Hydrophilic trans-cyclooctene (hyTCO) compounds, constructs and conjugates containing same

Abstract

The present invention relates to the field of bioconjugation of functional entities, such as payloads and targeting agents, mediated by novel trans-cyclooctene (TCO) compounds. More particularly, the present invention relates to novel trans-cyclooctene compounds, non-natural amino acids comprising such trans-cyclooctene compounds and their preparation. The novel trans-cyclooctene compounds and non-natural amino acids comprising such trans-cyclooctene compounds are characterized by high structural stability and hydrophilicity, and thus contribute to promoting conjugation of the functional entity. The invention further relates to the corresponding trans-cyclooctene functionalized constructs obtainable by binding said novel trans-cyclooctene compounds, in particular by covalence, to a targeting agent or a payload molecule, said functionalized constructs containing at least one functionalized residue comprising a monounsaturated trans-cyclooctene entity. The invention further relates to conjugates obtainable by covalent linkage, in particular by biorthogonal bioconjugation of Diels-Alder type cycloaddition, in which a first functionalized molecule, in particular selected from the group consisting of the trans-cyclooctene functionalized constructs, is linked to a second functionalized molecule, the second functionalized molecule comprises a docking group (DG) capable of reacting with the trans-cyclooctene-type functional group of the first functionalized molecule. The invention further relates to methods of making the constructs and conjugates. The invention also relates to such conjugates of the invention for use in medicine, corresponding pharmaceutical compositions and corresponding diagnostic and analytical kits.

Description

Technical Field

[0001] This invention relates to the field of bioconjugation of functional entities (such as payloads and targeting agents) mediated by trans-cyclooctene (TCO) compounds. More particularly, this invention relates to novel trans-cyclooctene compounds, non-natural amino acids comprising such trans-cyclooctene compounds, and their preparation. The novel trans-cyclooctene compounds and the non-natural amino acids comprising such trans-cyclooctene compounds are characterized by high structural stability and hydrophilicity, thus facilitating the conjugation of the functional entities.

[0002] The present invention further relates to corresponding trans-cyclooctene functionalized constructs obtainable by combining the novel trans-cyclooctene compound (particularly by covalent bonding) with a targeting agent or payload molecule, the functionalized construct containing at least one functionalized residue comprising a monounsaturated trans-cyclooctene entity.

[0003] The present invention further relates to conjugates obtainable by covalent linking (particularly by biorthogonal bioconjugation via Diels-Alder cycloaddition), wherein a first functionalized molecule (particularly selected from the trans-cyclooctene functionalized construct) is linked to a second functionalized molecule, the second functionalized molecule comprising a docking group (DG) capable of reacting with the trans-cyclooctene functional group of the first functionalized molecule.

[0004] The present invention further relates to methods for preparing the aforementioned constructs and conjugates.

[0005] The present invention also relates to the use of such conjugates of the present invention in medicine, corresponding pharmaceutical compositions, and corresponding diagnostic and analytical kits. Background Technology

[0006] The strain-promoted inverse electron demand Diels-Alder reaction (SPIEDAC) is one of the fastest bioorthogonal reactions and is widely used in various applications in chemical biology, biotechnology, cell biology, life sciences, materials science, and nuclear medicine.

[0007] The rapid reaction kinetics of this bioorthogonal click reaction enable its use at extremely low concentrations, such as in living systems like cells, and even for pre-targeting in organisms. Furthermore, it enables highly efficient bioconjugation reactions for the production of bioconjugates, largely unaffected by the buffer solution, pH, or added solvents used, for example, for the production of antibody-drug conjugates (ADCs) for drug delivery. Moreover, its extremely fast reaction kinetics, even compared to other click reactions such as CuAAC or SPAAC, enable its use in vivo click applications, such as pre-targeting in nuclear medicine.

[0008] ADCs are a rapidly growing class of cancer treatment drugs that have attracted widespread attention, as evidenced by the increasing number of approved ADC drugs and the continuous rise in the number of clinical trials.

[0009] The desired conjugation of each payload to a monoclonal antibody (mAb) is typically accomplished through random attachment. This often results in a heterogeneity in the current drug-to-antibody ratio (DAR), ranging from completely unmodified mAbs to unfavorable attachments of large amounts of cytotoxic molecules. This leads to batch-to-batch variability issues and can cause aggregation and associated side effects such as rapid clearance, immunogenicity, or hepatotoxicity.

[0010] The genetic encoding of strained cyclooctyne-lysine derivatives for click reactions into proteins has been reported (T. Plass, S. Milles, C. Koehler, C. Schultz, EA Lemke, Angew. Chem. Int. Ed. 2011, 50, 3878-81) and the synthesis and genetic encoding of ncAAs capable of (strain-promoted) anti-electron-demanding Diels-Alder cycloaddition (IEDDA) with 1,2,4,5-tetraazines have been reported (T. Plass, S. Milles, C. Koehler, J. Szymański, R. Mueller, et al., Angew. Chem. Int. Ed. 2012, 51, 4166-70; I. Nikić, T. Plass, O. Schraidt, J. Szymański, JAG Briggs, et al., Angew. Chem. Int. Ed. 2014, 53, 2245-9; E. Kozma, I. Nikić, BR Varga, IV Aramburu, JH Kang, et al., ChemBioChem 2016, 17, 1518-24; J.-E. Hoffmann, T. Plass, I. Nikić, IV Aramburu, C. Koehler, et al., Chem. Eur.J. 2015, 21, 12266-70). This extension of GCE technology leads to the specific introduction of these ncAA sites into unglycosylated immunoglobulins produced by insect cells, followed by subsequent modification via click chemistry (C. Koehler, PFSauter, M. Wawryszyn, GE Girona, K. Gupta, et al., Nat. Methods 2016, 13, 997-1000.).

[0011] Incorporating non-classical amino acids (ncAAs) into peptides through translation provides the most flexible and precise basis for biologically conjugated protein modifications via a variety of payloads.

[0012] This has been used for fluorescent labeling of proteins, RIC (also pre-targeted), and drug delivery in ADCs.

[0013] Among the various candidates that could be selected as the bioconjugation-mediated moiety, trans-cyclooctene (TCO) derivatives, such as ncAA-based TCO (TCO-ncAA), have faster reaction kinetics compared to other candidate molecules (NK Devaraj, et al 2009).

[0014] The widespread success of TCO-ncAAs is hampered by the fact that these compounds exhibit an intrinsic instability from click-reactive trans isomers to non-reactive cis isomers [(Béquignat et al., 2020; Cooket al., 2016; Darko et al., 2014; Keinänen et al., 2019; Lang et al., 2012; Meyer et al., 2017; Reinkemeier et al., 2021; Rondon & Degoul, 2020; Rossin et al., 2010, 2013; Ruivo et al., 2019; Zeglis et al., 2015)]. The mechanism of this phenomenon is believed to be due to the presence of thiol groups and the action of metalloproteins [(Darko et al., 2014; Rossin et al., 2013)].

[0015] It has been confirmed that this occurs not only during expression in cell culture media, but also in the blood circulation of organisms such as mice. Furthermore, previously introduced ncAAs, which have an amino acid handle at the 2-position, are prone to the removal of the amino acid moiety after click reactions, and are therefore unsuitable for the modification of biomolecules [(Fan et al., 2016)].

[0016] However, for in vivo labeling applications, very fast reaction kinetics are essential.

[0017] Most importantly, most TCOs or cyclooctyne compounds are lipophilic moieties, leading to nonspecific binding and longer wash times in fluorescent labeling applications, and adversely affecting the hydrophilicity of bioconjugates in targeted therapy, where increased hydrophilicity has been shown to have a beneficial effect on the properties of ADCs for targeted drug delivery.

[0018] Therefore, the full potential of this technology cannot yet be realized by incorporating ncAA-based TCOs.

[0019] Therefore, there is an urgent need for hydrophilic and particularly stable highly reactive TCO derivatives and ncAA improved through genetic code extension (GCE). Summary of the Invention

[0020] The aforementioned problems are surprisingly solved by providing novel hydrophilic hyTCOs that do not undergo trans-to-cis isomerization and allow for near-quantitative conjugation with diene derivatives or conjugates (such as 1,2,4,5-tetraazine derivatives). Furthermore, due to their increased hydrophilicity, the conjugation of more hydrophobic payloads provides better functional properties of such conjugates in vivo, in the case of pharmacologically active conjugates.

[0021] More specifically, the above problems are solved by providing hyTCO derivatives having a homopolar, particularly hydrophilic, substituent at the 3-position, combined with a polar, particularly hydrophilic moiety at the 2-position.

[0022] This invention enables the long-term use of hyTCO and hyTCO-ncAA under biologically relevant conditions, particularly the expression of proteins incorporating hyTCO-ncAA and their subsequent efficient use in biological conjugation.

[0023] It also enables the hyTCO-modified portion to be used for in vivo click applications, such as pre-targeting in nuclear medicine.

[0024] Furthermore, it helps optimize the hydrophilic properties of the ADC's bioconjugation handle, affecting its pharmacokinetics, solubility, and aggregation. Increased hydrophilicity should also help improve the elution properties of intracellular fluorescent labels and reduce nonspecific binding.

[0025] Surprisingly, despite the increased steric hindrance of the novel hyTCO derivatives, they exhibit very fast reaction kinetics, even exceeding those of previously known TCO-based compounds.

[0026] More specifically, the above problems are solved by providing the following solutions: a) Novel hyTCO derivatives (represented by general formula I as described below); b) Non-natural amino acids (represented by general formula II as described below) that contain this type of novel hyTCO compound; c) Novel hyTCO functionalized constructs, obtainable by conjugating (particularly covalently) the novel hyTCO compound to a targeting agent or payload molecule; and d) A conjugate, which can be obtained by covalently linking the novel hyTCO-functionalized construct to a second functionalized molecule containing a docking group (DG) capable of reacting with the hyTCO-type functional group of the first functionalized molecule. Attached Figure Description

[0027] Figure 1A) EIC of TCO-e incubated in DMEM at 37 °C for 2 days. Under these conditions, over 60% of trans-cyclooctene isomerized to cis-cyclooctene; B) Chemical structure of TCO-e. Figure 2 ELSD and EIC traces of compound 13 (N6-((2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)ethoxy)carbonyl)-L-lysine) incubated at 37 °C for 4 days in various culture media or mouse serum were compared with the EIC of compound 13 dissolved in DMEM. No changes in ELSD or EIC peaks were detected after this time period (within the normal range of variation).

[0028] Figure 3 Comparison of ELSD and EIC traces of compound 13 incubated at 37 °C for 4 days in DMEM with the same sample 5 min after the addition of tetrazine. No substance at m / z = 329 was detected after the click reaction, corresponding to trans or cis ncAA; only residual tetrazine and click products could be identified (shown next to the mass spectrometer).

[0029] Figure 4 This paper presents the TCO head group and related conjugates as model systems for calculating ClogP values. ChemDraw 20 was used to calculate the ClogP values.

[0030] Figure 5 A) Trastuzumab HCC eluted using a linear gradient with buffer B (0.1 M sodium citrate, pH 3.2) H 2K16 13 - LC elution curve; B) Trastuzumab HC C H 2K16 13 - SDS-PAGE of LC collection fractions; C) Trastuzumab HC C H 2K16 13 Elution curves and SDS-PAGE of the collected fractions by size exclusion chromatography using 16-LC (Superdex S200 Increase, Cytiva, 28990944).

[0031] Figure 6 A) Unconjugated and conjugated ADCs trastuzumab HCC H 2K16 13 -16-LC by DAR analysis of reduced reverse-phase LC; B) ADC trastuzumab HC CH2K16TCO DAR analysis of a-16-LC.

[0032] Figure 7A) Absorption spectrum of trastuzumab-Cy5, showing antibody absorption at 280 nm and dye at 650 nm; B) SDS-PAGE of trastuzumab-Cy5.

[0033] Figure 8 The plasmid map of expression plasmid pCK-HSA-trastuzumab HC-LC was modified by replacing the lysine codon "AAG" with the amber codon "TAG" at position K249 of the heavy chain sequence.

[0034] Figure 9 Preparation at different positions (V) in the heavy or light chain H P41; CH2K90; V L K45) contains trastuzumab of the present invention containing ncAA13 and conjugated with payload compound 16. Cytotoxicity was determined using Her2-positive SKBR-3 cells. Kadcyla® was used as a positive control.

[0035] Figure 10 The structural formulas of the effective payload molecules P1-P4 used in the fusion experiment.

[0036] Figure 11 The results of cytotoxicity experiments on Her2-positive SKBR-3 cells, using trastuzumab HCV... H P41 13 -LC (at position V) H P41 is conjugated with ncAA compound 13 of the present invention and further conjugated with payloads P1-P4 in a molar ratio of 1:3.

[0037] Figure 12 : Protein sequences of trastuzumab heavy and light chains and localization of potential mutation sites. Detailed Implementation

[0038] A. Abbreviation ADC = Antibody-drug conjugate aq. = containing water Bps = base pairs BCN = 2-amino-6-(9-bicyclo[6.1.0]non-4-ynylmethoxycarbonylamino)hexanoic acid BOC = 2-amino-6-(tert-butoxycarbonylamino)hexanoic acid, and in the examples, "BOC" specifically refers to (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoic acid = Boc- -Lys-OH = N-α-tert-Butyloxycarbonyl- -Lysine conc. = concentrated clogP = calculated logarithmic distribution coefficient DAR = Drug-Antibody Ratio DOL = Marking Degree 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 EIC = Extracted Ion Count ELSD = Evaporative Light Scattering Detector eq.= equivalent EtOH = ethanol GCE = Genetic Code Extension h = hours 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 = kilodalton Min = minutes MMAE = Monomethylauratestatin E (( S )- N -((3 R 4 S 5 S )-1-(( S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)- N 3-Dimethyl-2-(( S )-3-methyl-2-(methylamino)butyramido)butyramide(( S )- N -((3 R 4 S 5 S )-1-((S )-2-((1 R ,2 R )-3-(((1 S ,2 R )-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)butanamido(butanamide), an antitumor agent MeOH = Methanol ncAA = non-classical amino acid NES = Core Output Signal NLS = Nuclear Localization Signal O-tRNA = Orthogonal tRNA O-RS = Orthogonal RS PBS = Phosphate-buffered saline PMSF = Benzylsulfonyl fluoride PNP chloroformate = 4-nitrophenyl chloroformate POI = Peptide of Interest pRS = prokaryotic RS ptRNA = prokaryotic tRNA PylRS = Pyrrololysyl tRNA synthetase PylRS AF = Mutant methanogenic octopus containing amino acid substitutions for Y306A and Y384F ( M. mazei pyrrololysyl-tRNA synthetase 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-Lys = 2-amino-6-(cyclooct-2-yn-1-yloxycarbonylamino)hexanoic acid SDS-PAGE = Sodium dodecyl sulfate polyacrylamide gel electrophoresis SPIEDAC = Strain-Promoted Inverse Electron Demand Diels-Alder Cycloaddition 5-TAMRA = 5-Carboxytetramethylrhodamine 5-TAMRA-OSu = 5-Carboxytetramethylrhodamine N-succinimide ester, fluorophore TCO-Lys = N-ε-((trans-cyclooctyl-4-en-1-yloxy)carbonyl)-L-lysine TCO -Lys = N-ε-((trans-cyclooctyl-2-en-1-yloxy)carbonyl)-L-lysine TCO# -Lys = N-ε-((trans-cyclooctyl-3-en-1-yloxy)carbonyl)-L-lysine TCO-E-Lys = trans-cyclooct-4-ene-L-lysine TCO A-Lys = trans-cyclooct-2-ene-L-lysine hyTCO = the hydrophilic TCO of this invention hyTCO-ncAA = a non-classical amino acid whose side chain is functionalized with hyTCO. TFA = Trifluoroacetic acid THF = Tetrahydrofuran TLC = Thin-layer chromatography tRNA Pyl = tRNA with an anticodon can be acylated with pyrrolidone by wild-type or modified PylRS, and the anticodon is preferably the reverse complement of the selected codon in order to specifically incorporate the ncAA site into the POI.

[0039] The U6 promoter is the promoter that typically controls the expression of U6 RNA (a small nuclear RNA) in mammalian cells. UHPLC-MS = Ultra-high performance liquid chromatography / mass spectrometry

[0040] B. Definition

[0041] B.1 General Definition

[0042] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of terms shall be clear; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms.

[0043] As used herein, the terms “purified,” “substantially purified,” and “isolated” refer to a state free from other distinct compounds that are typically associated with the compounds of the present invention in their natural state. Therefore, “purified,” “substantially purified,” and “isolated” objects comprise at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% (by weight), of a given sample mass. In one embodiment, these terms mean that the compounds of the present invention comprise at least 95%, 96%, 97%, 98%, 99%, or 100% (by weight), of a given sample mass. As used herein, when referring to nucleic acids or proteins, the terms “purified,” “substantially purified,” and “isolated” also refer to a state of purification or concentration different from that of natural occurrence, such as in a prokaryotic or eukaryotic environment, for example in bacterial or fungal cells, or in mammalian organisms, particularly humans. Any degree of purification or concentration greater than that of natural occurrence, including (1) purification from other related structures or compounds, or (2) association with structures or compounds that are typically not associated with said prokaryotic or eukaryotic environment, falls within the meaning of “isolated.” Nucleic acids or proteins, or classes of nucleic acids or proteins, described herein can be isolated or associated with structures or compounds that are not typically associated with them in nature, based on various methods and processes known to those skilled in the art.

[0044] In the context of the specification provided herein and the appended claims, the use of "or" means "and / or" unless otherwise stated.

[0045] Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are interchangeable and are not used to restrict.

[0046] It should be further understood that when the description of various implementations uses the term "comprising", those skilled in the art will understand that in certain specific cases, the language "substantially consisting of" or "consisting of" can be used instead of describing the implementation.

[0047] The term “about” indicates a potential variation of ±25% in the value, particularly ±15%, ±10%, and even more particularly ±5%, ±2%, or ±1%.

[0048] The term “basically” describes a value range of approximately 80-100%, for example, 85-99.9%, especially 90-99.9%, even more especially 95-99.9%, or 98-99.9%, especially 99-99.9%.

[0049] "Mainly" refers to a percentage in the range of 50% or higher, such as in the range of 51-100%, especially in the range of 75-99.9%; even more so in the range of 85-98.5%, such as 95-99%.

[0050] If this disclosure relates to features, parameters, and ranges of varying degrees of preference (including general and non-preferred features, parameters, and ranges), then unless otherwise stated, the disclosure of this specification covers any combination of two or more such features, parameters, and ranges, regardless of their respective degrees of preference.

[0051] B.2 Chemical Definition

[0052] The term "halogen" refers to a fluorine, bromine, chlorine, or iodine radical, especially a fluorine radical.

[0053] The term "hydrocarbon group" refers to optionally substituted, straight-chain or branched, acyclic or cyclic, homogeneous or heterocarbon group having a hydrocarbon chain containing 1-50, 1-25, 1-12, or particularly 1-6 carbon atoms. A "heterocarbon group" may carry one or more, for example 1, 2, 3, 4, or 5, more particularly 1 or 2 identical or different heteroatoms within its hydrocarbon chain, especially selected from O, S, and NH. Specific examples of such hydrocarbon groups include optionally substituted, linear or branched (hetero)alkyl-, (hetero)alkenyl-, (hetero)ynyl-, (hetero)cycloalkyl-, and (hetero)cycloalkenyl- groups.

[0054] "Hydrocarbylene group" refers to optionally substituted, straight-chain or branched, homo- or heterocarbylene-bridging groups having a hydrocarbon chain containing 1-50, 1-25, 1-12, or particularly 1-6 carbon atoms. The "heterocarbylene" group may carry one or more, for example 1, 2, 3, 4, or 5, more particularly 1 or 2 identical or different heteroatoms within its hydrocarbon chain, especially selected from O, S, and NH. Specific examples of such hydrocarbylene groups include optionally substituted, linear or branched (hetero)alkylene-, (hetero)alkenyl-, (hetero)ynylene-, (hetero)cycloalkylene-, and (hetero)cycloalkenyl- groups.

[0055] In the context of this invention, "bridging group" should be interpreted as divalent or polyvalent, particularly divalent groups. Specific examples of "bridging group" include hydrocarbon groups.

[0056] "alkyl" or "alkanyl" refers to a straight-chain or branched hydrocarbon residue, which can be selected from methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, 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; n-Heptyl, n-Octyl, n-Nonyl, n-Decyl, n-Undecyl, n-Dodecyl, n-Tridecyl, n-Tetradecyl, n-Pentanediyl, n-Hexadecyl, n-Heptadecanyl, n-Octadecanyl, n-Nondecyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, n-Eicosyl, squalyl and their branched isomers.

[0057] The term "alkenyl" refers to monounsaturated analogs of the aforementioned alkyl groups comprising a single carbon-carbon double bond. As non-limiting examples, C2-C6-alkenyl groups may be mentioned, such as vinyl, 1-propenyl, 2-propenyl, 1-methylvinyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3- Butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl 1-Methyl-3-pentenyl, 2-Methyl-3-pentenyl, 3-Methyl-3-pentenyl, 4-Methyl-3-pentenyl, 1-Methyl-4-pentenyl, 2-Methyl-4-pentenyl, 3-Methyl-4-pentenyl, 4-Methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl -3-Butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl.

[0058] "Alkyne" refers to monounsaturated analogs of the alkenyl group described above, which contains a single chemical carbon-carbon triple bond.

[0059] The term "cycloalkyl" refers to carbocyclic residues having 3-20 carbon atoms, more specifically C3-C4. 12-Cycloalkyl, particularly cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl, cyclopropyl-methyl, cyclopropyl-ethyl, cyclobutyl-methyl, cyclobutyl-ethyl, cyclopentyl-methyl, cyclopentyl-ethyl and cyclohexyl-methyl.

[0060] The term "cycloalkenyl" refers to monounsaturated or polyunsaturated analogs of the aforementioned cycloalkyl residues; as a non-limiting example, monounsaturated residues having at least 5 carbon atoms, such as cyclopenten-1-yl, cyclopenten-3-yl, cyclohexen-1-yl, cyclohexen-3-yl, and cyclohexen-4-yl-, may be mentioned.

[0061] The term "alkanoyl" refers to the R (C=O)-type acyl analog of the above-mentioned alkyl group, where R represents an alkyl or cycloalkyl group as defined above.

[0062] The term “heteroalkyl” or “heteroalkanyl” refers to analogs of the alkyl group described above, which carry one or more, such as 1, 2, 3, 4 or 5, more particularly 1 or 2 identical or different heteroatoms, especially selected from O, S and NH.

[0063] The term "heteroalkenyl" refers to analogs of the alkenyl groups described above, which carry one or more, such as 1, 2, 3, 4 or 5, more particularly 1 or 2 identical or different heteroatoms within their hydrocarbon chain, especially selected from O, S and NH.

[0064] The term "heterynyl" refers to analogs of the ynyl group described above, which carry one or more, such as 1, 2, 3, 4 or 5, more particularly 1 or 2 identical or different heteroatoms within their hydrocarbon chain, especially selected from O, S and NH.

[0065] The term “heterocyclic alkyl” refers to analogs of the cycloalkyl groups described above, which carry one or more, such as 1, 2, 3, 4 or 5, more particularly 1 or 2 identical or different heteroatoms, especially selected from O, S and NH.

[0066] The term "heterocyclic alkenyl" refers to analogs of the cycloalkenyl groups described above, which carry one or more, such as 1, 2, 3, 4 or 5, more particularly 1 or 2 identical or different heteroatoms within their hydrocarbon chain, especially selected from O, S and NH.

[0067] The term "alkylene" refers to a straight-chain or branched hydrocarbon bridge having 1-10 or 1-7 carbon atoms; non-limiting examples include -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)2-CH(CH3)-, and -CH2-CH(CH3)-CH2-. -CH2-CH(CH3)-, -CH(CH3)-CH2-, -CH(CH3)-CH(CH3)-, -C(CH3)2-CH2-, -CH2-C(CH3)2-, -CH2-CH(CH2CH3)-, -CH(CH2CH3)-CH2-, - (CH2)5-, -C(CH3)2-CH(CH3)-, -CH(CH3)-C(CH3)2-, -(CH2)6, -(CH2)7-, -CH(CH3)-CH2-CH2-CH(CH3)- or -CH(CH3)-CH2-CH2-CH2-CH (CH3)-;-CH(CH2CH3)-CH(CH2CH3)-、-C(CH2CH3)2-CH2-、-CH2-C(CH2CH3)2-、-CH2-CH(n-propyl)-、-CH(n-propyl)-CH2-、-CH(n-propyl)-CH(CH3)-、-CH2-CH(n-butyl)-、-CH(n-butyl)-CH2-、-CH(CH3)-CH(CH2CH3)-、-CH(CH3)-CH(n-propyl)-、-CH(CH2CH3)-CH(CH3)-、-CH(CH3)-CH(CH2CH3)-。

[0068] "Alkenyl" refers to the monounsaturated analogues of the aforementioned alkylene groups.

[0069] "Cycloalkenyl" refers to monocyclic analogs of the aforementioned alkenyl groups.

[0070] "Alkoxy" refers to a group of the formula RO-, where R is a straight-chain or branched alkyl group as defined above. As defined herein, a particular alkoxy group has 1 to 6, particularly 1 to 4, such as 1, 2, or 3 carbon atoms. Non-limiting examples are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, isobutoxy, or tert-butoxy.

[0071] "Alkenyloxy group" refers to a group of the formula RO-, where R is a straight-chain or branched alkenyl group as defined above. As defined herein, a particular alkenyloxy group has 2 to 6, particularly 2 to 4, and more particularly 2 or 3 carbon atoms.

[0072] "Aryl" refers to a monovalent monocyclic or polycyclic aromatic moiety, particularly having 6-26, especially 6-14, cyclic carbon atoms, particularly phenyl, fluorenyl, naphthenyl, and phenantrenyl.

[0073] "Heteroaryl" refers to analogs of the aforementioned aryl groups, which additionally contain at least one, such as 1 to 10, more particularly 1 to 5 identical or different cyclic heteroatoms selected from O, S and N.

[0074] "Aryl group" refers to an aryl residue attached to an alkyl group, each as defined above. In particular, such aryl groups contain 7-24 carbon atoms. As non-limiting examples, benzyl or phenethyl may be mentioned.

[0075] "Heteroarylene" refers to heteroaryl residues attached to an alkyl group, each as defined above. In particular, such heteroarylene groups contain 6-24 carbon atoms. As non-limiting examples, pyridinyl-2-methyl or pyridinyl-2-ethyl may be mentioned.

[0076] "alkylaryl" refers to an alkyl residue attached to an aryl group, each as defined above. In particular, such alkylaryl groups contain 7-24 carbon atoms. As a non-limiting example, ortho, meta, or p-methylphenyl or ortho, meta, or p-ethylphenyl residues may be mentioned.

[0077] "alkyl heteroaryl" refers to an alkyl residue attached to a heteroaryl group, each as defined above. In particular, such alkyl heteroaryl groups contain 6-24 carbon atoms. As non-limiting examples, 2-methyl-6-pyridinyl- or 2-ethyl-6-pyridinyl- may be mentioned.

[0078] Unless otherwise stated, the term "substituent" is selected from halogen, C1-C4-alkyl, CN, CF3, hydroxyl, -O-CF3, C1-C4-alkoxy, C2-C4-alkanoyloxy, -amino or -N(C1-C4-alkyl)2.

