WO09 / 117531, WO2005 / 081711, WO04 / 010957, WO02 / 088172, and WO01 / 24763, and U.S. Patent Nos. 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,124,431, 6,034,065, 5,780,588, 5,767,232, and U.S. Patent Nos. 5,821,521, 5,821,521, and 5,821,521. Nos. 7, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,879,278, 4,816,444 and 4,486,414, the disclosures of which are incorporated herein by reference in their entireties.

[0272] Exemplary drugs include dolastatins and their analogs, such as dolastatin A (U.S. Pat. No. 4,486,414), dolastatin B (U.S. Pat. No. 4,486,414), dolastatin 10 (U.S. Pat. Nos. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, Nos. 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat. No. 4,986,988), dolastatin 14 (U.S. Pat. No. 5,138,036), dolastatin 15 (U.S. Pat. No. 4,879,278), dolastatin 16 (U.S. Pat. No. 6,239,104), dolastatin 17 (U.S. Pat. No. 6,239,104), and dolastatin 18 (U.S. Pat. No. 6,239,104), each of which is incorporated herein by reference in its entirety.

[0273] Exemplary maytansine, maytansinoids (e.g., DM-1 and DM-4), or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Pat. Nos. 4,424,219, 4,256,746, 4,294,757, 4,307,016, 4,313,946, 4,315,929, 4,331,598, 4,361,650, 4,362,651, 4,363,652, 4,364,653, 4,365,654, 4,366,655, 4,367,656, 4,368,657, 4,369,660, 4,370,661, 4,371,662, 4,372,663, 4,373,664, 4,374,665, 4,375,666, 4,376,667, 4,377,668, 4,378,669, 4,379,670, 4,379,671, 4,379,672, 4,379,673, 4,379,674, 4,379,675, 4,379,675, 4,379,676, 4,379,677, 4,379,678, 4,379,679, 4,379,679, 4,379,679, 4,379,679, 4,379,679, 4,3 Nos. 4,362,663, 4,364,866, 4,450,254, 4,322,348, 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 6,333,410, 6,441,163, 6,716,821, and 7,276,497.

[0274] Other examples include mertansine and ansamitocin. Pyrrolobenzodiazepines (PBDs) explicitly include dimers and analogs, including but not limited to those described in Denny, Exp.Opin.Ther.Patents,10(4):459-474(2000); Hartley et al., ExpertOpinInvestigDrugs.2011,20(6):733-44; Antonau et al., ChemRev.2011,111(4),2815-64.

[0275] Calicheamicins include, for example, the enediynes, esperamicins, and those described in US Pat. Nos. 5,714,586 and 5,739,116.

[0276] Examples of duocarmycins and their analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizeresin, carzelesin, seco-adozelesin. Other examples include those described in, for example, U.S. Patent Nos. 5,070,092, 5,101,092, 5,187,186, 5,475,092, 5,595,499, 5,846,545, 6,534,660, 6,548,530, 6,586,618, 6,660,742, 6,756,397, 7,049,316, 7,553,816, 8,815,226, U.S. Patent No. US20150104407 filed May 2, 2014, and U.S. Patent No. 62 / 010,972 filed June 11, 2014, the disclosures of each of which are incorporated herein in their entirety.

[0277] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos. 2002 / 0103136 and 2010 / 0305149, and U.S. Patent No. 7,303,749, the disclosures of which are incorporated by reference herein in their entireties.

[0278] Exemplary epothilone compounds include epothilones A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036, 6,989,450, 6,121,029, 6,117,659, 6,096,757, 6,043,372, 5,969,145, and 5,886,026, and International Publication Nos. WO 97 / 19086, WO 98 / 0884, and WO 99 / 01066. 9, WO98 / 22461, WO98 / 25929, W098 / 38192, WO99 / 01124, WO99 / 02514, WO99 / 03848, WO99 / 07692, WO99 / 27890, and W099 / 28324, the disclosures of which are incorporated herein by reference in their entireties.

[0279] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021, the disclosures of which are incorporated by reference herein in their entireties.

[0280] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, and tetraplatin.

[0281] Exemplary DNA binding or alkylating agents include CC-1065 and its analogs, anthracyclines, calicheamicin, dactinomycin, mithromycin, pyrrolobenzodiazepines, and the like.

[0282] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds such as paclitaxel, docetaxel, tesetaxel, carbazitaxel, maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0283] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs, and unnatural camptothecins, such as, for example, CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, ciratecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan, and S39625. Other camptothecin compounds that can be used in the present invention include, for example, those described in J. Med. Chem., 29:2358-2363 (1986), J. Med. Chem., 23:554 (1980), J. Med Chem., 30:1774 (1987).

[0284] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, and MetAP2 inhibitors.Representative VGFR inhibitors and PDGFR inhibitors include sorafenib, sunitinib, and vatalanib.Representative MetAP2 inhibitors include fumagillol analogs, i.e., compounds that contain the fumagillin core structure.

[0285] Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991, Rho kinase inhibitors such as, for example, AZD7762, Aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237, PLK inhibitors such as, for example, BI2536, BI6727, GSK461364, ON-01910, and KSP inhibitors such as, for example, SB743921, SB715992, MK-0731, AZD8477, AZ3146 and ARRY-520.

[0286] Representative P13K / m-TOR / AKT signaling pathway inhibitors include phosphoinositide 3-kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0287] Exemplary P13 kinases are disclosed in U.S. Pat. No. 6,608,053 and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, paromid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147, and XL765.

[0288] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0289] Representative MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf, and p38 MAPK inhibitors.

[0290] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,944 and include GDC-0973, GSKl120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330 and GDC-0973.

[0291] Representative B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0292] Exemplary Bp38 MAPK inhibitors include BIRB796, LY2228820, and SB202190. Exemplary receptor tyrosine kinase inhibitors include, but are not limited to, AEE788 (NVP-AEE788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), gefitinib (Iressa), AP24534 (ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (dovitinib), sunitinib (sutent), sorafenib (nexavar), and vatalinib.

[0293] Exemplary protein chaperone inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0294] Exemplary HDAC inhibitors include belinostat (PXD101), CUDC-101, droxinostat, ITF2357 (gibinostat, gabinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, dacinostat), LBH-589 (panobinostat), MC1568, MGCD0103 (mosetinostat), MS-275 (entinostat), PCI-24781, pyroxamide (NSC696085), SB939, trichostatin A, and vorinostat (SAHA). Representative PARP inhibitors include iniparib (BSI201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP9722, MK4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0295] Representative Wnt / hedgehog signaling pathway inhibitors include vismodegib, cyclopamine, and XAV-939.

[0296] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitin, beta-amanitin, gamma-amanitin, eta-amanitin, amanulin, amanurinic acid, amanisamide, amanone, and pro-amanin.

[0297] Representative cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and TNF.

[0298] Non-limiting examples of specific agents include auristatins, maytansinoids, PBDs, topoisomerase inhibitors, anthracyclines, and the like.

[0299] In another embodiment, a combination of two or more different agents as described above is used.

[0300] According to another embodiment, the biologically active compound may be selected from any synthetic or natural compound comprising one or more natural and / or non-natural, proteinogenic and / or non-proteinogenic amino acid residues, such as in particular oligo- or polypeptides or proteins.

[0301] A particular group of such compounds include immunoglobulin molecules such as, for example, antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g., bispecific and trispecific mAb fragments or derivatives), polyclonal antibodies or monoclonal antibodies (human, humanized, murine or chimeric antibodies).

[0302] Typical, non-limiting examples of antibodies for use in the present invention are selected from biologically, and in particular pharmacologically, active antibody molecules. Non-limiting examples are selected from the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01, labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC49), cG250, J591, MOv-18, farletuzumab (MORAb-003), 3F8, ch14,18, KW-2871. , hu3S193, lgN311, IM-2C6, CDP-791, etaracizumab, volociximab, nimotuzumab, MM-121, AMG102, METMAB, SCH900105, AVE1642, IMC-A12, MK-0646, R1507, CP751871, KB004, IIIA4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81C6, pinatuzumab, rifatuzumab, glenbatumumab, coltuximab, lorvotuzumab, indatuximab, anti-PSM A, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, avituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumumab, anetumab, apolizumab, arcitumumab, asclinvacumab, atezolizumab, bavituximab, bectumab, belimumab, bivatuzumab, brontixtuzumab, cantuzumab, capromab, catumaxomab, sitatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, darotuzumab Zumab, Daratumumab, Demcizumab, Denintuzumab, Depatuximab, Dellotuximab, Detumomab, Dinutuximab, Drozitumab, Durigotumab, Durvalumab, Dusigitumab, Eclomeximab, Edrecolomab, Ergemtumab, Emactuzumab, Enavatuzumab, Emivetuzumab, Enfortumab, Enoblituzumab, Ensituximab, Epratuzumab, Ertumaxomab, Etaracizumab, Faretuzumab, Ficlatuzumab, Figitumumab, Franvotumab, Futuximab, Galiximab,Ganitumab, Icrucumab, Igovomab, Imalumab, Ingatuzumab, Indusatumab, Inebilizumab, Intetumumab, Iratumumab, Izatuximab, Lexatuzumab, Rilotomab, Lintuzumab, Lirilumab, Lucatumumab, Lumuletuzumab, Margetuximab, Matuzumab, Mirvetuximab, Mitsumomab, Mogamulizumab, Moxetumomab, Nacolomab, Naptumomab, Narunatumumab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab, Obinutuzumab, Oka Latuzumab, ofatumumab, olaratuzumab, onartuzumab, ontucizumab, oportuzumab, oregovomab, otlertuzumab, pancomab, palsatuzumab, pasotuximab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, kirizumab, racotumab, ramucirumab, rilotumumab, lobatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, tapritumomab, Tarectumomab, tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tobetumab, tremelimumab, tucotuzumab, ublituximab, urocupulumab, urelumab, utomilumab, vadastuximab, bundletuzumab, vanticutuzumab, vanucizumab, varlilumab, veltuzumab, besencumab, volociximab, borsetuzumab, votumumab, zalutumumab, zatuxima, combinations and derivatives thereof, and CAI25, CAI5-3, CAI9 -9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA242, placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogene product.

[0303] 2.2 Labeling agents Labeling agents that can be used in accordance with the present invention can include any type of label known in the art that does not adversely affect the reactivity of the tetrazine moiety.

[0304] Labels of the present invention include, but are not limited to, dyes (e.g., fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon rhodamine, BODIPY, or cyanine dyes), chromophores (e.g., phytochromes, phycobilins, bilirubin, etc.), radiolabels (e.g., hydrogen, fluorine, carbon, phosphorus, such as tritium, fluorine-18, carbon-11, carbon-14, phosphorus-32, phosphorus-33, sulfur-33, sulfur-35, iodine-123, or iodine-125, etc.), and the like. radioactive forms of fluorine, sulfur, or iodine), MRI-sensitive spin labels, affinity tags (e.g., biotin, His-tag, Flag-tag, Strep-tag, sugars, lipids, sterols, PEG linkers, benzylguanine, benzylcytosine, or cofactors), polyethylene glycol groups (e.g., branched PEG, linear PEG, PEG of different molecular weights, etc.), photocrosslinkers (such as p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports.

[0305] In some embodiments, exemplary dyes may include NIR contrast agents that fluoresce in the near infrared region of the spectrum. Exemplary near infrared fluorophores include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) of about 630-1000 nm, e.g., about 630-800 nm, about 800-900 nm, about 900-1000 nm, about 680-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, or about 950-1000 nm. Fluorescers with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm may also be used in the methods described herein.

[0306] In some embodiments, exemplary fluorophores include 7-amino-4-methylcoumarin-3-acetic acid (AMCA), TEXAS RED TM(Molecular Probes Inc., Eugene, OR), 5-(and-6)-carboxy-X-rhodamine, Lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein-5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-3-indacenepropionic acid), eosin-5-isothiocyanate, erythrosine-5-isothiocyanate, CASCADE TM blue acetyl azide (Molecular Probes Inc., Eugene, OR) and ATTO dye.

[0307] Other suitable fluorophores are described, for example, in European Patent Application Publication No. EP 3572468 A1. Further indicators are 177-lutetium, 89-zirconium, 131-iodine, 68-gallium, 99m-technetium, 225-actinium, 213-bismuta, 90-yttrium, 212-lead, 111-indium, 64-copper, 67-copper, 124-iodine, 227-thorium and 188-rhenium.

[0308] 2.3 Chelating agents Below is a list of commonly applicable chelating agents and their abbreviations. The corresponding salts are also applicable.

[0309] Acetylacetone (ACAC), ethylenediamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylenetriamine (DIEN), iminodiacetic acid (IDA), triethylenetetramine (TRIEN), triaminotriethylamine, nitrilotriacetic acid (NTA) and its salts (such as Na3NTA or FeNTA), ethylenediaminotriacetic acid (TED), ethylenediaminetetraacetic acid (EDTA) and its salts (such as Na2EDTA and CaNa2EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-z tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), oxalate (OX), tartrate (TART), citrate (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline, 2,2'-bipyridine (BPY), 1,10-phenanthroline (PHEN), dimercaptosuccinic acid (DMSA), 1,2-bis(diphenylphosphino)ethane (DPPE), sodium salicylate, methoxysalicylic acid, British anti-Lewis acid or 2,3-dimercaprol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA);Siderophores secreted by microorganisms, such as desferrioxamine or deferoxamine B (also known as Deferal (Novartis)) produced by Streptomyces spp., deferoxamine (DFO), a trihydroxamic acid secreted by Streptomyces pilosus, phytochemicals such as curcuminoids and derivatives of mugineic acid (e.g. 3-hydroxymugineic acid, 2'-deoxymugineic acid), synthetic chelating agents (e.g. ibuprofen), catechols, hydroxamates, and derivatives of hydroxypyridinones (e.g. hydroxamate desferal, hydroxypyridinones deferiprone), and deferoxamines. Liplon (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one), D-penicillamine (DPA or D-PEN), which is β-β-dimethylcysteine ​​or 3-mercapto-D-valine, tetraethylenetetramine (TETA) or trientine and its two major metabolites N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT), hydroxyquinoline, clioquinol, a halogenated derivative of 8-hydroxyquinoline, and 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2);

[0310] 2.4 Photosensitizers / Proteolytic agents Non-limiting examples include PROTACs in general, which are a number of different E3 ligase-binding molecules combined with specific target proteins for degradation (see International Publication No. WO2017 / 201449A1). (Maneiro et al., ACSChem. Biol. 2020, 15, 6, 1306-1312).

