Hydrophilic azadibenzocyclooctyne derivatives and their metal-free click reactions with hydrophilic azadibenzocyclooctyne derivatives

Abstract

In a first aspect, the present invention relates to an azadibenzocyclooctyne derivative according to formula (I) having a specific substituent on the benzo ring of the DIBAC structure and a specific substituent bonded to the nitrogen atom of the DIBAC structure, or a salt thereof. 6 is the linker structure -C(=O)-[L] n The present invention relates to a complex of formula (II) which is linked to the N atom of the 8-membered ring of the DIBAC structure via -Z-. A third aspect of the present invention relates to a method for modifying a target molecule, comprising reacting a complex according to the second aspect with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group. In a fourth aspect, the present invention relates to the use of a complex according to the second aspect for bioorthogonal labeling and / or modification of a target molecule. A fifth aspect of the present invention relates to a modified target molecule comprising a reaction product of a complex according to the second aspect, and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group obtained or obtainable from the method of the third aspect. In a sixth aspect, the present invention relates to a kit comprising a modified target molecule according to the fifth aspect as a detection reagent and a suitable capture reagent.

Description

[Technical Field]

[0001] In a first aspect, the present invention relates to an azadibenzocyclooctyne derivative or a salt thereof according to formula (I) having a specific substituent on the benzo ring of the DIBAC structure and a specific substituent bonded to the nitrogen atom of the DIBAC structure. 6 is the linker structure -C(=O)-[L] n The present invention relates to a conjugate of formula (II) bonded to the N atom of the eight-membered ring of the DIBAC structure via -Z-. A third aspect of the present invention relates to a method for modifying a target molecule, comprising reacting a conjugate according to the second aspect with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group. In a fourth aspect, the present invention relates to the use of a conjugate according to the second aspect for bioorthogonal labeling and / or modification of a target molecule. A fifth aspect of the present invention relates to a modified target molecule comprising a reaction product of a conjugate according to the second aspect, and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group obtained or obtainable from the method of the third aspect. In a sixth aspect, the present invention relates to a kit comprising a modified target molecule according to the fifth aspect as a detection reagent and a suitable capture reagent. [Background technology]

[0002] Bioorthogonal reactions are widely used in modern chemical biology for the modification of biomolecules. Herein, the [3 + 2] cycloaddition of azides with alkynes, which leads to stable 1,2,3-triazoles, i.e., the so-called strain-promoted azide-alkyne cycloaddition (SPAAC), holds a prominent position because it is the most frequently used. Another approach is the so-called strain-promoted alkyne-nitrone cycloaddition (SPANC). While these reactions generally require copper catalysis, there are variants that exploit the inherent ring strain of cyclic octynes ​​(strain-promoted azide-alkyne cycloaddition, SPAAC), thereby circumventing the need for any catalyst.

[0003] International Publication No. 2014 / 189370 discloses substituted dibenzoazacyclooctyne (DIBAC) derivatives with specific substituents on the benzo ring. Debets et al. (Chem. Commun. 2010, 46, 97-99) also describe DIBAC derivatives with specific substituents on the N atom of the eight-membered ring of the DIBAC structure. Synthetic routes for preparing DIBAC analogs have also been disclosed by Debets et al. (Org. Biomol. Chem., 2014, 12, 5031-5037).

[0004] Nevertheless, known DIBAC derivatives are generally hydrophobic molecules, resulting in complexes that are poorly soluble in aqueous solutions and prone to undesirable hydrophobic interactions. To date, no sufficiently water-soluble derivatives have been reported.

[0005] Therefore, the technical problem underlying the present invention was the need for DIBAC derivatives with improved solubility in aqueous solutions and minimal hydrophobic interactions. Summary of the Invention

[0006] This problem is solved by the present invention with the features of the independent patent claims. Advantageous developments of the invention, which may be realized individually or in combination, are set out in the dependent patent claims and / or in the following description and detailed embodiments.

[0007] When used below, the terms "have", "comprise" or "include", or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms may refer both to a situation in which the entity described in this context has no further features other than those introduced by these terms, and to a situation in which one or more further features are present. As an example, the expressions "A has B", "A includes B", and "A comprises B" may refer both to a situation in which no other elements exist in A besides B (i.e., a situation in which A consists only of B), and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D, or further elements.