[0079] Unless otherwise stated, the term “substitution” means that a group is substituted with one, two, or three, especially one or two, substituents.

[0080] The compounds of this invention form a “bond” between two adjacent structural motifs, which, unless otherwise stated, is a chemical bond or a bond selected from any possible orientation of ether, thioether, ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkylene oxide, or linear or branched polyalkylene oxide bond.

[0081] The "ether" bond contains at least one group of the following type: (-O-).

[0082] The thioether bond contains at least one group of the following type: (-S-).

[0083] The "amide" bond contains at least one of the following types of groups: -C(=O)N(R)- or -(R)NC(=O)-.

[0084] The "carbamate" bond contains at least one of the following types of groups: -OC(=O)-N(R)- or -N(R)-C(=O)-O-.

[0085] The "hydrazine" bond contains at least one of the following types of groups: -NH-NH-.

[0086] The "alkylene oxide" linker contains at least one group of the following type: -(CH2) x -O-, -O-(CH2) x -O-、-(CH2) x -O-(CH2) n1 C(O)-, -O-(CH2) x -NR-, -O-(CH2) x -C(O)- or -O-(CH2) x - where x = 1, 2, 3 or 4, especially 1 or 2, n1 = 1, 2 or 3.

[0087] The "polyalkylene oxide" linker contains repeating units of the same or different alkylene oxide groups as defined above, and can be linear or branched, especially linear; for example, -((CH2). x -O) y -、-(O-(CH2) x ) y -O-、-((CH2) x -O) y -(CH2) n1 C(O)-、-(O-(CH2) x ) y -NR-、-(O-(CH2) x ) y -C(O)- or -(O-(CH2) x ) y - where n and x are as defined above for "alkyleneoxy group", and y is an integer from 1 to 20, preferably from 1 to 15, 1 to 10 or 1 to 4.

[0088] In the chemical formula of the specific linker mentioned above, the independent residues R can represent H or C1 to C4-alkyl.

[0089] "Cleavable group" encompasses any group that can be enzymatically or chemically cleaved, particularly under in vivo or in vitro conditions; enzymatic cleavage can be achieved, for example, by the action of a protease; chemical cleavage can be achieved, for example, by hydrolytic cleavage or reductive cleavage of the SS bond.

[0090] Drug-antibody ratio (DAR) refers to the distribution of different payloads, such as drug or cytotoxic group pairs, in antibody-drug conjugates (ADCs), or the average of different drug-to-antibody payloads of an ADC.

[0091] Unless otherwise defined, the “tetraazine” or “tetraazine group” according to the invention refers to a residue consisting of a six-membered aromatic ring containing four nitrogen atoms with the molecular formula -C2N4-, particularly derived from 1,2,4,5-tetraazine or s-tetraazine isomers, and connected to adjacent groups via ring carbon positions 3 and 6.

[0092] "Bioorthogonal" reactions or "click reactions" refer to any chemical reaction that can occur within a living system without reacting with natural components / substrates or interfering with natural biochemical processes.

[0093] A "docking group" is a chemically reactive moiety that reacts with a trans-cyclooctene group as defined herein, particularly through a so-called "bioorthogonal" or "click reaction." In particular, such docking groups are preferably trans-cyclooctene reactive groups selected from dienes. More specifically, they are selected from dienes capable of reacting with trans-cyclooctene groups in a biological environment. As a non-limiting example, groups comprising optionally substituted triazine or tetraazine residues and 1,2-quinone may be mentioned. These groups are well known in the prior art [Yang et al., Angew. Chem. Int. Ed. 2012, 51, 5222–5225; Fan et al., Angew. Chem. Int. Ed. 2016, 55, 14046–14050; Mao et al., Angew. Chem. Int. Ed. 2019, 58, 1106–1109; Quet al., Angew. Chem. Int. Ed. 2018, 57, 12057–12061; Eising et al., Bioconjugate Chem. 2018, 29, 3054–3059; Meng et al., J. Org. Chem. 2017, 82, 1676–1687; Xie et al., Angew. Chem. Int. Ed. 2020, 59, 16967–16973; Lambertet al., J. Am. Chem. Soc. 2019, 141, 17068-17074; Jemas et al., J. Am. Chem.Soc. 2022, 144, 1647-1662; Selvaraj et al., Tetrahedron Letters 2014, 55,4795–4797; Dowling et al., J. Org. Chem. 2018, 83, 4229-4238; Battisti etal., Bioconjugate Chem. 2022, 33, 4, 608–624; Karver et al., BioconjugateChem. 2011, twenty two, 2263-2270; Bender et al., Org. Lett. 2017, 19, 5693-5696; Kamber et al., J. Am. Chem. Soc.2015, 137, 8388-8391; Ros et al., Bioconjugate Chem. 2020, 31, 933-938; Ros et al., Chem. Commun., 2020, 56,11086; van Onzen et al., J. Am. Chem. Soc. 2020, 142, 10955-10963; Carlson et al., J. Am. Chem. Soc. 2018, 140, 3603-3612.]. .

[0094] "Tetraazine linkage" refers to the reaction of trans-cyclooctene and s-tetraazine in a reverse-demand Diels-Alder reaction, followed by a reverse Diels-Alder reaction to eliminate nitrogen (N2). This type of reaction proceeds at high speed, allowing for biomolecular modification at extremely low concentrations.

[0095] The “reverse electron-demanding Diels–Alder (IEDDA) cycloaddition” is a reaction between an electron-depleted diene and an electron-rich dienophile, representing only one example of a different type of “bioorthogonal reaction.” The diene used can be, for example, 1,2,4,5-tetraazine or 1,2,4-triazine. The dienophile encompasses a variety of molecules, including strained cyclic alkenes such as trans-cyclooctene (TCO, norbornene, cyclopropene, or aziridine). Among these, the reaction between tetraazine and TCO is the fastest reported to date and is suitable for in vivo applications (Smeek et al, Current Opinion in Chemical Biology Volume 60, February 2021, Pages 79-88).

[0096] The term "bioorthogonal" refers to any chemical reaction that can occur within a living system (i.e., in an aquatic environment) without interfering with natural biochemical processes. For example, a "tetraazine" linkage can be considered a bioorthogonal reaction. Bioorthogonal chemistry is typically performed in two steps. First, the cell substrate is modified with a bioorthogonal functional group (also called a chemical reporter), such as one of the hydrophilic trans-cyclooctene compounds identified herein. Cell substrates include, for example, immunoglobulins, such as natural or recombinant antibodies. The chemical reporter must not significantly alter the structure of the substrate to avoid affecting its biological activity. In the second step, a probe, such as a functionalized molecule containing a docking group, such as a complementary functional group, such as a tetraazine group, is introduced to react with and label the substrate.

[0097] The compounds of this invention are "acid or base addition salts," particularly addition salts of physiologically tolerant acids or bases. Physiologically tolerant acid addition salts can be formed by treating the basic form of the compounds of this invention with a suitable organic or inorganic acid. Compounds of this invention containing acidic protons can be converted to their non-toxic metal or amine addition salt forms by treating with a suitable organic or inorganic base. The compounds and salts of this invention also comprise their hydrated and solvation forms, such as hydrates, alcohols, etc.

[0098] "Physiological tolerance" refers to acids or bases that are tolerated by systems that incorporate first and second dienophiles (e.g., biological systems such as translation systems for preparing polypeptides with trans-cyclooctenyl or cyclooctyne groups), and are essentially non-toxic to living cells.

[0099] The compounds described herein may contain one or more asymmetric elements, such as a stereogenic center, stereogenic axis, etc., for example, an asymmetric carbon atom, and therefore these compounds may exist in different stereoisomeric forms. For example, these compounds may be racemic or optically active forms. All stereoisomers and diastereomers, including purified and mixed forms, are included. Therefore, all these forms are intended to be included when a compound is referred to by a specific name or when a class of compounds is referred to. In the case of the hyTCO residues of this invention, their Z- and E-forms are excluded.

[0100] The compounds described in this article can also exist as more than one structural isomer, also known as constituent isomers or regio isomers. These molecules differ only in the different sequences of their atoms or atomic groups, but have the same gross formula.

[0101] Therefore, unless otherwise stated, any such potential stereo or regio isomers, or mixtures of more than one stereo and / or regio isomers, are within the scope of this invention for each compound, biomolecule, and conjugate described herein.

[0102] B.3 Biochemical Definition

[0103] A "peptide" is any oligomer of amino acid residues (natural or non-natural, or a combination thereof) of any length, usually but not exclusively linked by covalent peptide bonds. Peptides can originate from any source, such as naturally occurring peptides, peptides produced through recombinant molecular genetics, peptides derived from cells or translation systems, or peptides produced through cell-free synthetic methods. A peptide is characterized by its amino acid sequence, for example, the primary structure of its constituent amino acid residues. As used herein, the amino acid sequence of a peptide is not limited to a full-length sequence and can be a partial or complete sequence. Furthermore, it is not intended to limit peptides to having or not having any particular biological activity.

[0104] As used herein, the term "protein" is synonymous with "peptide." The term "peptide" refers to small polypeptides, such as, but not limited to, those with a length of 2-25 amino acids.

[0105] The primary objective of this targeting agent is to form a covalent or non-covalent bond with a specific "target". The secondary objective of the targeting agent is to deliver the "payload molecule" to the target. To achieve this secondary objective, the targeting agent must bind (reversibly or irreversibly) to at least one "payload molecule".

[0106] The hyTCO-ncAA can be directly incorporated into the target peptide using any of the many methods known in the art. While many embodiments utilize orthogonal translation systems as a route for the direct incorporation of non-natural amino acids, other direct incorporation methods (e.g., in vitro translation systems, solid-phase synthesis, etc.) may be optionally used. It should be understood that, in the typical embodiments described herein, it is preferred to incorporate the non-natural amino acid into the target peptide, i.e., during the peptide construction process, rather than through post-translational chemical derivatization.

[0107] In some of the embodiments described herein, an "orthogonal tRNA / aminoacyl-tRNA synthetase pair" can be used to specifically incorporate non-natural amino acid sites into the target peptide with high efficiency and high fidelity.

[0108] The term "translation system" refers to the components necessary for the incorporation of amino acids into a growing polypeptide chain (protein). Components of a translation system can include, for example, ribosomes, tRNA, synthases, and mRNA. Translation systems can be in vivo or in vitro.

[0109] "In vitro translation systems" can be cell-free translation systems. Cell-free translation systems are systems that synthesize desired proteins by obtaining the protein factors required for mRNA translation, for example, in the form of cell extracts, and then reconstructing the reaction in vitro. Such cell-free systems and their uses in protein synthesis are known in the art. Examples include *Escherichia coli* (…). E. coli (Spirin and Swartz, Cell-free Protein Synthesis, Wiley VCH Verlag, Weinheim, Germany, 2008).

[0110] Aminoacyl-tRNA synthetase (RS) is an enzyme capable of acylating tRNA with amino acids or amino acid analogs. Conveniently, the RS used in the method of the present invention can acylate tRNA with non-natural amino acids.

[0111] The method of the present invention conveniently utilizes a "tRNA / aminoacyl-tRNA synthetase (tRNA / RS) pair". Preferably, the tRNA / RS pair used in the method of the present invention is orthogonal to the translation system.

[0112] As used herein, the term "orthogonal" refers to molecules (e.g., orthogonal tRNA (O-tRNA) and / or orthogonal aminoacyl tRNA synthetase (O-RS)) used by the translation system of interest (e.g., a cell) with reduced efficiency. Orthogonal means that the orthogonal tRNA or orthogonal aminoacyl tRNA synthetase cannot interact with the endogenous aminoacyl tRNA synthetase or endogenous tRNA of the translation system of interest, or the efficiency is reduced, for example, below 20%, below 10%, below 5%, or, for example, below 1%. For example, the efficiency of orthogonal tRNA in the translation system of interest being acylated by any endogenous aminoacyl tRNA synthetase of the translation system of interest is reduced or even zero compared to the acylation of endogenous tRNA by endogenous aminoacyl tRNA synthetase. In another example, the efficiency of orthogonal aminoacyl tRNA synthetase acylation of any endogenous tRNA in the translation system of interest is reduced or even zero compared to the acylation of endogenous tRNA by endogenous aminoacyl tRNA synthetase.

[0113] The orthogonal tRNA / RS pairs used in the method of the present invention preferably have the following characteristics: O-tRNA is preferentially acylated by O-RS with the non-natural amino acids of the present invention. Furthermore, the orthogonal pairs function in the translation system of interest, for example, a translation system that uses O-tRNA acylated with non-natural amino acids to incorporate the non-natural amino acids of the present invention into the peptide chain. Incorporation occurs in a site-specific manner; for example, O-tRNA recognizes a select codon in the mRNA encoding the polypeptide, such as an amber stop codon.

[0114] The term "preferential acylation" refers to the acylation of O-tRNA with non-natural amino acids at, for example, an efficiency of about 50%, about 70%, about 75%, about 85%, about 90%, about 95%, or about 99% or higher compared to the endogenous tRNA or amino acids of the translation system of interest. The non-natural amino acids are then incorporated into the growing polypeptide chain with high fidelity, for example, with an efficiency greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% or higher for a given selected codon.

[0115] The term "selector codon" refers to a codon that is recognized by O-tRNA during translation but not by endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on mRNA and incorporates its amino acid, for example, a non-natural amino acid, into that site in the polypeptide. Selector codons can include, for example, nonsense codons, such as stop codons, e.g., amber, ochre, and milky codons; four or more base codons; codons derived from natural or non-natural base pairs, etc. For a given system, the selector codon can also include one of the natural three-base codons (i.e., the natural triplet), wherein the endogenous system does not use said natural triplet, for example, in systems lacking tRNA that recognizes the natural triplet or in systems where the natural triplet is a rare codon.

[0116] An "anticodon" has the reverse complementary sequence of the corresponding codon.

[0117] O-tRNA / O-RS pairs include O-tRNA (e.g., repressive tRNA, etc.) and O-RS.

[0118] "Repressive tRNAs" are tRNAs that alter the reading of messenger RNA (mRNA) in a given translation system. Repressive tRNAs can read, for example, stop codons, tetrabase codons, or rare codons.

[0119] As described in this article, O-tRNA is not acylated by endogenous synthases and is able to decode selection codons.

[0120] O-RS recognizes O-tRNAs, for example, those with extended anticodon loops, and preferentially acylates O-tRNAs with non-natural amino acids.

[0121] The tRNA and RS used in the method of the present invention can be naturally occurring or derived from mutations of naturally occurring tRNA and / or RS from various organisms. In various embodiments, the tRNA and RS are derived from at least one organism. In another embodiment, the tRNA is derived from a naturally occurring or mutated naturally occurring tRNA of a first organism, while the RS is derived from a naturally occurring or mutated naturally occurring RS of a second organism.

[0122] Suitable tRNA / RS pairs can be selected from libraries of mutant tRNAs and RSs, for example, based on library screening results. Alternatively, suitable tRNA / RS pairs can be heterologous tRNA / synthesizer pairs introduced into the translation system from the source species. Preferably, the cell used as the translation system is different from the source species.

[0123] For example, suitable orthogonal O-tRNAs can be derived from archaea, such as *Methanococcus janthraceae* (…). Methanococcus jannaschii ), thermophilic autotrophic methanococci ( Methanobacterium thermoautotrophicum ), spp. (Salmonella) Halobacterium ) such as volcanic halophilic bacteria ( Haloferax volcanii ) and species of the genus *Halobacter* NRC-I, *Archaeoptera* ( Archaeoglobus fulgidus ), Vibrio parahaemolyticus ( Pyrococcus furiosus ), Fireball bacteria ( Pyrococcus horikoshii ), Agile thermophilic bacteria ( Aeuropyrum pernix ), Methanococcus marinum ( Methanococcus maripaludis ), Candler methanogen ( Methanopyrus kandleri ), Methanococcus martensii ( Methanosarcina mazei (Mm), Aerobic thermophilic bacteria ( Pyrobaculum aerophilum ), Deep-sea thermococcus ( Pyrococcus abyssi ), sulfur leaf fungus ( Sulfolobus solfataricus (Ss), Tokyo sulfur fungus ( Sulfolobus tokodaii ), acidophilus ( Thermoplasma acidophilum ), volcanic thermogenes ( Thermoplasma volcanium ) etc., or true bacteria, such as Escherichia coli ( Escherichia coli Thermostats ( ) Thermus thermophilus ), Bacillus subtilis ( Bacillus subtilis ), thermophilic steatobacterium ( Bacillus stearothermphilus Orthogonal O-RS can be derived from organisms or combinations of organisms, such as archaea, such as *Methanococcus janniae*, *Methanococcus thermophilus*, *Halobacter* species such as *Haloxylon volcanicus* and *Halobacter* species NRC-J, *Archaeopterygium scintillans*, *Fierococcus fibrilliosa*, *Fierococcus burkini*, *Aerothermotrophus agileus*, *Methanococcus spp.*, *Methanococcus candela*, *Methanococcus martensii*, and *Methanococcus pasteurellii*. Methanosarcina bakeri ); Methanococcus haematococcus ( Methanosarcina hafniense Examples of sources include: *Thermophyton floccosum*, *Thermococcus pyrenoidosa*, *Thermophyton stolonifer*, *Thermophyton moniliforme*, *Thermophyton volcanoglyphus*, etc., or eubacteria such as *Escherichia coli*, *Thermophyton floccosum*, *Bacillus subtilis*, *Bacillus stearothermophilus*, etc. In one embodiment, eukaryotic sources, such as plants, algae, protozoa, fungi, yeasts, and animals, such as mammals, insects, arthropods, etc., can also be used as sources of O-tRNA and O-RS.

[0124] For example, WO 02 / 085923 and WO 02 / 06075 describe methods for evolving tRNA / RS pairs.

[0125] Preferably, RS is a pyrrolidone-lysyl-tRNA synthetase (pylRS) capable of acylating tRNA with the non-natural amino acids of the present invention. The pyrrolidone-lysyl-tRNA synthetase used in the method of the present invention can be wild-type or genetically engineered pylRS. Examples of wild-type pylRS include, but are not limited to, pylRS derived from archaea and eubacteria, such as *Methanococcus martensii*, *Methanococcus pasteurellii*, and *Methanococcus burtonensis*. Methanococcoides burtonii ), Acetic acid-nutritional methanococcus ( Methanosarcina acetivorans ), thermophilic methanococcus ( Methanosarcina thermophila ) and Haffni desulfuric acid bacteria ( Desulfitobacterium hafniense ).

[0126] Pyrrolidone-lysyl-tRNA synthetase (PylRS) is an aminoacyl-tRNA synthetase (RS). RS is an enzyme capable of acylating tRNA with an amino acid or amino acid analogue. Conveniently, the PylRS of the present invention is enzymatically active, meaning it can acylate tRNA with a specific amino acid or amino acid analogue, preferably UNAA or its hydrochloride. Pyl ).

[0127] As used herein, the term "archaeobacterial pyrrolidyl-tRNA synthetase" (abbreviated as "archaeobacterial PylRS") refers to PylRS, wherein at least one segment or the entire amino acid sequence of PylRS has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence of naturally occurring PylRS from archaea, or with the amino acid sequence of the enzymatically active fragment of such naturally occurring PylRS.

[0128] The PylRS of the present invention may comprise mutated archaea PylRS, or its enzymatically active fragments.

[0129] Generally, "mutant archaea PylRS" or "mutant archaea PylRS" differs from the corresponding wild-type PylRS in that it contains the addition, substitution, and / or deletion of one or more amino acid residues. Preferably, these are modifications that improve the stability of PylRS, alter the substrate specificity of PylRS, and / or enhance the enzymatic activity of PylRS. Particularly preferred "mutant archaea PylRS" or "mutant archaea PylRS" are described in more detail below.

[0130] The term "nuclear export signal" (abbreviated as "NES") refers to an amino acid sequence that can direct the export of its contained polypeptide (such as the NES-containing PylRS of this 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). NES are known in the art. For example, the database ValidNESs (http: / / validness.ym.edu.tw / ) provides experimentally validated sequence information for proteins containing NES. In addition, NES databases such as NESbase 1.0 (www.cbs.dtu.dk / databased / NESbase-1.0 / ; see Le Cour et al., Nucl Acids Res 31(1), 2003) and NES prediction tools such as NetNES (www.cbs.dtu.dk / services / NetNES / ; see La Cour et al., La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004), NESpredictor (NetNES, http: / / www.cbs.dtu.dk / ; see Fu et al., Nucl Acids Res 41:D338-D343, 2013; La Cour et al., Protein Eng Des Sel 17(6):527-536, 2004)) and NESsential (The web interface in combination with ValidNESs) is publicly available. Hydrophobic leucine-rich NESs are the most common and represent the best-characterized group of NESs to date. Hydrophobic leucine-rich NESs are non-conserved motifs with 3 or 4 hydrophobic residues. Many of these NESs contain the conserved amino acid sequence pattern LxxLxL or LxxxLxL, 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).

[0131] The term "nuclear localization signal" (abbreviated as "NLS," also known in the art as "nuclear localization sequence") refers to an amino acid sequence that can guide a polypeptide containing it (e.g., wild-type archaea PylRS) into the nucleus of a eukaryotic cell. The output is believed to be mediated by the binding of the NLS-containing polypeptide to an input protein (also known as a karyopherin) to form a complex that passes through nuclear pores. NLS is known in the art. Many NLS databases and NLS prediction tools are publicly available, such as NLSdb (see Nair et al., Nucl Acids Res 31(1), 2003), cNLS Mapper (www.nls-mapper.aib.keio.ac.jp; see Kosugi et al., Proc Natl Acad Sci US A.106(25):10171-10176, 2009; Kosugi et al., J Biol Chem 284(1):478-485, 2009), SeqNLS (see Lin et al., PLoS One 8(10):e76864, 2013) and NucPred (www.sbc.su.se / ~maccallr / nucpred / ; see Branmeier et al., Bioinformatics 23(9):1159-60, 2007).

[0132] The mutant archaea PylRS of the present invention, as defined above, can be further modified by removing optional NLS from the naturally occurring PylRS, wherein the mutant is derived from the introduction of at least one NES and / or by the introduction of at least one NES. NLS in naturally occurring PylRS can be identified using known NLS detection tools such as cNLS Mapper.

[0133] Removing NLS and / or introducing NES from the archaea PylRS or its mutants when expressed in eukaryotic cells can alter the localization of such modified peptides, particularly by preventing or reducing peptide accumulation in the eukaryotic cell nucleus. Therefore, the localization of the PylRS mutant of the present invention expressed in eukaryotic cells can be altered compared to PylRS or PylRS mutants (which differ from the PylRS mutant of the present invention in that they (still) contain NLS but lack NES).

[0134] When the archaea PylRS of the present invention contains NES but (still) contains NLS, it is preferable to select NES such that the strength of NES exceeds that of NLS, thereby preventing the accumulation of PylRS in the eukaryotic cell nucleus.

[0135] Removing NLS from wild-type or mutant PylRS and / or introducing NES into wild-type or mutant PylRS to obtain the PylRS of the present invention does not abolish the enzymatic activity of PylRS. Preferably, the enzymatic activity of PylRS is maintained at substantially the same level, that is, 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 enzymatic activity of the corresponding wild-type or mutant PylRS.

[0136] The NES is conveniently located within the PylRS or mutant PylRS of the present invention, thus making the NES functional. For example, the NES can be linked to the C-terminus (e.g., the C-terminus of the last amino acid residue) or the N-terminus (e.g., between amino acid residue 1, the N-terminal methionine, and amino acid residue 2) of the wild-type or mutant archaea PylRS.

[0137] WO2018 / 06948 discloses mutated PylRS modified by incorporation of NES and / or deletion of NLS sequences, which are hereby explicitly cited and incorporated into this paper.

[0138] C. Specific Aspects and Implementation Plans

[0139] 1. The first aspect of the present invention

[0140] According to a first aspect, the present invention provides a hydrophilic TCO (hyTCO) compound of general formula I. in n is an integer between 0 and 1-20, especially between 1-10, and even more so between 1-5; A is -CR 1 R 2 -、-O-、-S-、-N(R 1 )-、>CH-OZ、>CH-SZ、>CH-NR 1 R 2 >CH-OR 3 >CH-CN、>CH-NO2、>CH-SO2R 4 Or >CH-SR 3 Especially -C(R) 1 R 2 -, -O-, -S-, -N(R) 1 )-、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3; B is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; especially -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; M is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; especially -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-R 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; Q is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 , >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; especially -CR 1 R 2 -, -O-, -S-, -N(R1 )-、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 ; D is -OR 3 -SR 3 -OZ or -SZ; especially -OR 3 or -SR 3 More specifically -OR 3 ; U is OZ, -SZ, -OR 3 -SR 3 or -NR 5 R 6 Especially -OR 3 -SR 3 or -NR 5 R 6 More specifically -OR 3 ; in Residue Z can be the same or different protecting groups independently; R 1 R 2 R 5 and R 6 They are independently the same or different from each other, and are selected from H and optionally substituted, saturated or unsaturated, linear or branched, acyclic or cyclic homo- or heteroalkyl groups, such as optionally substituted, linear or branched homo- or heteroalkyl groups and optionally substituted linear or branched homo- or heteroalkyl acyl groups. R 3 and R 4 Independently identical or different from each other, and selected from H, optionally substituted, saturated or unsaturated, linear or branched, acyclic or cyclic homogeneous or heteroalkyl groups, such as optionally substituted, linear or branched homogeneous or heteroalkyl groups, optionally substituted linear or branched homogeneous or heteroalkylyl groups, and C4-C 24 Homogeneous or heteroaryl; X is a bridge base; W is a saturated or unsaturated polar group; L is a linking group; and G is a terminal group; Or its salts, such as pharmaceutically acceptable salts, or solvates; each as a stereoisomer or a mixture of at least two stereoisomers, wherein the cis / trans isomers of the TCO-ring moiety are excluded.

[0141] According to its specific implementation plan, the following applies: If M and B independently represent -O-, -S-, or -N(R) 1 If A is -CR, then A is -CR. 1 R 2 -; or If A is selected from -O-, -S-, or -N(R) 1 If M and B independently represent -CR, then M and B represent -CR. 1 R 2 -

[0142] According to another specific implementation, Q is -CR 1 R 2 -, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 Especially -CR 1 R 2 -、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 .

[0143] According to a more specific implementation scheme, Q is -CR 1 R 2 -

[0144] According to another, more specific implementation scheme, Q is >CH-NR 1 R 2 .

[0145] According to another, more specific implementation, Q is > CH-OR 3 .

[0146] According to another, more specific implementation, Q is >CH-CN.

[0147] According to another, more specific implementation, Q is >CH-NO2.

[0148] According to another, more specific implementation scheme, Q is > CH-SO2R 4 .

[0149] According to another, more specific implementation scheme, Q is >CH-SR 3 .

[0150] According to another specific implementation, A is -CR1 R 2 -、-O-、-S-、-N(R 1 )-、>CH-OZ、>CH-SZ、>CH-NR 1 R 2 >CH-OR 3 >CH-CN、>CH-NO2、>CH-SO2R 4 Or >CH-SR 3 Especially -C(R) 1 R 2 -, -O-, -S-, -N(R) 1 )-、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 .

[0151] According to a more specific implementation scheme, A is -CR 1 R 2 -

[0152] According to another, more specific implementation, A is -O-.