[0311] 3. Conjugation of payload molecule Y to the hydrophilic tetrazine

[0312] 3.1 Cleaving part A Suitable cleavable moieties A are well known from the prior art. You can refer to the following. Berg et al., ChemSocRev 2019, 48(16), 4361-4374; Poreba, FEBSJ 2020, 287(10), 1936-1969 and Salomon et al., MolPharmaceutics 201916, (12), 4817-4825;

[0313] Further examples include β-glucuronide linkers, which have a trigger residue derived from β-glucuronic acid. Non-limiting examples are as follows: [ka]

[0314] 3.2 Self-immolative group X Suitable self-immolative groups X are well known from the prior art. You can refer to the following. Santi et al., J Med Chem 2014, 57(6), 2303-2314; Arouan et al., Deutsche Kammerschutz 2015, 54(26), 7492-7509; Kolakowski et al., Deutsche Kammerschutz 2016, 128(28), 8080-8083

[0315] 4. (Bio)complex formation The bioconjugates of the present invention are provided by bioconjugation, where a suitably functionalized biomolecule reacts with a tetrazine compound of formula I to form a covalent bond between the functionalized biomolecule and the tetrazine. The biomolecule acts as a "targeting agent" that targets the payload moiety Y, which is part of the tetrazine compound of formula I, to a specific location of interest ("target"). Depending on the type of such "target", a respective appropriate "targeting agent" can be selected. Once targeted, the bioconjugate containing the payload moiety may further act on the target or on a biological structure containing the target.

[0316] 4.1 Targeting agents and their targets The primary purpose of such targeting agents is to form a covalent or non-covalent bond with a specific "target". The secondary purpose of the targeting agent is to transport a "payload molecule" to the target. To achieve this secondary purpose, the POI needs to be conjugated (reversibly or irreversibly) with at least one payload molecule. For this purpose, the POI needs to be functionalized by introducing at least one ncAA. The functionalized POI carrying at least one ncAA can then be bound to at least one payload molecule by bioconjugation via the ncAA residue. This ncAA is reactive with the payload molecule, and the payload molecule has a corresponding moiety that reacts with at least one ncAA residue of the POI. The bioconjugate thus obtained, i.e. the targeting agent, allows the transport of the payload molecule to the intended target.

[0317] For example, a "target" can be any molecule present within and / or on an organism, tissue, or cell. Such targets can be non-specific or specific to a particular organism, tissue, or cell. Targets include cell surface targets (receptors, glycoproteins, glycans, carbohydrates, etc.), structural proteins (such as amyloid plaques), abundant extracellular targets (such as the stroma), extracellular matrix targets (growth factors, proteases, etc.), intracellular targets (such as the surface of the Golgi apparatus, the surface of mitochondria, RNA, DNA, enzymes, components of cell signaling pathways, etc.), and / or foreign bodies (such as pathogens or parts thereof, such as viruses, bacteria, fungi, yeasts, etc.).

[0318] Examples of targets include compounds, such as proteins, whose presence or expression levels correlate with a particular tissue or cell type, or whose expression levels are up- or down-regulated in a particular disorder.

[0319] In particular, such targets are proteins such as receptors (internalizing or non-internalizing).

[0320] The target may be selected from any suitable target within the human or animal body, or on a pathogen or parasite.

[0321] Non-limiting examples of suitable targets include cellular components such as cell membranes and cell walls, receptors such as cell membrane receptors, intracellular structures such as the Golgi apparatus and mitochondria, enzymes, receptors, DNA, RNA, viruses or virus particles, macrophages, tumor-associated macrophages, antibodies, proteins, carbohydrates, monosaccharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptors, monoamine oxidase, muscarinic receptors, myocardial sympathetic nervous system, leukotriene receptors, leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptors, apoptosis markers, (anti-)angiogenic markers, gastrin receptors, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opioid receptors, GPIIb / IIIa receptors and other thrombosis-related receptors, fibrin, calcitonin receptors, tuftsin receptors, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptors, CCK receptors, sigma receptors, interleukin receptors, and the like. receptor, herpes simplex virus tyrosine kinase, human tyrosine kinase, integrin receptor, fibronectin target, AOC3, ALK, AXL, C242, CA-125, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD21, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44v6, CD45, CD51, CD52, CD54 , CD56, CD62E, CD62P, CD62L, CD70, CD72, CD74, CD79-B, CD80, CD105, CD125, CD138, CD141, CD147, CD152, CD154, CD174, CD227, CD326, CD340, VEGF / EGF and VEGF / EGF receptor, VEGF-A, VEGFR2, VEGFR1, TAG72, CEA, MUC1, MUC16, GPNMB, PSMA, Crypto, Tenascin-C, Melanocortin-1 receptor, G250, HLADR, ED-B, TMEFF2, EphB2, EphB4, EphA2, FAP, mesothelin, GD2, GD3, CAIX, 5T4, aggregation factor, CTLA-4, CXCR2, FGFRl, FGFR2 , FGFR3, FGFR4, NaPi2b, NOTCHl, NOTCH2, NOTCH3, NOTCH4, ErbB2, ErbB3, EpCAM, FLT3, HGF, HER2, HER3, HMI24 , ICAM, ICOS-L, IGF-1 receptor, TRPV1, CFTR, gdNMB, CA9, c-KIT, c-MET, ACE, APP, adrenergic receptor beta 2, claudin 3, RON, RORl, PD-Ll, PD-L2, B7-H3, B7-H4, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins, IFN-alpha, IFN-gamma, IgE, IGF-1 receptor , IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-22, IL-23, interferon receptors, ITGB2 (CD18), LFA-1 (CD11a), L-selectin, P-selectin, E-selectin, mucin, myostatin, NCA-90, NGF, PDGFRalpha, prostate cancer cells, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, Rh factor, SLAMF7, sphingosine-1-phosphate, TGF-1, TGFbeta2, TGFbeta, TNFalpha, TRAIL-R1, TRAIL-R2, CTAA16.88, vimentin, matrix metalloproteases (MMPs) such as MMP2, MMP9, MMP14, LDL receptor, endoglin, polysialic acid and their corresponding lectins. Examples of fibronectin targets include the alternatively spliced ​​extra domain A (ED-A) and extra domain B (ED-B) of fibronectin. Non-limiting examples of targets in the stroma are described in V. Hofmeister, D. Scrama, and J.C. Becker, Cancer Immun.; Immunother. 2008, 57, 1, the contents of which are incorporated herein by reference.

[0322] More specifically, to enable (specific) targeting of said targets, the targeting agent can comprise a compound comprising an ncAA-functionalized peptide sequence. Such compounds include, but are not limited to, antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g., bispecific and trispecific mAb fragments or derivatives), proteins, peptides (e.g., octreotide and derivatives), VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin, peptidomimetics, receptor agonists and antagonists, cytokines, hormones, steroids, toxins.

[0323] According to certain embodiments, the target is a receptor, and a targeting agent is used that can specifically bind to the target.Suitable targeting agents include, but are not limited to, the ligand of such receptor or its part that binds to the receptor (for example, the receptor-binding peptide in the case of receptor-binding protein ligand).

[0324] Other examples of targeting agents of a proteinaceous nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudins, apolipoprotein E, affibody molecules (e.g. ABY-025), ankyrin repeat proteins, ankyrin-like repeat proteins, interferons (e.g. alpha, beta, gamma interferon), interleukins, lymphokines, colony stimulating factors, and protein growth factors (e.g. alpha, beta tumor growth factors), platelet-derived growth factor (PDGF), uPAR targeting proteins, apolipoproteins, LDL, annexin V, endostatin, and angiostatin.

[0325] Examples of antibody-like peptide molecules used as targeting agents include LHRH receptor targeting peptides, EC-1 peptides, RGD peptides, HER2 targeting peptides, PSMA targeting peptides, somatostatin targeting peptides, bombesin, etc. Other examples of targeted drugs include lipocalins, such as anticalins.

[0326] In certain embodiments, Affibodies TM and multimers and derivatives.

[0327] In a particular embodiment, an antibody is used to form the targeting agent.Antibodies or immunoglobulins derived from IgG antibodies are particularly suitable for use in the present invention, but any class or subclass of immunoglobulin may be selected, such as IgG, IgA, IgM, IgD, and IgE.Suitably, the immunoglobulin is of class IgG, including but not limited to IgG subclasses (IgG1, 2, 3, and 4), or class IgM, which can specifically bind to a specific epitope on an antigen.Antibodies can be complete immunoglobulins obtained from natural or recombinant sources, and can be the immunoreactive portion of complete immunoglobulins. Antibodies include, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotypic antibodies, multispecific antibodies, antibody fragments (Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, etc.), single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, Nanobody TM (also called ), chimeric antibodies, chimeric antibodies containing at least one human constant region, dual affinity retargeting proteins (DARTs) TM), and multimers and derivatives, such as bivalent or multivalent single chain variable fragments (e.g., di-scFv, tri-scFv) and multivalent antibodies, including but not limited to minibodies, diabodies, tribodies, tribodies, tetrabodies, etc. See [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], [Cane. Gen. Prot. 201310, 1-18], [BioDrugs 2014, 28, 331-343], the contents of which are incorporated herein by reference.

[0328] "Antibody fragment" refers to at least a portion of the variable region of an immunoglobulin that binds to a target, i.e., the antigen-binding region.

[0329] In other embodiments, antibody mimetics, such as, but not limited to, affimers, anticalins, avimers, alphabodies, affibodies, DARPins, and multimers and derivatives thereof, are used as targeting agents. See [Trends in Biotechnology 2015, 33, 2, 65], the contents of which are incorporated herein by reference.

[0330] For the avoidance of doubt, in the context of the present invention, the term "antibody" is intended to encompass all antibody variants, fragments, derivatives, fusions, analogues and mimetics as outlined in this paragraph, unless otherwise specified.

[0331] In preferred embodiments, the targeting agent is selected from antibodies and antibody derivatives, such as antibody fragments, fragment fusions, proteins, peptides, peptidomimetics derived agents.

[0332] In another preferred embodiment, the targeting agent is selected from agents derived from antibody fragments, fragment fusions, proteins, and other antibody derivatives that do not contain an Fc domain.

[0333] Exemplary, non-limiting examples of antibody molecules that are further modified to form the ncAA-modified targeting agents of the present invention are selected from biologically, and in particular pharmacologically, active antibody molecules. Non-limiting examples are selected from the group consisting of trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01, labetuzumab, hua33, pemtumomab, oregovomab, minletumomab (CC49), cG250, J591, MOv-18, farletuzumab (MORAb-003), 3F8, ch14,18, KW-2871, hu3S193, lgN31 1, IM-2C6, CDP-791, etaracizumab, volociximab, nimotuzumab, MM-121, AMG102, METMAB, SCH900105, AVE1642, IMC-A12, MK-0646, R1507, CP751871, KB004, IIIA4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81C6 , pinatuzumab, rifastuzumab, glenbatumumab, cortuximab, lorvotuzumab, indatuximab, anti-PSMA, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, avituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, ask Rimvacumab, Atezolizumab, Bavituximab, Bectumomab, Belimumab, Bivatuzumab, Brontixumab, Cantuzumab, Capromab, Catumaxomab, Sitaxumab, Cixutumumab, Crivatuzumab, Codrituzumab, Conatumumab, Dacetuzumab, Darotuzumab, Daratumumab, Demcizumab, Denintuzumab, Depatuxizumab, Dellotuximab, Detuximab, Momab, dinutuximab, drozitumab, durigotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab, elgemtumab, emactuzumab, enavatuzumab, emibetuzumab, enfortumab, enoblitzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, faretuzumab, ficlatuzumab, figitumumab,framvotumab, futuximab, galiximab, ganitumab, icrucumab, igovomab, imalumab, imgatuzumab, indusatumab, inebilizumab, intetumumab, iratumumab, izatuximab, lexatuzumab, rilotomab, lintuzumab, lirilumab, lucatumumab, lumuletuzumab, margetuximab, matuzumab, mirvetuximab, mitumomab, mogamulizumab, moxetumomab, nacolomab, naptumomab, narunatumab, necitumumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab , obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontucizumab, oportuzumab, oregovomab, otlertuzumab, pancomab, palsatuzumab, pasotuzumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, kirisumab, racotumomab, ramucirumab, rilotumumab, lobatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, softuzumab, tacatuzumab, tapritumomab , tarectumab, tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tobetumab, tremelimumab, tucotuzumab, ublituximab, urocupulumab, urelumab, utomilumab, vadastuximab, bundletuzumab, vanticutuzumab, vanucizumab, varlilumab, veltuzumab, besencumab, volociximab, borsetuzumab, votumumab, zalutumumab, zatuxima, combinations and derivatives thereof, and CAI25, CAI5-3, CAI9-9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA242, placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and other monoclonal antibodies targeting the Neu oncogene product.

[0334] According to further specific embodiments of the present invention, targets and targeting agents are selected to provide specific targeting or enhanced targeting of tissues or diseases such as cancer, inflammation, infectious diseases, cardiovascular diseases (e.g., thrombus, arteriosclerotic lesions), hypoxic sites (e.g., stroke), tumors, cardiovascular disorders, brain disorders, apoptosis, angiogenesis, organs, and reporter genes / enzymes. This can be achieved by selecting targets with tissue, cell, or disease-specific expression.

[0335] As an example, a targeting agent specifically binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, of a particular cell population. When the targeting agent specifically binds to or complexes with the receptor, the agent enters the cell.

[0336] As used herein, a targeting agent that "specifically binds or complexes" or "targets" a cell surface molecule, extracellular matrix target, or other target, preferentially binds to the target via intermolecular forces. For example, a ligand can preferentially bind to a target with a dissociation constant (Kd or KD) of less than about 50 nM, less than about 5 nM, or less than about 500 pM.

[0337] 4.3 Dienophile Targeting agents, especially biomolecules, usually need to be functionalized to allow covalent attachment of the tetrazine compounds of general formula I.

[0338] Methods of functionalization are well known in the art.

[0339] The various classes of dienophiles suitable for reacting with the tetrazine moiety are well known in the art. In general, monocyclic or polycyclic, particularly monocyclic and bicyclic, unsaturated dienophiles are applicable.

[0340] Such dienophiles can react via Diels-Alder type cycloaddition reactions, such as cyclooctynyl dienophiles, transcyclooctenyl dienophiles, norbornenyl dienophiles, cyclopropenyl dienophiles, cyclobutenyl dienophiles, spirohexenyl dienophiles, BCN dienophiles, or azetine dienophiles.

[0341] These are described, for example, in the following prior art, the disclosures of which are incorporated herein by reference: Oliveira, B. L. et al., ChemSocRev 2017, 46, 4895; Kosma, Yi, ChemBioChem2017,18,486; Siegle, S.J. et al., ChemEurJ2018,24,2426; Ramil, C. P. et al., JAmChemSoc2017, 139, 13376 Liu, K. et al., ChemComm, 2017, 53, 10604 International Publication No. WO2015 / 107064A1 in the name of the European Molecular Biology Laboratory International Publication No. WO2012 / 104422A1 U.S. Patent Application Publication No. US2013137763A1.

[0342] 4.4 Bioorthogonal Bioconjugation Targeting agents, particularly targeting agents that include a polypeptide moiety and that include one or more UNAA residues, can be prepared according to the present invention using a suitable translation system, particularly an in vivo translation system. The in vivo translation system can be a cell, such as a prokaryotic cell or a eukaryotic cell. The cell can be a bacterial cell, such as E. coli, a fungal cell, such as a yeast cell, such as Saccharomyces cerevisiae or a methylotrophic yeast, a plant cell, or an animal cell, such as an insect cell or a mammalian cell, such as a HEK cell or a HeLa cell. The eukaryotic cell used for polypeptide expression can be a single cell or part of a multicellular organism.