[0008] Furthermore, it should be noted that the terms "at least one," "one or more," or similar expressions indicating that a feature or element may be present one or more times are typically used only once when introducing each feature or element. Hereinafter, in most cases, when referring to each feature or element, the expressions "at least one" or "one or more" will not be repeated, despite the fact that each feature or element may be present one or more times.

[0009] Furthermore, as used below, the terms "preferably," "more preferably," "particularly," "more particularly," "particularly," "more particularly," or similar terms are used in conjunction with optional features without limiting the possibility of substitution. Features introduced by these terms are therefore optional features and are not intended to limit the scope of the claims in any way. The present invention can be implemented by using alternative features, as those skilled in the art will recognize. Similarly, features introduced by "in an embodiment of the present invention" or similar expressions are intended to be optional features, without any limitations regarding alternative embodiments of the invention, without any limitations regarding the scope of the invention, and without any limitations regarding the possibility of combining features introduced in such a way with other optional or non-optional features of the invention.

[0010] First Aspect—Azadibenzocyclooctyne Derivatives In a first aspect, the present invention provides a compound of formula (I) [ka] (In the formula, R 1 , R 2 is, independently, -[(CH2) a CR x R y ] b R z a group (wherein a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - Based on: -Rz (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - Not a group, c is 0; and --[CR r R s ] d -R t a group wherein d is an integer selected from the range of 1 to 10, and R r represents a hydrogen atom, a hydroxyl group, and -[CR'(OH)] e -H groups, and R s is a hydrogen atom or - -[CR”(OH)] f -H group, and R t represents a hydrogen atom, a C1-C5 alkyl group, and -[CR"'(OH)] g -H groups (each R', R", and R"' is independently a hydrogen atom or a -[CH(OH)] h -H groups, and each of d, e, f, g and h is R t is a hydrogen atom or a C1-C5 alkyl group, R r , R s are independently integers selected from the range of 1 to 10, provided that at least one of is not a hydrogen atom; R 3 , R 4 are independently selected from the group consisting of a hydrogen atom, a C1-C3-alkyl group, a halogen atom, and an —O—C1-C3-alkyl group; R 5 is a carboxyl group, an activated carboxyl group and -NHR 5a R is selected from the group consisting of 5ais a hydrogen atom or a C1-C5 alkyl group; L comprises a chain of covalently bonded atoms (linker) forming the backbone and having a length in the range of 1 to 100 atoms; n is R 5 is either 0 or 1 if it is a carboxyl group or an activated carboxyl group, and R 5 Ga-NHR 5a group, the value is 1.) or a salt thereof.

[0011] When the index "n" of the linker L is 0, the linker L is absent and C=O and R 5 The C atoms of R are directly connected by a single bond. 1 , R 2 - [CR r R s ] d -R t The group represents a linear or branched polyhydroxyl structure. 1 , R 2 -[(CH2) a CR x R y ] b R z The group represents the residue of at least one sulfonic acid group.

[0012] The azadibenzocyclooctyne derivatives according to formula (I) or their salts have significantly improved solubility in aqueous solutions and therefore have wide suitability for use in aqueous systems. r R s ] d -R t The azadibenzocyclooctyne derivative according to formula (I) having a group or a salt thereof can now be converted into a [(CH2) a CR x R y ] b R zThe hydrophilicity of the compounds of formula (I) having a 1,3-dipolar group is further improved. Furthermore, the azadibenzocyclooctyne derivatives or salts thereof according to formula (I) can avoid undesirable hydrophobic interactions such as protein aggregation, thereby avoiding potential non-specific binding in diagnostic assays. The azadibenzocyclooctyne derivatives or salts thereof according to formula (I) described herein optionally have additional substituents, such as the complexes of formula (II) described in more detail herein below, and when reacted with target molecules containing 1,3-dipole groups or 1,3-(hetero)diene groups, avoid the need for a copper catalyst for cycloaddition.