[0153] According to another, more specific implementation, A is -S-.

[0154] According to another, more specific implementation, A is -N(R) 1 )-.

[0155] According to another, more specific implementation scheme, A is >CH-NR 1 R 2 .

[0156] According to another, more specific implementation scheme, A is >CH-OR 3 .

[0157] According to another, more specific implementation, A is >CH-CN.

[0158] According to another, more specific implementation, A is >CH-NO2.

[0159] According to another, more specific implementation, A is >CH-SO2R 4 .

[0160] According to another, more specific implementation scheme, A is >CH-SR 3 .

[0161] According to another specific implementation, B is -CR 1 R 2-、-O-、-S-、-N(R 1 )-、>CH-OZ、>CH-SZ、>CH-NR 1 R 2 >CH-OR 3 >CH-CN、>CH-NO2、>CH-SO2R 4 Or >CH-SR 3 Especially -C(R) 1 R 2 -, -O-, -S-, -N(R) 1 )-、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 .

[0162] According to a more specific implementation plan, B is -CR 1 R 2 -

[0163] According to another, more specific implementation, B is -O-.

[0164] According to another, more specific implementation, B is -S-.

[0165] According to another, more specific implementation, B is -N(R) 1 )-.

[0166] According to another, more specific implementation scheme, B is >CH-NR 1 R 2 .

[0167] According to another, more specific implementation scheme, B is >CH-OR 3 .

[0168] According to another, more specific implementation scheme, AB is >CH-CN.

[0169] According to another, more specific implementation, B is >CH-NO2.

[0170] According to another, more specific implementation scheme, B is >CH-SO2R 4 .

[0171] According to another, more specific implementation scheme, B is >CH-SR 3 .

[0172] According to another specific implementation, M is -CR 1 R 2 -、-O-、-S-、-N(R1 )-、>CH-OZ、>CH-SZ、>CH-NR 1 R 2 >CH-OR 3 >CH-CN、>CH-NO2、>CH-SO2R 4 Or >CH-SR 3 Especially -C(R) 1 R 2 -, -O-, -S-, -N(R) 1 )-、>CH-NR 1 R 2 >CH-OR 3 ,>CH-CN,>CH-NO2,>CH-SO2R 4 Or >CH-SR 3 .

[0173] According to a more specific implementation scheme, M is -CR 1 R 2 -

[0174] According to another, more specific implementation, M is -O-.

[0175] According to another, more specific implementation, M is -S-.

[0176] According to another, more specific implementation, M is -N(R) 1 )-.

[0177] According to another, more specific implementation, M is >CH-NR 1 R 2 .

[0178] According to another, more specific implementation, M is >CH-OR 3 .

[0179] According to another, more specific implementation, M is >CH-CN.

[0180] According to another, more specific implementation, M is >CH-NO2.

[0181] According to another, more specific implementation, M is > CH-SO2R 4 .

[0182] According to another, more specific implementation scheme, M is >CH-SR 3 .

[0183] According to another specific embodiment, a compound of general formula I is provided, wherein residues A, B, M, and Q are identical and each represents -C(R) 1 R 2 )-, where R1 and R 2 Alkyl groups, which independently represent H or optionally substituted, linear or branched, have 1 to 10 carbon atoms, particularly 1 to 5 carbon atoms, and more particularly 1 or 2 carbon atoms.

[0184] According to another specific embodiment, a compound of general formula I is provided, wherein residues A, B, M, and Q are identical and each represents -C(R) 1 R 2 )-, where R 1 and R 2 Each of the following can be independently represented by H or an unsubstituted linear or branched alkyl group having 1-10 carbon atoms, particularly 1-5 carbon atoms, and more particularly 1 or 2 carbon atoms.

[0185] According to another specific embodiment, a compound of general formula I is provided, wherein residues A, B, M, and Q are identical and each represents -C(R) 1 R 2 )-, where R 1 and R 2 Represents H.

[0186] According to another specific embodiment, a compound of general formula I is provided, wherein residues U and D each represent -OR. 3 , where R 3 Each of the following is an independent representation of H or a linear or branched alkyl group having 1-10 carbon atoms, particularly 1-5 carbon atoms, and more particularly 1 or 2 carbon atoms.

[0187] According to another specific embodiment of the above-mentioned compound, residue Z is a thiol protecting group or an alcohol protecting group, particularly a cleavable protecting group, and more particularly, is independently selected from acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), methoxytriphenylmethyl (MT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), triphenylmethyl (triphenylmethyl, Tr), silyl ether, methyl ether, and ethoxyethyl ether (EE) residues.

[0188] According to another specific embodiment of the above-described compound, X is selected from: a) Linear or branched lower alkylene groups, especially -(CH2). n1 -, where n1 is an integer from 1 to 4, specifically methylene or ethylene; b) -O-, -S-, >CH-OZ, >CH-SZ, >CH-OR 3 Or >CH-SR 3 ; in Z and R 3 As defined above; c) Linear or branched mono- or polyalkylene oxide moieties, particularly selected from the linear moieties -((CH2) x -O) y -、-(O-(CH2) x ) y - and -(O-(CH2) x ) y -O-, and its branched or monounsaturated or polyunsaturated analogues; in x represents an integer selected from 1, 2, 3, or 4 independently; specifically 1 or 2; and y independently represent integers selected from 1-20, especially 1-15, 1-10, or 1-4; or According to a more specific implementation scheme, X is as defined in a).

[0189] According to another, more specific implementation, X is as defined in b).

[0190] According to another, more specific implementation, X is as defined in c).

[0191] According to another specific embodiment, a compound of general formula I is provided, wherein X is selected from methylene or ethylene and part of -((CH2) x -O) y -or-(O-(CH2)) x ) y - where x is 1 and 2, and y is 1 or 2.

[0192] According to another specific embodiment of the above compound, the polar group W is selected from: a) -C(O)-, -OC(O)-, -C(O)-O-; b) -O-[C(O)-[CH2] x1 ] x2 C(O)-;-O-[C(S) [CH2] y1 ] y2 C(O)- in x1, x2, y1, and y2 independently represent integers selected from 1 to 6, especially 1 to 4, and even more especially 1 to 2; c) -SC(S)-, -OC(S)-, -C(S)-, -C(S)-O-, -C(S)-S-; -NR 7 -C(O)-、-C(O)-NR 7 -、-C(NR7 -NR 7 -C(NR 7 )-、-NR 7 -C(O)NR 7 -、-NR 7 -C(NR 7 )-;or d) -NR 8 -[C(O) [CH2) x1 ] x2 C(O)-;NR 8 -[C(S) [CH2) y1 ] y2 C(O)-; in x1, x2, y1, and y2 independently represent integers selected from 1 to 6, especially 1 to 4, and even more especially 1 to 2; and R 7 and R 8 Each can be independently represented by H or a lower alkyl group, especially H or a C1-C4 alkyl group.

[0193] According to a more specific implementation scheme, W is as defined in a).

[0194] According to another, more specific implementation, W is as defined in (b).

[0195] According to another, more specific implementation, W is as defined in c).

[0196] According to another, more specific implementation scheme, W is as defined in (d).

[0197] According to another specific embodiment, a compound of general formula I is provided, wherein W is selected from C(O)-, -OC(O)- and -C(O)-O-.

[0198] According to another specific embodiment of the above compounds, each L is independently selected from: a) Linear or branched alkylene, linear or branched alkenyl, cycloalkylene, cycloalkenyl, or analogs thereof containing one or more heteroatoms in their carbon skeleton; particularly linear or branched C1-C 10 -alkylene, linear or branched C2-C 10 -Alkenyl, C3-C8-cycloalkylene, C3-C8-cycloalkylene, or analogs thereof containing one or more heteroatoms in their carbon skeleton; more particularly linear or branched C1-C4-alkylene, linear or branched C2-C4-alkylene, C3-C6-cycloalkylene, C3-C6-cycloalkylene, or analogs thereof containing one or more heteroatoms in their carbon skeleton; most particularly methylene, ethylene, and propylene; (partially La) b) Linear or branched mono- or polyalkylene oxide moieties, particularly selected from the linear moieties -((CH2) x3 -O) y3 -、-(O(CH2) x3 ) y3 -、-(O-(CH2) x3 ) y3 -O-、-((CH2) x3 -O) y3 -(CH2) n3 C(O)-、-(O(CH2) x3 ) y3 -NR 7’ -、-(O-(CH2) x3 ) y3 -C(O)- and its branched analogues; in x3 represents an integer selected from 1, 2, 3, or 4 independently; specifically 1 or 2; y3 represents integers selected from 1-20, especially 1-15, 1-10, or 1, 2, 3 and 4, independently of each other; n3 is an integer chosen from 2 and 3; and R 7 'Represents H or a lower alkyl group, especially H or C1-C4-alkyl, and more particularly methyl or ethyl; (Partial Lb) The condition is that, when two or more identical or different linker portions La and / or Lb exist, these elements can be directly linked to each other by chemical bonds, or by a combination of -C(O)-, -OC(O)-, -C(O)-O-, -SC(S)-, -OC(S)-, -C(S)-, -C(S)-O-, -C(S)-S-; -NR 7 -C(O)-、-C(O)-NR 7 -、-C(NR 7 -NR 7 -C(NR 7 )-, -S(O)-, -S(O)2-, -P(O)OR 7 -、-OP(O)OR 7 -、-P(O)OR 7 -O- The same or different coupling parts are indirectly connected to each other; where R 7 As defined above.

[0199] According to a more specific implementation scheme, L is as defined in a).

[0200] According to another, more specific implementation, L is as defined in (b).

[0201] According to another specific embodiment, a compound of formula I is provided, where n = 0 or 1.

[0202] According to another specific embodiment, a compound of formula I is provided, wherein L represents the moiety La, selected from methylene, ethylene, and propylene; or represents the moiety Lb, selected from -((CH2) x3 -O) y3 -or-(O(CH2)) x3 ) y3 - where x3 and y3 are independently selected from integers 1 and 2.

[0203] According to another specific embodiment of the above compound, G has one of the following meanings: a) G is H or R 7 Furthermore, if n ≠ 0, then G can also be -OR. 7 -C(O)OR 7 -NR 7 2 or -C(O)NR 7 2; especially R 7 It represents H or C1-C4-alkyl.

[0204] b) G is a leaving group E, selected from halogens, pentafluorophenyl (Pfp), tetrafluorophenyl (Tfp), oxazolone, succinimide (Su), sulfosuccinimide, trifluoroacetyl, azide, p-nitrophenyl (PNP) and nitro-containing aromatic groups. c) G is the thiol reaction part of the following general formula. in J is selected from -NH-, -S-, or -O-, or may not exist; r is an integer from 1 to 4, or 0 if J does not exist; and M1 is the thiol reactive moiety, more particularly selected from M2-M16 (e.g., as described in WO 2022 / 058395): in: X 3 It is H, halogen, PhS, MeS; X 4 It is halogen, PhS, MeS; X 6 Is it H or C1-C? 12 Alkyl groups, preferably H or C 1-6 alkyl; X 5 It is H, C1-C 12 Alkyl, C6-C 12 Aryl, C7-C 12 Alkyl or C7-C 12 Aryl alkyl group, preferably H or p-methylphenyl; and The aromatic rings of (M6) and (M8) may optionally be heteroaromatic rings, such as phenyl or pyridine rings; d) G is -NH-(CH2)2)C≡CH, -NH-(CH2)2-N3, -O-aryl or -HN-aryl, H, -OH, -NH2 halogen, R 9 -CH=C(R) 9 2. -C≡CR 9 -[C(R) 9 )2C(R 9 )2O] q -R 9 , -CN, -N3, -NCL, -LCN, -LR 9 - + N(R 9 )2、-N(R 9 3、-C(L)N(R) 9 )2、C(R 9 )2L R 9 -C(L) R 9 -C(L)LR 9 -S(O)R 9 -S(O) )2 R 9 -S(O)OR 9 -S(O)2OR 9 -S(O)N(R) 9 )2、-S(O)2N(R 9 )2、-OS(O)R 9 -OS(O)2R 9 -OS(O)OR 9 -OS(O)2OR 9 -P(O)(R 9 (OR) 9 -P(O)(OR) 9 )2、-OP(O)(OR 9 )2、-Si(R 9 3. -LC(L)R 9 -LC(L)LR 9 -LC(L)N(R) 9 )2、-N(R 9 )C(L)R9 -N(R) 9 )C(L)LR 9 and -N(R) 9 )C(L)N(R 9 )2, in q is in the range of 1-200, especially 1-20, even more so 1-10, and especially 1-5. L is oxygen or sulfur, and R 9 Independently selected from hydrogen, halogen, C1-C 24 Alkyl groups, such as C1-C 10 Alkyl or C1-C3 alkyl, C6-C 24 (Miscellaneous) aryl, C7-C 24 Alkyl (hetero)aryl and C7-C 24 (Hetero)arylalkyl; e) G is -OZ, -SZ, -OR 1 -C(O)OR 1 -N(R) 1 )2、-C(O)N(R 1 )2、-SR 1 or -R 1 , Among them, Z and R 1 As defined above; and f) G is a cyclic or acyclic strain-promoted acetylene-azidocycloaddition (SPAAC) reactive group, especially, i. alkynyl-terminated group ii. DBCO-derived residues of the following general formula in X 3 It is N, >CH- or >CH-O-; or iii. Residues of the following formula derived from BCN iv. Cyclooctyne-derived residues, especially SCO-derived residues of the following general formula. in X 4 It is -O- or -O-(CO)-; v. or residues of formulas M20-M38 (e.g., as described in WO 2022 / 058395). in B- It is an anion, or in: R 15 Independently selected from hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) C1-C 24 Alkyl, C6-C 24 (Miscellaneous) aryl, C7-C 24 Alkyl (hetero)aryl and C7-C 24 (Hetero)arylalkyl, wherein the alkyl, (hetero)aryl, alkyl(hetero)aryl, and (hetero)arylalkyl are optionally substituted, wherein two substituents R 15 They can be linked together to form optionally substituted annulated cycloalkyl groups or optionally substituted annulated (hetero)aryl substituents, wherein R 16 Independently selected from hydrogen, halogens, C1-C 24 Alkyl, C6-C 24 (Miscellaneous) aryl, C7-C 24 Alkyl (hetero)aryl and C7-C 24 (Hetero)arylalkyl; in Y 2 It is C(R) 15 )2, O, S or NR 15 ; u is 0, 1, 2, 3, 4 or 5; u' is 0, 1, 2, 3, 4, or 5, where u + u' = 4, 5, 6, 7, or 8; and v is an integer in the range of 8-16.

[0205] According to a more specific implementation scheme, G is as defined in a).

[0206] According to another, more specific implementation, G is as defined in (b).

[0207] According to another, more specific implementation, G is as defined in (c).

[0208] According to another, more specific implementation, G is as defined in (d).

[0209] According to another, more specific implementation, G is as defined in (e).

[0210] According to another, more specific implementation, G is as defined in f).

[0211] According to another, more specific implementation, G is as defined in f) i).

[0212] According to another, more specific implementation, G is as defined in f) ii).

[0213] According to another, more specific implementation, G is as defined in f) iii).

[0214] According to another, more specific implementation, G is as defined in f) iv).

[0215] According to another, more specific implementation, G is as defined in f) v).

[0216] According to another specific embodiment, the compound has formulas I.1 to I.12. Wherein D and U are as defined above; or their salts, such as pharmaceutically acceptable salts, or solvates; each as a stereoisomer or a mixture of at least two stereoisomers, wherein the cis / trans isomers of the TCO-ring moiety are excluded.

[0217] According to another specific embodiment of the above compound, D is -OH and U is -OAc, or D is -OH and U is -OH.

[0218] 2. The second aspect of the invention

[0219] According to a second aspect of the invention, ncAA derivatives of general formula II are provided. in n, A, B, M, Q, D, U, X, W, L are as defined above for compounds of general formula I. E 1 It is an H or amine protecting group; and G 1 Selected from -NH-, -NH-C(=NH)NH-, -S-, -O-, -O-aryl-, -NH-(CH2) m -O-aryl-, where m is an integer from 1 to 10; -HN-aryl- or part of the following formula or Any side chain residue selected from natural or non-natural amino acids, which is permitted to conjugate with part W, optionally via part (L) of the compound of formula II.n And specifically selected from -S-CH2-, -O-CH2-, -Op-(phenylene)-CH2-, -O-CH(CH3)-, -O-(C=O)-CH2-,)-; -O-(C=O)-CH2-CH2-, -N-(C=O)-CH2-,)-; -N-(C=O)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or Or its salts, such as pharmaceutically acceptable salts, or solvates; each as a stereoisomer or a mixture of at least two stereoisomers, wherein the cis / trans isomers of the TCO-ring moiety are excluded.

[0220] According to a first specific embodiment, a compound of general formula II is provided, wherein residues A, B, M, and Q are identical and each represents -C(R) 1 R 2 )-, where R 1 and R 2 Each of these terms independently represents an optionally substituted, linear or branched alkyl group having 1 to 10 carbon atoms, particularly 1 to 5 carbon atoms, and more particularly 1 or 2 carbon atoms.

[0221] According to another specific embodiment, a compound of general formula II is provided, wherein residues A, B, M, and Q are identical and each represents -C(R) 1 R 2 )-, where R 1 and R 2 Represents H.

[0222] According to another specific embodiment, a compound of general formula II is provided, wherein residues U and D each represent -OR. 3 , where R 3 Each of the following is an independent representation of H or a linear or branched alkyl group having 1-10 carbon atoms, particularly 1-5 carbon atoms, and more particularly 1 or 2 carbon atoms.

[0223] According to another specific embodiment, a compound of general formula II is provided, wherein X is selected from methylene or ethylene and part of -((CH2) x -O) y -or-(O-(CH2)) x ) y - where x is 1 and 2, and y is 1 or 2.

[0224] According to another specific embodiment, a compound of general formula II is provided, wherein W is selected from C(O)-, -OC(O)- and -C(O)-O-.

[0225] According to another specific embodiment, a compound of formula II is provided, wherein n = 1.

[0226] According to another specific embodiment, a compound of formula II is provided, wherein L represents the moiety La, which is selected from methylene, ethylene, and propylene; or represents the moiety Lb, which is selected from -((CH2) x3 -O) y3 -or-(O(CH2)) x3 ) y3 - where x3 and y3 are independently selected from integers 1 and 2.

[0227] According to another specific embodiment, a compound of general formula II is provided, wherein residue E 1 It's H.

[0228] According to another specific embodiment, a compound of general formula II is provided, wherein residue G 1 It is -S-CH2-, -O-CH2-, -Op-(phenylene)-CH2-, -O-CH(CH3)-, -O-(C=O)-CH2-,)-; -O-(C=O)-CH2-CH2-, -N-(C=O)-CH2-,)-; -N-(C=O)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or

[0229] According to a specific embodiment of the compound of formula II above, residue E 1 It is an H or amine protecting group, selected from 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), acetyl (Ac), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), or toluenesulfonyl (Ts) group.

[0230] According to another specific embodiment, the compound has any one of formulas II.1 to II.3. in E 1 It is H or an amine protecting group as defined above; Or its salts, such as pharmaceutically acceptable salts, or solvates; each as a stereoisomer or a mixture of at least two stereoisomers, wherein the cis / trans isomers of the TCO-ring moiety are excluded.

[0231] According to a specific embodiment of the compound of formula II, D is -OH and U is -OAc, or D is -OH and U is -OH.

[0232] 3. The third aspect of the invention

[0233] According to a third aspect of the present invention, a method for preparing a compound of formula VIII is provided. M, A, B, D, Q, and U are as defined above; The method includes the following steps: a) Provide compounds of formula III b) Converting compounds of formula III into compounds of formula IV. Where D1 is O, S or N; in particular, if D1 is oxygen, then an organic peroxyacid, especially 3-chloroperoxybenzoic acid, is used, which is a compound of epoxidized form III. c) Reacting a compound of formula IV with a nucleophile UH (especially a low-molecular-weight aliphatic carboxylic acid, such as acetic acid or propionic acid) in the presence of an organometallic catalyst (e.g., especially tetrakis(triphenylphosphine)palladium(O)) to obtain a compound of formula V. d) Oxidation of the compound of formula V in the presence of a suitable oxidizing agent (if D is -OH, then for example, especially pyridinium chlorochromate) to obtain the compound of formula VI. e) Reacting the compound of formula VI with a silyl-protected enol compound (e.g., particularly 1-(tert-butyldimethylsilyloxy)-1-methoxyethylene), especially after activation with a BF3 source, to obtain the compound of formula VII. as well as f) Isomerize the compound of formula VII, especially photoisomerize it, to obtain the compound of formula VIII.

[0234] According to a specific implementation of the method, D1 is an oxygen atom.

[0235] According to another specific embodiment of the method, at least one of steps a) to f) is carried out in a liquid reaction medium, particularly in an organic liquid reaction medium.

[0236] According to another specific embodiment of the method, step b) is carried out in the presence of an oxidant selected from 3-chloroperoxybenzoic acid, potassium peroxymonosulfate (oxone), other peroxy acids, H2O2, tert-butyl peroxide, other peroxides, hypochlorite, persulfate, O2, dimethyldioxane, other dioxanes, particularly 3-chloroperoxybenzoic acid.

[0237] According to another specific embodiment of the method, step c) is carried out in the presence of a palladium catalyst selected from tetrakis(triphenylphosphine)palladium(O), Pd(dppe)2, Pd2(dba)3, Pd(COD)Cl2, especially tetrakis(triphenylphosphine)palladium(O).

[0238] According to another specific embodiment of the method, step d) is carried out in the presence of an oxidant selected from pyridinium chlorochromate, pyridinium dichromate, chromium oxide, dichromate chromate, DMP, IBX, peridinanes, DMSO and oxalyl chloride, MnO2, KMnO4, H2O2, O2, and especially pyridinium chlorochromate.

[0239] According to another specific embodiment of the method, step e) is carried out in the presence of the following substances. a. Lewis acids selected from BF3∙OEt2, TiCl4, Ti(OR)4, AgOTf, TMSOTf, Cu(OTf)2, SiCl4, transition metal salts, especially BF3∙OEt2; and b. Silyl-protected enol esters selected from silylketene acetals, N,O-silylketene acetals, silylketene ethers, and especially (tert-butyldimethylsilyloxy)-1-methoxyethylene.

[0240] According to another specific embodiment of the method, step f) is carried out in the presence of UV light, or in the absence of a photosensitizer, which is capable of absorbing UV light and producing a physicochemical change in adjacent molecules such as substituted aryl groups, wherein the substituted aryl groups are selected from methyl benzoate, benzene, toluene, xylene, benzophenone, acetophenone, cumene, phthalimide, phthalate ester, terephthalate ester, benzamide, and benzonitrile.

[0241] According to another specific implementation of the method, the total reaction time for any one of steps a) to f) is 15 min to 48 h, or 10 to 24 h, or 24 h or 12 h, particularly 180 min.

[0242] According to another specific embodiment of the method, any one of steps a) to f) is performed at a temperature of about -100 °C to about 100 °C or about -80 °C to about 70 °C.

[0243] According to another specific implementation of the method a) M, A, and B are each -CH2-; b) In the compounds of formulas IV and VI, D1 represents O; c) D represents -OH; and d) U represents OAc or OH.

[0244] 4. Fourth aspect of the invention

[0245] According to the fourth aspect, a method for preparing compound X is provided. include a) By removing the ester group, the compound of formula VIII above Where M, A, B, and Q are each -CH2-, and D and U are each -OH, converting to compounds of formula IX, and b) Converting compounds of formula IX into compounds of formula X in the presence of N,N,N',N'-tetramethyl-O-(N-succinimide)ureon tetrafluoroborate.

[0246] 5. The fifth aspect of the invention

[0247] According to a fifth aspect of the present invention, a method for preparing a compound of formula XII is provided. Includes the following steps: a) The compound of formula VIII above M, A, B, and Q are each -CH2-, and D and U are each -OH. Compounds converted to formula XI as well as b) The compound of formula XI, particularly under temperature control, between -10 and +20 °C o The compound of formula XII is converted by 1,1'-carbonyldiimidazole at a temperature of C.

[0248] 6. The sixth aspect of the invention

[0249] According to a sixth aspect of the present invention, a method for preparing a compound of formula XIV is provided. Includes the following steps: a) Converting compounds of formula VIII above, especially in the presence of Fmoc Lys-OH and organic nucleophiles (e.g., particularly DIPEA), into compounds of formula XIII. as well as b) Converting compounds of formula XIII into compounds of formula XIV, particularly by deprotection in the presence of a secondary base (such as piperidine).

[0250] 7. The seventh aspect of the invention

[0251] According to a seventh aspect of the present invention, a method for preparing a compound of formula XVI is provided. Includes the following steps: a) Converting compounds of formula XII, especially in the presence of Fmoc Lys-OH and organic nucleophiles (e.g., particularly DIPEA), into compounds of formula XV. as well as b) Converting compounds of formula XV into compounds of formula XVI, particularly by deprotection in the presence of a secondary base (such as piperidine).

[0252] 8. The eighth aspect of the invention - hyTCO-functionalized construct

[0253] According to an eighth aspect of the invention, a hydrophilic trans-cyclooctene (hyTCO)-functionalized construct is provided, which can be obtained by binding a compound according to any one of the first or second aspects of the invention to a targeting agent (TA) or a payload molecule (PM), particularly by covalent binding, wherein the functionalized construct contains at least one functionalized residue (FR) comprising a monounsaturated trans-cyclooctene entity of general formula (II'). in n, A, B, D, U, M, Q, X, and W are as defined above; G 1 As defined above, or does not exist; and L 1 The L in the connector section has the meaning of or represents a branch section as defined above.

[0254] A “branched portion” represents at least a trivalent group that allows for functionalization in a single step using more than one, for example, two of the three hyTCO moieties. Non-limiting examples of such branched groups include trivalent cyclic aliphatic or cyclic aromatic residues of the following general formula. Or a trivalent, acyclic, aliphatic group or tertiary amino group as shown in the following formula. According to a specific embodiment, the functionalized construct is a functionalized target (functionalized TA), wherein the target is selected from viruses, whole cells, bacteriophages, liposomes, biomolecules, and low or high molecular weight compounds, such as, in particular, immunoglobulins, such as antibodies, antibody derivatives, antibody fragments, fusion molecules containing at least one antibody or antibody fragment, enzymes, proteins, peptides, peptide-like molecules, carbohydrates, monosaccharides, polysaccharides, oligonucleotides or polynucleotides, especially DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutic agents, receptor agonists and receptor antagonists, cytokines, hormones, steroids, toxins and their derivatives.

[0255] According to another specific embodiment, the functionalized construct is a functionalized payload molecule (functionalized PM), wherein the payload molecule is selected from bioactive compounds, labeling agents, protein degrading agents, particularly payloads suitable for protein hydrolysis-targeted chimeras (PROTAC), photosensitizers, and chelating agents.