[0343] The applied cell line comprises (e.g., is provided with) at least one unnatural amino acid or salt thereof that corresponds to the UNAA residue of the targeting agent to be prepared. (i) PylRS and tRNA of the present invention Pyl where PylRS is a tRNA Pyl can be (preferably selectively) acylated with UNAA or a salt thereof. (ii) a polynucleotide encoding a targeting agent, wherein any position in the targeting agent occupied by a UNAA residue is selected from the group consisting of tRNA Pyl The sequence is encoded by a codon (eg, a selector codon) that is the reverse complement of the anticodon of the sequence.

[0344] The cell line is cultured to allow translation of the polynucleotide (ii) encoding the targeting agent, thereby producing the targeting agent.

[0345] According to the methods of the present invention, to produce a targeting agent, translation in step (b) can be achieved by culturing the cell line under appropriate conditions, preferably in the presence of UNAA or a salt thereof (e.g., in a culture medium containing UNAA or a salt thereof), for a suitable time to allow translation at the cellular ribosomes. A polynucleotide encoding a targeting agent (and optionally PylRS, tRNA Pyl Depending on the type of gene that is being used, it may be necessary to induce expression by adding a compound that induces transcription, such as arabinose, isopropyl β-D-thiogalactoside (IPTG) or tetracycline. Pyl The codon (containing one or more codons that are the reverse complement of the anticodon contained in the tRNA) is bound to the ribosome. A polypeptide is then formed by the stepwise addition of amino acids and UNAAs at the positions coded for by the codons that are recognized (bound) by each aminoacyl-tRNA. Thus, the UNAAs are bound to the tRNA Pyl is incorporated into the targeting agent at a position encoded by a codon that is the reverse complement of the anticodon contained in

[0346] The cell line comprises a polynucleotide sequence encoding the PylRS of the present invention, thereby enabling the cell to express the PylRS. Pyl is a tRNA contained in cells. Pyl The polynucleotide sequence encoding PylRS and the tRNA Pyl The polynucleotide sequences encoding the may be present on the same polynucleotide or on separate polynucleotides.

[0347] Thus, in one embodiment, the invention provides a method of making a targeting agent that contains one or more UNAA residues, the method comprising the steps of: (a) providing a cell line comprising a polynucleotide sequence encoding: - at least one PylRS of the invention, - at least one tRNA that can be acylated by PylRS (tRNA Pyl ), and - at least one targeting agent (any position occupied by a UNAA residue in the targeting agent is Pyl (encoded by a codon that is the reverse complement of the anticodon in (b) enabling translation of the polynucleotide sequence by the cell line in the presence of UNAA or a salt thereof, thereby translating PylRS, tRNA Pyl and generate POIs.

[0348] The cell lines used to prepare targeting agents comprising one or more unnatural amino acid residues described herein are PylThe targeting agent can be prepared by introducing into a (host) cell a polynucleotide sequence encoding the targeting agent, which may be located on the same or separate polynucleotides and introduced into the cell by methods known in the art (e.g., viral-mediated gene delivery, electroporation, microinjection, lipofection, etc.).

[0349] After translation, the targeting agent prepared according to the present invention can be optionally recovered and purified to partial or substantial homogeneity according to procedures generally known in the art. Unless the targeting agent is secreted into the culture medium, recovery usually requires cell disruption. Methods of cell disruption are well known in the art and include physical disruption, such as (ultrasonic) sonication, liquid shear disruption (e.g., by French press), mechanical methods (e.g., using a blender or grinder), or freeze-thaw cycles, as well as chemical lysis using agents that disrupt lipid-lipid, protein-protein and / or protein-lipid interactions (such as detergents), and combinations of physical disruption techniques and chemical lysis. Standard procedures for purifying polypeptides from cell lysates or culture medium are also well known in the art and include, for example, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis, and the like. Protein refolding steps can be used, if necessary, in creating a correctly folded mature protein. High performance liquid chromatography (HPLC), affinity chromatography, or other suitable methods can be used in the final purification step where high purity is required. Antibodies generated against the polypeptides of the present invention can be used as purification reagents, i.e., affinity-based purification of the polypeptides. Various purification / protein folding methods are well known in the art, including, for example, Scopes, Protein Purification, Springer, Berlin (1993) and Deutscher, Methods in Enzymology, Vol. 182: A Guide to Protein Purification, Academic Press (1990) and references cited therein.

[0350] As mentioned above, one skilled in the art will recognize that after synthesis, expression and / or purification, a polypeptide may have a conformation that differs from the desired conformation of the associated polypeptide. For example, polypeptides produced by prokaryotic systems are often optimized by exposure to chaotropic agents to achieve proper folding. For example, during purification from lysates from E. coli, the expressed polypeptide is denatured and then renatured as necessary. This is accomplished, for example, by solubilizing the protein with a chaotropic agent such as guanidine HCl. In general, it may be desirable to denature and reduce the expressed polypeptide and then allow the polypeptide to refold into a preferred conformation. For example, guanidine, urea, DTT, DTE, and / or chaperonins can be added to the translation product of interest. Methods for reducing, denaturing, and renature proteins are well known to those skilled in the art. The polypeptide can be refolded in a redox buffer that includes, for example, oxidized glutathione and L-arginine.

[0351] The targeting agents thus prepared can then be converted into the respective bioconjugates by reaction with tetrazine compounds of general formula I above.

[0352] 5. Pharmaceutical Compositions The conjugates or bioconjugates of the invention, such as APCs, and particularly the ADCs of the invention (i.e., active agents or components), are generally provided as "pharmaceutical compositions" comprised of a therapeutically and / or prophylactically or diagnostically effective amount of at least one such active component, or a pharma- ceutical acceptable salt thereof, and, optionally, at least one pharma- ceutical acceptable excipient.

[0353] The pharmaceutical compositions may be delivered by a suitable route of administration, such as oral, rectal, transmucosal, topical, ophthalmic, otic, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0354] Depending on the nature or method of administration and the dosage form, said composition, said at least one additional pharmaceutical excipient may vary.

[0355] An "excipient" is a substance that is formulated with an active ingredient for a variety of purposes, including long-term stabilization, bulking up a solid formulation that contains a small amount of a potent active ingredient (hence they are often called "bulking agents," "fillers," or "diluents"), or imparting a therapeutic effect to the active ingredient in the final dosage form (e.g., facilitating drug absorption, reducing viscosity, increasing solubility, etc.). Excipients not only aid in in vitro stability, such as preventing denaturation and aggregation over the expected shelf life, but also aid in the manufacturing process of the pharmaceutical composition by helping to deal with problems with the active ingredient, such as promoting powder flowability and non-stickiness. The selection of appropriate excipients depends on the particular active ingredient and other factors, as well as the route of administration and dosage form.

[0356] The excipients may be selected from among immune adjuvants, anti-adherents, binders, coating agents, colorants, disintegrants, flavors, glidants, lubricants, preservatives, adsorbents, sweeteners, and fillers.

[0357] Non-limiting examples of excipients include diluents, preservatives, stabilizers, emulsifiers (such as emulsifying polymers such as polysorbates and poloxamers), antioxidants, anti-irritants, chelating agents, stabilizing salts (such as chlorides, sulfates, phosphates, diphosphates, hydrobromides, nitrates, etc.), suspending agents, antibacterial agents, antifungal agents, etc. In addition, buffers (such as buffer systems of low molecular weight organic acids and their respective salts) or inorganic buffer substances (such as phosphate buffers) can also be used. Further suitable ingredients are also known from the relevant pharmacological standard literature. The proportions of the various ingredients also vary depending on the nature of the specific ingredients used and are generally known to those skilled in the art (Remington´s Pharmaceutical science ("Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), edited by A. Wade and P. J. Weller, or Remington's Pharmaceutical Sciences, Mack Publishing Co. (edited by A. R. Gennaro, 1985)).

[0358] As used herein, a pharmaceutical composition may be provided in the form of a "dosage form" or "unit dose" and may contain one or more APCs, particularly ADCs, as described herein. Thus, as used herein, a pharmaceutical composition may provide, for example, two active agents mixed in a unit dose, or provide two active agents combined in a dosage form in which the active agents are physically separated.

[0359] Furthermore, the pharmaceutical composition may be administered in a targeted drug delivery system, such as in a liposome coated with an endothelial cell-specific antibody.

[0360] The pharmaceutical compositions of the present invention can be manufactured in a manner known per se, for example, by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or combinations thereof. Proper formulation depends upon the route of administration chosen.

[0361] As used herein, the phrase "pharmacologically acceptable" is used to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk / benefit ratio.

[0362] The present invention includes all "pharmaceutical acceptable salt forms" of the active ingredients. Pharmaceutically acceptable salts are those in which the counter ion does not contribute significantly to the physiological activity or toxicity of the compound and functions as a pharmacological equivalent. These salts can be prepared according to common organic techniques using commercially available reagents. Anionic salt forms include acetate, acetate, besylate, bromide, chloride, citrate, fumarate, glucuronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, xinofoate, and the like. Cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, zinc, and the like.

[0363] A "therapeutically effective amount" and / or a "prophylactically effective amount" means an amount effective to provide a therapeutic and / or prophylactic benefit when administered to a human or non-human patient. More specifically, a "therapeutically effective amount" is an amount of an active ingredient or a combination of two or more active ingredients disclosed herein that completely or partially inhibits the progression of a disease state or at least partially alleviates one or more symptoms of a disease state.

[0364] By "diagnostically effective amount" is meant an amount effective to obtain diagnostically valuable information regarding the state or progression of a medical condition from a patient.

[0365] The therapeutic benefit is the alleviation of symptoms in a patient with a disease, for example, an amount effective to reduce symptoms in a patient with a disease. In certain circumstances, the patient may not show symptoms of the disease being treated. Thus, a prophylactically effective amount of a compound is also an amount sufficient to have a significant positive effect on the symptoms of a disease, disorder, or condition, for example, an amount sufficient to significantly reduce the frequency and severity of the symptoms of the disease.

[0366] A therapeutically effective amount is also a prophylactically effective amount.

[0367] As used herein, a "patient" refers to a human or non-human animal, particularly a human animal.

[0368] A "dosage form" refers to a unitary administration of one or more active ingredients ("unit dosages") described herein.

[0369] The term "therapy" or "treatment" refers to the following: (i) Preventing the onset of a disease, disorder, or condition in a patient who may be susceptible to, but has not yet been diagnosed with, the disease, disorder, or condition. (ii) inhibiting the disease, disorder, or condition, i.e., halting its progression; (iii) Relieving the disease, disorder, or condition, i.e., causing the regression of the disease, disorder, or condition, particularly including prophylactic or therapeutic treatment, or a combination thereof.

[0370] The "frequency" of administration depends on the compound used and the type of infection being treated. It can be administered once a day. In some cases, it may be more effective to administer the active ingredient several times a day, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 times.

[0371] It will be understood, however, that the specific dosage level and frequency for a particular patient will depend on a variety of factors, including the activity of the particular compound used, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease in the patient being treated. Patients may typically be monitored for therapeutic or prophylactic effectiveness using assays appropriate for the condition being treated or prevented, which will be familiar to those skilled in the art.

[0372] Particular examples of pharmaceutical compositions according to the invention are formulations in liquid form, such as solutions, suspensions, emulsions, etc., which contain a therapeutically effective amount of at least one APC, in particular an ADC component as defined above, optionally together with at least one further pharma- ceutically acceptable excipient as defined above, and which can be administered by any suitable route.

[0373] Further examples of pharmaceutical compositions according to the present invention include solid dosage forms such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.

[0374] Numerous possible variations, which will be readily apparent to those of skill in the art upon consideration of the disclosure herein, are also within the scope of the present invention.

[0375] The following examples are for illustrative purposes only and are not intended to limit the scope of the embodiments described herein. EXAMPLES

[0376] A) Materials and methods Reagents were purchased from commercial sources and used without purification. All solvents, including anhydrous solvents, were used as received from commercial sources. Air- and water-sensitive reagents and reactions were typically handled under an argon atmosphere.

[0377] The progress of the reaction was monitored by TLC on Merck silica gel plates 60F254 or by UHPLC-MS. TLC detection was performed by UV light at 254 nm or potassium permanganate staining.

[0378] Flash chromatography purification was performed on a Biotage IsoleraOne purification system using silica gel (0.060-0.200 mm), KP-Sil cartridges.

[0379] Preparative HPLC purification was performed on an Agilent Infinity 1260 series instrument consisting of an Agilent 1260 preparative pump, a 1260 preparative autosampler, a 1260 fraction collector, and a 1260 multi-wavelength detector VL. The preparative column used was a Waters X-BridgePrep C18 column: 5 μm, 19 x 150 mm, operated with a linear gradient of H2O containing 0.1% TFA and acetonitrile as solvent.

[0380] Nuclear magnetic resonance spectra were recorded on a Bruker Avance (400 MHz) NMR system at room temperature. Chemical shifts (δ) are given in parts per million (ppm), coupling constants (J) are given in Hertz (Hz), and multiplicities are reported using standard abbreviations.

[0381] UHPLC-MS analysis was performed on an Agilent Infinity 1290 series instrument consisting of an Agilent 1290 quaternary pump, 1290 sampler, 1290 thermostat column compartment, and 1290 diode array detector VL+ with quadrupole LC / MS 6120 and Infinity 1260 ELSD. The analytical column used was an Acquity UPLCBEHC18 column: 1.7 μm, 2.1x50 mm, operated with a linear gradient of H2O and acetonitrile containing 0.1% TFA as solvent.

[0382] B) Synthesis of compounds Example 1 Synthesis of diethyl ((6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (1) [ka]

[0383] To a solution of 4-aminomethylbenzonitrile (169 mg, 1.00 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (651 μL, 709 mg, 4.00 mmol, 4.00 eq) in EtOH (0.5 mL) was added 3-mercaptopropionic acid (87.0 μL, 106 mg, 1.00 mmol, 1.00 eq) followed by hydrazine monohydrate (776 μL, 801 mg, 16.0 mmol, 16.0 eq) at 0 °C. The mixture was stirred overnight at room temperature. Then NaNO2 (1.38 g, 20.0 mmol, 20.0 eq) in H2O was added and acidified to pH ~ 3 by dropwise addition of 1 M HClaq.

[0384] Purification by HPLC gave 1 as a pink oil (300 mg) which was used directly in the next step.

[0385] Example 2 Synthesis of ((6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (2) [ka]

[0386] To a solution of 1 (300 mg) in DMF was added trimethylsilyl bromide (530 μL, 614 mg, 4.01 mmol, 5.00 equiv) at 0° C. The mixture was stirred at room temperature overnight, then diluted with MeOH and HO and the solvent was evaporated.

[0387] Purification by HPLC afforded 2 as a pink powder (30.0 mg, 8% for two steps).