[0013] The linker L in Formula (I) comprises, and preferably consists of, a chain of atoms forming a backbone, the backbone having a length ranging from 1 to 100 atoms, preferably from 4 to 50 atoms, more preferably from 5 to 20 atoms, and more preferably from 6 to 15 atoms. All atoms forming the backbone are covalently bonded to each other. In one embodiment, the backbone consists of carbon atoms and one or more heteroatoms selected from O, N, and S, and optionally contains at least one aryl, heteroaryl, substituted aryl, or substituted heteroaryl group (e.g., a phenylene ring occupies a length of 4 atoms). Heteroatoms at internal positions are unsubstituted or have one or more substituents selected from the group consisting of hydrogen atoms, C1-C5 alkyl, and =O. In some embodiments, one or more heteroatoms are part of a linkage, and this linkage is preferably selected from the group consisting of an amide bond, an ester bond, an ether bond, a carbamate bond, and a urea bond. Carbon atoms in the backbone chain are substituted with one or more substituents selected from the group consisting of hydrogen atoms and C1-C10 alkyl groups. The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight-chain, branched-chain, or cyclic hydrocarbon radical, or combination thereof, having the specified number of carbon atoms (i.e., C1-C10 means 1 to 10 carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of n-pentyl, n-hexyl, and the like. In one embodiment, the backbone is composed of two or more straight-chain alkyl chain segments with one or more heteroatoms between the segments. Preferably, the backbone is composed of two or more straight-chain alkyl chain segments, preferably unsubstituted, having a length ranging from 6 to 15 atoms, with one or more heteroatoms between the segments, the one or more heteroatoms being selected from O and N.More preferably, the backbone consists of two linear alkyl chain segments having a length in the range of 6 to 10 atoms, preferably unsubstituted, and the bond between the segments is selected from the group consisting of an ether bond, a urea bond, a carbamate bond and an amide bond.

[0014] Salts of azadibenzocyclooctyne derivatives R 1 and / or R 2 Ga-SO3 - When the group is present, the group is present in deprotonated form and the negative charge is preferably mediated by an alkali metal cation, preferably Na + or K + , and trialkylammonium cation NR k R p R q is compensated by a suitable cation selected from k , R p , R q are independently a C1 to C6 alkyl group, and more preferably R k , R p , R q are the same and each is a C1 to C6 alkyl group. 5 is a carboxyl group, the group may exist in either its protonated or its deprotonated form, and the negative charge is preferably borne by an alkali metal cation, preferably Na + or K + , and trialkylammonium cation NR k R p R q is compensated by a suitable cation selected from k , R p , R q are independently a C1 to C6 alkyl group, and more preferably R k , R p , R q are the same and each is a C1 to C6 alkyl group. A preferred trialkylammonium cation is N,N,N-triethylammonium.

[0015] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 3 , R 4 are independently a hydrogen atom or a methyl group, and preferably R 3 , R 4 are identical and are each a hydrogen atom.

[0016] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 In each of the formulas, a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - It is a base; -R z (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - It is not a group, and c is 0.

[0017] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 In each of the formulas, a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x and R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R y , is a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - wherein c is an integer ranging from 1 to 4; R x , R y , R z At least one of the following is (CH2) c SO3 - It is based (However, R x (CH2) c SO3 - group, and when c is 0, R z (CH2) c SO3 - - group, provided that c is not 0.

[0018] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 wherein, independently, a is 0 or an integer ranging from 1 to 4, b is 1, and Rx , R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R y is a water atom, a C1-C3 alkyl group, and (CH2) c SO3 - wherein c is an integer ranging from 1 to 4; R x , R y , R z At least one of (CH2) c SO3 - It is based (However, R x (CH2) c SO3 - group, and when c is 0, R z (CH2) c SO3 - - group, provided that c is not 0.

[0019] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 wherein, independently, a is 0 or an integer ranging from 1 to 4, b is 1, and R x , R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R y is a hydrogen atom, R x , R z At least one of the following is (CH2) c SO3 - It is based (However, Rx (CH2) c SO3 - group, and when c is 0, R z (CH2) c SO3 - - group, provided that c is not 0.

[0020] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 In each of the formulas, a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R z is a hydrogen atom and (CH2) c SO3 - wherein c is either 0 or an integer ranging from 1 to 4; R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - It is the base. -R z (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - It is not a group, and c is 0.

[0021] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R1 , R 2 are the same, and both are [(CH2) a CR x R y ] b R z is a group, and b is 0 or 1. The indices "a" and R x , R y , R z is as defined above.