[0256] According to a more particular embodiment of the functionalized construct a) Bioactive compounds are selected from biomolecules and low or high molecular weight compounds, such as, in particular, immunoglobulins, such as antibodies, antibody derivatives, antibody fragments, fusion molecules containing at least one antibody or antibody fragment, enzymes, proteins, peptides, peptide-like molecules, carbohydrates, monosaccharides, polysaccharides, oligonucleotides or polynucleotides, especially DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutic agents, receptor agonists and receptor antagonists, cytokines, hormones, steroids, toxins and their derivatives, more particularly selected from small organic, inorganic or organometallic molecules, drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, and polypeptides, peptide-like molecules, amino acids, nucleotides, oligonucleotides or polynucleotides, nucleosides, DNA, RNA, toxins, glycans and immunoglobulins; b) The labeling agent is selected from dyes, radioactive labels and fluorophores, MRI-sensitive spin labels, affinity tags, polyethylene glycol groups, photocrosslinking agents, NMR probes, X-ray probes, pH probes, IR probes, resins and solid supports; c) The chelating agent is selected from acetylacetone (ACAC), ethylenediamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylenetriamine (DIEN), iminodiacetic acid (IDA), triethylenetetramine (TRIEN), triaminotriethylamine, nitrotriacetic acid (NTA) and its salts such as Na3NTA or FeNTA, ethylenediaminetriacetic acid (TED), ethylenediaminetetraacetic acid (EDTA) and its salts such as Na2EDTA and CaNa2EDTA, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-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 agent or 2,3-dimercaptopropanol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA); siderophores secreted by microorganisms, such as deferrioxamine or deferoxamine B, also known as Deferral (Novartis), by Streptomyces genus ( Streptomyces spp Produced by *Streptomyces pubescens*; deferoxamine (DFO), produced by *Streptomyces pubescens* (…). Streptomyces pilosus Trihydroxamic acid secreted by the body; derivatives of phytochemicals such as curcuminoids and mugineic acid, such as 3-hydroxy-mugineic acid and 2'-deoxy-mugineic acid; synthetically produced chelating agents, such as ibuprofen; derivatives of catechols, hydroxamic acids, and hydroxypyridinones, such as deferroamine hydroxamic acid and deferroamine hydroxypyridinone; deferoxone (L1 or 1,2-dimethyl-3-hydroxypyridin-4-one); D-penicillamine (DPA or D-PEN), which is β-β-dimethylcysteine ​​or 3-mercapto-D-valine; tetraethylenetetramine (TETA) or tricentine and its two major metabolites, N1-acetyltriethylenetetramine (MAT) and N1,N 10 - Diacetyltriethylenetetramine (DAT); hydroxyquinoline; chloroiodohydroxyquinoline, which is a halogenated derivative of 8-hydroxyquinoline; and 5,7-dichloro-2-[(dimethylamino)methyl]quinoline-8-ol (PBT2); d) The photosensitizer / protein degrader is selected from payloads suitable for protein hydrolysis-targeted chimeras (PROTAC).

[0257] According to another specific embodiment of the functionalized construct, the functionalized residue (FR) of Formula II' is linked (directly or via a cleavable or incleavable portion) to a target agent (TA) or payload molecule (PM), particularly an amino acid residue of the TA or PM, thereby forming a functionalized TA of general formula (XX.1). in TA is the target region as defined above; and FR is the functionalized residue of formula II' above. Or the functionalized PM of general formula (XX.2) in a is 0 or represents an integer selected from 1 and 2. b is 0 or represents an integer selected from 1 and 2. c represents selecting an integer that is at least 1, specifically chosen from 1 and 2. Y 1 Represents the cuttable portion. Y 2 Represents the self-destruction portion. PM is the payload molecule as defined above, and FR is the functionalized residue of equation II' above, where L is the residue when c represents an integer greater than 1. 1 This represents the branch, where n is 1, and G1 does not exist.

[0258] More specifically Y 1 Represents an enzyme- or chemically cleavable linker group, selected from... a) Peptide groups, especially dipeptide, tripeptide, or tetrapeptide groups; b) Formula - (CR) 7’ R 8’ ) n’ -SS-(CR 7’ R 8’ ) n’ -X5- disulfide group in n' is 1-4 Residue R 7’ and R 8’ They are independently selected from H or lower alkyl groups, especially methyl; or two residues R 7’ and R 8’ Together with the carbon atoms attached to them, they form cyclic C4- to C8-alkyl groups; and Part of X5 is selected from -C(O)- and -O-; c) A hydrazone group, selected from >C=NN(R9 ')- and -N(R 9 ')-N=C< in R 9 ' is H or a lower alkyl group; and d) β-glucuronidase-sensitive cleavable linker groups, especially those carrying β-glucuronide-derived trigger residues. and / or Y 2 Represents the self-destructive portion, which is selected from a) The following formula contains a p-amino-benzyl alcohol derivative group -NH-p-phenylene-CH2-O- or -O-CH2-p-phenylene-NH-, or -NH-p-phenylene-CH2-N + R2- b) -OC(O)-O-; c) -OC(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’ - in R2 represents lower alkyl groups, especially C1 to C4 alkyl groups. Z' represents an integer selected from 1 to 6, especially 1 to 4; R 10’ and R 11’ Each can independently represent H or a lower alkyl group; R 12’ and R 13’ Each can independently represent H, methyl, or ethyl, especially H or methyl, especially H; and X 1’ and X 2’ Each can be represented independently of O, S, or NR. 10 d) The following formula contains methylene alkoxycarbamate (MAC) type linkages. -OC(O)-NR 13’ -C(R 14’ R 15’ )-(O)- -OC(O)-NR13’ -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)- in R 13’ R 14’ R 15’ and R 16’ Each can be independently represented by H or a lower alkyl group, especially C1 to C4-alkyl groups.

[0259] According to another specific implementation, the functionalized construct is a functionalized TA of formula XX.1. in TA is a target agent as defined above; FR is a functionalized residue of formula II' in A, B, M, and Q are -CR 1 R 2 -, where R 1 and R 2 As defined above; D and U are -OR 3 , where R 3 As defined above; and n, L 1 X and W are as defined above; and G 1 As defined above, in particular any side chain residue selected from natural or non-natural amino acids, which is permitted to conjugate with part W, optionally through part (L) of the compound of formula II. n And specifically selected from -S-CH2-, -O-CH2-, -Op-(phenylene)-CH2-, -O-CH(CH3)-, -O-(C=O)-CH2-,)-; -O-(C=O)-CH2-CH2-, -N-(C=O)-CH2-,)-; -N-(C=O)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or More specifically, the functionalized construct has the general formula XXI. in X is methylene or ethylene; W is -C(O)-, –C(O)-O- or –OC(O)- G 1 Selected from -S-CH2-, -O-CH2-, -Op-(phenylene)-CH2-, -O-CH(CH3)-, -O-(C=O)-CH2-; -O-(C=O)-CH2-CH2-, -N-(C=O)-CH2-; -N-(C=O)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or Especially -Op-(phenylene)-CH2- and -N-(CH2)4-, L 1 And n is as defined above; TA is the target part as defined above.

[0260] According to a very specific embodiment of the functionalized construct, TA is the immunoglobulin portion.

[0261] According to another specific embodiment of the functionalized construct, the functionalized TA has formula XX.1 in TA is the target portion as defined above. FR is the functionalized residue of the following formula in L 1 And n is as defined above, A, B, M, and Q are -CR 1 R 2 -, D and U are -OR 3 , X and W are as defined above; G 1 The absence or representation of a portion formed by conventional coupling reactions, particularly click reactions…, for example, especially in J is selected from -NH-, -S-, or -O-, or may not exist; or More specifically, the functionalized construct has the general formula XXII in L 1 And n is as defined above, X is methylene or ethylene; W is -C(O)-, -C(O)-O- or -OC(O)-; TA is the target region.

[0262] According to a very specific embodiment of the functionalized construct, TA is the antibody portion.

[0263] According to another specific implementation, the functionalized construct is a functionalized PM of formula XX.2. in a is 0 or an integer selected from 1 and 2. b is 0 or an integer selected from 1 and 2. c represents selecting an integer that is at least 1, specifically chosen from 1 and 2. Y 1 This represents the divisible portion as defined above; and Y 2 This represents the self-destructive portion as defined above. PM is the payload portion as defined above. FR is a functionalized residue of formula II' in A, B, M, and Q are -CR 1 R 2 -, where R 1 and R 2 As defined above; D and U are -OR 3 , where R 3 As defined above; and n, L 1 X and W are as defined above; The condition is that, When c represents an integer greater than 1, then L 1 This represents the branch, where n is 1.

[0264] According to another specific embodiment, the functionalized construct is a functionalized PM of formula XXIII. in X, W, n, a, b, L1 Y 1 and Y 2 As defined above.

[0265] 9. Ninth aspect of the invention - Conjugate (TA-PM)

[0266] According to a ninth aspect of the invention, a conjugate is provided, which can be obtained by covalently linking a first functionalized molecule selected from a hydrophilic trans-cyclooctene (hyTCO)-functionalized construct as defined above with a second functionalized molecule comprising a docking group (DG) capable of reacting with the trans-hyTCO-functionalized group of the first molecule; particularly by a bisorthogonal bioconjugate via a Diels-Alder cycloaddition reaction of the two functionalized molecules; more particularly, wherein the DG is selected from optionally substituted triazine or optionally substituted tetraazine, capable of covalently reacting with the trans-cyclooctene group in a copper strain-free, reverse electron-demanding Diels-Alder cycloaddition (SPIEDAC).

[0267] According to a specific embodiment of the conjugate, the hydrophilic trans-cyclooctene (hyTCO)-functionalized construct is selected from the functionalized target agent TA of general formula (XX.1). in FR is a functionalized residue of formula II', and TA is the target portion as defined above; Or the functionalized PM of general formula (XX.2) in a is 0 or represents an integer selected from 1 and 2. b is 0 or represents an integer selected from 1 and 2. c represents selecting an integer that is at least 1, specifically chosen from 1 and 2. Y 1 Represents the cuttable portion. Y 2 Represents the self-destruction portion. FR is the functionalized residue of formula II', where L1 represents the branch part when c represents an integer greater than 1, n is 1, and G1 does not exist; and PM is the payload molecule as defined above.

[0268] According to another specific embodiment of the conjugate, the second functionalized molecule containing the docking group (DG) is selected from... Functionalized targeting agent TA of general formula XX.3 Or the functionalized effective load molecule PM of general formula (XX.4) in a, b, c, and Y 1 and Y 2 As defined above; TA is the target region as defined above, and PM is the payload molecule as defined above, and DG is the docking group as defined above.

[0269] In compounds of formula XX.3, the DG can be chemically linked to the TA in a manner known per se via suitable amino acid side chains, for example, by reacting a DG with an active ester group with a lysine side chain or by reacting a DG with a thiol reactive group such as a maleimide group with a cysteine ​​side chain. Conventional enzymatic reactions, such as glycan remodeling, are also suitable.

[0270] According to another specific embodiment, the conjugate is obtained through the following... i) By functionalizing the targeting agent TA in FR is a functionalized residue of formula II', and TA is the target portion as defined above; It reacts with the functionalized loaded molecule PM of general formula XX.4 (by clicking reaction). in a, b, c, and Y 1 and Y 2 As defined above; PM is the payload molecule as defined above, and DG is the docking group as defined above.

[0271] and conjugates obtained by optional separation

[0272] or

[0273] ii) By functionalizing the payload molecule PM of general formula (XX.2) in a, b, c, and Y 1 and Y 2 As defined above; FR is the functionalized residue of formula II', where L1 represents the branch part when c represents an integer greater than 1, n is 1, and G1 does not exist; TA is the target region as defined above, and PM is the payload molecule as defined above; Reaction with the functionalized target agent TA of general formula XX.2 (by clicking reaction) in TA is the target region as defined above, and DG is the docking group as defined above.

[0274] According to another specific embodiment of the conjugate, the DG is a diene containing a group selected from tetraazine or triazine, which is capable of covalently reacting with the trans-cyclooctene group of Formula II' above in a copper strain-free, reverse electron-demanding Diels-Alder cycloaddition (SPIEDAC).

[0275] According to another specific embodiment of the conjugate, the TA is an immunoglobulin molecule, particularly a monoclonal antibody, its derivative or fragment, carrying at least one non-classical amino acid residue (ncAA) in the polypeptide chain, wherein the ncAA carries a hyTCO-type side chain as defined in either the first or second aspect above.

[0276] According to another specific embodiment of the conjugate, the PM is as defined above.

[0277] 10. The tenth aspect of the invention

[0278] According to a tenth aspect of the invention, a pharmaceutical composition is provided comprising at least one conjugate as defined above in a pharmaceutically acceptable carrier, or a diagnostic composition is provided comprising at least one conjugate as defined above in a diagnostically acceptable carrier.

[0279] 11. The eleventh aspect of the present invention

[0280] According to the eleventh aspect of the invention, a conjugate, particularly an ADC as defined above, is provided for medical use, such as for diagnosis and treatment.

[0281] More specifically, an antibody payload conjugate (APC), particularly an ADC as defined above, is provided for the diagnosis or treatment of cancers such as breast cancer, gastric cancer, or other Her2-overexpressing tumors, such as ovarian, endometrial, bladder, lung, colon, and head and neck tumors.

[0282] 12. The twelfth aspect of the invention

[0283] According to the twelfth aspect, a diagnostic or analytical kit is provided, comprising at least one hyTCO type compound or conjugate as defined above.

[0284] D. Further implementation plans

[0285] 1. Payload molecule (PM)

[0286] 1.1 Bioactive compounds

[0287] Bioactive compounds include, but are not limited to, the following:

[0288] The bioactive compounds applicable to this invention include, but are not limited to: small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptide-like substances, amino acids, oligonucleotides or polynucleotides, nucleosides, DNA, RNA, toxins, polysaccharides and immunoglobulins.

[0289] Exemplary categories of bioactive compounds that can be used in the practice of this invention include, but are not limited to, hormones, cytotoxins, antiproliferative / antitumor agents, antiviral agents, antibiotics, cytokines, anti-inflammatory agents, antihypertensive agents, chemosensitizers, photosensitizers and radiosensitizers, anti-AIDS substances, antiviral agents, immunosuppressants, immunostimulants, enzyme inhibitors, anti-Parkinson's agents, neurotoxins, channel blockers, cell-extracellular matrix interaction modulators (including cell growth inhibitors and anti-adhesion molecules), DNA, RNA or protein synthesis inhibitors, steroid or non-steroidal anti-inflammatory agents, anti-angiogenic factors, and anti-Alzheimer's agents.

[0290] In some embodiments, the bioactive compound is a low to medium molecular weight compound (e.g., about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0291] Exemplary cytotoxic drugs, particularly those used in cancer treatment, generally include DNA damaging agents, antimetabolites, natural products and their analogues, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidine synthase inhibitors, DNA binding agents, DNA alkylating agents, radiation sensitizers, DNA intercalating agents, DNA cleaving agents, microtubule stabilizers and destabilizers, and topoisomerase inhibitors. Examples include, but are not limited to, platinum-based drugs, anthracycline family drugs, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, taxanes, lexitropsins, pteridine family drugs, diynenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxol. Particularly useful members in these categories include, for example, auristatins, maytansines, maytansine compounds, calicheamicins, dactinomycins, duocarmycins, CC1065 and its analogues, camptothecin and its analogues, SN-38 and its analogues; DXd, tubulolysin M, cryptomycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBD), pyridinobenzodiazepines (PDD), and indobenzobenzodiazepines (IBD). (See US20210206763A1), methotrexate, methotrexate, dichloromethhotrexate, 5-fluorouracil, DNA minor groove binders, 6-mercaptopurine, cytarabine, melphalan, leurosine, leurosideine, actinomycins, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, PNU-159682 (see US 10,288,745 B2.) and their analogues), mitomycin C, mitomycin A, caminomycin, aminopterin, tamethasone, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, paclitaxel, taxotereretinoic acid (acid), butyric acid, N8-acetylsemine, astrone, colchicine, camptothecin, esperamicin, ene-diynes and their analogues, hemiasterlin and its analogues.

[0292] Other exemplary drug classes include angiogenesis inhibitors, cell cycle progression inhibitors, PI3K / 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).

[0293] Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP, and auristatin AQ. Suitable auristatin derivatives are also found in U.S. Publications 2003 / 0083263, 2011 / 0020343 and 2011 / 0070248; PCT Publications WO09 / 117531, WO2005 / 081711, WO04 / 010957; WO02 / 088172 and WO01 / 24763; and U.S. Patents 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; and 5,780,5 88; 5,767,237; 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, all of which are incorporated herein by reference in their entirety.

[0294] Exemplary drugs include dolalastatin and analogues, including: dolalastatin A (US Patent No. 4,486,414), dolalastatin B (US Patent No. 4,486,414), dolalastatin 10 (US Patent 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, 6,034,065, 6,323,315), dolalastatin 13 (US Patent No. 4,986,988), dolalastatin 14 (US Patent No. 5,138,036), and dolalastatin 15. Dolasatin 16 (US Patent No. 4,879,278), Dolasatin 17 (US Patent No. 6,239,104), and Dolasatin 18 (US Patent No. 6,239,104), each of which is incorporated herein by reference in its entirety.

[0295] U.S. Patent 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; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,20 Exemplary maytansine, maytansine-like compounds such as DM-1 and DM-4, ​​or maytansine-like compound analogues, including maytanol and maytanol analogues, are described in 8,020; 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.

[0296] Other examples include maytansine DM1 and anserine; pyrrolobenzodiazepines (PBDs), which 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., Expert Opin Investig Drugs. 2011, 20(6):733-44], and Antonow et al., Chem Rev. 2011, 111(4), 2815-64].

[0297] Calicutomycin includes, for example, enediynes, esperamycin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116.

[0298] Examples of duocarmycins and analogues include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, DU-86, KW-2189, duocarmycin, duocarmycin, duocarmycin, and seco-duocarmycin. Other examples include, for instance, those described in 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; US20150104407; 61 / 988,011 filed May 2, 2014 and 62 / 010,972 filed June 11, 2014; the disclosure of each patent is incorporated herein by reference in its entirety.

[0299] Exemplary vinca alkaloids include vincristine, vinca alkaloid, vindesine, and navelbine, as well as those disclosed in U.S. Publications 2002 / 0103136 and 2010 / 0305149 and U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety.

[0300] Exemplary epothilone compounds include epothilones A, B, C, D, E, and F, and their derivatives. Suitable epothilone compounds and their derivatives 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 WO97 / 19086; WO98 / 08849; WO98 / 22461; WO98 / 25929; WO98 / 38192; WO99 / 01124; WO99 / 02514; WO99 / 03848; WO99 / 07692; WO99 / 27890; and WO99 / 28324, the entire disclosure of which is incorporated herein by reference.

[0301] Exemplary cryptomycin is described in U.S. Patent Nos. 6,680,311 and 6,747,021; the disclosures thereof are incorporated herein by reference in their entirety.

[0302] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, isopropylplatin, omaliplatin, and tetraplatin.

[0303] Exemplary DNA-binding or alkylating agents include CC-1065 and its analogues, anthracyclines, calichiomycin, actinomycin, mitomycines, pyrrolobenzodiazepines, etc.

[0304] Exemplary microtubule stabilizers and destabilizers include taxane compounds such as paclitaxel, docetaxel, testrate, and cabazitaxel; maytansine compounds, aurestatin and its analogues, vinca alkaloid derivatives, epothilone, and cryptomycin.

[0305] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs, and non-natural camptothecin, such as CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimaritecan, canenatinib, silatecan, letopecan, ixenocarb, DXd, diflometotecan, belotetane, letopecan, and S39625. Other camptothecin compounds that may be used in this 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).

[0306] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, and MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib, and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, which are compounds comprising the fumagillin core structure.

[0307] Exemplary cell cycle progression inhibitors include CDK inhibitors, such as BMS-387032 and PD0332991; Rho-kinase inhibitors, such as AZD7762; aurora kinase inhibitors, such as AZD1152, MLN8054, and MLN8237; PLK inhibitors, such as BI 2536, BI6727, GSK461364, and ON-01910; and KSP inhibitors, such as SB 743921, SB715992, MK-0731, AZD8477, AZ3146, and ARRY-520.

[0308] Exemplary P13K / m-TOR / AKT signaling pathway inhibitors include phosphoinositol 3-kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0309] Exemplary P13 kinases, including BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomida 529, pirivoxin, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147, and XL765, are disclosed in U.S. Patent No. 6,608,053.

[0310] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0311] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf, and p38 MAPK inhibitors.

[0312] Exemplary MEK inhibitors, including GDC-0973, GSK1 120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330 and GDC-0973, are disclosed in U.S. Patent No. 7,517,944.

[0313] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0314] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820, and SB 202190. Exemplary receptor tyrosine kinase inhibitors include, but are not limited to, AEE788 (NVP-AEE 788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), gefitinib (Iressa), AP24534 (Panatinib), ABT-869 (linifanib), AZD2171, CHR-258 (dovirtinib), sunitinib (Sutent), sorafenib (Nexavar), and vatalinib (Vatalinib).

[0315] Exemplary protein chaperone inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0316] Exemplary HDAC inhibitors include belistat (PXD101), CUDC-101, droxinostat, ITF2357 (givinostat, Gavinostat)), JNJ-26481585, LAQ824 (NVP-LAQ824, danostat), LBH-589 (pabistat), MC1568, MGCD0103 (mocetinostat)), MS-275 (entinostat), PCI-24781, pyroxamide (NSC696085), SB939, trichostatin A, and vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0317] Exemplary inhibitors of the Wnt / Hedgehog signaling pathway include vemodilution, cyclopamine, and XAV-939.

[0318] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include α-amatoxinine, β-amatoxinine, γ-amatoxinine, η-amatoxinine, amatoxin-free cyclic peptide, amanullic acid, amanisamide, amanon, and proamanullin.

[0319] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, and TNF.

[0320] As non-limiting examples of specific drugs, auristatins, maytansine compounds, PBD, topoisomerase inhibitors, and anthracyclines may be mentioned.

[0321] In another embodiment, a combination of two or more different drugs as described above is used.

[0322] According to another embodiment, the bioactive compound may be selected from any synthetic or naturally occurring compound containing one or more natural and / or non-natural, proteogenic and / or non-proteogenic amino acid residues, such as oligosaccharides or peptides or proteins.

[0323] A specific group of such compounds includes immunoglobulin molecules such as antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g., bispecific or trispecific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human, humanized, mouse, or chimeric antibodies.

[0324] Typical, non-limiting examples of antibodies used in this invention are selected from biological, and particularly 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, and minretumomab. (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, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, III A4, Mapatumumab, HGS-ETR2, CS-1008, Denosumab, Sibrotuzumab, F19, 81 C6, Pinatuzumab, Lifastuzumab, Glembatumumab, Coltuximab, Lovotuzumab, Indatuximab, Anti-PSMA, MLN-0264, ABT-414, Milatuzumab, Ramucirumab, Abagovomab, Abituzumab, Adecatumumab, AfutuzumabAtumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, ascrinvacumab, atezolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, brontictuzumab, cantozumab Cantuzumab, capromab, catumaxomab, citatuzumab, cixutuzumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, dallotuzumab, daratumumab, demcizuma, denintuzumab Depatuxizuma, derlotuximab, detumomab, dinutuximab, drozitumab, duligotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab, elgemtumab, emactuzumab, enav (List of Chinese characters follows, likely related to a specific type of abbreviation or similar)Galiximab, Ganitumab, Icrucumab, Igovomab, Imarumab, Imgatuzumab, Indusatumab, Inbilizumab, Intetumumab, Iratumumab, Isatuximab, Lexatuzumab, Lilotomab, Lintuzumab b) Lirilumab, Lucarumumab, Lumentuzumab, Margetuximab, Matuzumab, Mirvetuximab, Mitumomab, Mogamulizumab, Moxetumoma, Nacolomab, Naptumomab, Narnatumab, Necitumumab Nesvacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontuxizumab, oportuzumab, oregovomab, otlertuzumab Pankomab, parsatuzumab, pastuxizumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, quilizumab, racotumomab, ramucirumabRilotumumab, Robatumuma, Sacituzumab, Samalizumab, Satumomab, Seribantumab, Siltuximab, Sofituzumab, Tacatuzumab, Taplitumomab, Tarextumab, Tenatumoma, Teprotumumab, Tetulomab, Ticilimumab, Tigatuzumab, Tositumomab, Tovetumab, Trimelimumab melimumab, tucotuzumab, ublituximab, ulocuplumab, urelumab, utomiluma, vadastuximab, vandortuzumab, vantictumab, vanucizumab, varlilumab, veltuzumab, vesencumab, volociximab, vorsetuzumab, votumumab, zalutumumab, zatuximab, their combinations and derivatives, and other CAIs. 25. Monoclonal antibodies against CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostate 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 products.

[0325] 1.2 Labeling agents

[0326] The labeling agents that can be used according to the present invention may contain any type of labeling known in the art that will not negatively affect the reactivity of the tetrazine moiety.

[0327] The markings of this invention include, but are not limited to, dyes (e.g., fluorescent, luminescent, or phosphorescent dyes, such as dansyl, coumarin, fluorescein, acridine, rhodamine, silico-rhodamine, BODIPY, or cyanine dyes), chromophores (e.g., photosensitive pigments, phycobilins, bilirubin, etc.), and radioactive markings (e.g., radioactive forms of hydrogen, fluorine, carbon, phosphorus, sulfur, or iodine, such as tritium, fluorine-18, carbon-11, carbon-14, phosphorus-32, phosphorus-33, sulfur-33, sulfur-35, iodine-123, or iodine-125). MRI-sensitive spin labels, affinity tags (e.g., biotin, His-tag, Flag-tag, strep-tag, sugars, lipids, sterols, PEG-connectors, benzylguanine, benzylcytosine, or cofactors), polyethylene glycol groups (e.g., branched PEG, linear PEG, PEG of different molecular weights, etc.), photocrosslinkers (e.g., p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, and solid supports.

[0328] In some embodiments, exemplary dyes may include NIR contrast agents that fluoresce in the near-infrared region of the spectrum. Exemplary near-infrared fluorophores may include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) between about 630 and 1000 nm, for example, between about 630 and 800 nm, between about 800 and 900 nm, between about 900 and 1000 nm, between about 680 and 750 nm, between about 750 and 800 nm, between about 800 and 850 nm, between about 850 and 900 nm, between about 900 and 950 nm, or between about 950 and 1000 nm. Fluorophores with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm may also be used in the methods described herein.

[0329] In some embodiments, exemplary fluorophores include 7-amino-4-methylcoumarin-3-acetic acid (AMCA), TEXAS RED™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, 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)-carboxamide]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazaindole-3-propionic acid (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-dioxane-3-propionic acid). diaza-3-indacenepropionic acid), eosin-5-isothiocyanate, erythrosine-5-isothiocyanate, and CASCADE™ blue acetyl azide (Molecular Probes, Inc., Eugene, Oreg.) and ATTO dyes.

[0330] Other suitable fluorophores are described, for example, in EP3572468A1. Other labeling agents include 177-lutetium, 89-zirconium, 131-iodine (Iod), 68-gallium, 99m-technetium, 225-actinium, 213-bismuth, 90-yttrium, 212-lead, 111-indium, 64-copper, 67-copper, 124-iodine, 227-thorium, and 188-rhenium.