[0388] Example 3 Synthesis of 4-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzoic acid (3) [ka]

[0389] To a solution of 4-cyanobenzoic acid (588 mg, 4.00 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (2.60 mL, 2.84 g, 16.0 mmol, 4.00 eq) in EtOH (2 mL) was added 3-mercaptopropionic acid (348 μL, 424 mg, 4.00 mmol, 1.00 eq) followed by hydrazine monohydrate (3.10 mL, 3.20 g, 64.0 mmol, 16.0 eq) at 0 °C. The mixture was stirred overnight at room temperature. Then NaNO2 in H2O (5.52 g, 80.0 mmol, 20.0 eq) was added and acidified to pH ~ 3 by dropwise addition of 1 M HClaq. The pink precipitate was filtered and washed with 0.1 M HClaq.

[0390] Purification by flash chromatography (dichloromethane / MeOH 20:1) afforded 3 as a pink powder (304 mg, 22%).

[0391] Example 4 Synthesis of 2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((4-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamoyl)benzoate (4) [ka]

[0392] To a solution of 2 (5.00 mg, 17.8 μmol, 1.00 eq) in DMF (0.2 mL), DIPEA (12.4 μL, 9.20 mg, 71.2 μmol, 4.00 eq) and 5-TAMRA-OSu (10.3 mg, 19.6 μmol, 1.10 eq) were added and the mixture was stirred at room temperature overnight. The crude reaction mixture was directly purified by HPLC.

[0393] Purification by HPLC gave 4 as a red-brown powder (4.0 mg, 32%).

[0394] Example 5 Synthesis of ((6-(4-(((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (5) [ka]

[0395] To a solution of 3 (5.57 mg, 15.8 μmol, 1.00 eq) was added DIPEA (6.88 μL, 5.11 mg, 39.5 μmol, 2.50 eq) and HATU (9.01 mg, 23.7 μmol, 1.50 eq) and the mixture was stirred at room temperature for 30 min. Then MMAE (12.5 mg, 17.4 μmol, 1.10 eq) was added and the mixture was stirred at room temperature overnight. Diluted with dichloromethane and washed with HO, the organic phase was dried and the solvent was evaporated under reduced pressure.

[0396] The crude product was dissolved in DMF (0.5 mL) and trimethylsilyl bromide (10.4 μL, 12.1 mg, 79.0 μmol, 5.00 equiv) was added at 0° C. The mixture was stirred at room temperature overnight and directly purified by HPLC.

[0397] Purification by HPLC gave 5 as a pink powder (5.0 mg, 32% for two steps).

[0398] Example 6 Synthesis of 2,5-dioxopyrrolidin-1-yl 4-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzoate (6) [ka]

[0399] To a solution of 3 (100 mg, 284 μmol, 1.00 eq) in dichloromethane (2.8 mL) was added N-hydroxysuccinimide (49.0 mg, 426 μmol, 1.50 eq) and EDC hydrochloride (81.6 mg, 426 μmol, 1.50 eq) and the mixture was stirred at room temperature for 1 h.

[0400] Purification by flash chromatography (dichloromethane / MeOH 20:1) afforded 6 as a pink solid (118 mg, 93%).

[0401] Example 7 Synthesis of (4-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzoyl)-L-valyl-L-alanine (7) [ka]

[0402] To a solution of 6 (118 mg, 263 μmol, 1.00 eq) in DMF (2.6 mL) was added H-Val-Ala-OH (74.1 mg, 394 μmol, 1.50 eq) and NEt3 (72.8 μL, 525 μmol, 2.00 eq) and the mixture was stirred at room temperature. It was diluted with dichloromethane and washed with H2O and 1M HClaq. The organic phase was dried and the solvent was evaporated under reduced pressure.

[0403] Purification by flash chromatography (dichloromethane / MeOH 20:1 to 10:1) afforded 7 as a pink solid (135 mg, 98%).

[0404] Example 8 Synthesis of diethyl ((6-(4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (8) [ka]

[0405] To a solution of 7 (116 mg, 222 μmol, 1.00 eq) in DMF (2.2 mL), p-aminobenzyl alcohol (41.0 mg, 333 μmol, 1.50 eq), HATU (127 mg, 333 μmol, 1.50 eq), and DIPEA (96.7 μL, 555 μmol, 2.50 eq) were added and the mixture was stirred at room temperature. It was diluted with dichloromethane and washed with H2O and 1M HClaq. The organic phase was dried and the solvent was evaporated under reduced pressure.

[0406] Purification by flash chromatography (dichloromethane / MeOH 20:1 to 10:1) afforded 8 as a pink solid (131 mg, 94%).

[0407] Example 9 Synthesis of 4-((S)-2-((S)-2-(4-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl)carbonate (9) [ka]

[0408] To a solution of 8 (131 mg, 209 μmol, 1.00 eq) in dichloromethane (2.1 mL) was added PNP chloroformate (63.1 mg, 313 μmol, 1.50 eq) and DIPEA (54.5 μL, 313 μmol, 1.50 eq) and the mixture was stirred at room temperature. After 3 h, 20 mg of PNP chloroformate was added and the mixture was stirred for another 1 h. It was diluted with dichloromethane and washed with saturated NaHCO3aq. The organic phase was dried and the solvent was evaporated under reduced pressure.

[0409] Purification by flash chromatography (dichloromethane / MeOH 20:1 to 10:1) afforded 9 as a pink solid (101 mg, 61%).

[0410] Example 10 Synthesis of 4-((S)-2-((S)-2-(4-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzamido)-3-methylbutanamido)propanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (10) [ka]

[0411] To a solution of 9 (20.0 mg, 25.2 μmol, 1.00 eq) and MMAE (18.1 mg, 25.2 μmol, 1.00 eq) in DMF (0.2 mL) was added pyridine (0.1 mL), HOBt monohydrate (0.773 mg, 5.05 μmol, 0.200 eq) and DIPEA (4.39 μL, 25.2 μmol, 1.00 eq) and the mixture was stirred at room temperature overnight. It was diluted with dichloromethane and washed with HO. The organic phase was dried and the solvent was evaporated under reduced pressure.

[0412] The crude product 10 (34.0 mg, 98%) was used directly in the next step.

[0413] Example 11 ((6-(4-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxo Synthesis of ethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (11) [ka]

[0414] To a solution of crude 10 (34.0 mg, 24.8 μmol, 1.00 eq) in DMF (0.5 mL) was added trimethylsilyl bromide (32.7 μL, 248 μmol, 10.0 eq) and the mixture was stirred at room temperature overnight. It was diluted with MeOH and HO and the mixture was directly purified by HPLC.

[0415] Purification by HPLC gave 11 (2.1 mg, 6%) as a pink solid.

[0416] Example 12 Synthesis of ((6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (12) [ka]

[0417] To a solution of 6 (30.8 mg, 68.5 μmol, 1.00 eq) in dichloromethane (0.7 mL) was added trimethylsilyl bromide (27.1 μL, 206 μmol, 3.00 eq) at 0° C. and the mixture was stirred at this temperature for 3 h. DMF (0.7 mL) was added and the mixture was stirred at room temperature for 3 days. It was diluted with MeOH and HO and the solvent was evaporated under reduced pressure.

[0418] Purification by HPLC gave 12 as a pink powder (17.4 mg, 65%).

[0419] Example 13 ((6-(4-(((2S)-1-(((2S)-1-((4-((((2S)-1-(((14S,32S,33R,2S,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxiran-8(1,3)-benzo[b]phenyl] Synthesis of dicyclohexyltetradecaphane-10,12-dien-4-yl)oxy-1-oxopropan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (13) [ka]

[0420] To a solution of H-Val-Ala-PAB-maytansinoid 28 (12.5 mg, 12.9 μmol, 1.00 eq) and 12 (11.2 mg, 28.4 μmol, 2.20 eq) in DMF (0.5 mL) was added NEt3 (16.1 μL, 116 μmol, 9.00 eq), and the mixture was stirred at room temperature overnight. It was diluted with HClaq, and the mixture was directly purified by HPLC.

[0421] Purification by HPLC gave 13 (7.8 mg, 48%) as a pink powder.

[0422] Example 14 Synthesis of 5-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)isophthalic acid (14) [ka]

[0423] To a solution of 5-cyanoisophthalic acid (287 mg, 1.50 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (972 μL, 6.00 mmol, 4.00 eq) in EtOH (1.5 mL) was added 3-mercaptopropionic acid (131 μL, 1.50 mmol, 1.00 eq) followed by hydrazine monohydrate (1.17 mL, 24.0 mmol, 16.0 eq) at 0 °C. The mixture was stirred overnight at room temperature. Then NaNO2 (2.07 g, 30.0 mmol, 20.0 eq) in H2O was added and acidified to pH ~ 1 by dropwise addition of 1 M HClaq. The pink precipitate was filtered and washed with 0.1 M HClaq.

[0424] The crude product 14 (594 mg) was used directly in the next step without further purification.

[0425] Example 15 Synthesis of bis(2,5-dioxopyrrolidin-1-yl)5-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)isophthalate (15) [ka]

[0426] To a mixture of 14 (594 mg, 1.50 mmol, 1.00 eq) in dichloromethane (15 mL) was added N-hydroxysuccinimide (691 mg, 6.00 mmol, 4.00 eq) and EDC hydrochloride (1.15 g, 6.00 mmol, 4.00 eq) and the mixture was stirred at room temperature.

[0427] Purification by flash chromatography (dichloromethane / MeOH 20:1) afforded 15 as a pink solid (750 mg, 85% for two steps).

[0428] Example 16 Synthesis of 1,1'-(5-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)-1,3-phenylene)bis(1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosan-29-oic acid) (16) [ka]

[0429] To a mixture of 15 (300 mg, 508 μmol, 1.00 eq) in DMF (5 mL), NEt3 (140 μL, 103 mg, 1.02 mmol, 2.00 eq) and 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (449 mg, 1.02 mmol, 2.00 eq) were added and the mixture was stirred at room temperature. It was diluted with HO and the mixture was directly purified by HPLC.

[0430] Purification by HPLC gave 16 as a pink oil (83 mg, 13%).

[0431] Example 17 Synthesis of 2-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)acetic acid (17) [ka]

[0432] To a solution of cyanoacetic acid (3.40 g, 40.0 mmol, 4.00 eq) and diethyl cyanomethylphosphonate (1.62 mL, 10.0 mmol, 1.00 eq) in EtOH (10 mL) was added 3-mercaptopropionic acid (871 μL, 10.0 mmol, 1.00 eq) at 0 °C, followed by hydrazine monohydrate (7.76 mL, 160 mmol, 16.0 eq). The mixture was stirred overnight at room temperature. Then NaNO2 in H2O (13.8 g, 200 mmol, 20.0 eq) was added and acidified to pH ~ 1 by dropwise addition of 1 M HClaq. It was extracted with ethyl acetate, the organic phase was dried and the solvent was evaporated under reduced pressure.

[0433] Two purifications by flash chromatography (dichloromethane / MeOH 50:1→10:1 and 20:1→10:1) gave 17 as a pink oil (260 mg, 9%).

[0434] Example 18 Synthesis of diethyl ((6-(5-aminopyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (18) [ka]

[0435] To a solution of 5-amino-2-pyridinecarbonitrile (1.20 g, 10.1 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (6.52 mL, 7.14 g, 40.3 mmol, 4.00 eq) in EtOH (10 mL) was added 3-mercaptopropionic acid (878 μL, 1.07 g, 10.1 mmol, 1.00 eq) followed by hydrazine monohydrate (7.82 mL, 8.07 g, 161 mmol, 16.0 eq) at 0° C. The mixture was stirred at room temperature overnight.

[0436] The solvent was removed by rotary evaporation and the residue was purified by reverse-phase flash chromatography. The fractions containing the dihydrotetrazine intermediate were oxidized in air overnight, and the solvent was evaporated to give the crude product 18.

[0437] Purification by flash chromatography (dichloromethane / MeOH 20:1) afforded 18 as a dark red solid (555 mg, 17%).

[0438] Example 19 Synthesis of ((6-(5-aminopyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (19) [ka]

[0439] To a solution of 18 (78.0 mg, 241 μmol, 1.00 eq) in dichloromethane / DMF (2:1, 3 mL) was added trimethylsilyl bromide (159 μL, 184 mg, 1.20 mmol, 5.00 eq) at 0° C. The mixture was stirred at room temperature overnight. It was then diluted with MeOH and HO and the solvent was evaporated.

[0440] Purification by HPLC afforded 19 as a red-pink powder (36.0 mg, 56%).

[0441] Example 20-Synthesis of diethyl((6-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (20) [ka]

[0442] A mixture of 4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)benzonitrile (980 mg, 3.17 mmol, 5.00 eq), diethyl cyanomethylphosphonate (103 μL, 112 mg, 634 μmol, 1.00 eq), hydrazine monohydrate (1.54 mL, 1.59 g, 31.7 mmol, 50.0 eq) and Zn(OTf)2 (11.5 mg, 31.7 μmol, 0.0500 eq) was stirred at 60 °C for 30 min. Then, an aqueous solution of NaNO2 (875 mg, 12.7 mmol, 20.0 eq) was added and acidified to pH ~ 3 by dropwise addition of 1 M HClaq. This was extracted with DCM and the solvent was evaporated.

[0443] Purification by flash chromatography (dichloromethane / MeOH 40:1 to 10:1) afforded 20 as a dark pink oil (9.0 mg, 3%).

[0444] Example 21 Synthesis of ((6-(4-(13-hydroxy-2,5,8,11-tetraoxatridecyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (21) [ka]

[0445] To a solution of 20 (9.0 mg, 17.5 μmol, 1.00 eq) in DMF (0.2 mL) was added trimethylsilyl bromide (11.6 μL, 87.5 μmol, 5.00 eq) at 0° C., and the mixture was stirred at room temperature for 90 min. The reaction was quenched with MeOH and HO, and the mixture was directly subjected to HPLC purification.

[0446] Purification by HPLC gave 21 as a dark pink oil (1.7 mg, 21%).

[0447] Example 22 Synthesis of 4-(6-(2,5,8,11,14,17,20,23,26,29,32,35,38-Tridecaoxanonatriacontyl)-1,2,4,5-tetrazin-3-yl)benzoic acid (22) [ka]

[0448] To a mixture of 2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxatetracontane-40-nitrile (1.20 g, 2.00 mmol, 1.00 eq), 4-cyanobenzoic acid (1.18 g, 8.00 mmol, 4.00 eq) in EtOH (2 mL) was added 3-mercaptopropionic acid (174 μL, 212 mg, 2.00 mmol, 1.00 eq) followed by hydrazine monohydrate (1.55 mL, 1.60 g, 32.0 mmol, 16.0 eq) at 0 °C. The mixture was stirred overnight at room temperature. Then, an aqueous solution of NaNO2 (5.52 g, 80.0 mmol, 20.0 eq) was added and the mixture was acidified to pH ~ 3 by dropwise addition of 1 M HClaq. Extracted with DCM and evaporated the solvent.

[0449] Purification by flash chromatography (dichloromethane / MeOH 20:1 to 10:1) afforded 22 as a pink oil (352 mg, 23%).