[0331] 1.3 Chelating agents

[0332] The following is a list of typical applicable chelating agents and their abbreviations; the corresponding salts are also applicable:

[0333] Acetylacetone (ACAC), ethylenediamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylenetriamine (DIEN), iminodiacetic acid (IDA), triethylenetetramine (TRIEN), triaminotriethylamine, nitrotriacetic acid (NTA) and its salts such as Na3NTA or FeNTA, ethylenediaminetriacetic acid (TED), ethylenediaminetetraacetic acid (EDTA) and its salts such as Na2EDTA and CaNa2EDTA, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclododecane-1,4,7,10-tetraacetic acid, Cyclononane-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 agent or 2,3-dimercaptopropanol (BAL), meso-2,3-dimercaptosuccinic acid (DMSA); siderophores secreted by microorganisms, such as deferral or deferral B, also known as deferral (Novartis) produced by *Streptomyces* genus; deferoxamine (DFO), a trihydroxyoxime acid secreted by *Streptomyces* var. *milli*; derivatives of phytochemicals such as curcumin and maltose, such as 3-hydroxy-maltose and 2'-deoxy-maltose; synthetically produced chelating agents, such as ibuprofen; derivatives of catechols, hydroxamic acids, and hydroxypyridinones, such as deferoxamine hydroxamic acid and deferoxamine hydroxypyridinone; deferoxone (L1 or 1,2-dimethyl-3-hydroxypyridin-4-one); D-penicillamine (DPA or D-PEN), which is β-β-dimethylcysteine ​​or 3-mercapto-D-valine; tetraethylenetetramine (TETA) or tricentine and its two major metabolites, N1-acetyltriethylenetetramine (MAT) and N1,N 10 - Diacetyltriethylenetetramine (DAT); hydroxyquinoline; chloroiodohydroxyquinoline, which is a halogenated derivative of 8-hydroxyquinoline; and 5,7-dichloro-2-[(dimethylamino)methyl]quinoline-8-ol (PBT2).

[0334] 1.4 Protein Degrading Agent

[0335] PROTAC can be mentioned as a non-limiting example, but there are many different E3 ligase-binding molecules that co-degrade specific target proteins (vgl WO2017201449A1). (Maneiro, M. et al ACS Chem. Biol.2020, 15, 6, 1306–1312) With the discussion of protein degradation, compounds suitable for targeted protein degradation (TPD) should be mentioned. A major class of molecules is called proteolytic-targeting chimeras (PROTACs). See Békés, M., Langley, DR & Crews, CMPROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181–200 (2022). These chimeras are heterobifunctional small molecules composed of two ligands linked by a linker. One ligand recruits and binds to the protein of interest (POI), while the other ligand recruits and binds to an E3 ubiquitin ligase. PROTAC simultaneously binds to the POI and the ligase, inducing ubiquitination of the POI and its subsequent degradation by the ubiquitin-proteasome system. PROTAC is then recycled to target another copy of the POI.

[0336] 2. Targeting agents (TAs) and their targets

[0337] The primary objective of this targeting agent is to form a covalent or non-covalent bond with a specific "target." A secondary objective is to target and transport the "payload molecule" to the target. To achieve this second objective, the POI must (reversibly or irreversibly) bind to at least one payload molecule. This requires functionalizing the POI by introducing at least one ncAA. The functionalized POI carrying the at least one ncAA can then be linked to the at least one payload molecule via a bioconjugation of the ncAA residues. The ncAA reacts with the payload molecule, which then carries the corresponding portion that reacted with the at least one ncAA residue of the POI. The resulting bioconjugation, or targeting agent, allows the payload molecule to be transferred to the intended target.

[0338] For example, a "target" can be any molecule present in an organism, tissue, or cell and / or on it. Such targets can be non-specific or specific to a particular organism, tissue, or cell. Targets include cell surface targets, such as receptors, glycoproteins, glycans, and carbohydrates; structural proteins, such as amyloid plaques; abundant extracellular targets, such as those in the matrix, and extracellular matrix targets such as growth factors and proteases; intracellular targets, such as the surface of the Golgi apparatus, the surface of mitochondria, RNA, DNA, enzymes, and components of cell signaling pathways; and / or foreign substances, such as pathogens such as viruses, bacteria, fungi, yeast, or parts thereof.

[0339] Examples of targets include compounds such as proteins whose presence or expression levels are associated with a certain tissue or cell type, or whose expression levels are upregulated or downregulated in a certain disease.

[0340] In particular, such targets are proteins such as (internalized or non-internalized) receptors.

[0341] The target can be selected from any suitable target in the human or animal body or on pathogens or parasites.

[0342] Non-limiting examples of suitable targets include, but are not limited to: cellular components such as cell membranes or cell walls, receptors such as cell membrane receptors, intracellular structures such as the Golgi apparatus or mitochondria, enzymes, receptors, DNA, RNA, viruses or viral particles, macrophages, tumor-associated macrophages, antibodies, proteins, carbohydrates, monosaccharides, polysaccharides, cytokines, hormones, steroids, somatostatin receptors, monoamine oxidases, muscarinic receptors, cardiac sympathetic nervous system, leukotriene receptors (e.g. on leukocytes), urokinase plasminogen activator receptor (uPAR), folate receptors, apoptosis markers, etc. (Anti-)angiogenic markers, gastrin receptor, dopaminergic system, serotonergic system, GABAergic system, adrenergic system, cholinergic system, opioid receptors, GPIIb / IIIa receptors and other thrombosis-related receptors, fibrin, calcitonin receptor, phagocytic peptide receptor, P-glycoprotein, neurotensin receptor, neuropeptide receptor, substance P receptor, NK receptor, CCK receptor, sigma receptor, interleukin receptor, herpes simplex virus tyrosine kinase, human tyrosine kinase, integrin receptor, fibronectin target, AOC3, AL K, AXL, C242, CA-125, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CD20, CD21, CD22, CD23, CD 25. CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44v6, CD45, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD7 0, 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, Cripto, tendin C, melanocortin-1 receptor, G250, HLADR, ED-B, TMEFF2, EphB2, EphB4, EphA2, FAP, mesothelin, GD2, GD3, CAIX, 5T4, aggregation factor, CTLA-4, CXCR2, FGFR1, FGFR2, FGFR3, FGFR4, NaPi2b, NOTCH1, 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 β2, Claudine 3. RON, ROR1, PD-L1, PD-L2, B7-H3, B7-H4, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrin, IFN-α, IFN-γ, IgE, IGF-1 receptor, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-22, IL-23, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin, P-selectin, E-selectin, mucin, myostatin, NCA-90, NGF, PDGFR α, prostate cancer cells, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, Rh factor, SLAMF7, sphingosine-1-phosphate, TGF-1, TGFβ2, TGFβ, TNFα, TRAIL-R1, TRAIL-R2, CTAA 16.88. Vimentin, matrix metalloproteinases (MMPs) such as MMP2, MMP9, MMP14, LDL receptor, endothelial glycoproteins, polysialic acid and their corresponding lectins. Examples of fibronectin targets include the alternative splicing extra domain-A (ED-A) and extra domain-B (ED-B) of fibronectin. Non-restrictive examples of targets in the matrix can be found in V. Hofmeister, D. Schrama, JC Becker, CancerImmun. ;Immunother. 2008, 57, 1, the contents of which are incorporated herein by reference.

[0343] More specifically, to allow for (specific) targeting of the aforementioned targets, the targeting agent may comprise a compound containing 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 such as octreotide and its derivatives, VIP, MSH, LHRH, chemokines, bufotenoids, elastin, peptide mimics, receptor agonists and antagonists, cytokines, hormones, steroids, and toxins.

[0344] Depending on the specific aspect, the target is a receptor, and a targeting agent capable of binding specifically to the target is used. Suitable targeting agents include, but are not limited to, ligands of the receptor or portions thereof that can still bind to the receptor, such as receptor-binding peptides in the case of receptor-binding protein ligands.

[0345] Other examples of protein-based targets include insulin, transferrin, fibrinogen-γ fragment, platelet-reactive protein, tight junction protein (claudin), apolipoprotein E, affibody molecules such as ABY-025, ankyrin repeat protein, ankyrin-like repeat protein, interferons such as α, β and γ interferons, interleukins, lymphokines, colony-stimulating factors and protein growth factors such as tumor growth factors such as α and β tumor growth factors, platelet-derived growth factor (PDGF), uPAR-targeting proteins, apolipoproteins, LDL, annexin V, endostatin and angiostatin.

[0346] Examples of peptide molecules used in targeted therapies, such as antibodies, include LHRH receptor-targeting peptides, EC-1 peptides, RGD peptides, HER2-targeting peptides, PSMA-targeting peptides, somatostatin-targeting peptides, and toad peptides. Other examples of targeted therapies include lipid transport proteins, such as anticalcin.

[0347] One particular implementation uses Affibodies™ and polymers and derivatives.

[0348] In one particular embodiment, the antibody is used to form a target. While antibodies or immunoglobulins derived from IgG antibodies are particularly suitable for this invention, they can be selected from any class or subclass of immunoglobulins, such as IgG, IgA, IgM, IgD, and IgE. Suitably, the immunoglobulin belongs to the IgG class, including but not limited to IgG subclasses (IgG1, 2, 3, and 4), or the IgM class capable of specifically binding to specific epitopes on an antigen. The antibody can be a complete immunoglobulin derived from natural or recombinant sources, and can be an immunoreactive portion of a complete immunoglobulin. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, camelified single-domain antibodies, recombinant antibodies, anti-idiotypic antibodies, multispecific antibodies, antibody fragments such as Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single-chain variable fragment antibodies (scFv), tandem / bi-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as trisomy, camel antibodies, microantibodies, nanoantibodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single-domain antibodies (sdAb, also known as Nanobody™), chimeric antibodies, chimeric antibodies containing at least one human constant region, and biphile antibodies such as biphile-redirected proteins (DART™). And their polymers and derivatives, such as divalent or multivalent single-chain variable fragments (e.g., di-scFvs, tri-scFvs), including but not limited to microantibodies, diisosomes, triabodies, triisosomes, tetraisosomes, etc., as well as multivalent antibodies. References to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and [Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331-343], the contents of which are incorporated herein by reference.

[0349] "Antibody fragment" refers to at least a portion of the variable region where an immunoglobulin binds to its target, i.e., the antigen-binding region.

[0350] Other implementations use antibody mimics as targeting agents, such as, but not limited to, affimers, anticalcitonins, affinity multimers, alphabodies, affibodies, DARPins, and their multimers and derivatives; see [Trends in Biotechnology 2015, 33, 2, 65], the contents of which are incorporated herein by reference.

[0351] To avoid ambiguity, in the context of this invention, the term "antibody" means to encompass all antibody variants, fragments, derivatives, fusions, analogs and mimics outlined in this paragraph, unless otherwise stated.

[0352] In a preferred embodiment, the target agent is selected from substances derived from antibodies and antibody derivatives, such as antibody fragments, fragment fusions, proteins, peptides, and peptide mimics.

[0353] In another preferred embodiment, the target agent is selected from substances derived from antibody fragments, fragment fusions, and other antibody derivatives that do not contain an Fc domain.

[0354] Typical, non-limiting examples of antibody molecules that require further modification to form the ncAA-modified target of the present invention are selected from biological, and particularly pharmacologically active, antibody molecules. Non-limiting examples are selected from the group consisting of: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, oflambumab, gemtuzumab, verbentuximab, teimozumab, tosimomumab, pertuzumab, adenomyumab, IGN101, INA01, labezizumab, hua33, petumozumab, ogovumab, minremuzumab (CC49), cG250, J591, MOv-18, favuzumab (MORAb-003), 3F8, ch14,18, KW-2871, hu3S193, lgN311, IM-2C6, CDP-791, irexizumab, voloximab, nimotuzumab, MM-121, AMG. 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, III A4, Mapamumumab, HGS-ETR2, CS-1008, Denosumab, Sirolizumab, F19, 81 C6, Pinatuzumab, Lifatuzumab, Gabatuzumab, Cortuximab, Lovotuzumab, Indanetoxicum, Anti-PSMA, MLN-0264, ABT-414, Milazumab, Ramucirumab, Abavomarab, Abitutuzumab, Ademumab, Atorizumab, Pentatexate Atomumab, Ametuzumab, Anamozab, Anatozumab, Apocizumab, Asimumab Avazolam, Atezolizumab, Bavitiximab, Betumomab, Belimumomab, Bivaruzumab, Brontozumab, Cantozumab, Carosumab, Caputoxumab, Sitaturab, Cetuximab, Critrub, Cotrastuzumab, Konatumumab, Darcyzab, Darottozab, Datuximab, Densizumab, Dietuximab, Dilotuximab, Demozumab Denutoximab, Traziltoximab, Duligorutuzumab, Duvalilumab, Dustaurumab, Emeximab, Ezetroxumab, Egantoximab, Emitorutuzumab, Enerutuzumab, Enerutuzumab, Entoximab, Eprazolamab, Ertoxetine, Iressizumab, Fatauzolamab, Filatauzolamab, Fentaurumab, Vortoximab Garliximab, Garnituzumab, Irucurumab, Igvovomab, Imalumab, Imatrozumab, Induruzumab, Insolizumab, Intolimumab, Itolimumab, Isaruximab, Lesamumab, Rilotuzumab, Lintoruzumab, Lirelurumab, Lucarumab, Lutoruzumab, Magiruximab, Mituximab, Mitomozumab, Mitumozumab, Mogliruzumab, MosetumumabNacrolimus, Napotumab, Napotumab, Nexavar, Nevasu, Nitozumab, Nivolumab, Nofetolimus, Obituzumab, Ocalatumab, Ofamumab, Olazumab, Ontoxizolimus, Ontoxizolimus, Obotoxizolimus, Ogovo, Oleotoxizolimus, Pancolimus, Persatuzumab, Pertoxizolimus, Pertrastuzumab, Petubrumumab, Pidtilimab, Pintoxizolimus, Viportuzumab, Putomomumab, Quinolizumab, Ritumomumab, Ramolizumab, Rituxizolimus, Rotoxizolimus, Saxizumab, Samalizumab, Saxizumab Morimab, Seretuzumab, Celetuximab, Sofoetuzumab, Tacalutuzumab, Tapriltuzumab, Tasetuzumab, Tetumumab, Tetuximab, Tatuximab, Tetuximab, Tetuximab, Tetuximab, Tetuximab, Tosimob, Tovitumab, Trimelimumab, Cimo-Interleukin-1, Utuximab, Ulotuximab, Urelimumab, Utoluumab, Vardatoxib, Vantoxib, Vanetuximab, Varucine, Vileluumab, Vileluumab, Vileluumab, Vileluumab, Vixenkumab, Voloxib, Voseltuzumab, Votemob, Zalumab, Zaletuximab, their combinations and derivatives, and other targeted CAIs. 25. Monoclonal antibodies against CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostate 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 products.

[0355] According to another specific embodiment of the invention, targets and targeting agents are selected to result in tissue or disease specificity or increased targeting, such as cancer, inflammation, infection, cardiovascular diseases (e.g., thrombosis, atherosclerotic lesions), hypoxic sites (e.g., stroke), tumors, cardiovascular diseases, brain diseases, apoptosis, angiogenesis, organs, and reporter genes / enzymes. This can be achieved by selecting targets that have tissue, cell, or disease-specific expression.

[0356] For example, for a given cell population, the target agent specifically binds to or complexes with cell surface molecules (such as cell surface receptors or antigens). After the target agent specifically binds to or complexes with the receptor, the drug enters the cell.

[0357] As used herein, a target agent that "specifically binds to or co-conjugates" or "targets" cell surface molecules, extracellular matrix targets, or another target preferably associates with the target via intermolecular forces. For example, the ligand may preferably associate with the 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.

[0358] 3. hyTCO functionalized constructs

[0359] The hyTCO portion of this invention is part of the functionalized residues of formula II'. in n, A, B, D, U, M, Q, X, and W are as defined above; G 1 As defined above, or does not exist; and L 1 The L-shaped connector, as defined above, represents a branch section. Suitable for forming specific hyTCO functionalized constructs. In particular: Functionalized TA of general formula (XX.1) in TA is the target region as defined above; and FR is the functionalized residue of formula II' above. Or the functionalized PM of general formula (XX.2) in a is 0 or represents an integer selected from 1 and 2. b is 0 or represents an integer selected from 1 and 2. c represents selecting an integer that is at least 1, specifically chosen from 1 and 2. Y 1 Represents the divisible portion. Y 2 Represents the self-destruction portion. PM is the payload molecule as defined above, and FR is the functionalized residue of equation II' above, where L is the residue when c represents an integer greater than 1. 1 This represents the branch, where n is 1, and G1 does not exist.

[0360] 3.1 Enzymatically or chemically cleavable fractions (Y 1 )

[0361] The term "cleavable moiety" encompasses any group that can be enzymatically or chemically cleaved, particularly under in vivo or in vitro conditions. For example, enzymatic cleavage can be achieved through the action of proteases. Chemical cleavage can be achieved, for example, through hydrolytic or reductive cleavage of the S-S bond.

[0362] Suitable slit portions are well known in the prior art. See Bargh et al., ChemSoc Rev 2019, 48(16), 4361-4374; Poreba, FEBS J 2020, 287(10), 1936-1969; and Salomon et al., Mol Pharmaceutics 2019 16,(12), 4817–4825.

[0363] According to a specific embodiment of the present invention, the portion Y 1 It is an enzyme- or chemically cleavable linker group, selected from... a) Peptide groups, especially dipeptide, tripeptide, or tetrapeptide groups; 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 in n2 represents an integer from 1 to 4. Residue R 7 and R 8 They are independently selected from H or lower alkyl groups, especially methyl; or two residues R 7 and R 8 Together with the carbon atoms attached to them, they form cyclic C4- to C8-alkyl groups; and Part of X5 is selected from -C(O)- and -O-; Part X 5´ Selected from -C(O)- and -(O)C-(CH2)-NH-; c) A hydrazone group, selected from >C=NN(R 9 - and -N(R) 9 )-N=C< in R 9 It is H or a lower alkyl group; and d) β-glucuronidase-sensitive cleavable linker groups (glucuronide-linker groups), especially those carrying β-glucuronide-derived trigger residues; According to its specific implementation, the cleavable connector is based on the peptide group of feature a).

[0364] According to another specific embodiment, the cuttable connector is based on the glucuronic acid-connector group of feature d).

[0365] As a further example, the β-glucuronic acid linker, which carries a β-glucuronic acid-derived trigger residue, can be mentioned.

[0366] Non-restrictive examples include: or

[0367] 3.2 Self-destruction portion (Y 2 )

[0368] Suitable self-immolative portion Y 2 This is well known in the prior art. See: Santi et al., J Med Chem 2014, 57(6), 2303–2314; Alouane et al., Angew ChemInt Ed 2015, 54(26), 7492-7509; and Kolakowski et al., Angew Chem 2016, 128(28), 8080-8083.

[0369] 4. The conjugate of the present invention

[0370] The present invention also relates to conjugates synthesized by forming a covalent bond between a first functionalized molecule and a second functionalized molecule, wherein the first functionalized molecule is the hyTCO-functionalized construct as described above, and the second functionalized molecule contains a docking group (DG) capable of reacting with the hyTCO functional group of the first molecule.

[0371] Methods for preparing such conjugates are well known in principle in the art. Biorthogonal bioconjugation via a Diels-Alder cycloaddition reaction of the two molecules can be performed according to prior art teachings (see, for example, Oliveira et al, Chem Soc Rev, 2017, 46, 4895-4950). More specifically, the docking group (DG) is selected from optionally substituted triazine or optionally substituted tetraazine, capable of covalently reacting with the hyTCO group of the present invention in a copper strain-free, reverse electron-demanding Diels-Alder cycloaddition (SPIEDAC).

[0372] In principle, the hyTCO-functionalized construct is selected from the functionalized target agent TA of general formula (XX.1). As defined above Or the functionalized PM of general formula (XX.2) As defined above.

[0373] The hyTCO-functionalized construct and the functionalized target agent TA selected from general formula (XX.3) Or the functionalized effective load molecule PM of general formula (XX.4) The corresponding DG-functionalized counterparts are conjugated, as defined above, to form constructs suitable for PM targeted delivery.

[0374] Suitable triazine or tetraazine docking groups are also known from existing technologies. For example, specific instances are described in WO2023 / 10494 or various scientific articles [Yang et al., Angew. Chem. Int. Ed. 2012, 51, 5222–5225; Fan et al., Angew. Chem. Int. Ed. 2016, 55, 14046–14050; Mao et al., Angew. Chem. Int. Ed. 2019, 58, 1106–1109; Qu et al., Angew. Chem. Int. Ed.2018, 57, 12057–12061; Eising et al., Bioconjugate Chem. 2018, 29, 3054–3059; Meng et al., J. Org. Chem. 2017, 82, 1676–1687; Xie et al., Angew.Chem. Int. Ed. 2020, 59, 16967–16973; Lambert et al., J. Am. Chem. Soc. 2019,141, 17068-17074; Jemas et al., J. Am. Chem. Soc. 2022, 144, 1647-1662; Selvaraj et al., Tetrahedron Letters 2014, 55, 4795–4797; Dowling et al., J.Org. Chem. 2018, 83, 4229-4238; Battisti et al., Bioconjugate Chem. 2022, 33,4, 608–624; Karver et al., Bioconjugate Chem. 2011, 22, 2263-2270; Bender etal., Org. Lett. 2017, 19, 5693-5696; Kamber et al., J. Am. Chem. Soc. 2015,137, 8388-8391; Ros et al., Bioconjugate Chem. 2020, 31, 933-938; Ros et al. al.,Chem. Commun., 2020, 56, 11086; van Onzen et al., J. Am. Chem. Soc.2020,142, 10955-10963; Carlson et al., J. Am. Chem. Soc. 2018, 140, 3603-3612.]. .

[0375] WO2023 / 10494 discloses the following phosphonate-terminated DG in n3 represents an integer selected from 1 or 2. Sp 1 and Sp 2 Are they the same or different spacer groups? The α, β and γ bonds are independently selected from chemical bonds, or ether, thioether, ester, amide, carbonyl (especially ketone), urethane, diurethane, carbonate, hydrazine, urea, alkylene oxide, or linear or branched polyalkylene oxide bonds; Z represents phosphorus containing a hydrophilic group, especially (R 1 O)2P(O)-、(R 1a O)2P(O)-O- and (R 2 O)3P-O-; in R 1 R 1a and R 2 They may be the same or different, and independently represent H or lower alkyl groups, especially methyl or ethyl; more particularly H; and R represents H or a chemical group capable of forming a chemical bond, or a chemical group capable of forming an ether, thioether, ester, such as an active ester like succinimide- or pentafluorophenyl-ester, amide, carbamate, dicarbamate, carbonate, hydrazine, urea, alkyleneoxy, or a linear or branched polyalkyleneoxy bond.

[0376] As an example of a suitable spacer group, one can mention a) A monocyclic or polycyclic aromatic moiety having 6-14 ring carbon atoms, optionally monosubstituted or polysubstituted, particularly 1,2-phenylene, 1,3-phenylene, or 1,4-phenylene; wherein one or more optional substituents are independently selected from -halogen, -CHal3, -OH, -SH, -NR'2, NO2, -CN, -C(=O)R'', -C(=O)OR''', alkyl, alkenyl, alkynyl, and alkoxy; in R', R'', and R''' are independently selected from H and C1– to C4-alkyl groups; (partially M1); b) Heterocyclic residues of general formula X in One, two, or three of the ring portions X1 to X4 represent N, and the other represents >CH; (part M2); c) Linear or branched lower alkylene groups, especially –(CH2). n1 -, Where n1 is an integer from 1 to 4; more specifically, methylene; (part of M3); d) Linear or branched polyalkylene oxide moieties, particularly selected from linear moieties -((CH2) x1 -O) y1 -or-(O-(CH2)) x1 ) y1 -and its branched analogues; in x1 independently represents an integer selected from 1, 2, 3, or 4; specifically 1 or 2; and y1 represents 1-20 independently; in particular, integers 1-4; (part of M4); Sp 1 Selected from M1, M2, M3 or a combination thereof; and Sp 2 Selected from M1 to M4 or a combination thereof.

[0377] The following shows a particularly preferred structure for compounds of general formula II. In this formula, residue R 1 They are H or lower alkyl groups, especially methyl or ethyl; more particularly H.

[0378]

[0379] Other specific instances of intermediates of general formula II include:

[0380] Other tetrazines suitable as DGs may be analogs of the specific compounds mentioned above, wherein the phosphonate group is missing and replaced by H, or wherein the tetrazine moiety is replaced by the 1,2,4-triazine moiety.

[0381] 5. Biological orthogonal biological conjoints

[0382] According to the present invention, using a suitable translation system, particularly an in vivo translation system, targeted agents, especially targeted agents comprising a polypeptide moiety containing one or more UNAA residues, can be prepared. The in vivo translation system can be a cell, such as a prokaryotic or eukaryotic cell. The cell can be a bacterial cell, such as *Escherichia coli*. E. coli ); fungal cells, such as yeast cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). S. cerevisiae Eukaryotic cells used for polypeptide expression can be methylotrophic yeast; plant cells; or animal cells, such as insect cells, mammalian cells, such as HEK cells or HeLa cells. Eukaryotic cells used for polypeptide expression can be unicellular or part of a multicellular organism.

[0383] The applied cell system comprises (e.g., supplied) at least one non-natural amino acid or a salt thereof corresponding to a UNAA residue of the target agent to be prepared. The cell system further comprises: (i) The PylRS and tRNA of the present invention Pyl The PylRS described therein is capable (preferably selectively) of using UNAA or its hydrochloride-acylated tRNA. Pyl ;and (ii) A polynucleotide encoding a target, wherein any position of the target occupied by UNAA residues is tRNA. Pyl The inverse complementary codon of the anticodon is encoded by the codon (e.g., the chosen codon).

[0384] The cell culture system allows for the translation of the target agent, encoding a polynucleotide (ii), thereby producing the target agent.

[0385] To generate the target agent, according to the method of the invention, the translation in step (b) can be accomplished by culturing cells under suitable conditions, preferably in the presence of UNAA or its salts (e.g., in a culture medium containing UNAA or its salts), at a suitable time for ribosomal translation in the cells. This is based on the polynucleotide encoding the target agent (and optionally PylRS, tRNA). Pyl Expression may need to be induced by adding transcription-inducing compounds, such as arabinose, isopropyl β-D-thiogalactoside (IPTG), or tetracycline. Ribosomes bind to mRNA encoding the target (and contain one or more RNA molecules). PylThe codons contain the anticodons and their inverse complementary codons. Then, amino acids and UNAAs are sequentially linked at the positions encoded by the codons recognized (bound) by each aminoacyl-tRNA to form a polypeptide. Therefore, in tRNA... Pyl The location of the codon encoding the inverse complement of the included anticodon will incorporate UNAA into the target agent.

[0386] The cellular system may contain a polynucleotide sequence encoding the PylRS of the present invention, which allows for the expression of PylRS by cells. Similarly, expression of tRNA encoded by cells may also be possible via the cellular system. Pyl The polynucleotide sequence produces tRNA Pyl The polynucleotide sequence encoding PylRS and the tRNA encoding PylRS Pyl The polynucleotide sequence can be located on the same polynucleotide or on different polynucleotides.

[0387] Therefore, in one embodiment, the present invention provides a method for preparing a targeting agent comprising one or more UNAA residues, wherein the method comprises the following steps: (a) Provide a cellular system containing a polynucleotide sequence encoding the following: - At least one PylRS of the present invention, - At least one tRNA that can be acylated by PylRS (tRNA Pyl ),and - At least one target, wherein any position of the target occupied by the UNAA residue is determined by tRNA. Pyl The codon encoding of the inverse complement of the anticodon; and (b) Allows the translation of polynucleotide sequences via cellular systems in the presence of UNAA or its salts, thereby producing PylRS and tRNA. Pyl And POI.