[0450] Example 23 Synthesis of 4-((S)-2-((S)-2-(4-(6-(2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxanonatriacontyl)-1,2,4,5-tetrazin-3-yl)benzamido)-3-methylbutanamido)propanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R )-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (23) [ka]

[0451] To a solution of H-Val-Ala-PAB-MMAE (27) (11.0 mg, 10.6 μmol, 1.00 eq), 22 (9.86 mg, 12.7 μmol, 1.20 eq), HATU (6.05 mg, 15.9 μmol, 1.50 eq) and DIPEA (4.62 μL, 3.42 mg, 26.5 μmol, 2.50 eq) were added and the mixture was stirred at room temperature for 1 h. The crude reaction mixture was directly subjected to HPLC purification.

[0452] Purification by HPLC gave 23 as a pink powder (3.9 mg, 20%).

[0453] Example 24 Synthesis of ethyl hydrogen ((6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (24) [ka]

[0454] To a mixture of 4-aminomethylbenzonitrile (169 mg, 1.00 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (1.62 mL, 1.77 g, 10.0 mmol, 10.0 eq) was added hydrazine monohydrate (2.43 μm, 2.50 g, 50.0 mmol, 50.0 eq) and Zn(OTf)2 (18.2 mg, 50.0 μmol, 0.0500 eq). The mixture was stirred overnight at room temperature. Then NaNO2 in HO (1.38 g, 20.0 mmol, 20.0 eq) was added and the mixture was acidified to pH ~ 3 by dropwise addition of 1 M HClaq.

[0455] Purification by HPLC gave 24 as a pink powder (26.1 mg, 30%).

[0456] Example 25 Synthesis of ((6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (25) [ka]

[0457] To a solution of 24 (94.0 mg, 272 μmol, 1.00 eq) in DMF was added trimethylsilyl bromide (382 μL, 416 mg, 2.72 mmol, 10.0 eq) at 0° C. The mixture was stirred at room temperature for 5 h. It was then diluted with MeOH and HO and the solvent was evaporated.

[0458] Purification by HPLC gave 2 as a pink powder (26.1 mg, 30%).

[0459] Example 26 Synthesis of 4-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)benzoic acid (26) [ka]

[0460] To a solution of 4-cyanobenzoic acid (649 mg, 4.37 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (3.15 mL, 3.45 g, 30.0 mmol, 6.86 eq) was added hydrazine monohydrate (7.28 mL, 7.51 g, 150 mmol, 34.3 eq) and Zn(OTf)2 (54.5 mg, 150 μmol, 0.0500 eq). The mixture was stirred at room temperature for 4 h. Then NaNO2 (5.52 g, 80.0 mmol, 20.0 eq) in H2O was added and acidified to pH ~ 3 by dropwise addition of 1 M HClaq. The pink precipitate was filtered and washed with 0.1 M HClaq to give a crude mixture of 3 and 4-(6-((ethoxy(hydroxy)phosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)benzoic acid.

[0461] To a solution of the crude precipitate in DMF was added trimethylsilyl bromide (2.64 mL, 3.06 g, 20.0 mmol, 5.00 equiv) at 0° C. The mixture was stirred at room temperature for 5 h. It was then diluted with MeOH and HO and the solvent was evaporated.

[0462] Purification by HPLC gave 26 as a pink powder (64.5 mg, 5%).

[0463] Example 27 Synthesis of H-Val-Ala-PAB-MMAE (4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate) (27) [ka]

[0464] To a solution of Fmoc-Val-Ala-PAB-PNP (34.1 mg, 50.1 μmol, 1.00 eq) and MMAE (36.0 mg, 50.1 μmol, 1.00 eq) in DMF (0.5 mL) was added pyridine (0.25 mL), HOBt (0.768 mg, 5.01 μmol, 0.100 eq) and DIPEA (8.73 μL, 6.48 mg, 50.1 μmol, 1.00 eq) and the mixture was stirred at room temperature for 8 h. After complete conversion, piperidine (0.125 mL) was added and the mixture was stirred for an additional 5 min before being directly submitted to HPLC purification.

[0465] Purification by HPLC gave 27 (44.3 mg, 85%) as a white solid.

[0466] Example 28 Synthesis of H-Val-Ala-PAB-Maytansinoid ((14S,32S,33R,2S,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxiran-8(1,3)-benzenecyclotetradecaphane-10,12-dien-4-yl N-(((4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-N-methyl-L-alaninate) (28) [ka]

[0467] To a solution of Fmoc-Val-Ala-PAB-PNP (41.0 mg, 63.0 μmol, 1.00 eq) and maytansinoid (42.9 mg, 63.0 μmol, 1.00 eq) in DMF (0.6 mL) was added pyridine (0.28 mL), HOBt (0.965 mg, 6.30 μmol, 0.100 eq) and DIPEA (10.9 μL, 8.14 mg, 63.0 μmol, 1.00 eq) and the mixture was stirred at room temperature for 8 h. After complete conversion, piperidine (0.15 mL) was added and the mixture was stirred for an additional 5 min before being directly subjected to HPLC purification.

[0468] Purification by HPLC gave 28 (25.8 mg, 42%) as a white solid.

[0469] Example 29 Synthesis of diethyl (6-cyanopyridin-3-yl)phosphonate (29) [ka]

[0470] To a mixture of diethyl phosphite (567 μL, 608 mg, 4.40 mmol, 1.10 eq), NEt3 (610 μL, 445 mg, 4.40 mmol, 1.10 eq) and Pd(PPh3)4 (231 mg, 200 μmol, 0.0500 eq), 5-bromo-2-cyanopyridine (732 mg, 4.00 mmol, 1.00 eq) and 1 mL of toluene were added and the mixture was stirred at 90 °C for 1 h.

[0471] Purification by flash chromatography (cyclohexane / ethyl acetate 1:1) afforded 29 as a colorless oil (650 mg, 68%).

[0472] Example 30 Synthesis of 3-(6-(4-(hydroxy(methoxy)phosphoryl)phenyl)-1,2,4,5-tetrazin-3-yl)propanoic acid (30) [ka]

[0473] To a solution of dimethyl(4-cyanophenyl)phosphonate (54.0 mg, 256 μmol, 1.00 eq) and 3-cyanopropionic acid (87.0 mg, 878 μmol, 3.43 eq) in EtOH (0.26 mL) was added hydrazine monohydrate (170 μL, 176 mg, 3.51 mmol, 13.7 eq) followed by 3-mercaptopropionic acid (19.1 μL, 23.3 mg, 220 μmol, 0.859 eq). The mixture was stirred overnight at room temperature. Sodium nitrite (303 mg, 4.39 mmol, 20.0 eq) was then added and the solution was acidified by dropwise addition of 1 M HClaq. It was extracted with ethyl acetate (3 times) and the combined organic phases were dried over sodium sulfate and evaporated under reduced pressure.

[0474] Purification by reverse-phase flash chromatography (0-50% MeOH in H2O) afforded 30 as a pink solid (40.4 mg, 49%).

[0475] Example 31 Synthesis of 3-(6-(4-phosphonophenyl)-1,2,4,5-tetrazin-3-yl)propanoic acid (31) [ka]

[0476] To a solution of 30 (15.4 mg, 47.5 μmol, 1.00 eq) in DMF (0.5 mL) was added trimethylsilyl bromide (31.3 μL, 36.4 mg, 237 μmol, 5.00 eq) at 0° C. The mixture was stirred at room temperature for 30 min. Water was added and the mixture was directly purified.

[0477] Purification by reverse-phase flash chromatography (0-50% MeOH in H2O) afforded 31 as a pink solid (12.3 mg, 83%).

[0478] Example 32 Synthesis of (4-(6-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)-1,2,4,5-tetrazin-3-yl)phenyl)phosphonic acid (32) [ka]

[0479] To a solution of 31 (9.50 mg, 30.6 μmol, 1.00 eq) in DMF (0.3 mL) was added NEt3 (5.09 μL, 3.72 mg, 36.8 μmol, 1.20 eq) and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (11.1 mg, 36.8 μmol, 1.20 eq). The mixture was stirred at room temperature. After 1.5 h, another 10 mg of TSTU and 4 μL of NEt3 were added and the mixture was stirred for another 30 min. It was diluted with water and the mixture was directly purified by HPLC.

[0480] Purification by HPLC gave 32 as a pink solid (2.2 mg, 18%).

[0481] Example 33 Synthesis of 2-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)acetic acid (33) [ka]

[0482] To a solution of 17 (30.0 mg, 103 μmol, 1.00 eq) in DMF (0.5 mL) was added trimethylsilyl bromide (136 μL, 158 mg, 1.03 mmol, 10.0 eq) at 0° C. The mixture was stirred at room temperature for 3.5 h. Water was added and the mixture was directly purified by HPLC.

[0483] Purification by HPLC gave 33 as a pink solid (2.8 mg, 12%).

[0484] Example 34 Synthesis of diethyl (4-(6-(5-aminopyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)phosphonate (34) [ka]

[0485] To a solution of diethyl(4-cyanophenyl)phosphonate (398 mg, 256 μmol, 2.00 eq) and 2-cyano-5-aminopyrimidine (100 mg, 833 μmol, 1.00 eq) in EtOH (0.8 mL) was added hydrazine monohydrate (646 μL, 667 mg, 13.3 mmol, 16.0 eq) followed by 3-mercaptopropionic acid (218 μL, 265 mg, 2.50 mmol, 3.00 eq). The mixture was stirred at room temperature overnight. The reaction was diluted with water and extracted with dichloromethane (2 times). The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure.

[0486] The residue was dissolved in 5 mL of dichloromethane, 150 mg of p-benzoquinone was added, and the mixture was stirred for 5 min and directly subjected to column chromatography.

[0487] Purification by flash chromatography (dichloromethane / MeOH, gradient from 20:1 to 10:1) afforded 34 as an orange-brown oil (139 mg, 43%).

[0488] Example 35 Synthesis of (4-(6-(5-aminopyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)phosphonic acid (35) [ka]

[0489] To a solution of 34 (22.8 mg, 58.9 μmol, 1.00 eq) in DMF (0.6 mL) was added trimethylsilyl bromide (155 μL, 180 mg, 1.18 mmol, 20.0 eq) at 0° C. The mixture was stirred at room temperature for 2 h. Water was added and the mixture was stirred for 5 min. The formed precipitate was filtered and washed with water and acetone.

[0490] 35 was isolated as a red solid (2.8 mg, 12%).

[0491] Example 36 Synthesis of diethyl (6-(6-(5-aminopyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)phosphonate (36) [ka]

[0492] To a solution of 29 (240 mg, 999 μmol, 1.00 eq) and 2-cyano-5-aminopyridine (476 mg, 4.00 mmol, 4.00 eq) in EtOH (1 mL), hydrazine monohydrate (775 μL, 800 mg, 13.3 mmol, 16.0 eq) was added, followed by 3-mercaptopropionic acid (87.1 μL, 106 mg, 999 μmol, 1.00 eq). The mixture was stirred overnight at room temperature. Then, 1 mL of DMF was added to dissolve the formed precipitate, and the mixture was stirred at 60 °C for 4 h. The mixture was directly purified by reverse phase column chromatography.

[0493] The isolated dihydrotetrazine compound (9.9 mg) was dissolved in 0.5 mL DMF / MeOH, 2.7 mg of p-benzoquinone was added, and the mixture was stirred for 5 minutes. The mixture was directly purified by HPLC.

[0494] Purification by HPLC gave 36 as an orange solid (7.3 mg, 2%).

[0495] Example 37 Synthesis of (6-(6-(5-aminopyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)phosphonic acid (37) [ka]

[0496] To a solution of 36 (3.8 mg, 9.81 μmol, 1.00 eq) in DMF (0.1 mL) was added trimethylsilyl bromide (12.9 μL, 15.0 mg, 98.1 μmol, 10.0 eq) at 0 °C. The mixture was stirred at room temperature overnight. Then, 12 μL of TMS-Br was added and the mixture was stirred at room temperature for 5 h. Then, 20 μL of TMS-Br was added and the mixture was stirred at room temperature for 6 h. Water was added and the mixture was directly purified by HPLC.

[0497] Purification by HPLC gave 37 as an orange solid (1.8 mg, 55%).

[0498] Example 38 Synthesis of (6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)glycine (38) [ka]

[0499] To a solution of (6-cyanopyridin-3-yl)glycine (340 mg, 1.92 mmol, 1.00 eq) and diethyl cyanomethylphosphonate (621 μL, 680 mg, 3.84 mmol, 2.00 eq) in EtOH (2 mL) was added hydrazine monohydrate (1.49 mL, 1.54 g, 30.7 mmol, 16.0 eq) followed by 3-mercaptopropionic acid (502 μL, 611 mg, 5.76 mmol, 3.00 eq). The mixture was stirred at room temperature overnight. Afterwards, the volatile components were removed under reduced pressure and the residue was purified by reverse phase column chromatography.

[0500] The isolated dihydrotetrazine compound (320 mg) was dissolved in 10 mL of DMF / MeOH, 108 mg of p-benzoquinone was added, and the mixture was stirred for 5 min.

[0501] The mixture was directly purified by column chromatography to give 35 mg of product 38. The isolated mixed fraction was further purified by HPLC.

[0502] Purification by HPLC gave 38 as a red solid (130 mg, 18%).

[0503] Example 39 Synthesis of (6-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)glycine (39) [ka]

[0504] To a solution of 38 (35.0 mg, 91.5 μmol, 1.00 eq) in DMF (1 mL) was added trimethylsilyl bromide (109 μL, 126 mg, 824 μmol, 9.00 eq) at 0° C. The mixture was stirred at room temperature for 4 h. Water was added and the mixture was stirred for 30 min. It was extracted with ethyl acetate and the aqueous phase was lyophilized. The resulting oil was precipitated from acetone and filtered.

[0505] 39 was isolated as a red solid (30.0 mg, 90%).

[0506] Example 40 Synthesis of Boc-Val-Cit-PAB-MMAE 4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (40) [ka]

[0507] To a solution of Boc-Val-Cit-PAB-PNP (18.0 mg, 27.9 μmol, 1.00 eq) and MMAE (20.0 mg, 27.9 μmol, 1.00 eq) in DMF (0.25 mL), pyridine (0.125 mL), HOBt (0.428 mg, 2.79 μmol, 0.100 eq) and DIPEA (4.86 μL, 3.61 mg, 27.9 μmol, 1.00 eq) were added and the mixture was stirred at room temperature for 2 days. The mixture was then directly subjected to HPLC purification.

[0508] Purification by HPLC afforded 40 (25.2 mg, 74%) as a white solid.

[0509] Example 41 Synthesis of H-Val-Cit-PAB-MMAE 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (41) [ka]

[0510] To a solution of 40 (25.2 mg, 20.6 μmol, 1.00 equiv) in dichloromethane (0.8 mL) was added trifluoroacetic acid (0.2 mL) and the mixture was stirred at room temperature for 15 min, after which the solvent was removed under reduced pressure.

[0511] Purification by HPLC afforded 41 (17.0 mg, 73%) as a white solid.