[0388] Cellular systems for preparing targeting agents containing one or more non-natural amino acid residues, as described herein, can be developed by using cell systems encoding PylRS, tRNA, etc. Pyl The preparation involves introducing the polynucleotide sequence of the target agent into (host) cells. The polynucleotide sequence may be located on the same or different polynucleotides and can be introduced into cells using methods known in the art (e.g., virus-mediated gene delivery, electroporation, microinjection, liposome transfection, etc.).

[0389] Following translation, the targeting agent prepared according to the invention can optionally be recovered or purified, partially or substantially homogenized, according to methods known in the art. Recovery typically requires cell lysis unless the targeting agent is secreted into the culture medium. Methods of cell lysis are known in the art and include physical lysis, such as by sonication, pure liquid lysis (e.g., by French pressure), mechanical methods (such as those using stirrers or grinders), 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 lysis techniques and chemical lysis. Standard methods for purifying peptides from cell lysates or culture media are also known in the art, including, for example, ammonium sulfate or ethanol precipitation, acid or alkali extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis, etc. When necessary, a protein refolding step can be used to prepare a properly folded mature protein. When high purity is desired, high performance liquid chromatography (HPLC), affinity chromatography, or other suitable methods can be used for the final purification step. Antibodies prepared against the peptides of this invention can be used as purification reagents, i.e., for affinity-based purification of peptides. 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: Guide to Protein Purification, Academic Press (1990); and those shown in the references cited therein.

[0390] As previously described, those skilled in the art will recognize that, after synthesis, expression, and / or purification, a polypeptide can have a conformation different from the desired conformation of the related polypeptide. For example, polypeptides produced via prokaryotic systems are typically optimized for proper folding by exposure to a dissociative agent. During purification from lysates, for example, derived from *E. coli*, the expressed polypeptide is optionally denatured and then renatured. This is done, for example, by dissolving the protein in a dissociative agent such as guanidine HCl. Generally, it is occasionally necessary to denature and reduce the expressed polypeptide, and then refold the polypeptide to a preferred conformation. For example, guanidine, urea, DTT, DTE, and / or chaperone proteins can be added to the translation product of interest. Methods for reducing, denaturing, and renaturing proteins are well known to those skilled in the art. Polypeptides can be refolded in a redox buffer containing, for example, oxidized glutathione and L-arginine.

[0391] The target agent thus prepared can then be converted into the corresponding bioconjugate by reacting with a tetrazine compound of the above general formula I.

[0392] 6. Pharmaceutical Composition

[0393] The conjugates or bioconjugates of the present invention, such as APCs, and particularly the ADCs (i.e., active agents or ingredients) of the present invention, are generally provided as “pharmaceutical compositions” comprising at least one such active ingredient or a pharmaceutically acceptable salt thereof in a therapeutically and / or preventively effective or diagnostically effective amount, and optionally at least one pharmaceutically acceptable excipient.

[0394] The pharmaceutical composition may be delivered via a suitable route of administration, such as oral, rectal, mucosal, local, ocular, ear, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection, as appropriate.

[0395] Depending on the properties of the composition or the mode of administration and dosage form, the at least one additional pharmaceutical excipient may be different.

[0396] An excipient is a substance formulated with an active ingredient for various purposes, such as for long-term stability, filling solid dosage forms containing small amounts of the active ingredient (hence often called a "bulking agent," "filler," or "diluent"), or imparting therapeutic enhancements to the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, or increasing solubility. Excipients can also be useful in the manufacture of pharmaceutical compositions to assist in addressing issues related to the active substance, for example, by improving powder flowability or non-stick properties, and further assisting in in vitro stability, such as preventing denaturation or aggregation within the expected shelf life. The selection of a suitable excipient depends not only on the route of administration and dosage form but also on the specific active ingredient and other factors.

[0397] Excipients may be selected from the following categories: immune adjuvants, anti-adhesion agents, adhesives, coatings, colorants, disintegrants, flavorings, gliding agents, lubricants, preservatives, adsorbents, sweeteners, and mediators.

[0398] Non-limiting examples of excipients include diluents, preservatives, stabilizers, emulsifiers such as emulsified polymers such as polysorbate or poloxamer, antioxidants; anti-irritants, chelating agents and stabilizing salts such as chlorides, sulfates, phosphates, diphosphates, hydrobrominates and nitrates, suspending agents, antibacterial agents or antifungal agents. Additionally, buffers, such as buffer systems of low molecular weight organic acids with their corresponding salts, or inorganic buffers such as phosphate buffers, may be used. Other suitable components are also known from relevant pharmacological standard literature. Furthermore, the proportions of various components will vary depending on the nature of the specific component 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 PJ Weller or in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. RGennaro edit. 1985)).

[0399] Pharmaceutical compositions as used herein may be presented in the form of a “dosage form” or a “unit dose” and may contain one or more APCs, particularly ADCs as described herein. Therefore, pharmaceutical compositions as used herein may, for example, provide two active agents mixed together in a unit dose, or provide two active agents combined in a dosage form, wherein the active agents are physically separated.

[0400] In addition, drug compositions can be administered in targeted drug delivery systems, for example, in liposomes coated with endothelial cell-specific antibodies.

[0401] The pharmaceutical compositions of the present invention can be prepared in ways known per se, for example by conventional mixing, dissolving, emulsifying, encapsulating, embedding, or combinations thereof. A suitable formulation depends on the chosen route of administration.

[0402] As used in this article, “pharmaceutical acceptable” means that a compound, material, composition, and / or dosage form is suitable for contact with a patient’s tissues, within reasonable medical judgment, and without excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable risk / benefit ratio.

[0403] This invention includes all “pharmaceutically acceptable salt forms” of the active ingredient. A pharmaceutically acceptable salt is a salt in which the counterion does not significantly contribute to the physiological activity or toxicity of the compound and functions as a pharmacological equivalent. These salts can be prepared using commercially available reagents according to common organic techniques. Some anionic salt forms include acetates, stearates, benzenesulfonates, bromides, chlorides, citrates, fumarates, glucurons, hydrobroms, hydrochlorides, hydroiodates, iodides, lactates, maleates, methanesulfonates, nitrates, papoate, phosphates, succinates, sulfates, tartrates, p-toluenesulfonate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

[0404] "Therapeutic effective amount" and / or "preventive effective amount" means an effective amount that provides any therapeutic and / or preventive benefit when administered to a human or non-human patient. More specifically, "therapeutic effective amount" is an amount of the active ingredient disclosed herein, or a combination of two or more such active ingredients, that completely or partially inhibits the progression of a disease condition, or at least partially alleviates one or more symptoms of a disease condition.

[0405] "Diagnostic effective quantity" refers to the effective quantity of diagnostically valuable information that can be obtained from a patient regarding the state or progression of the disease.

[0406] Therapeutic benefits can include improving symptoms in patients, for example, effectively reducing the amount of symptoms. In some cases, patients may not experience symptoms of the disease condition they are treating. Therefore, a preventatively effective amount of a compound is also sufficient to have a significant positive effect on any indicator of the disease, symptom, or disease condition, such as a significant reduction in the frequency and severity of disease symptoms.

[0407] The effective dose for treatment can also be the effective dose for prevention.

[0408] As used in this article, "patient" refers to humans or non-humans, especially humans and animals.

[0409] "Dosage form" is any unit of administration ("unit dose") of one or more active agents as described herein.

[0410] The term "treating" or "treatment" means: (i) preventing a patient who is susceptible to a disease, condition, and / or disease status but has not yet been diagnosed from developing that disease, condition, or disease status; (ii) suppressing a disease, condition, or disease status, i.e., preventing its development; and (iii) alleviating a disease, condition, or disease status, i.e. causing its remission. In particular, it encompasses preventive or therapeutic treatments or combinations thereof.

[0411] The frequency of dosing can vary depending on the compound used and the specific type of infection being treated. A once-daily dosing regimen is feasible. Dosing regimens in which the active agent is administered multiple times daily, such as 2–10 times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, may sometimes be more helpful.

[0412] However, it should be understood that the specific dosage level and frequency for any particular patient will depend on a variety of factors, including the activity of the particular compound used, age, weight, general health condition, sex, diet, timing of administration, route of administration, excretion rate, drug combination, and the severity of the patient's specific disease. The effectiveness of treatment or prevention can generally be monitored using assays appropriate to the disease condition being treated or prevented, as is familiar to those skilled in the art.

[0413] Specific examples of pharmaceutical compositions according to the invention include liquid forms such as solutions, suspensions and emulsions, and contain a therapeutically effective amount of at least one APC, particularly an ADC component as defined above, optionally together with at least one other pharmaceutically acceptable excipient as defined above, and can be administered via any suitable route.

[0414] Other examples of pharmaceutical compositions according to the invention include solid forms such as powders, tablets, pills, capsules, pouches, suppositories, and dispersible granules.

[0415] Many possible variations that will immediately become apparent to those skilled in the art upon consideration of the disclosure provided herein also fall within the scope of this invention.

[0416] Example

[0417] The following examples are illustrative only and are not intended to limit the scope of the embodiments described herein.

[0418] Experimental Section

[0419] A) Materials and Methods

[0420] Reagents were purchased from commercial suppliers and used directly without further purification. All solvents, including anhydrous solvents, were commercially available. Air- and water-sensitive reagents and reactions were generally carried out under an argon atmosphere.

[0421] The reaction progress was monitored by TLC on Merck silica gel plates 60 F254 or by UHPLC-MS. TLC detection was performed by 254 nm UV light or potassium permanganate staining.

[0422] Rapid chromatographic purification was performed using silica gel (0.060-0.200 mm) and a KP-Sil column on a Biotage Isolera One purification system.

[0423] Preparative HPLC purification was performed on an Agilent Infinity 1260 series instrument, which consists 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-Bridge Prep C18 column: 5 µm; 19 x 150 mm, operated under a linear gradient of H₂O and acetonitrile, both containing 0.1% TFA as solvent.

[0424] Nuclear magnetic resonance spectra were recorded at room temperature on a Bruker Avance (400 MHz) NMR system. Chemical shifts (δ) are given in parts per million (ppm), coupling constants (J) are given in Hertz (Hz), and multiplicity is reported using standard abbreviations.

[0425] UHPLC-MS analysis was performed on an Agilent Infinity 1290 series instrument, which consists of an Agilent 1290 quaternary pump, a 1290 injector, a 1290 constant-temperature column oven, and a 1290 diode array detector VL+ equipped with a quadrupole LC / MS 6120 and an Infinity 1260 ELSD. The analytical column used was an Acquity UPLC BEH C18 column: 1.7 µm; 2.1 x 50 mm, operated under a linear gradient of H₂O and acetonitrile, both containing 0.1% TFA as solvent.

[0426] The photoisomerization of cis-cyclooctene was performed using a custom-designed continuous flow photoreactor, which consisted of: a fan-ventilated, opaque aluminum-walled UV unit housing housing, housing three low-pressure Hg lamps (40 mm x 533 mm, two HNS L 55W 2G11 and one HNS L 96W 2G11, Osram Licht AG) and a quartz tube (OD 16 mm, ID 14 mm, QSIL GmbH), the quartz tube being sealed with a tightly fitting Teflon adapter containing an O-ring and threaded holes (in-house manufactured) for connecting the flanged Teflon tube via a threaded PEEK adapter; an electrical unit allowing independent switching of the variable intensity (2 x 55 W and 1 x 95 W) of the low-pressure Hg lamps to control the intensity of the UV light; and an HPLC pump (Model P402), purchased from Laterk Labortechnik-Geräte GmbH & Co. Teflon tubing includes connections with external and internal thread Luer fittings for connecting silicone cartridges (Biotage SNAP) of different sizes.

[0427] B) Synthesis of compounds

[0428] Example 1: Synthesis of (Z)-9-oxabicyclo[6.1.0]non-2-ene (1)

[0429] Under stirring, a solution of 3-chloroperoxybenzoic acid (12.7 g, 73.4 mmol, 1.00 eq) in CH3Cl (220 mL) was added dropwise to 1,3-cyclooctadiene (11.4 mL, 9.93 g, 91.8 mmol, 1.25 eq) over 3 h. After complete addition, the mixture was stirred overnight at room temperature (rt). The precipitate was filtered off, and the filter cake was washed several times with CH2Cl2. The filtrate was washed with an aqueous solution of NaSO3, followed by a wash with a saturated aqueous solution of NaHCO3. The organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure.

[0430] Purification by rapid chromatography (cyclohexane / EtOAc 20:1→10:1) yielded a colorless oily product 1 (8.93 g, 98%).

[0431] Example 2: Synthesis of (Z)-8-hydroxycyclooct-2-en-1-yl acetate (2)

[0432] 1 (8.40 g, 67.6 mmol, 1.00 eq) was added dropwise to a solution of acetic acid (5.80 mL, 6.09 g, 101 mmol, 1.50 eq) and tetrakis(triphenylphosphine)palladium(0) (7.82 g, 6.76 mmol, 0.100 eq) in 190 mL of THF at 0 °C. After complete addition, the mixture was stirred at this temperature for 2 h. The reaction was terminated by adding 5 mL of H₂O₂ (30% in water). A saturated aqueous solution of NaHCO₃ was added, and the mixture was extracted with CH₂Cl₂. The organic phase was dried over Na₂SO₄, and the solvent was removed under reduced pressure.

[0433] Purification by rapid chromatography (cyclohexane / EtOAc 10:1→1:1) yielded a colorless oily product 2 (10.9 g, 87%).

[0434] Example 3: Synthesis of (Z)-8-oxocyclooct-2-en-1-yl acetate (3)

[0435] Add pyridinium chlorochromate (17.5 g, 81.1 mmol, 1.50 eq) to a CH2Cl2 (540 mL) solution (9.96 g, 54.1 mmol, 1.00 eq) and stir the mixture at rt for 8 h. Filter the reaction mixture through a silica stopper and remove the solvent under reduced pressure.

[0436] Purification by rapid chromatography (cyclohexane / EtOAc 20:1→5:1) yielded a colorless oily product 3 (7.11 g, 72%).

[0437] Example 4: Synthesis of (Z)-2-(2-acetoxy-1-hydroxycyclooct-3-en-1-yl)acetic acid methyl ester (4)

[0438] BF3∙OEt2 (9.63 mL, 11.1 g, 78.0 mmol, 2.00 eq) was added to a solution of 3 (7.11 g, 39.0 mmol, 1.00 eq) of CH2Cl2 (390 mL) at -78 °C. Then, 1-(tert-butyldimethylsiloxy)-1-methoxyethylene (10.2 mL, 8.82 g, 46.8 mmol, 1.20 eq) was added dropwise, and the reaction was stirred at this temperature for 1 h. The reaction was terminated by adding a saturated aqueous solution of NaHCO3. The phases were separated, and the aqueous phase was extracted with CH2Cl2. The combined organic phases were dried over Na2SO4, and the solvent was removed under reduced pressure.

[0439] Purification by rapid chromatography (cyclohexane / EtOAc 20:1→1:1) yielded a colorless oily 4 (8.16 g, 82%), a mixture of trans and cis isomers (4:1).

[0440] Example 5: Synthesis of (E)-2-(2-acetoxy-1-hydroxycyclooct-3-en-1-yl)acetate (5)

[0441] A two-necked 500 mL flask was connected to a continuous flow photoreactor. A 100 g Biotage SNAP column topped with 10% AgNO3 silica gel (58.4 g, 34.4 mmol, 1.30 eq) was loaded. The photoisomerization system was equilibrated for 20 min at a flow rate of 100 mL / min through a column connected to Et2O / hexane (500 mL). Then, 4 (6.78 g, 26.5 mmol, 1.00 eq) dissolved in a minimum amount of Et2O and methyl benzoate (6.67 mL, 7.20 g, 52.9 mmol, 2.00 eq) were added to the two-necked flask, and the system was reequilibrated for 20 min while cooling at 0 °C. Two 55 W UV lamps were then turned on, and photoisomerization was carried out for 30 h at a continuous flow (100 mL / min) while cooling at 0 °C. The system was then rinsed with 500 mL of Et2O and the column was vented. AgNO3 silica was transferred to an Erlenmeyer flask, CH2Cl2 and 25% NH4OH aqueous solution were added, and the mixture was stirred vigorously for 5 min.

[0442] Filter out the silica, and wash the filter cake several times with 25% NH4OH and CH2Cl2. Separate the phases, and extract the aqueous phase with CH2Cl2. Wash the combined organic phases with water, dry on Na2SO4, and remove the solvent under reduced pressure.

[0443] Purification by rapid chromatography (cyclohexane / EtOAc 20:1→3:1) yielded a separable mixture (3.02 g, 45%) of trans and cis isomers (anti-5 and syn-5) as a colorless crystalline solid (cis isomer) and a colorless oil (trans isomer).

[0444] Example 6: Synthesis of (E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetic acid (anti-6, syn-6)

[0445] Add 1 M NaOH to a 45 mL solution of anti-5 (1.17 g, 4.57 mmol, 1.00 eq) in MeOH. aq (45.7 mL, 45.7 mmol, 10.0 eq), and the mixture was stirred at rt for 1.5 h. Then, 1 M HCl was added. aq The mixture was acidified and extracted twice with EtOAc. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure. Co-evaporation with toluene was performed to remove residual AcOH impurities, yielding a colorless crystalline solid of anti-6 (837 mg, 92%). Add 1 M NaOH to a MeOH (5.85 mL) solution of syn-5 (150 mg, 585 µmol, 1.00 eq). aq (5.85 mL, 5.85 mmol, 10.0 eq), and the mixture was stirred at rt for 1 h. Then, 1 M HCl was added. aq The mixture was acidified and extracted twice with EtOAc. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure. Co-evaporation with toluene was performed to remove residual AcOH impurities, yielding syn-6 (79.0 mg, 67%) as a colorless crystalline solid.

[0446] Example 7: Synthesis of 2,5-dioxopyrrolidone-1-yl(E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetate (7)

[0447] N,N,N',N'-tetramethyl-O-(N-succinimide)ureon tetrafluoroborate (2.17 g, 7.19 mmol, 1.20 eq) and DIPEA (1.3 mL, 968 mg, 7.49 mmol, 1.25 eq) were added to a DMF (8 mL) solution of anti-6 (1.20 g, 5.99 mmol, 1.00 eq), and the reaction was stirred at rt for 1 h. The reaction was terminated with H2O and extracted with CH2Cl2. The combined organic phases were dried over Na2SO4, and the solvent was removed under reduced pressure.

[0448] Purification by rapid chromatography (cyclohexane / EtOAc 9:1→1:1) yielded a colorless solid 7 (1.34 g, 75%).

[0449] Example 8: Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetyl)-L-lysine (8)

[0450] Add Fmoc-Lys-OH (1.85 g, 5.02 mmol, 1.20 eq) and DIPEA (910 µL, 675 mg, 5.23 mmol, 1.25 eq) to the crude solution of 7 (1.24 g, 4.18 mmol, 1.00 eq) in DMF (42 mL), and stir the reaction at rt for 3 h. Then, add 1 M HCl. aq The mixture was acidified and extracted twice with EtOAc. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure.

[0451] Purification by reversed-phase rapid chromatography (H2O / MeOH 0%→100%) yielded a colorless solid 8 (2.11 g, 2 steps 92%).

[0452] Example 9: Synthesis of N6-(2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetyl)-L-lysine (9)

[0453] Add piperidine (9.5 mL) to the crude solution of 7 (2.11 g, 3.83 mmol, 1.00 eq) in DMF (38 mL), and stir the reaction at rt for 15 min. Remove the solvent under reduced pressure, absorb the residue in water, and filter. Wash the filter cake with water.

[0454] Purification was performed by reversed-phase rapid chromatography (H2O / MeOH 0%→100%) to obtain a colorless solid of 9 (984 mg, 78%).

[0455] Example 10: Synthesis of (E)-1-(2-hydroxyethyl)cyclooct-3-ene-1,2-diol (10)

[0456] Anti-5 in THF (1 mL) (100 mg, 390 µmol, 1.00 eq) was added to a suspension of LiAlH4 (29.6 mg, 780 µmol, 2.00 eq) in Et2O (1 mL) at 0 °C, and the mixture was stirred at rt for 2 h. Water was then added and extracted with EtOAc. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure.

[0457] Purification by rapid chromatography (cyclohexane / EtOAc 20:1→1:1) yielded 10 (35.0 mg, 48%) of a colorless oil.

[0458] Example 11: Synthesis of 2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)ethyl 1H-imidazol-1-carboxylic acid ester (11)

[0459] 1,1'-carbonyldiimidazole (75.7 mg, 467 µmol, 1.10 eq) was added to a 1 mL solution of DMF containing 10 (79.0 mg, 424 µmol, 1.00 eq) at 0 °C, and the reaction was stirred for 1 h. The solution containing crude product 11 was used directly for the next step.

[0460] Example 12: Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-((2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)ethoxy)carbonyl)-L-lysine (12)

[0461] Add Fmoc-Lys-OH (234 mg, 636 µmol, 1.50 eq) and DIPEA (111 µL, 82.2 mg, 636 µmol, 1.50 eq) to the crude solution of 11 (119 mg, 424 µmol, 1.00 eq) in DMF (1 mL), and stir the reaction at rt for 5 h. Then add water to dissolve the remaining Fmoc-Lys-OH, and stir the mixture overnight at 60 °C. The solution was then dissolved in 1 M HCl. aq The mixture was acidified and extracted twice with EtOAc. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure.

[0462] Purification was performed by reversed-phase rapid chromatography (H2O / MeOH 0%→100%) to obtain 12 (17.0 mg, 7% in 2 steps) as a colorless solid.

[0463] Example 13: Synthesis of N6-((2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)ethoxy)carbonyl)-L-lysine (13)

[0464] Piperidine (75.0 µL) was added to a crude solution of 12 (17.0 mg, 29.3 µmol, 1.00 eq) in DMF (0.3 mL), and the reaction was stirred at rt for 15 min. The reaction mixture was directly subjected to reversed-phase rapid chromatography (H2O / MeOH 0%→100%) to give 13 (6.10 mg, 58%) as a colorless solid.

[0465] Example 14: Synthesis of 2,5-dioxopyrrolidone-1-yl 4-(1,2,4,5-tetraazine-3-yl)benzoate (14)

[0466] Add N-hydroxysuccinimide (80.0 mg, 686 µmol, 1.50 eq) and EDC (132 mg, 686 µmol, 1.50 eq) to a solution of 4-(1,2,4,5-tetraazine-3-yl)benzoic acid (92.5 mg, 458 µmol, 1.00 eq) in dichloromethane (4.6 mL) and stir the mixture overnight at room temperature.

[0467] Purification by rapid chromatography (dichloromethane / MeOH 20:1) yielded 14 (108 mg, 79%) as a pink solid.

[0468] Example 15: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(3-aminopropamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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 (15)

[0469] HOBt (0.904 mg, 5.91 µmol, 0.100 eq) and DIPEA (10.3 µL, 7.64 mg, 59.1 µmol, 1.00 eq) were added to a solution of (2S,3S,4S,5R,6S)-methyl-6-(2-(3-(((9H-fluoren-9-yl)methoxy)carbonyl-amino)propamido)-4-(((4-nitrophenoxy)carbonyloxy)methyl)phenoxy)-3,4,5-triacetoxy-tetrahydro-2H-pyran-2-carboxylic acid ester (54.0 mg, 59.1 µmol, 1.00 eq) and monomethylaurestatin E (42.4 mg, 59.1 µmol, 1.00 eq) in DMF / pyridine (2:1, 0.9 mL), and the mixture was stirred overnight at room temperature. Then 1 M NaOH was added. aq (591 µL, 591 µmol, 10.0 eq), and the mixture was stirred at rt for 1 h.

[0470] Purified by HPLC, 15 (46.7 mg, 70%) was obtained as a white solid.

[0471] Example 16: (2S,3S,4S,5R,6S)-6-(2-(3-(4-(1,2,4,5-tetraazine-3-yl)benzamido)propionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1 Synthesis of (16) phenylpropyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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

[0472] NEt3 (5.67 µL, 4.14 mg, 40.9 µmol, 1.20 eq) was added to solutions of 15 (38.5 mg, 34.1 µmol, 1.00 eq) and 14 (20.4 mg, 68.1 µmol, 2.00 eq) in DMF / pyridine (4:1, 0.7 mL), and the mixture was stirred overnight at room temperature. Then 1 M NaOH was added. aq (591 µL, 591 µmol, 10.0 eq), and the mixture was stirred overnight at rt.

[0473] Purified by HPLC, 16 (7.9 mg, 18%) was obtained as a pink solid.

[0474] Example 17: Synthesis of 2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl)acetamide (17)

[0475] DIPEA (50.4 µL, 289 µmol, 2.00 eq) was added to a DMF (1 mL) solution of 7 (43.0 mg, 145 µmol, 1.00 eq) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione (51.1 mg, 289 µmol, 2.00 eq), and the mixture was stirred at rt for 2 h. The reaction mixture was purified directly by HPLC.

[0476] 17 (11.0 mg, 24%) was purified by HPLC to obtain a white powder.

[0477] Example 18: Synthesis of 2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)-N-(prop-2-yn-1-yl)acetamide (18)

[0478] DIPEA (23.4 µl, 135 µl, 2.00 eq) was added to a DMF (0.5 mL) solution of 7 (20.0 mg, 67.3 µmol, 1.00 eq) and propargyl-1-amine (5.17 µL, 80.7 µmol, 1.20 eq), and the mixture was stirred at rt for 30 min. The reaction mixture was purified directly by HPLC.

[0479] 18 (3.7 mg, 23%) was obtained by HPLC purification.

[0480] Example 19: Synthesis of N-(2-azidoethyl)-2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetamide (19)

[0481] DIPEA (23.4 µl, 135 µl, 2.00 eq) was added to a DMF (0.5 mL) solution of 7 (20.0 mg, 67.27 µmol, 1.00 eq) and 2-azidoethylamine hydrochloride (9.89 mg, 80.7 µmol, 1.20 eq), and the mixture was stirred at rt for 30 min. The reaction mixture was purified directly by HPLC.

[0482] 19 (1.9 mg, 11%) was obtained by HPLC purification.

[0483] Example 20: Synthesis of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetamyl)phenyl)propionic acid (20)

[0484] DIPEA (15.7 mg, 21.1 µL, 121 µmol, 1.20 eq) was added to a DMF (1 mL) solution of (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-aminophenyl)propionic acid (44.7 mg, 111 µmol, 1.10 eq) and 7 (30.0 mg, 101 µmol, 1.00 eq), and the mixture was stirred at rt.

[0485] 20 (4.3 mg, 7%) was obtained by HPLC purification.

[0486] Example 21: Synthesis of methyl (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(2-aminoethoxy)phenyl)propionate (21)

[0487] DIAD (102 mg, 99.0 μL, 503 μmol, 1.05 eq) was added dropwise to a THF (1 mL) solution of Fmoc-Tyr-OMe (200 mg, 479 μmol, 1.00 eq), (2-hydroxyethyl)carbamate tert-butyl ester (81.1 mg, 503 μmol, 1.05 eq), and triphenylphosphine (132 mg, 503 μmol, 1.05 eq) at 0 °C. The mixture was stirred at rt.

[0488] After the reaction was complete, the reaction mixture was directly purified by column chromatography (cyclohexane / EtOAc 20:1→1:1), and the isolated Boc-protected intermediate was deprotected in dichloromethane / TFA (3:1, 4 mL). The solvent was evaporated under reduced pressure.