[0512] Example 42 Synthesis of 4-((S)-2-((S)-2-(2-((6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((( 1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (42) [ka]

[0513] To a solution of 41 (17.0 mg, 15.1 μmol, 1.00 eq) in DMF (0.3 mL) was added 38 (5.79 mg, 15.1 μmol, 1.00 eq), EDC (3.48 mg, 18.2 μmol, 1.20 eq) and NEt3 (3.15 μL, 2.30 mg, 22.7 μmol, 1.50 eq) and the mixture was stirred at room temperature for 4 h. Then, an additional 10 mg of 38, 8 mg of EDC and 38 μL of NEt were added and the mixture was stirred for another 4 h. The mixture was directly subjected to HPLC purification.

[0514] Purification by HPLC gave 42 (2.4 mg, 11%) as a pale red solid.

[0515] Example 43 ((6-(5-((2-(((S)-1-(((S)-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-di Synthesis of isopropyl 1-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)amino)pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (43) [ka]

[0516] To a solution of 42 (2.4 mg, 1.61 μmol, 1.00 eq) in DMF (0.1 mL) was added TMS-Br (10.6 μL, 12.3 mg, 80.7 μmol, 50.0 eq) and the mixture was stirred at room temperature for 2 days. Water was added and the mixture was extracted with ethyl acetate. The aqueous phase was lyophilized.

[0517] 43 was isolated as a pale red solid (1 mg, 43%).

[0518] Example 44 Synthesis of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (44) [ka]

[0519] To a solution of MMAE (19.6 mg, 27.4 μmol, 1.00 eq) and β-D-glucuronide-PNP-carbonate (25.0 mg, 27.4 μmol, 1.00 eq) in DMF (0.5 mL) was added pyridine (0.25 mL), HOBt (0.419 mg, 2.74 μmol, 0.100 eq) and DIPEA (4.77 μL, 27.4 μmol, 1.00 eq). The mixture was stirred at room temperature overnight. Then 1M NaOHaq (0.274 mL, 274 μmol, 10.0 eq) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by HPLC.

[0520] Purification by HPLC gave 44 (17.0 mg, 55%).

[0521] Example 45 Synthesis of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (45) [ka]

[0522] To a solution of exatecan mesylate (14.5 mg, 27.4 μmol, 1.00 eq) and β-D-glucuronide-PNP-carbonate (25.0 mg, 27.4 μmol, 1.00 eq) in DMF (0.5 mL) was added pyridine (0.25 mL), HOBt (0.419 mg, 2.74 μmol, 0.100 eq) and DIPEA (4.77 μL, 27.4 μmol, 1.00 eq). The mixture was stirred at room temperature overnight. Then 1M NaOHaq (0.274 mL, 274 μmol, 10.0 eq) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by HPLC.

[0523] Purification by HPLC gave 45 (3.0 mg, 13%).

[0524] Example 46 Synthesis of 4-((S)-4-amino-2-((S)-2-((S)-2-aminopropanamido)propanamido)-4-oxobutanamido)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (46) [ka]

[0525] To a solution of MMAE (28.1 mg, 39.1 μmol, 1.00 eq) and Fmoc-Ala-Ala-Asn-PAB-PNP (30.0 mg, 39.1 μmol, 1.00 eq) in DMF (0.5 mL) was added pyridine (0.25 mL), HOBt (0.598 mg, 3.91 μmol, 0.100 eq) and DIPEA (6.81 μL, 39.1 μmol, 1.00 eq). The mixture was stirred at room temperature overnight. Then, 1 M NaOHaq (0.391 mL, 391 μmol, 10.0 eq) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was directly purified by HPLC.

[0526] Purification by HPLC gave 46 (16.6 mg, 38%).

[0527] Example 47 Synthesis of (3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-15,16-dithia-5,8,11-triazanonadecan-19-oic acid (47)

[0528] [ka]

[0529] To a solution of 3,3'-dithiodipropionic acid (29.3 mg, 139.3 μmol, 2.00 eq) and HATU (26.5 mg, 69.6 μmol, 1.00 eq) in DMF (0.5 mL) was added DIPEA (12.1 μL, 69.6 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, MMAE (50.0 mg, 69.6 μmol, 1.00 eq) and DIPEA (12.1 μL, 69.6 μmol, 1.00 eq) were added and the reaction mixture was stirred at room temperature overnight and directly subjected to HPLC purification.

[0530] Purification by HPLC gave 47 (43.0 mg, 68%).

[0531] Example 48 Synthesis of tert-butyl (2-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate (48) [ka]

[0532] A solution of tert-butyl N-(2-cyanoethyl)carbamate (500 mg, 2.94 mmol, 1.00 eq), diethyl cyanomethylphosphonate (950 μL, 5.88 mmol, 2.00 eq), and 3-mercaptopropionic acid (768 μL, 8.813 mmol, 3.00 eq) in EtOH / DMF (1:1, 10 mL) was added to hydrazine monohydrate (2.28 mL, 47.0 mmol, 16.0 eq) and the mixture was stirred at room temperature overnight. Afterwards, NaNO2 in H2O (4.99 g, 58.8 mmol, 20.0 eq) was added and the pH was acidified to about 3 by dropwise addition of 1 M HClaq. The formed precipitate was filtered and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure. Purification by reverse phase column chromatography gave 48 (360 mg, 33%).

[0533] Example 49 Synthesis of diethyl ((6-(2-aminoethyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (49)

[0534] [ka]

[0535] To a solution of 48 (160 mg, 0.426 mmol, 1.00 eq) in DCM (2 ml) was added TFA (328 μl, 4.26 mmol, 10.0 eq) and the mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure.

[0536] Evaporation gave crude 49 (192 mg), which was used crude in subsequent reactions.

[0537] Example 50 Synthesis of ((6-(2-aminoethyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (50) [ka]

[0538] To a solution of crude 49 (100 mg, 257 μmol, 1.00 equiv) in DMF (5.1 mL) was added trimethylsilyl bromide (170 μL, 1.28 mmol, 5.00 equiv) at 0 °C and the mixture was stirred at room temperature overnight. Water was added and the solvent was evaporated under reduced pressure. Purification by reverse phase chromatography (HO / MeOH, 0-50%, 10 g Sfaer C18)

[0539] Purification by reverse phase chromatography gave 50 (12.5 mg, 22%).

[0540] Example 51 Synthesis of diethyl ((6-(5-aminopyrimidin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (51) [ka]

[0541] A solution of 5-amino-2-pyrimidinecarbonitrile (200 mg, 1.67 mmol, 1.00 eq), diethyl cyanomethylphosphonate (0.539 mL, 3.33 mmol, 2.00 eq), and 3-mercaptopropionic acid (435 μL, 5.00 mmol, 3.00 eq) in EtOH / DMF (1:1, 5 mL) was added to hydrazine monohydrate (1.29 mL, 26.6 mmol, 16.0 eq) and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure. Purification by reverse phase column chromatography gave mixed fractions. The combined fractions containing the product were purified by flash chromatography.

[0542] Purification by flash chromatography (MeOH in DCM, following a gradient from 0% to 15%) afforded 51 (97.0 mg, 18%).

[0543] Example 52 Synthesis of diethyl((6-(5-aminopyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (52) [ka]

[0544] To a solution of 51 (97.0 mg, 296 μmol, 1.00 eq) in THF / MeOH (9:1, 3 mL) was added p-benzoquinone (39.4 mg, 365 μmol, 1.23 eq) and the mixture was stirred at room temperature for 5 min. It was diluted with DCM and quenched with H2O and a saturated aqueous solution of NaHCO3. It was extracted with ethyl acetate (3 times), the combined organic phases were dried over sodium sulfate and the solvent was evaporated under reduced pressure.

[0545] Purification by flash chromatography (MeOH in DCM, following a gradient from 0% to 30%) afforded 52 (33.0 mg, 34%).

[0546] Example 53 Synthesis of ((6-(5-aminopyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (53) [ka]

[0547] To a solution of 52 (100 mg, 307 μmol, 1.00 eq) in DMF (1 mL) was added trimethylsilyl bromide (203 μL, 1.54 mmol, 5.00 eq) at 0° C. and the mixture was stirred at room temperature overnight. Water was added and the solvent was evaporated under reduced pressure. Purification by reverse phase column chromatography gave 53 (36.0 mg, 44%).

[0548] Example 54 Synthesis of ((6-(5-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-15,16-dithia-5,8,11-triazanonadecan-19-amido)pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (54) [ka]

[0549] To a solution of 47 (10.0 mg, 13.2 μmol, 1.00 eq) and HATU (4.17 mg, 11.0 μmol, 1.00 eq) in DMF (0.5 mL) was added triethylamine (1.5 μL, 11.0 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, 53 (4.60 mg, 13.2 μmol, 1.20 eq) and triethylamine (1.53 μL, 11.0 μmol, 1.00 eq) were added and the mixture was stirred at room temperature overnight. The reaction mixture was directly subjected to HPLC purification.

[0550] Purification by HPLC gave 54 (5.0 mg, 39%).

[0551] Example 55 Synthesis of 2-(2-((6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-2-oxoethoxy)acetic acid (55) [ka]

[0552] To a solution of 18 (64.8 mg, 200 μmol, 1.00 eq) and diglycolic anhydride (27.8 mg, 240 μmol, 1.20 eq) in DMF (2 mL) was added 4-(dimethylamino)pyridine (2.44 mg, 20.0 μmol, 0.100 eq) and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure.

[0553] Purification by reverse-phase column chromatography afforded 55 (72.0 mg, 82%) as a pink oil.

[0554] Example 56 Synthesis of 4-((6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-4-oxobutanoic acid (56) [ka]

[0555] A solution of 18 (60.0 mg, 185 μmol, 1.00 eq) and succinic anhydride (22.2 mg, 222 μmol, 1.20 eq) in CHCl3 (0.4 mL) was stirred overnight at 60 °C. The reaction mixture was diluted with DCM. The pink precipitate was filtered and washed with DCM.

[0556] 56 (69.0 mg, 88%) was isolated as a pink solid.

[0557] Example 57 Synthesis of tert-butyl (2-((6-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-2-oxoethyl)carbamate (57) [ka]

[0558] To a solution of N-Boc-glycine (486 mg, 2.78 mmol, 2.00 eq) in THF (5 mL), N-methylmorpholine (763 μL, 6.94 mmol, 5.00 eq) and isobutyl chloroformate (360 μL, 2.78 mmol, 2.00 eq) were added at 0 °C and the mixture was stirred for 5 min. Then, 18 (450 mg, 1.39 mmol, 1.00 eq) was added at 0 °C and the mixture was stirred overnight at room temperature. Water and ethyl acetate were added, the phases were separated and the aqueous phase was extracted with ethyl acetate (2 times). The combined organic phases were washed with saturated aqueous NaHCO3, dried over Na2SO4 and the solvent was evaporated under reduced pressure.

[0559] Purification by reverse phase chromatography gave 57 (300 mg, 45%).

[0560] Example 58 Synthesis of ((6-(5-(2-aminoacetamido)pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (58) [ka]

[0561] To a solution of 57 (200 mg, 415 μmol, 1.00 eq) in DMF (1 mL) was added trimethylsilyl bromide (274 μL, 2.08 mmol, 5.00 eq) at 0° C. and the mixture was stirred at room temperature overnight. MeOH and water were added and the solvent was evaporated under reduced pressure.

[0562] Purification by reverse phase column chromatography gave 58 (66.0 mg, 49%).

[0563] Example 59 Synthesis of ((6-(5-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,19-tetraoxo-2-oxa-15,16-dithia-5,8,11,20-tetraazadocosan-22-amido)pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (59) [ka]

[0564] To a solution of 47 (10.0 mg, 11.0 μmol, 1.00 eq) and HATU (4.17 mg, 11.0 μmol, 1.00 eq) in DMF (0.5 mL) was added triethylamine (1.53 μL, 11.0 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, 58 (5.35 mg, 13.2 μmol, 1.20 eq) and triethylamine (1.53 μL, 11.0 μmol, 1.00 eq) were added and the mixture was stirred at room temperature overnight and directly submitted to HPLC purification.

[0565] Purification by HPLC gave 59 (1.7 mg, 13%).

[0566] Example 60 Synthesis of tert-butyl (2-((2-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)pyrimidin-5-yl)amino)-2-oxoethyl)carbamate (60) [ka]

[0567] To a solution of N-Boc-glycine (255 mg, 1.46 mmol, 2.00 eq) in THF (5 mL), N-methylmorpholine (0.401 mL, 3.64 mmol, 5.00 eq) and isobutyl chloroformate (0.189 mL, 1.46 mmol, 2.00 eq) were added at 0 °C and the mixture was stirred for 5 min. Then, 52 (237 mg, 0.729 mmol, 1.00 eq) was added and the mixture was stirred at room temperature overnight. Water and ethyl acetate were added, the phases were separated and the aqueous phase was extracted with ethyl acetate (2 times). The combined organic phases were washed with saturated aqueous NaHCO3, dried over Na2SO4 and the solvent was evaporated under reduced pressure.

[0568] Purification by reverse phase column chromatography gave 60 (152 mg, 43%).

[0569] Example 61 Synthesis of diethyl ((6-(5-(2-aminoacetamido)pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (61) [ka]

[0570] To a solution of 60 (130 mg, 0.269 mmol, 1.00 eq) in DCM (2 mL) was added TFA (208 μl, 2.70 mmol, 10.0 eq) at room temperature and the mixture was stirred overnight. The solvent was removed under reduced pressure.

[0571] Evaporation gave 61 (79 mg, 59%).

[0572] Example 62 Synthesis of ((6-(5-(2-aminoacetamido)pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (62) [ka]

[0573] To a solution of 61 (67.0 mg, 135 μmol, 1.00 eq) in DMF (0.5 mL) was added TMS-Br (178 μL, 1.35 mmol, 10.0 eq) at 0° C. and the mixture was stirred at room temperature overnight. MeOH and water were added and the solvent was evaporated under reduced pressure.

[0574] Purification by reverse phase column chromatography gave 62 (92 mg) as a mixture with DMF, which was used directly in the next reaction.

[0575] Example 63 Synthesis of ((6-(5-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,19-tetraoxo-2-oxa-15,16-dithia-5,8,11,20-tetraazadocosan-22-amido)pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (63) [ka]

[0576] To a solution of 47 (10.0 mg, 11.0 μmol, 1.00 eq) and HATU (4.17 mg, 11.0 μmol, 1.00 eq) in DMF (0.5 mL) was added triethylamine (1.53 μL, 11.0 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then 62 (5.37 mg, 13.18 μmol, 1.20 eq) triethylamine (1.53 μL, 11.0 μmol, 1.00 eq) was added and the mixture was stirred at room temperature overnight and directly submitted to HPLC purification.

[0577] Purification by HPLC gave 63 (1.2 mg, 9%).

[0578] Example 64 Synthesis of (6-(6-(5-((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2-oxa-15,16-dithia-5,8,11-triazanonadecan-19-amido)pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)phosphonic acid (64) [ka]

[0579] To a solution of 47 (2.75 mg, 3.02 μmol, 1.00 eq) and HATU (1.15 mg, 3.02 μmol, 1.00 eq) in DMF (0.5 mL) was added triethylamine (0.42 μL, 3.02 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Afterwards, 37 (1.00 mg, 3.02 μmol, 1.00 eq) and triethylamine (0.42 μL, 3.02 μmol, 1.00 eq) were added and the mixture was stirred at room temperature overnight and directly submitted to HPLC purification.

[0580] Purification by HPLC gave 64 (1 mg, 27%).

[0581] Example 65 (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8- Synthesis of diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(3-(6-(4-phosphonophenyl)-1,2,4,5-tetrazin-3-yl)propanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (65) [ka]

[0582] To a solution of 32 (2.00 mg, 4.91 μmol, 1.00 eq) and 44 (4.44 mg, 3.93 μmol, 0.800 eq) in DMF (0.3 mL) was added triethylamine (0.753 μL, 5.40 μmol, 1.10 eq), and the mixture was stirred at room temperature for 3 h and directly subjected to HPLC purification.

[0583] Purification by HPLC gave 65 (4.4 mg, 63%).

[0584] Example 66 Synthesis of (2S,3S,4S,5R,6S)-6-(4-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)-2-(3-(4-(6-(phosphonomethyl)-1,2,4,5-tetrazin-3-yl)benzamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (66) [ka]

[0585] A solution of 12 (0.928 mg, 2.36 μmol, 1.00 eq) and 45 (2.00 mg, 2.36 μmol, 1.00 eq) was added with triethylamine (0.362 μL, 2.60 μmol, 1.10 eq), stirred at room temperature for 3 h, and directly subjected to HPLC purification.

[0586] Purification by HPLC gave 66 (0.6 mg, 23%).

[0587] Example 67 ((6-(4-(((S)-1-(((S)-1-(((S)-4-amino-1-((4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl Synthesis of diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (67) [ka]

[0588] A solution of 12 (1.40 mg, 3.56 μmol, 1.00 eq) and 46 (4.00 mg, 3.56 μmol, 1.00 eq) in DMF (0.5 mL) was added with triethylamine (0.545 μL, 3.91 μmol, 1.10 eq), stirred at room temperature for 3 h, and directly subjected to HPLC purification.

[0589] Purification by HPLC gave 67 (1.0 mg, 20%).

[0590] Example 68 Synthesis of 3-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)propanoic acid (68) [ka]

[0591] A solution of 3-cyanopropionic acid (400 mg, 4.04 mmol, 1.00 eq), diethyl cyanomethylphosphonate (2.61 mL, 16.1 mmol, 4.00 eq) and 3-mercaptopropionic acid (1.06 mL, 12.1 mmol, 3.00 eq) in EtOH (1 mL) was added to hydrazine monohydrate (3.13 mL, 64.6 mmol, 16.0 eq) at 50 °C and the mixture was stirred at this temperature for 5 h. Afterwards, the reaction mixture was cooled to 0 °C, NaNO2 in H2O (6.86 g, 80.7 mmol, 20.0 eq) was added and acidified to pH ~ 3 by dropwise addition of 1 M HClaq. The formed precipitate was filtered and the aqueous phase was extracted with ethyl acetate (2 times). The combined organic phases were dried over Na2SO4 and the solvent was evaporated under reduced pressure.

[0592] Purification by reverse phase column chromatography gave mixed fractions. The fractions containing the product were combined, the solvent was evaporated under reduced pressure and the residue was dissolved in water (5 mL). The precipitate formed was filtered and the filtrate was lyophilized.

[0593] 68 (140 mg, 11%) was isolated as a pink oil.

[0594] Example 69 Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)propanoate (69) [ka]

[0595] To a solution of 68 (120 mg, 0.394 mmol, 1.00 eq) and TSTU (142 mg, 0.473 mmol, 1.20 eq) in DCM (0.5 mL) was added DIPEA (82.4 μL, 0.473 mmol, 1.20 eq) and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure.

[0596] Purification by reverse phase column chromatography gave 69 (88.0 mg, 56%).

[0597] Example 70 Synthesis of 4-((S)-4-amino-2-((S)-2-((S)-2-(3-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)propanamide)propanamide)propanamide)-4-oxobutanamide)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1 S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (70) [ka]

[0598] To a solution of 69 (1.43 mg, 3.56 μmol, 1.00 eq) and 46 (4.00 mg, 3.56 μmol, 1.00 eq) in DMF (0.3 mL) was added triethylamine (0.992 μL, 7.12 μmol, 2.00 eq), and the mixture was stirred at room temperature for 3 h and directly subjected to HPLC purification.

[0599] Purification by HPLC gave 70 (1.8 mg, 36%).

[0600] Example 71 (2S,3S,4S,5R,6S)-6-(4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8- Diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(3-(6-((diethoxyphosphoryl)methyl)-1,2,4,5-tetrazin-3-yl)propanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (71) [ka]

[0601] To a solution of 69 (1.56 mg, 3.89 μmol, 1.10 eq) and 45 (4.00 mg, 3.54 μmol, 1.00 eq) in DMF (0.3 mL) was added triethylamine (0.986 μL, 7.08 μmol, 2.00 eq), and the mixture was stirred at room temperature for 3 h and directly subjected to HPLC purification.

[0602] Purification by HPLC gave 71 (1.2 mg, 24%).

[0603] Example 72 Synthesis of ((6-(3,5-bis(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (72) [ka]

[0604] To a solution of 15 (135 mg, 229 μmol, 1.00 eq) in DMF (2.2 mL) was added TMS-Br (151 μL, 1.14 mmol, 5.00 eq) at 0 °C, and the mixture was stirred at room temperature overnight. Water and MeCN were added, and the mixture was directly purified by reverse phase column chromatography.

[0605] Purification by reverse phase column chromatography afforded 72 (11.2 mg, 9%) as a pink solid.

[0606] Example 73 Synthesis of diethyl ((6-(3,5-bis(((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,15,18,21,24,27,30,33,36-nonaoxa-5,8,11-triazaoctatriacontan-38-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (73) [ka]

[0607] To a solution of 16 (23.3 mg, 18.7 μmol, 1.00 eq) and MMAE (32.3 mg, 45.0 μmol, 2.40 eq) in DMF (0.2 mL), HATU (21.4 mg, 56.2 μmol, 3.00 eq) and triethylamine (13.0 μL, 93.7 μmol, 5.00 eq) were added and the mixture was stirred at room temperature for 2 h. Water was then added and the mixture was directly subjected to HPLC purification.

[0608] Purification by HPLC gave 73 (22.5 mg, 45%) as a pink oil.

[0609] Example 74 Synthesis of ((6-(3,5-bis(((3R,4S,7S,10S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12-trioxo-2,15,18,21,24,27,30,33,36-nonaoxa-5,8,11-triazaoctatriacontan-38-yl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (74) [ka]

[0610] To a solution of 73 (22.0 mg, 8.32 μmol, 1.00 eq) in DMF (0.2 mL) was added TMS-Br (11.0 μL, 83.2 μmol, 10.0 eq) at 0° C. and the mixture was stirred at room temperature for 6 h. Then, 20 μL of TMS-Br was added and the mixture was stirred overnight. Then, 40 μL of TMS-Br was added and the mixture was stirred for 8 days. Water was added and the mixture was directly purified by HPLC.

[0611] Purification by HPLC gave 74 (3.5 mg, 16%) as a pink powder.

[0612] Example 75 Synthesis of diethyl ((6-(3,5-bis((2-aminoethyl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonate (75) [ka]

[0613] To a solution of 1,2-diaminoethane (44.8 μL, 671 μmol, 6.00 eq) in DMF (0.6 mL) was added a solution of 15 (66.0 mg, 112 μmol, 1.00 eq) in DMF (0.6 mL), and the mixture was stirred at room temperature for 15 min. Then, 0.5 mL of 1 M HCl was added, and the mixture was directly purified by HPLC.

[0614] Purification by HPLC gave 75 (8.0 mg, 15%) as a pink oil.

[0615] Example 76 Synthesis of ((6-(3,5-bis((2-aminoethyl)carbamoyl)phenyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (76) [ka]

[0616] To a solution of 75 (8.00 mg, 16.7 μmol, 1.00 eq) in DMF (0.2 mL) was added TMS-Br (44.0 μL, 333 μmol, 20.0 eq) at 0° C. and the mixture was stirred at room temperature for 2 h. Water was added and the mixture was directly purified by HPLC.

[0617] Purification by HPLC afforded 76 (4.0 mg, 57%) as a pink solid.

[0618] Example 77 Synthesis of 3-((3-(((S)-1-(((14S,16S,32R,33S,2S,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenecyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)disulfanyl)propanoic acid (77) [ka]

[0619] To a solution of 3,3'-dithiodipropionic acid (19.4 mg, 92.3 μmol, 2.00 eq) and HATU (17.5 mg, 46.1 μmol, 1.00 eq) in DMF (0.5 mL) was added DIPEA (8.04 μL, 46.1 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, maytansinol-Ala (30.0 mg, 46.1 μmol, 1.00 eq) and DIPEA (8.04 μL, 46.1 μmol, 1.00 eq) were added and the reaction mixture was stirred at room temperature overnight and directly subjected to HPLC purification.

[0620] Purification by HPLC gave 77 (15.2 mg, 68%).

[0621] Example 78 Synthesis of ((6-(5-(3-((3-(((S)-1-(((14S,16S,32R,33S,2S,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenecyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)disulfanyl)propanamido)pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (78) [ka]

[0622] To a solution of 77 (8.00 mg, 9.50 μmol, 1.00 eq) and HATU (3.61 mg, 9.50 μmol, 1.00 eq) in DMF (0.50 mL) was added triethylamine (1.32 μL, 9.50 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, 53 (3.82 mg, 14.25 μmol, 1.50 eq) and triethylamine (1.32 μL, 9.50 μmol, 1.00 eq) were added and the mixture was stirred at room temperature for 3 h. The reaction mixture was directly subjected to HPLC purification.

[0623] Purification by HPLC afforded 78 (mixture with free 77) (3.20 mg (75% pure), 22%).

[0624] Example 79 Synthesis of ((6-(2-(3-((3-(((S)-1-(((14S,16S,32R,33S,2S,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenecyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)disulfanyl)propanamido)ethyl)-1,2,4,5-tetrazin-3-yl)methyl)phosphonic acid (79) [ka]

[0625] To a solution of 77 (7.20 mg, 8.55 μmol, 1.00 eq) and HATU (3.25 mg, 8.55 μmol, 1.00 eq) in DMF (0.50 mL) was added triethylamine (1.19 μL, 8.55 μmol, 1.00 eq) and the mixture was stirred at room temperature for 1 h. Then, 50 (3.82 mg, 14.25 μmol, 1.50 eq) and triethylamine (1.19 μL, 8.55 μmol, 1.00 eq) were added and the mixture was stirred at room temperature for 3 h. The reaction mixture was directly subjected to HPLC purification.

[0626] Purification by HPLC gave 79 (0.90 mg, 10%).

[0627] C) Preparation of ADCs and cytotoxicity assays Preparation Example 1 Trastuzumab A132TCO * Preparation of A Trastuzumab A132TCO * A was expressed in insect cells (Spodopterafrugiperda cells, Sf21) utilizing a baculovirus-based transduction system. Thus, the trastuzumab heavy chain gene containing an amber stop codon at position A132 and a C-terminal 6-His tag was cloned into the first multiple cloning site of the pACEBac-DUAL plasmid (described, for example, in WO2017 / 093254). The trastuzumab light chain gene was cloned into the second multiple cloning site of the plasmid without further modifications.

[0628] The resulting plasmid, pACEBacDUAL-TrastuzumabHeavyA132TAG-6His-Light, contains the NES-PylRS AF (e.g., as described in WO2018 / 069481) and an expression cassette for U6(Sf21)-tRNA Pyl The vector was transformed into DH10MultiBac-TAG cells containing a Bacmid with the backbone of the vector (described for example in WO2017 / 093254). Transformation results in the incorporation of the plasmid into the Bacmid-DNA. After preparation of the Bacmid-DNA, insect cells (Sf21 cells) were transfected with the Bacmid-DNA. After 3 days, the V0 virus was harvested and transduced into a fresh batch of Sf21 cells to generate the V1 virus. This virus was used to transduce a large-scale expression culture (liter scale). TCO * Expression was allowed to proceed for 4 days after addition of A-Lys. Cells were harvested at 500rcf using a Beckman rotor (JLA8.1000) for 1 hour at 4°C.

[0629] Cells were resuspended in lysis buffer (4xPBS, 0.2mM TCEP, 1mM PMSF, 5mM imidazole, pH 8) and sonicated three times for 30 seconds on ice. After centrifugation at 27143.1 RCF in a fixed angle rotor (JA25.50, Beckman) for 1 hour at 4°C, the cleared lysate was incubated on nickel beads for 1 hour at 4°C on a rocker. Nickel beads were collected on a polypropylene (PP) column (Qiagen, Cat. No: 34964) and washed with lysis buffer containing 10nM imidazole. Trastuzumab was eluted from the nickel beads using 500mM imidazole in lysis buffer. The eluted fraction was loaded onto a MabSelect Prism A column equilibrated with buffer (0.02M Na2PO4, 0.15M NaCl, pH 7.2). After a washing step with buffer A, trastuzumab was eluted from the column using a gradient up to 100% buffer B (0.1 M sodium citrate, pH 3.2). Fractions containing 1 M Tris pH 10 were collected to neutralize the eluted sample. After analyzing the fractions by SDS-PAGE, fractions containing trastuzumab were pooled and concentrated using an Amicon filter device (30 kDa cut-off). The sample was further purified using a Superdex S200 (10 / 30) column equilibrated with 1x PBS buffer. Fractions were collected and analyzed by SDS-PAGE. After concentrating the corresponding fractions, the sample was used for labeling with cytotoxic payloads.

[0630] Preparation Example 2 Trastuzumab-TCO * Preparation of A-5 10 nmol of trastuzumab A132TCO *A was incubated with 40 nmol of phosphonate-tetrazine-MMAE (5) in 1xPBS at 37°C with shaking at 600 rpm for 1 h. The reaction mix was washed with 1xPBS on a filter device (Amicon filter device, 30 kDa cutoff) to remove unreacted drug. The ADC was further purified by size-exclusion chromatography using a Superdex Increase S200 column equilibrated with 1xPBS (Figure 2A). Collected fractions were analyzed by SDS-PAGE and stained with Coomassie blue (Figure 2B). Fractions containing the ADC were pooled and concentrated on an Amicon filter device.

[0631] Preparation Example 3 Trastuzumab-TCO * Preparation of A-11 10nmol trastuzumab A132TCO * A was incubated with 40 nmol phosphonate-tetrazine-Val-Ala-PAB-MMAE (11) in 1xPBS with shaking at 600 rpm for 1 h at 37°C. The reaction mix was washed with 1xPBS on a filter device (Amicon filter device, 30 kDa cutoff) to remove unreacted drug. The ADC was further purified by size-exclusion chromatography using a Superdex Increase S200 column equilibrated with 1xPBS (Figure 3A). Collected fractions were analyzed by SDS-PAGE and stained with Coomassie blue (Figure 3B). Fractions containing the ADC were pooled and concentrated on an Amicon filter device.