[0489] 21 (47.0 mg, 17%) was obtained by HPLC purification.

[0490] Example 22: Synthesis of methyl propionate (2S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-(4-(2-(2-(((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetamamido)ethoxy)phenyl)propionate (22)

[0491] DIPEA (12.7 mg, 17.1 μL, 98.2 μmol, 1.20 eq) was added to DMF (0.8 mL) solutions of 7 (24.3 mg, 81.8 μmol, 1.00 eq) and 21 (47.0 mg, 81.8 μmol, 1.00 eq), and the mixture was stirred at rt.

[0492] The solvent was evaporated under reduced pressure, and the crude product 22 was used in the next step.

[0493] Example 23: Synthesis of (S)-2-amino-3-(4-(2-(2-((E)-1,2-dihydroxycyclooct-3-en-1-yl)acetamyl)ethoxy)phenyl)propionic acid (23)

[0494] Add 1 M NaOH to a 0.8 mL solution of MeOH (51.4 mg, 80.0 μmol, 1.00 eq). aq (32.0 mg, 800 μL, 800 μmol, 10.0 eq), and the mixture was stirred at rt for 1 h. Then, 1 M HCl was added. aq The neutralization reaction was followed by purification of the mixture directly by reversed-phase rapid chromatography.

[0495] 23 (16.7 mg, 51%) was purified by reversed-phase rapid chromatography.

[0496] Example 24: Synthesis of (Z)-2-(2-acetoxy-1-((tert-butyldimethylsilyl)oxy)cyclooct-3-en-1-yl)acetate (24):

[0497] 1,1,1-trifluoro-N-trifluoromethanesulfonylmethanesulfonamide (15.4 mg, 11.3 μL, 0.005 Eq, 54.9 μmol) was added to a solution of 3 (2.00 g, 1 Eq, 11.0 mmol) in diethyl ether (22.0 mL) at 20 °C under a nitrogen atmosphere. The reaction mixture was cooled to –20 °C, at which temperature tert-butyl[(1-methoxyvinyl)oxy]dimethylsilane (2.17 g, 2.39 mL, 1.05 Eq, 11.5 mmol) was slowly added, and the mixture was then placed at -20 °C and stirred for 30 min. The ice was removed, and the reaction was allowed to proceed overnight at 20 °C. The crude reaction mixture was evaporated under vacuum, and the mixture was purified by silica gel column chromatography (eluent: cyclohexane / ethyl acetate 0–10%). The fractions were evaporated under vacuum to give a colorless oil, 24 (1.90 g, 5.13 mmol, 47%).

[0498] Example 25: Synthesis of (E)-2-(2-acetoxy-1-((tert-butyldimethylsilyl)oxy)cyclooct-3-en-1-yl)acetate (25):

[0499] A two-necked flask was connected to a continuous flow photoreactor. A 100 g Biotage Sfaer SiOH column topped with 10% AgNO3 silica gel (35.8 g, 10% Wt, 2.6 eq, 21.0 mmol) was used. The photoisomerization system was equilibrated for 20 min at a flow rate of 100 mL / min through a column connected to Et2O / hexane (300 mL). Methyl benzoate (1.16 g, 1.07 mL, 1.05 eq, 8.50 mmol) and methyl benzoate (3.00 g, 1 eq, 8.10 mmol) in 30 mL of hexane / Et2O (6:4) were added to the two-necked flask, and the system was reequilibrated for 20 min while cooling at 0 °C. A 55 W UV lamp was then turned on, and photoisomerization was carried out at a continuous flow (100 mL / min) for 47 h while cooling at 0 °C. The system was then flushed with Et2O and the column was ventilated. AgNO3 silica was transferred to an Erlenmeyer flask, CH2Cl2 and a 25% NH4OH aqueous solution were added, and the mixture was vigorously stirred for 5 min. The mixture was filtered through diatomaceous earth, and the filter cake was washed three times with 25 ml of NH4OH (1x) and then with 50 ml of DCM (10% MeOH).

[0500] Separate the layers and extract twice with 100 ml of DCM. Dry the combined DCM layers with Na2SO4, filter, and evaporate. Purify the crude product by silica gel column chromatography (elution buffer: cyclohexane / ethyl acetate 0-10%). Evaporate the fractions under vacuum to give 25 (260 mg, 702 μmol, 9%) as a colorless solid.

[0501] Example 26: Synthesis of methyl 2-((1S,E)-1,2-dihydroxycyclooct-3-en-1-yl)acetate (26):

[0502] Sodium methoxide (806 mg, 853 μL, 0.200 eq, 3.7 mmol) was added to a methanol (15.0 mL) solution of compound 5 (4.78 g, 1.0 eq, 18.7 mmol) prepared according to Example 5 above at 25 °C, and the reaction mixture was stirred for 1 hour. The reaction mixture was diluted with CH2Cl2 and washed with brine. The organic layer was dried on Na2SO4 and concentrated under reduced pressure to give a yellow oily compound 26 (3.88 g, 18.1 mmol, 97%).

[0503] Example 27: Synthesis of (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetic acid methyl ester (27):

[0504] Iodimethane (5.02 g, 5.0 eq, 35.4 mmol) was added to a solution of 26 (1.52 g, 1.0 eq, 7.1 mmol) in DMF (5.0 mL) under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C. A suspension of sodium hydride (311 mg, 1.1 eq, 7.8 mmol) in DMF (50.0 mL) was slowly added to the reaction mixture, and the mixture was stirred at room temperature for 3 h. The reaction mixture was terminated with saturated NH4Cl. The organic layer was extracted with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 2–20%). The product fractions were combined and concentrated to give a colorless oily 27 (0.77 g, 3.39 mmol, 48%).

[0505] Example 28: Synthesis of (E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetic acid (28):

[0506] Add 1 M NaOH to a 27 g (0.77 g, 1.0 eq, 3.4 mmol) methanol (10.0 mL) solution. aq (1.36 g, 10.0 eq, 33.9 mL, 33.9 mmol), and the reaction mixture was stirred at room temperature for 2 h. Then, 1 M HCl solution was added until the mixture was acidic. The organic layer was extracted with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure to give a colorless oily 28 (0.72 g, 3.35 mmol, 99%).

[0507] Example 29: Synthesis of 2,5-dioxopyrrolidone-1-yl(E)-2-(1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetate (29):

[0508] DIPEA (541 mg, 1.3 eq, 720 μL, 4.2 mmol) was added to a solution of 28 (0.72 g, 1.0 eq, 3.3 mmol) and 2-(2,5-dioxopyrrolidone-1-yl)-1,1,3,3-tetramethylisoureamonium tetrafluoroborate (1.21 g, 1.2 eq, 4.0 mmol) in DMF (8.0 mL), and the reaction mixture was stirred at room temperature for 2 h.

[0509] The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 5-100%). The product fractions were combined, concentrated, and ground with diethyl ether to remove residual impurities. The product was dried under vacuum to give 29 (0.88 g, 2.83 mmol, 85%) as a white powder.

[0510] Example 30: Synthesis of (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetic acid methyl ester (30):

[0511] Iodomethane (335 mg, 5.0 eq, 147 μL, 2.4 mmol) was added to a 2.0 mL solution of DMF (100 mg, 1.0 eq, 471 μmol) at 28°C under a nitrogen atmosphere. This mixture was then added dropwise to a cooled suspension of sodium hydride (226 mg, 12.0 eq, 5.7 mmol) in 3.0 mL of DMF at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 h.

[0512] The reaction mixture was terminated with saturated NH4Cl. The organic layer was extracted with diethyl ether, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 5-60%). The product fractions were combined and concentrated to give 30 (5.3 mg, 218 μmol, 46%) as a colorless oil.

[0513] Example 31: Synthesis of (E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetic acid (31):

[0514] Add 1 M NaOH to 30 (70.0 mg, 1.0 eq, 289 μmol) of methanol (3.0 mL). aq (116 mg, 10.0 eq, 2.89 mL, 2.89 mmol), and the reaction mixture was stirred at room temperature for 2 h. Then, 1 M HCl solution was added until the mixture was acidic. The organic layer was extracted with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure to give a colorless oil, 31 (49.2 mg, 216 μmol, 75%).

[0515] Example 32: Synthesis of 2,5-dioxopyrrolidone-1-yl(E)-2-(1,2-dimethoxycyclooct-3-en-1-yl)acetate (32):

[0516] DIPEA (34.0 mg, 1.3 eq, 0.3 mmol) was added to a DMF (2.0 mL) solution of 31 (48.0 mg, 1.0 eq, 0.2 mmol) and 2-(2,5-dioxopyrrolidone-1-yl)-1,1,3,3-tetramethylisoureamonium tetrafluoroborate (76.0 mg, 1.2 eq, 0.3 mmol), and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 5-100%). The product fractions were combined, concentrated, and ground with diethyl ether to remove residual impurities. The product was dried under vacuum to give 32 (34.2 mg, 0.1 mmol, 50%) as a white powder.

[0517] Example 33: (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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxo Synthesis of (33)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(2-((1S,2S,E)-1-hydroxy-2-methoxycyclooctyl-3-en-1-yl)acetamyl)propamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid:

[0518] Add 6-(2-(3-aminopropamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-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 (39.9 mg, 1.1 mL) to 29 (10.0 mg, 1.0 eq, 32.1 μmol) of DMF (0.1 mL). A solution of DMF (0.3 mL, eq, 35.3 μmol) was added. Triethylamine (6.5 mg, 2.0 eq, 8.9 μL, 64.2 μmol) was then added, and the reaction mixture was stirred at room temperature for 1 h. The crude reaction mixture was purified by preparative HPLC. The fractions were combined and lyophilized to give a white powder of 33 (25.8 mg, 19.4 μmol, 61%).

[0519] Example 34: (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-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxo Synthesis of (34)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-(3-(2-((1S,2S,E)-1,2-dimethoxycyclooctyl-3-en-1-yl)acetamyl)propamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid:

[0520] Add 6-(2-(3-aminopropamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triaza)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (38.2 mg, 1.1 mL) to a solution of 32 (10.0 mg, 1.0 eq, 30.7 μmol) of DMF (0.1 mL). A solution of 0.3 mL DMF (33.8 μmol eq) was added. Triethylamine (24.9 mg, 8.0 eq, 34.3 μL, 246 μmol) was then added, and the reaction mixture was stirred at room temperature for 4 h. The crude reaction mixture was purified by preparative HPLC. The fractions were combined and lyophilized to give a white powder of 34 (23.3 mg, 17.4 μmol, 56%).

[0521] Example 35: Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-((E)-1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetyl)-L-lysine (35):

[0522] To a solution of 29 (48.5 mg, 1.0 eq, 156 μmol) in 3.0 mL of DMF, (((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine (68.9 mg, 1.2 eq, 187.0 μmol) and triethylamine (18.9 mg, 1.2 eq, 26.1 μL, 187 μmol) were added, and the reaction mixture was stirred at room temperature for 4 h. Then, 1 M HCl solution was added until the mixture was acidic. The organic layer was extracted with ethyl acetate, dried over Na₂SO₄, and concentrated under reduced pressure. The crude mixture was purified by rapid RP chromatography (MeCN in water: 10-100%). The fractions were combined and evaporated under vacuum to give a colorless oil, 35 (66.0 mg, 117 μmol, 75%).

[0523] Example 36: Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-((E)-1-hydroxy-2-methoxycyclooct-3-en-1-yl)acetyl)-L-lysine (36):

[0524] To a solution of 35 (66.0 mg, 1.0 eq, 117.0 μmol) in DMF (0.6 mL), piperidine (124.0 mg, 12.5 eq, 144.0 μL, 1.5 mmol) was added, and the reaction mixture was stirred for 20 min. The reaction mixture was diluted with water and filtered. The filtrate was evaporated under vacuum. The crude product was dissolved in water and purified by rapid RP chromatography (MeCN in water: 10-100%). The fractions were combined and evaporated under vacuum to give 36 (22.8 mg, 66.6 μmol, 57%) as a white solid.

[0525] Comparative Example 37: Synthesis of N6-(2-((E)-1-hydroxycyclooct-3-en-1-yl)acetyl)-L-lysine (44) - an analogue of the compound described in WO2022 / 133225:

[0526] Synthesis of step a) (Z)-cyclooct-3-en-1-ol (37):

[0527] A lithium aluminum hydride solution (1 mL in THF) (394 mg, 0.5 eq, 10.4 mL, 10.4 mmol) was added dropwise to 1 (2.58 g, 1.0 eq, 20.8 mmol) of anhydrous THF (20.0 mL) at 0 °C, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was carefully stopped with water (3 mL) and filtered. The filtrate was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 2–20%). The product fractions were combined and evaporated under vacuum to give a colorless oil, 37 (1.52 g, 12.0 mmol, 58%).

[0528] Step b) (Z)-Cyclooct-3-en-1-one (38):

[0529] Add (Z)-cyclooct-3-en-1-ol (1.37 g, 1.0 eq, 10.8 mmol) in anhydrous DCM (20.0 mL) to a flask. Add 3-oxo-1lambda5-benzo[d][1,2]iodaoxole-1,1,1(3H)-triyl triacetate (5.15 g, 1.1 eq, 12.15 mmol), and stir the reaction mixture overnight at room temperature. Dilute the reaction mixture with diethyl ether and wash with saturated NaHCO3. Extract the organic layer, dry it on Na2SO4 and diatomaceous earth, and filter. Concentrate the crude product under reduced pressure and purify it by silica gel column chromatography (ethyl acetate in cyclohexane: 2% at 1V, 2-10% at 15V). The product fractions were combined and evaporated under vacuum to give a colorless oily product 38 (1.06 g, 8.5 mmol, 79%).

[0530] Step c) (Z)-2-(1-hydroxycyclooct-3-enyl)methyl acetate (39):

[0531] At -78 °C, boron trifluoride diethyl ether (2.10 g, 2.0 eq, 1.80 mL, 14.0 mmol) was added to 38 (0.90 g, 1.0 eq, 7.2 mmol) in 120 mL of anhydrous DCM, followed by dropwise addition of tert-butyl((1-methoxyvinyl)oxy)dimethylsilane (1.6 g, 1.2 eq, 1.8 mL, 8.7 mmol). The reaction mixture was stirred overnight at room temperature and then terminated with saturated NaHCO3. The organic layer was extracted with DCM and dried over Na2SO4. The crude product was concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate in cyclohexane: 3–30%). The product fractions were combined and evaporated under vacuum to give 39 (0.86 g, 4.4 mmol, 60%) as a colorless oil.

[0532] Step d) Synthesis of (E)-2-(1-hydroxycyclooctyl-3-enyl)acetic acid methyl ester (40):

[0533] A two-necked flask was connected to a continuous flow photoreactor. A 25 g Biotage Sfaer SiOH column topped with 10% AgNO3 silica gel (9.2 g, 10% Wt, 1.3 eq, 5.4 mmol) was used. The photoisomerization system was equilibrated for 20 min at a flow rate of 100 mL / min through a column connected to Et2O / hexane (300 mL). Methyl benzoate (567 mg, 0.52 mL, 1.0 eq, 4.16 mmol) and 39 (0.83 g, 1.00 eq, 4.16 mmol) in 100 mL of hexane / Et2O (1:1) were added to the two-necked flask, and the system was reequilibrated for 20 min while cooling at 0 °C. Two 55 W UV lamps were then turned on, and photoisomerization was carried out for 20 h at a continuous flow rate (100 mL / min) while cooling at 0 °C. The system was then rinsed with Et2O and the column was purged with gas. AgNO3 silica was transferred to an Erlenmeyer flask, CH2Cl2 and a 25% NH4OH aqueous solution were added, and the mixture was vigorously stirred for 5 min. The mixture was filtered through diatomaceous earth, and the filter cake was washed three times with 25 ml NH4OH (1x) and 50 ml DCM (10% MeOH). The layers were separated, extracted twice with 100 ml DCM, and the combined organic layers were dried over Na2SO4, filtered, and evaporated. The crude brown oil was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 10-40%). The product fractions were combined and evaporated under vacuum to give 40 (0.12 g, 595 μmol, 14%) as a white solid.

[0534] Step e) Synthesis of (E)-2-(1-hydroxy-cyclooct-3-en-1-yl)acetic acid (41):

[0535] Add 1 M NaOH to a 2.0 mL solution of 40 (54.6 mg, 1.0 eq, 0.28 mmol) of methanol. aq (54.6 mg, 10.0 eq, 2.8 mL, 2.8 mmol), and the reaction mixture was stirred at room temperature for 2 h. Then, 1 M HCl solution was added until the mixture was acidic. The organic layer was extracted with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure to give a colorless oily 41 (20.8 mg, 0.11 mmol, 41%).

[0536] Step f) Synthesis of 2,5-dioxopyrrolidone-1-yl(E)-2-(1-hydroxycyclooct-3-en-1-yl)acetate (42):

[0537] DIPEA (18.2 mg, 1.3 eq, 24.3 μL, 0.14 mmol) was added to a DMF (1.0 mL) solution of 41 (20.8 mg, 1.0 eq, 0.11 mmol) and 2-(2,5-dioxopyrrolidone-1-yl)-1,1,3,3-tetramethylisoureamonium tetrafluoroborate (40.8 mg, 1.2 eq, 0.14 mmol), and the reaction mixture was stirred at room temperature for 3.5 h. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate in cyclohexane: 5-100%). The product fractions were combined, concentrated, and ground with diethyl ether. The product was dried under vacuum to give 42 (29.2 mg, 0.11 mmol, 92%) as a white powder.

[0538] Step g) Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(2-((E)-1-hydroxycyclooct-3-en-1-yl)acetyl)-L-lysine (43):

[0539] To a solution of 42 (29.2 mg, 1.0 eq, 0.10 μmol) in 3.0 mL of DMF, (((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine (45.9 mg, 1.2 eq, 125 μmol) and triethylamine (12.6 mg, 1.2 eq, 17.4 μL, 125 μmol) were added, and the reaction mixture was stirred overnight at room temperature. Then, 1 M HCl solution was added until the mixture was acidic. The organic layer was extracted with ethyl acetate, dried over Na₂SO₄, and concentrated under reduced pressure. The crude mixture was purified by rapid RP chromatography (MeCN in water: 10-100%). The fractions were combined and evaporated under vacuum to give a colorless oil, 43 (12.1 mg, 22.6 μmol, 22%).

[0540] Step h) Synthesis of N6-(2-((E)-1-hydroxycyclooct-3-en-1-yl)acetyl)-L-lysine (44):

[0541] To a solution of 43 (12.0 mg, 1.0 eq, 22.4 μmol) in 1.0 mL of DMF, piperidine (23.9 mg, 12.5 eq, 27.7 μL, 0.28 mmol) was added. The reaction mixture was stirred for 20 min, then diluted with water and filtered. The filtrate was evaporated under vacuum. The crude product was dissolved in water and purified by rapid RP chromatography (MeCN in water: 10-100%). The fractions were combined and evaporated under vacuum to give 44 (3.50 mg, 11.2 μmol, 50%) as a white solid.

[0542] Example 38: Synthesis of perfluorophenyl (E)-2-(1,2-dihydroxycyclooct-3-en-1-yl)acetate (45) (active ester):

[0543] Add 3-(((ethylimino)methylene)amino)-N,N-dimethylpropyl-1-amine hydrochloride (283 mg, 1.5 eq, 1.5 mmol) and N,N-dimethylpyridin-4-amine (24.0 mg, 0.2 eq, 0.2 mmol) to a DCM (10.0 mL) solution of anti-6 (prepared according to Example 6 above) (197 mg, 1.0 eq, 0.98 mmol) and 2,3,4,5,6-pentafluorophenol (217 mg, 1.2 eq, 1.18 mmol), and stir the reaction mixture for 2 h.

[0544] The reaction mixture was terminated with saturated NH4Cl and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude mixture was purified by rapid chromatography (ethyl acetate in cyclohexane: 2-35%). The fractions were combined and evaporated under vacuum to give 45 (223 mg, 608 μmol, 62%) as a colorless oil.

[0545] C) Test Results 1

[0546] 1. Stability test

[0547] To assess the stability of the ncAA of the present invention under physiological conditions, compound 13 (obtained according to Example 13 above) was dissolved in various cell culture media (LB, DMEM, FreeStyle HEK, FreeStyle CHO) or mouse serum to a final concentration of 2 mmol / L, and the solution was incubated at 37 °C for 4 days.

[0548] LC-MS analysis was performed using the extracted ion count (EIC) at m / z = 329, corresponding to a mass of 13. No significant changes were observed in the detected substance at m / z = 329 (see [link to analysis]). Figure 2 This is contrary to the previously described ncAA (Reinkemeier et al., Chem. Eur. J. 2021, 27, 6094; see also...). Figure 1 ).

[0549] Therefore, 13 is completely stable under physiological conditions for at least 4 days. DAR analysis of ADCs incorporating 13 also confirms this. Figure 6 ).

[0550] To confirm that no non-reactive substances with the same retention time were formed during the experiment, a slight excess of diethyl ((6-(5-aminopyridin-2-yl)-1,2,4,5-tetraazine-3-yl)methyl)phosphonate was added, and the same LC-MS analysis was performed after 5 min at room temperature to determine the EIC at m / z = 329. For all samples tested, the substance at m / z = 329 disappeared, and only the corresponding click product could be identified. Figure 3 The data shown are representative of 13 incubated in DMEM.

[0551] 2. Hydrophilicity test

[0552] A comparison of the clogP- hydrophilicity values ​​of different TCO compounds in this invention shows the improved hydrophilicity of these derivatives (see [link]). Figure 4 Furthermore, the head group and the final ncAA are soluble in pure water, unlike most other reported TCOs.

[0553] 3. Genetic code amplification experiment

[0554] 3.1 Trastuzumab HCC H 2K16 13 -LC expression

[0555] Trastuzumab HCC H 2K16 13 -LC is a trastuzumab variant carrying a component integrated into its heavy chain (c H The ncAA in position K249 of the 2-domain structure, as described in Example 13 above. The preparation of different trastuzumab variants of suitable cloning and expression constructs is described in the applicant's WO2023 / 094525, which is incorporated herein by reference.

[0556] Freestyle™ 293-F cells (Thermo Fisher Scientific, R79007) were cultured in a shaking incubator at 37°C, 8% CO2, and 120 rpm using Freestyle™ 293 expression medium (Thermo Fisher Scientific, 12338026). One day before transfection, the required cell volume was divided into 0.5 x 102 cells. 6 Cells were cultured at a density of [number] cells / ml and incubated for another 24 hours. On the day of transfection, cells were harvested by centrifugation at 100 rcf for 10-20 minutes and resuspended in fresh culture medium to obtain 1 x 10[number] cells / ml. 6 A density of cells / ml was achieved. 1 µg DNA / ml expression medium was added (see [link]). Figure 8 The plasmid map of expression plasmid pCK-HSA-trastuzumab HC-LC (modified by replacing the lysine codon "AAG" with the amber codon "TAG" at position K249 of the heavy chain sequence) was mixed with 40 µl / ml expression medium. PEI Max (Polysciences, 49553-93-7) was added at a 1:4 ratio (DNA: PEI Max).

[0557] Vortex the mixture 3 x 5 seconds, incubate in a protective enclosure for 15 minutes, and add dropwise to the cells. Dissolve compound 13 in water, filter aseptically, and add to the cells to obtain a final concentration of 500 µM in the expression culture.

[0558] 3.2 Trastuzumab HCC H 2K16 13 -LC purification

[0559] The expression culture was harvested by centrifugation at 4500 rpm for 30 minutes, and the supernatant was filtered through a 0.45 µm filter. The volume of the clear supernatant was measured, and 10 x Buffer A (0.2 M Na2PO4, 1.5 M NaCl, pH 7.2) was added to obtain a final concentration of 1 x Buffer A (1 / 10 of the total volume). This mixture was loaded onto a HiTrap Fibro™ PrismA column (Cytiva, 17549855) equilibrated in Buffer A (0.02 M Na2PO4, 0.15 M NaCl, pH 7.2). After washing away unbound material, the antibody was eluted with a linear gradient using Buffer B (0.1 M sodium citrate, pH 3.2). Figure 5 A). The gradient was collected and analyzed on SDS-PAGE. Figure 5 (B) The fractions containing antibodies were pooled, concentrated, and the buffer was exchanged for 1 x PBS using a 30 kDa cutoff amicon filter (Millipore, UFC903008). Concentrations were determined using a DeNovix DS-11 spectrometer.

[0560] 4. Joining and stability experiments

[0561] 4.1 Trastuzumab HCC H 2K16 13 -LC conjugation with drug compound 16

[0562] 0.243 nmol of trastuzumab HC CH2K16 13 -LC was mixed with 973 nmol of compound 16 (see Example 16) to a total volume of 36 µl and incubated at RT for 4 hours. The reaction was purified by size exclusion chromatography (Superdex S200Increase, Cytiva, 28990944), peaks were collected fractionally, and the desired fractions were analyzed and concentrated by SDS-PAGE. Figure 5 C).

[0563] 4.2 Trastuzumab HCC H 2K16 13 DAR analysis of -16-LC

[0564] Following the standard protocol described in the product manual, the final ADC was deglycosylated using a fixed PNGase F (Genovis, G1-PF6-010). The drug-antibody ratio (DAR) was analyzed by reductive reversed-phase liquid chromatography (LC). Therefore, the deglycosylated antibody was reduced by incubating with DTT in PBS (50 mmol / L) at 37 °C for 30 min, followed by LC analysis using an AdvanceBio RP-mAb C4 column (Agilent) at 80 °C. The antibody was then analyzed by conjugation (I... con ) and unjoined (I uncon DAR (Delta Arithmetic Calculation) of Matter:

[0565] based on Figure 6 The chromatogram shown in Figure A is for trastuzumab CH2K16. 13 The DAR of -16-LC was determined to be 1.7-1.8. (Compared to the doping of TCO) A-Lys instead of compound 13, the ADC trastuzumab HC CH2K16TCO Comparison of the corresponding DAR analysis of a-16-LC showed that the latter's stability was significantly improved during protein expression. Due to TCO During expression, isomerization occurs, and the major portion of the antibody remains unconjugated. Figure 6 B).

[0566] 4.3 Conjugation of active ester 7 with trastuzumab

[0567] 1.5 mg trastuzumab (0.01 μmol) was dissolved in PBS (1.50 mL), and the pH was adjusted to pH = 8 by adding 0.5 mL of 0.1 M NaHCO3 solution. 10 µL of a 25 mmol / L solution of compound 7 in DMF / H2O (1:1) was added to the antibody solution, and the mixture was incubated at RT with shaking for 1 h.

[0568] The mixture was diluted with PBS to a final volume of 2.5 mL and purified by elution with PBS using a PD-10 column (Sephadex G25-M). The 2.5 mL flow-through was discarded, and the eluent was collected in 0.5 mL fractions. The fractions were analyzed by SDS-PAGE, and the antibody-containing fractions were pooled and concentrated to yield 1.03 mg of trastuzumab-7.

[0569] 4.4 Determination of conjugation and labeling degree (DOL) of trastuzumab-7 with Cy5-tetraazine

[0570] Dissolve 0.15 mg of trastuzumab-7 (1 nmol) in 150 µL of PBS, then add 2 µL of a 10 mmol / L solution of Cy5-tetraazine (also known as sulfo-Cy5-tetraazine, a fluorophore derivative with a tetrazine group for TCO-linked labeling; CAS-Nr.: 1801695-57-7, Click Chemistry Tools) in DMSO / H2O (20 nmol) and incubate the mixture at RT for 20 min.