[0632] D) Cytotoxicity assay Assay Example 1 Cytotoxicity Assay Cells of the breast cancer cell line SK-BR-3 were seeded at 5000 cells / well in black 96-well plates 2 days before ADC application. The concentration of the various ADCs or Abs (as negative controls) was adjusted to 5 μM. Serial dilutions were prepared ranging from 0 to 100 nM. The medium was aspirated from the 96-well plates and replaced with the dilutions of ADCs or Abs. As ADCs, trastuzumab A132TCO * A-5 and trastuzumab A132TCO* In addition to A-11, unmodified trastuzumab WT was also tested. After 5 days of incubation, the plate was removed from the incubator, warmed at room temperature for 30 minutes, and 100 μl of CellTiter-Glo® 2.0 Cell (Promega) was added to each well. The plate was shaken on a rocker at 50 rpm for 2 minutes and incubated at room temperature for 10 minutes. The luminescence signal was then read using a plate reader. The luminescence signal was normalized to the measurement at time point 0 nM (negative control). The plot in Figure 4 shows the normalized luminescence signal of the two ADCs and trastuzumab WT at different concentrations (M).

[0633] The contents of documents cross-referenced in this description are incorporated by reference.

Claims

1. Tetrazine compounds of general formula I, 【Chemical Engineering 102】 m is 0 or 1; n represents an integer selected from 1 and 2; o represents an integer selected from 0, 1, or 2; A represents a cleavable linker moiety; Sp １ and Sp ２ each independently represents a spacer moiety, or Sp 1 is absent; X represents a self-immolative moiety; Y represents a small organic drug moiety, a labeling agent, or a chelating moiety; Z represents a phosphorus-containing hydrophilic group, (R １ O) ２ P(O)- and (R １ａ O) ２ P(O)—O—; where residue R １ and R1a represents H; Or, the compound is a salt form of said phosphorus-containing hydrophilic moiety.

2. The spacer Sp １ does not exist, or more specifically, a) a monocyclic or polycyclic, optionally mono- or polysubstituted, aromatic moiety having 6 to 14 ring carbon atoms, in particular 1,4-phenylene, The one or more optional substituents may be, independently of each other, -halogen, -C(halogen) ３ , -OH, -SH, -NR' ２ , NO ２ , —CN, —C(═O)R″, —C(═O)OR′″, alkyl, alkenyl, alkynyl, and alkoxy; where R', R'' and R''' are independently H and C １ ~C ４ alkyl (moiety M1); b) heterocyclic residues of general formula X, 【Chemistry 103】 ring part １ ~X ４ one, two or three, more particularly one or two, of these represent N and the others represent >CH (moiety M2), c) linear or branched C 1 -C 4 alkylene, in particular —(CH ２ ) ｎ１ - and n1 is an integer from 1 to 4, more particularly methylene (moiety M3); d) a combination of at least two identical or more particularly different moieties selected from M1, M2, and M3; and / or Spacer Sp ２ teeth, a) monocyclic or polycyclic, optionally mono- or polysubstituted, aromatic moieties having 6 to 14 ring carbon atoms, in particular 1,2-phenylene, 1,3-phenylene or 1,4-phenylene, or a group of the formula 【Chemical 104】 is a monocyclic part of More specifically, it is 1,4-phenylene. The one or more optional substituents may be, independently of each other, -Hal, -CHal ３ , -OH, -SH, -NR' ２ , NO ２ , —CN, —C(═O)R″, —C(═O)OR′″, alkyl, alkenyl, alkynyl, and alkoxy; R', R'' and R''' are independently H and C １ ~C ４ alkyl (moiety M1); b) heterocyclic residues of general formula X, 【Chemistry 105】 Here, the ring moiety X １ ~X ４ one, two or three, more particularly one or two, of these represent N and the others represent >CH (moiety M2), c) linear or branched C 1 -C 4 alkylene, in particular —(CH ２ ) ｎ１ -, where n1 is an integer from 1 to 4, more particularly methylene (moiety M3); d) a linear or branched polyalkylene oxide moiety, in particular a linear moiety -((CH ２ ) ｘ１ -O) ｙ１ - or -(O-(CH ２ ) ｘ１ ) ｙ１ - and branched analogs thereof, x1 independently represent an integer selected from 1, 2, 3 or 4, in particular 1 or 2; y1 independently represent an integer from 1 to 20, in particular from 1 to 4 (moiety M4), e) a heteroatom-containing moiety, -N(R'''')-, -(CH ２ ) ｘ２ -N(R’’’’)-、 -N(R’’’’)-(CH ２ ) ｘ３ -C(O)O-、 -N(R’’’’)-(CH ２ ) ｘ３ -C(O)-、 -N(R’’’’)-(CH ２ ) ｘ４ -N(R’’’’)-、 -N(R’’’’)-C(O)-(CH ２ ) ｘ４ -N(R’’’’)-、 - (CH ２ ) ｘ４ —C(O)O—, and -(CH ２ ) ｘ４ -C(O)- is selected from R"" are independently H and C １ ~C ４ alkyl, x2 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2; x3 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2; x4 represents an integer selected from 1, 2, 3 or 4, in particular 1 or 2 (moiety M5); or f) a combination of at least two identical or more specifically different moieties selected from M1, M2, M3, M4 and M5; The compound of claim 1.

3. The linker group A is an enzymatically or chemically cleavable linker group selected from the following a) to d): a) a peptidyl group, in particular a di-, tri- or tetra-peptidyl group; b) Formula - (CR ７ R ８ ) ｎ２ -S-S-(CR ７ R ８ ) ｎ２ -X ５ - or X ５’ - (CR ７ R ８ ) ｎ２ -S-S-(CR ７ R ８ ) ｎ２ -X ５ - disulfide group, where n2 represents an integer from 1 to 4; residue R ７ and R ８ are independently selected from H or C 1 -C 4 alkyl, in particular methyl, or two residues R ７ and R ８ together with the carbon atoms to which they are attached form a ring C ４ ~C ８ Forming an alkyl group, Part X ５ is selected from —C(O)— and —O—; Part X ５’ is -C(O)- and -(O)C-(CH ２ )-NH-; c) A hydrazone group is >C=N-N(R ９ )- and -N(R ９ )-N=C<; where: R ９ is H or C 1 -C 4 alkyl; d) β-glucuronidase-sensitive cleavable linker groups, particularly those having a β-glucuronic acid-derived trigger residue; The compound of claim 1.

4. The self-immolative group X is selected from the following a) to d): a) a compound of the formula —NH-p-phenylene-CH ２ —O— or —O—CH ２ -p-phenylene-NH- or -NH-p-phenylene-CH ２ -N ＋ (R ２０ ) ２ - a group derived from p-aminobenzyl alcohol, b) -O-C(O)-O-, c) -O-C(O)-NR １０ - (CR １２ R １３ ) ｚ -NR １１ —C(O)—O—, or -X １ -C(O)-NR １０ -(CR １２ R １３ ) ｚ -NR １１ -C(O)-X ２ -、 wherein z represents an integer selected from 1 to 6, in particular from 1 to 4, R ２０ represent, independently of one another, H or a C 1 -C 4 alkyl group, R １０ and R １１ represent, independently of one another, H or a C 1 -C 4 alkyl group, R １２ and R １３ represent, independently of one another, H, methyl or ethyl, in particular H or methyl, in particular H, and X １ and X ２ are independently O, S or NR １０ represents d) methylene alkoxycarbamate (MAC) type bonds of the formula: -OC(O)-NR １３ -C(R １４ R １５ )-(O)- -OC(O)-NR １３ -C(R １４ R １５ )-(S)- —OC(O)—NR １３ -C(R １４ R １５ )-(NR １６ )-or -OC(O)-NR １３ -C(R １４ R １５ )-(NR １６ -C(O)O)- and Here, R １３ , R １４ , R １５ , and R １６ are each independently H or C 1 -C 4 alkyl, in particular C １ ~C ４ represents alkyl, The compound of claim 1.

5. 2. The compound of claim 1, wherein the payload residue Y is selected from small organic drug moieties, labeling agents, such as in particular dyes, radiolabels and fluorophores, proteolytic agents, in particular payloads applicable to proteolytic targeting chimeras (PROTACs), photosensitizers, and chelators.

6. Sp １ teeth -M1-M3-, -M2-M3-, -M3-M1-, -M3-M2- is selected from one of the combinations of parts wherein the bonds between said moieties M1, M2, M3 are independently selected from a chemical bond, an ether, a thioether, an ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine or a urea, and an alkylene oxide or a linear or branched polyalkylene oxide bond; and / or Sp ２ teeth, -M1-M3-, -M1-M4-, -M2-M3-, -M2-M4-, -M2-M5-, -M3-M1-, -M3-M2-, -M3-M4-, -M1-M3-M4-, -M1-M4-M3-, -M2-M3-M4-, -M2-M4-M3-, -M3-M2-M4-, -M3-M4-M2-, -M2-M5-M4- is selected from one of the combinations of parts 2. The compound of claim 1, wherein the bonds between the moieties M1, M2, M3, M4, and M5 are independently a chemical bond, an ether, a thioether, an ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine, or a urea, and an alkylene oxide or a linear or branched polyalkylene oxide bond.

7. A conjugate obtained by reacting a functionalized targeting agent with a tetrazine compound of general formula I according to claim 1 via a Diels-Alder type cycloaddition reaction between the dienophile moiety of the functionalized targeting agent and the tetrazine compound of formula I, In particular, said functionalized targeting agent is a conjugate selected from viruses, whole cells, phages, liposomes, biomolecules, and low or high molecular weight compounds, in particular antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions, enzymes, proteins, peptides, peptidomimetics, carbohydrates, monosaccharides, polysaccharides, oligonucleotides or polynucleotides, in particular DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins, and derivatives thereof.

8. 8. The conjugate of claim 7, wherein the functionalized targeting agent comprises, as a functional group, at least one dienophile moiety that reacts with the tetrazine moiety of the compound of Formula I.

9. The functionalized targeting agent is a conjugate comprising at least one polypeptide sequence having at least one unnatural amino acid residue within its amino acid sequence, the unnatural amino acid residue comprising at least one dienophile moiety conjugated to the tetrazine moiety of the compound of formula I, and in particular, the functionalized biomolecule is a polyclonal or monoclonal immunoglobulin molecule, in particular a monoclonal antibody or a fragment thereof.

10. 8. The conjugate of claim 7, formed by biorthogonal bioconjugation via a Diels-Alder type cycloaddition reaction between a tetrazine compound of formula I and a biomolecule functionalized with a dienophile moiety.

11. the functional group of the functionalized biomolecule reacting via a Diels-Alder type cycloaddition reaction is (iii) a transcyclooctenyl dienophile group of the following formula: 【Chemistry 106】 and where R １ is hydrogen, halogen, C １ -C ４ - alkyl, (R ａ O) ２ P(O)O-C １ -C ４ - alkyl, (R ｂ O) ２ P(O)-C １ -C ４ -Alkyl, CF ３ , CN, hydroxyl, C １ -C ４ -alkoxy, -O-CF ３ , C ２ -C ５ -Alkenoxy, C ２ -C ５ -alkanoyloxy, C １ -C ４ - alkylaminocarbonyloxy or C １ -C ４ -Alkylthio, C １ -C ４ -Alkylamino, di-(C １ -C ４ -alkyl)amino, C ２ -C ５ -alkenylamino, C ２ -C ５ -alkenyl-C １ -C ４ -alkyl-amino or di-(C ２ -C ５ -alkenyl)amino, R ａ , R ｂ are independently hydrogen or C ２ -C ５ -alkanoyloxymethyl, or (iv) a cyclooctynyl dienophile group of the formula: 【Chemistry 107】 And, where: R ２ is hydrogen, halogen, C １ -C ４ - alkyl, (R ｃ O) ２ P(O)O-C １ -C ４ - alkyl, (R ｄ O) ２ P(O)-C １ -C ４ -Alkyl, CF ３ , CN, hydroxyl, C １ -C ４ -alkoxy, -O-CF ３ , C ２ -C ５ -Alkenoxy, C ２ -C ５ -alkanoyloxy, C １ -C ４ - alkylaminocarbonyloxy or C １ -C ４ -Alkylthio, C １ -C ４ -Alkylamino, di-(C １ -C ４ -alkyl)amino, C ２ -C ５ -alkenylamino, C ２ -C ５ -alkenyl-C １ -C ４ -alkyl-amino or di-(C ２ -C ５ -alkenyl)amino, R ｃ , R ｄ are independently hydrogen or C ２ -C ５ 11. The complex of claim 10, wherein the alkanoyl group is -alkanoyloxymethyl.

12. A method for preparing the bioconjugate of claim 7, comprising reacting a tetrazine compound of any one of claims 1 to 7 with a functionalized biomolecule having a functional dienophile group in an aqueous, optionally buffered reaction medium, and carrying out a Diels-Alder type cycloaddition reaction between the molecules.

13. A tetrazine intermediate of general formula II, 【Chemistry 108】 where: n3 represents an integer selected from 1 or 2; Sp １ and Sp ２ is as defined in claim 2, bonds α, β and γ are independently selected from chemical bonds; Z is a phosphorus-containing hydrophilic group (R １ O) ２ P(O)- and (R １ａ O) ２ P(O)—O—; where: R １ , and R １ａ respectively represent H, R represents H or a chemical group capable of forming a chemical bond, or a chemical group capable of forming an ether, a thioether, an ester (such as an active ester like succinimidyl ester or pentafluorophenyl ester, an amide, a carbamate, a dicarbamate, a carbonate, a hydrazine, a urea, an alkylene oxide or a linear or branched polyalkylene oxide bond), more particularly, R represents an amino group or a carboxyl group, with the proviso that R does not represent a chemical protecting group, in particular does not represent a cleavable protecting group, more particularly does not represent an N-, O-, or S-protecting group.

14. A method for preparing a tetrazine intermediate of general formula II according to claim 13, comprising the steps of: a) (i) a first cyano compound of general formula III 【Chemistry 109】 (Wherein Z and Sp １ is as defined above, and the hydroxyl group of residue Z is optionally provided in the form of a protected i.p. alkoxy; (ii) a second cyano compound of general formula IV 【Chemical 110】 (Wherein R and Sp ２ and n3 is as defined above. (iii) reacting in the presence of hydrazine hydrate; b) subsequent oxidation; c) optionally isolating the resulting tetrazine compound; d) optionally deprotecting the hydroxyl group of residue Z.

15. A conjugate according to any one of claims 7 to 11 for use in medicine, in particular in diagnosis and / or therapy.

16. A pharmaceutical composition comprising at least one conjugate according to any one of claims 7 to 11 in a pharmaceutically acceptable carrier.

17. A diagnostic or analytical kit comprising at least one tetrazine compound according to any one of claims 1 to 6.

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