[0571] Dilute the mixture with PBS to a final volume of 2.5 mL and purify by elution with PBS using a PD-10 column (Sephadex G25-M). Discard the 2.5 mL flow-through and collect the eluent in 0.5 mL fractions. Collect and concentrate the visible blue fractions (fractions 2–7) to separate trastuzumab-Cy5.

[0572] DOL was assessed using UV / Vis spectroscopy with a DS-11+ spectrophotometer at level 5, assuming quantitative conversion. The determined DOL value was 3.8.

[0573] The results are as follows Figure 7 As shown.

[0574] D) Test Results 2

[0575] 1. Further stability experiments

[0576] To further evaluate the stability of the ncAA of the present invention under physiological conditions, compound 36 (obtained according to Example 36 above) was dissolved in LB medium to a final concentration of 2 mmol / L, and the solution was incubated at 37 °C.

[0577] The following existing technologies ncAA DOTCO-Lys, OxTCO-Lys, sOxTCO-Lys

[0578] Compared with the preparations described above, ncAA44 was also dissolved in LB medium and incubated at 37 °C. For DOTCO-Lys, OxTCO-Lys, and sOxTCO-Ly, the respective TCO structures disclosed in the prior art (see Kozma, E., et al. Chembiochem (2016) 17, No. 16 1518-1524; or WO 2016 / 025480) were linked to the amino acid lysine for comparison with the ncAA of the present invention.

[0579] LC-MS analysis was performed using extracted ion counts (EICs) to determine the mass of each compound. After only 2 days of incubation, ncAA based on the existing TCOs became significantly unstable (DOTCO-Lys = 0% trans, OxTCO-Lys = 55% trans, sOxTCO-Lys = 45% trans remaining) and completely isomerized after 6 days. In comparison, ncAA44 also completely isomerized after 6 days, while ncAA36 retained >85% trans even after 6 days in LB medium at 37 °C. This highlights the need for a combination of geminal 3-substituents and 2-substituents to increase the stability of these derivatives.

[0580] 2. Genetic code expansion experiments and ADC preparation

[0581] 2.1 Expression of trastuzumab mutants containing ncAA 13 at different positions

[0582] Genetic code expansion technology was used in Expi293F suspension cells (Thermo Fisher) to perform experiments at different locations (V) in the heavy or light chains. H P41; C H 2K90; V L K45) contains the expression of different trastuzumabs containing ncAA13 (see Example 13 above). Briefly, cells have been transfected with trastuzumab expression plasmids containing the amber STOP codon at the desired position, such as in the variable heavy chain (VH), constant heavy chain (CH), or variable light chain (VL). Furthermore, PylRS and tRNA encoding *Methanococcus martensii* were expressed. Pyl The plasmid was used for transfection to ensure that HTCO was incorporated into the trastuzumab antibody throughout the expression process. Seven days after expression, trastuzumab was purified from the supernatant of the expression culture using protein A.

[0583] 2.2 Conjugation of trastuzumab mutant with payload 16

[0584] The trastuzumab mutant was mixed with compound 16 (see Example 16 above) in PBS and incubated at RT for 4 hours. The reaction was purified by size exclusion chromatography (Superdex S200 Increase, Cytiva, 28990944), peaks were collected fractionally, and the desired fractions were analyzed and concentrated on SDS-PAGE.

[0585] 2.3 Cytotoxicity assay of conjugated trastuzumab mutant

[0586] SKBR-3 (Her2-positive cells) were seeded at a density of 5000 cells / ml in 96-well plates, and ADC was added at different concentrations. Kadcyla was used as a control. After 5 days of incubation, CellTiter-Glo® 2.0 cell viability assay (Promega) was added to the cells, and luminescence was measured using a microplate reader. Data were analyzed using a Graph Pad Prism. Results are as follows. Figure 9 As shown.

[0587] The data demonstrate that the isomerization stability of ncAA in this invention is independent of the conjugation position within Ab.

[0588] 2.4 Trastuzumab HCV H P41 13- LC conjugation with different payloads and cytotoxicity assays

[0589] Trastuzumab HCV H P41 13 -LC (at position V) H P41 was conjugated with compound 13 at a molar ratio of 1:3 with the effective loads P1-P4 (see...). Figure 10 After incubation overnight at 37 °C, ADC was purified by size exclusion chromatography. For cytotoxicity assays, SKBR-3 (Her2-positive cells) were seeded at a density of 5000 cells / ml in 96-well plates, and ADC was added at different concentrations. After 5 days of incubation, CellTiter-Glo® 2.0 cell viability assay (Promega) was added to the cells, and luminescence was measured using a microplate reader. Data were analyzed using a Graph Pad Prism. Results are shown below. Figure 11 As shown.

[0590] 2.5 Trastuzumab HCV H P41 36 -LC expression

[0591] Expi293-F cells (Thermo Fisher Scientific, R79007) were cultured in a shaking incubator at 37°C, 8% CO2, and 120 rpm using Expi™ 293 expression medium (Thermo Fisher Scientific, A1435101). The day before transfection, the required cell volume was divided into 3-4 x 102 cells. 6 Cells were cultured at a density of [number] cells / ml and incubated for another 24 hours. On the day of transfection, the cells were divided into 3 x 10 [cells / ml] groups. 6Cells / ml. Mix 1 µg DNA / ml expression culture with 1000 µl / ml OptiPro SFM medium. Add PEIMax (Polysciences, 49553-93-7) at a 1:4 ratio (DNA: PEIMax) for transfection. Vortex the mixture 3 x 5 seconds, incubate under a protective shield for 15 minutes, and add dropwise to the cells. Dissolve the corresponding amount of Compound 36, which yields a final concentration of 1 mM in the expression culture, in H2O, aseptically filter, and add to the cells.

[0592] The expression culture was harvested by centrifugation at 4500 rpm for 30 minutes, and the supernatant was filtered through a 0.22 µm filter. The volume of the clear supernatant was measured, and 10 x Buffer A (PBS pH 7.4) was added to obtain a final concentration of 1 x Buffer A (1 / 10 of the total volume). This mixture was loaded onto a HiTrap Fibro™ PrismA column (Cytiva, 17549855) equilibrated in Buffer A (1xPBS, pH 7.4). After washing away unbound material, the antibody was eluted with a linear gradient using Buffer B (0.1 M sodium citrate, pH 3.4) at position V. H P41 incorporates compound 36 into trastuzumab HC V H P41 36 -LC). Fractions were collected throughout the gradient, neutralized with 10M Tris pH10, and analyzed on SDS-PAGE. Fractions containing antibodies were pooled, concentrated, and the buffer was exchanged for 1 x PBS using a 30 kDa cutoff amicon filter (Millipore, UFC903008). Concentrations were determined using a DeNovix DS-11 spectrometer.

[0593] 2.6 Trastuzumab HCV H P41 36 -LC combined with payload 16

[0594] 1.713 nmol of trastuzumab HC V H P41 36 -LC was mixed with 5.14 nmol of payload 16 (see Example 16) to a total volume of 50 µl and incubated overnight at 37 °C with shaking at 300 rpm. The reaction was purified by size exclusion chromatography (Superdex S200 Increase, Cytiva, 28990944), peaks were collected fractionally, and the desired fractions were analyzed and concentrated on SDS-PAGE. The DAR was determined to be 1.8.

[0595] This further illustrates the isomerization stability of the ncAA of the present invention.

[0596] Referring to SEQ ID NO:1 (trastuzumab heavy chain) and SEQ ID NO:2 (trastuzumab light chain), the above terms C H 2K16 refers to position K249 of SEQ ID NO:1. C H 2K90 refers to position K323 of SEQ ID NO:1. V H P41 refers to position P41 of SEQ ID NO:1, and V L K45 refers to position K45 of SEQ ID NO:2.

[0597] All publicly available information from any documents cited in this article is incorporated herein by reference.

Claims

1. Compound of the general formula I wherein n is 0 or an integer from 1 to 20, in particular from 1 to 10, more particularly from 1 to 5; A is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 , >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; B is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; M is -CR 1 R 2 -, -O-, -S-, -N(R 1 )-, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; Q is -CR 1 R 2 -, >CH-OZ, >CH-SZ, >CH-NR 1 R 2 , >CH-OR 3 >CH-CN, >CH-NO2, >CH-SO2R 4 or >CH-SR 3 ; with the proviso that If M and B independently of one another represent -O-, -S- or -N(R 1 )-, then A is -CR 1 R 2 - or If A is selected from -O-, -S- or -N(R 1 )-, then M and B independently of one another represent -CR 1 R 2 -, D is -OR 3 , -SR 3 , -OZ or -SZ; U is OZ, -SZ, -OR 3 , -SR 3 or -NR 5 R 6 wherein the residues Z are independently of each other identical or different protecting groups; R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently of each other identical or different and are selected from H and linear or branched lower alkyl; X is a bridging group; W is a saturated or unsaturated polar group; L is a linking group; and G is a terminal group; or a salt, such as a pharmaceutically acceptable salt, or a solvate thereof; each as a stereoisomer or a mixture of at least two stereoisomers.

2. Compound according to claim 1, wherein the residues Z are thiol protecting groups or alcohol protecting groups, in particular cleavable protecting groups, more particularly independently of each other selected from the group consisting of acetyl (Ac), benzoyl (Bz), benzyl (Bn), beta-methoxyethoxymethyl ether (MEM), methoxy methyl ether (MOM), methoxytrityl (MT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), trityl (triphenylmethyl, Tr), silyl ether, methyl ether and ethoxyethyl ether (EE) residues; and / or wherein X is selected from the group consisting of: a) linear or branched lower alkylene, in particular -(CH2) n1 wherein n1 is an integer from 1 to 4, in particular methylene or ethylene; b) -O-, -S-, >CH-OZ, >CH-SZ, >CH-OR 3 or >CH-SR 3 ; wherein Z and R 3 As defined above; c) C1-C4-alkylene, in particular C1-C3-alkylene, more particularly methylene or ethylene; d) linear or branched mono- or polyalkyleneoxy moieties, in particular selected from linear moieties -((CH2) x -O) y -, -(O-(CH2) x ) y - and -(O-(CH2) x ) y -O-, and branched or mono- or polyunsaturated analogues thereof; wherein x independently of each other represents an integer selected from 1, 2, 3 or 4; in particular 1 or 2; and y independently of each other represents an integer selected from 1 to 20, in particular from 1 to 15, from 1 to 10 or from 1 to 4; or and / or wherein the polar group W is selected from the group consisting of a) -C(O)-, -O-C(O)-, -C(O)-O-; b) -O-[C(O)-[CH2] x1 ] x2 C(O)-; -O-[C(S) [CH2] y1 ] y2 C(O)- wherein x1, x2, y1 and y2 independently of each other represent an integer selected from 1 to 6, in particular from 1 to 4, more particularly from 1 to 2; c) -S-C(S)-, -O-C(S)-, -C(S)-, -C(S)-O-, -C(S)-S-; -NR 7 -C(O)-, -C(O)-NR 7 -, 7 , 7 , 7 , 7 -C(O)NR 7 -, 7 -C(NR 7 )-; or d) -NR 8 -[C(O) [CH2] x1 ] x2 C(O)-; NR 8 -[C(S) [CH2] y1 ] y2 C(O)-; wherein x1, x2, y1 and y2 independently of each other represent an integer selected from 1 to 6, in particular from 1 to 4, more particularly from 1 to 2; and R 7 and R 8 independently of one another represent H or lower alkyl, in particular H or C1-C4-alkyl. and / or wherein each L is independently selected from the group consisting of a) linear or branched alkylene, linear or branched alkenylene, cyclic alkylene, cyclic alkenylene or an analogue containing one or more heteroatoms in the carbon skeleton; in particular linear or branched C1-C 10 alkylene, linear or branched C2-C 10 alkylene, C3-C8-cyclic alkylene, C3-C8-cyclic alkenylene or an analogue containing one or more heteroatoms in the carbon skeleton; more particularly linear or branched C1-C4-alkylene, linear or branched C2-C4-alkenylene, C3-C6-cyclic alkylene, C3-C6-cyclic alkenylene or an analogue containing one or more heteroatoms in the carbon skeleton; most particularly methylene, ethylene and propylene; (part La) b) linear or branched mono- or polyalkyleneoxy moieties, in particular selected from the group consisting of linear moieties -((CH2) x3 -O) y3 -, -(O(CH2) x3 ) y3 -, -(O-(CH2) x3 ) y3 -O-, -((CH2) x3 -O) y3 -(CH2) n3 C(O)-, -(O(CH2) x3 ) y3 -NR 7’ -, -(O-(CH2) x3 ) y3 -C(O)- and branched analogues thereof; wherein x3 independently of each other represents an integer selected from 1, 2, 3 or 4; in particular 1 or 2; y3 independently of each other represents an integer selected from 1 to 20, in particular from 1 to 15, from 1 to 10 or from 1, 2, 3 and 4; n3 is an integer selected from 2 and 3; and R 7 ' represents H or lower alkyl, in particular H or C1-C4-alkyl, more particularly methyl or ethyl; (part Lb) The condition is that, when two or more identical or different connector portions La and / or Lb exist, these elements can be directly connected by chemical bonds, or by a combination of -C(O)-, -OC(O)-, -C(O)-O-, -SC(S)-, -OC(S)-, -C(S)-, -C(S)-O-, -C(S)-S-; -NR 7 -C(O)-、-C(O)-NR 7 -、-C(NR 7 -NR 7 -C(NR 7 )-, -S(O)-, -S(O)2-, -P(O)OR 7 -、-OP(O)OR 7 -、-P(O)OR 7 -O- The same or different coupling parts are indirectly connected to each other; where R 7 As defined above; and / or wherein G has one of the following meanings: i. G is H or R 7 ; and G can also be -OR if n ≠ 0 7 , -C(O)OR 7 , -NR 7 2 or -C(O)NR 7 2; wherein R7 is as defined above; ii. a leaving group E selected from halogen, pentafluorophenyl (Pfp), tetrafluorophenyl (Tfp), oxazolone, succinimidyl (Su), sulfosuccinimidyl, trifluoroacetyl, azido, p-nitrophenyl (PNP) and a nitro-containing aromatic group; iii. a thiol reactive moiety of the general formula wherein J is selected from -NH-, -S- or -O- or is absent; r is an integer selected from 1 to 4 or 0 when J is absent; and M1 is a thiol reactive moiety, more particularly selected from M2 to M16: wherein: X 3 is H, halogen, PhS, MeS; X 4 is halogen, PhS, MeS; X 6 is H or CrC 12 alkyl, preferably H or CrC 1-6 alkyl; X 5 is H, CrC 12 alkyl, C6-C 12 aryl, C7-C 12 alkylaryl or C7-C 12 aralkyl, preferably H or p-methylphenyl; and wherein the aromatic ring of (M6) and (M8) can optionally be a heteroaromatic ring, such as a phenyl or pyridine ring; iv. -NH-(CH2)2)C≡CH, -NH-(CH2)2-N3, -O-aryl or -HN-aryl, H, -OH, -NH2halogen, R 9 , -CH=C(R 9 )2, -C≡C R 9 , -[C(R 9 )2C(R 9 )2O] q -R 9 , -CN, -N3, -NCL, -LCN, -L R 9 , - + N(R 9 )2, -N(R 9 )3, -C(L)N(R 9 )2, C(R 9 )2L R 9 , -C(L) R 9 , -C(L)L R 9 , -S(O)R 9 , -S(O )2 R 9 , -S(O)OR 9 , -S(O)2OR 9 , -S(O)N(R 9 )2, -S(O)2N(R 9 )2, -OS(O)R 9 , -OS(O)2R 9 , -OS(O)OR 9 , -OS(O)2OR 9 , -P(O)(R 9 )(OR 9 ), -P(O)(OR 9 )2, -OP(O)(OR 9 )2, -Si(R 9 )3, -LC(L)R 9 , -LC(L)LR 9 , -LC(L)N(R 9 )2, -N(R 9 )C(L)R 9 , -N(R 9 )C(L)LR 9 and -N(R 9 )C(L)N(R 9 )2, wherein q is in the range of 1 to 200, in particular 1 to 20, more particularly 1 to 10, especially 1 to 5, L is oxygen or sulfur, and M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 R 9 independently selected from hydrogen, halogen, C1-C 24 alkyl, such as C1-C 10 alkyl or C1-C3 alkyl, C6-C 24 aryl, C7-C 24 alkyl(aryl) and C7-C 24 (aryl)alkyl; v. -OZ, -SZ, -OR 1 , -C(O)OR 1 , -N(R 1 )2, -C(O)N(R 1 )2, -SR 1 , or -R 1 , wherein Z and R 1 as defined above; and vi. strained cyclo or acyclic alkyne-azide cycloaddition (SPAAC)-reactive groups, in particular, i. alkyne-terminating groups ii. DBCO-derived residues of the following general formula wherein X 3 is N, >CH- or >CH-O-; or iii. BCN-derived residues of the following formula iv. cyclooctyne-derived residues, in particular SCO-derived residues of the following general formula wherein X 4 is -O- or -O-(CO)-; v. or residues of the formula M20-M38 wherein B - is an anion, or wherein: R 15 is independently selected from the group consisting of hydrogen, halogen, -OR 16 , -NO2, -CN, -S(O)2R 16 , -S(O)3 (-) , C1-C 24 alkyl, C6-C 24 (hetero)aryl, C7-C 24 alkyl(hetero)aryl and C7-C 24 (hetero)arylalkyl, wherein alkyl, (hetero)aryl, alkyl(hetero)aryl and (hetero)arylalkyl are optionally substituted, wherein two substituents R 15 may be linked together to form an optionally substituted fused cycloalkyl or an optionally substituted fused (hetero)aryl substituent, and wherein R 16 is independently selected from the group consisting of hydrogen, halogen, C1-C 24 alkyl, C6-C 24 (hetero)aryl, C7-C 24 alkyl(hetero)aryl and C7-C 24 (hetero)arylalkyl; Y 2 is C(R 15 )2, O, S or NR 15 ; u is 0, 1, 2, 3, 4 or 5; u’ is 0, 1, 2, 3, 4 or 5, wherein u + u’ = 4, 5, 6, 7 or 8; and v is an integer in the range of 8-16.

3. The compound according to any one of the preceding claims, wherein the compound is selected from any one of the formulae I.1-I.12 wherein D and U are as defined above; or a salt, such as a pharmaceutically acceptable salt, or solvate thereof; each as a stereoisomer or in a mixture of at least two stereoisomers, wherein, in particular, a) D is -OH and U is -OAc, or b) D is -OH and U is -OH.

4. A compound of the general formula II wherein n, A, B, M, Q, D, U, X, W, L are as defined above for the compound of the general formula I; E 1 is H or an amine protecting group; and G 1 -NH-, -NH-C(=NH)NH-, -S-, -O-, -O-aryl-, -NH-(CH2) m -O-aryl-, wherein m is an integer from 1 to 10; -HN-aryl- or a moiety of the following formula or any side chain residue selected from natural or non-natural amino acids, which allows conjugation to moiety W, optionally through moiety (L) of the compound of formula II n and in particular selected from -S-CH2-, -0-CH2-, -0-p-(phenylene)-CH2-, -0-CH(CH3)-, -0-(C=0)-CH2-, -0-(C=0)-CH2-CH2-, -N-(C=0)-CH2-, -N-(C=0)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or or a salt, such as a pharmaceutically acceptable salt, or solvate thereof; each as a stereoisomer or in a mixture of at least two stereoisomers.

5. The compound according to claim 4, wherein the compound is selected from any one of the formulae II.1-II.4 wherein E 1 is H or an amine protecting group as defined above; or salts, such as pharmaceutically acceptable salts, or solvates thereof; each as a stereoisomer or in a mixture of at least two stereoisomers; and wherein, in particular a) D is -OH and U is -OAc, or b) D is -OH and U is -OH.

6. A hydrophilic trans-cyclooctene (hTCO)-functionalized construct obtainable by binding the compound according to any one of claims 1-5 to a targeting agent (TA) or a payload molecule (PM), in particular by covalent binding, wherein the functionalized construct contains at least one functionalized residue (FR) comprising a monounsaturated trans-cyclooctene entity of the general formula (II’) wherein n, A, B, D, U, M, Q, X and W are as defined above; G 1 as defined above or is absent; and L 1 has the meaning or represents a branching moiety as defined above.

7. The functionalized construct according to claim 6, which is a functionalized targeting agent (TA), wherein the targeting agent is selected from the group consisting of viruses, whole cells, phages, liposomes, biomolecules and low or high molecular weight compounds, such as, in particular, immunoglobulins, such as antibodies, antibody derivatives, antibody fragments, fusion molecules comprising at least one antibody or antibody fragment, enzymes, proteins, peptides, peptidomimetics (pseudopeptides), carbohydrates, monosaccharides, polysaccharides, oligo- or polynucleotides, in particular DNA, RNA, PNA and LNA molecules, aptamers, drugs, glycoproteins, glycans, lipids, polymers, chemotherapeutics, receptor agonists and receptor antagonists, cytokines, hormones, steroids, toxins and derivatives thereof. or which is a functionalized payload molecule (PM), wherein the payload molecule is selected from the group consisting of biologically active compounds, labeling agents, protein degrading agents, in particular payloads suitable for proteolysis-targeting chimer (PROTAC), photosensitizers and chelators.

8. The functionalized construct according to any one of claims 6 and 7, wherein the functionalizing residue (FR) of formula II’ is linked, in particular directly or via a cleavable or non-cleavable moiety, to a targeting agent (TA) or a payload molecule (PM), in particular to an amino acid residue of said TA or PM, thereby forming a functionalized TA of general formula (XX.1) wherein TA is a targeting moiety as defined above; and FR is a functionalizing residue of formula II’ above or a functionalized PM of general formula (XX.2) wherein a is 0 or represents an integer selected from 1 and 2, b is 0 or represents an integer selected from 1 and 2, c represents an integer selected to be at least 1, in particular selected from 1 and 2, Y 1 represents a cleavable moiety, Y 2 represents a self-cleaving moiety, PM is a payload molecule as defined above, and FR is a functionalizing residue of formula II’ above, wherein, when c represents an integer greater than 1, then L 1 represents a branched moiety, n is 1 and G1is absent.

9. The functionalized construct according to claim 8, which is a functionalized TA of formula XX.1, wherein TA is a targeting agent as defined above; FR is a functionalizing residue of formula II’ wherein A, B, M and Q are -CR 1 R 2 -, wherein R 1 and R 2 are as defined above; D and U are -OR 3 wherein R 3 as defined above; and n, L 1 X and W are as defined above; and G 1 as defined above, in particular any side chain residue selected from natural or non-natural amino acids, which allows conjugation to moiety W, optionally via a moiety (L) of said compound of formula II n and in particular selected from -S-CH2-, -0-CH2-, -0-p-(phenylene)-CH2-, -0-CH(CH3)-, -0-(C=0)-CH2-, -0-(C=0)-CH2-CH2-, -N-(C=0)-CH2-, -N-(C=0)-CH2-CH2-, -N-(CH2)4-, -N-(CH2)5-, or or which is a functionalized TA of formula (XX) wherein TA is a targeting moiety as defined above, FR is a functionalizing residue of formula wherein L 1 and n is as defined above, A, B, M and Q are -CR 1 R 2 - D and U are -OR 3 , X and W are as defined above; G 1 is absent or represents a moiety formed by a conventional coupling reaction, in particular a click reaction, such as in particular wherein J is selected from -NH-, -S- or -O- or is absent; or or which is a functionalized PM of formula XX.2, wherein a is 0 or represents an integer selected from 1 and 2, b is 0 or represents an integer selected from 1 and 2, c represents an integer selected to be at least 1, in particular selected from 1 and 2, Y 1 represents a cleavable moiety as defined above; and Y 2 represents a self-cleaving moiety as defined above, PM is a payload moiety as defined above, FR is a functionalizing residue of formula II’ wherein A, B, M and Q are -CR 1 R 2 -, wherein R 1 and R 2 are as defined above; D and U are -OR 3 wherein R 3 as defined above; and n, L 1 X and W are as defined above; with the proviso that when c represents an integer greater than 1, then L 1 represents a branched moiety, n is 1.

10. A conjugate prepared by covalently linking a first functionalizing molecule selected from the hyTCO-functionalized constructs of any one of claims 6-9 with a second functionalizing molecule comprising a docking group (DG) capable of reacting with the trans-cyclooctene type functional group of the first molecule; in particular obtainable by double ortho-bioconjugation via Diels-Alder type cycloaddition of said two functionalizing molecules; more particularly wherein the docking group (DG) is selected from an optionally substituted triazinyl or an optionally substituted tetrazinyl group, capable of covalently reacting with the trans-cyclooctene group in a copper-strain promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC).

11. The conjugate according to claim 10, wherein the hyTCO-functionalized construct is selected from a functionalized targeting agent TA of general formula (XX.1) wherein FR is a functionalizing residue of formula II’, and TA is a targeting moiety as defined above; or a functionalized PM of general formula (XX.2) wherein a is 0 or represents an integer selected from 1 and 2, b is 0 or represents an integer selected from 1 and 2, c represents an integer selected to be at least 1, in particular selected from 1 and 2, Y 1 represents a cleavable moiety, Y 2 represents a self-cleaving moiety, FR is a functionalized residue of formula II', wherein, when c represents an integer greater than 1, then L1 represents a branching moiety, n is 1, and G1 is absent; and PM is a payload molecule as defined above.

12. Conjugate according to any one of claims 10 and 11, wherein the second functionalized molecule comprising a docking group (DG) is selected from a functionalized targeting agent TA of general formula (XX.3) or a functionalized payload molecule PM of general formula (XX.4) wherein a, b and c and Y 1 and Y 2 as defined above; TA is a targeting moiety as defined above, and PM is a payload molecule as defined above, and DG is a docking group as defined above.

13. Conjugate according to any one of claims 10 to 12, wherein the docking group (DG) group is a diene comprising a group selected from tetrazine or triazine, which group is capable of covalently reacting with the trans-cyclooctene group of formula II’ above in a copper-strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC).

14. Conjugate according to any one of claims 10 to 13, wherein the TA is an immunoglobulin molecule, in particular a monoclonal antibody, or a derivative or antigen binding fragment thereof, carrying at least one non-canonical amino acid residue (ncAA) in the polypeptide chain, wherein the ncAA carries a hyTCO type side chain as defined in any one of claims 1 to 5.

15. A pharmaceutical composition comprising at least one conjugate as defined in any one of claims 10 to 14 in a pharmaceutically acceptable carrier, or a diagnostic composition comprising at least one conjugate as defined in any one of claims 10 to 14 in a diagnostic suitable carrier.

16. Conjugate as defined in any one of claims 10 to 14 for use in medicine, in particular for use in diagnosis and therapy.

17. Conjugate as defined in any one of claims 10 to 14, which is an ADC, for use in diagnosis or therapy of cancer, in particular breast cancer, gastric cancer or other Her2 overexpressing tumors, such as ovarian, endometrial, bladder, lung, colon and head and neck tumors.

18. A diagnostic or analytical kit comprising at least one hyTCO type compound construct or conjugate as defined in any one of claims 1 to 14.

Images