[0022] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are respectively -[CR r R s ] d -R t group, and each R 1 , R 2 Regarding, independently, R r represents a hydrogen atom, a hydroxyl group, and -[CH(OH)] e -H, and R s is a hydrogen atom or -[CH(OH)] f -H group, and R t represents a hydrogen atom, C1-C5 alkyl, and -[CH(OH)] g -H groups, and each of d, e, f, and g is selected from the group consisting of R t is a hydrogen atom or a C1-C5 alkyl group, R r , R s are independently an integer selected from the range of 1 to 10, provided that at least one of them is not a hydrogen atom.

[0023] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R 1 , R 2 are identical and are -[CH(OH)] d It is a —H group, and d is an integer selected from the range of 1 to 10, preferably from the range of 1 to 5, and more preferably 2 or 3.

[0024] According to one embodiment of the azadibenzocyclooctyne derivative or salt thereof, R5 is an activated carboxyl group, and R 5 is selected from the group consisting of a 4-nitrophenyl group, a pentafluorophenyl group and an N-succinimidyl group, and is preferably an N-succinimidyl group.

[0025] R 5 is a carboxyl group, the group may be activated in situ by, for example, HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), HBTU (3-[bis(dimethylamino)methyliumyl]-3H-benzotriazole-1-oxide hexafluorophosphate), a carbodiimide preferably selected from the group consisting of N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), or a phosphonium salt, preferably benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

[0026] According to an embodiment of the azadibenzocyclooctyne derivative or salt thereof, L has the structure -(CH) p -(X) m -(CH2) q - and p and q are independently integers selected from the range of 2 to 10; X is selected from the group consisting of -C(=Y)-NH-, -NH-C(=Y)-, -C(=Y)-O- and -OC(=Y)-, where Y is an oxygen atom or a sulfur atom; m is 0 or 1. [When m is 0, X is absent, and (CH2) p , (CH2) q are directly connected by a single bond].

[0027] According to one embodiment of the azadibenzocyclooctyne derivative or a salt thereof, m is 1, X is a —C(═O)—NH— group, and / or p and q are the same and are both integers selected from the range of 2 to 5, preferably 2 or 3.

[0028] According to one embodiment, the azadibenzocyclooctyne derivative or salt thereof has the formula (Ia), (Ib) or (Ic): [ka] [ka] [ka] (Wherein L, n and R 5 is as defined above).

[0029] According to one embodiment, the azadibenzocyclooctyne derivative or salt thereof has the formula (Ia-1), (Ib-1) or (Ic-1): [ka] [ka] [ka] In the formula, R 5 is as defined above).

[0030] According to one embodiment, the azadibenzocyclooctyne derivative or salt thereof has the formula (Ia-1) or (Ib-1), preferably (Ia-1). [ka] [ka]

[0031] Second embodiment - complex In a second aspect, the present invention provides a compound of formula (II) [ka] (In the formula, L, R 1 , R 2 , R 3 , R 4 and n is as defined above in relation to the first aspect for the azadibenzocyclooctyne derivative of formula (I) or a salt thereof; R 6 is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide; preferably a metal complex; Z is -C(=O)-O-, C(=O)-NR 7 -, and -NR 7 -C(=Y)-, R 7 is a hydrogen atom or a C1 to C5 alkyl group, and Y is an oxygen atom or a sulfur atom, preferably an oxygen atom.

[0032] All definitions given above in the section relating to the first aspect also apply in relation to the conjugate of the second aspect.

[0033] According to one embodiment of the conjugate of formula (II), R 6 is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a metal complex, a radioisotope, an active pharmaceutical ingredient (drug), a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide and a polynucleotide, preferably a metal complex, and a further linker selected from the group consisting of a chain, a polypropylene glycol chain and a mixed polyethylene / polypropylene glycol chain is connected between Z and R 6 It either exists or does not exist between.

[0034] The metal complex is preferably a ruthenium(II) or iridium(III)-based complex, more preferably a ruthenium(II) or iridium(III)-based electrochemiluminescent complex. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a highly sensitive and selective method. ECL combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. In general, ruthenium(II) complexes, especially [Ru(bpy)3], are regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface. 2+ (which emits photons at about 620 nm) is used as an ECL label. Ruthenium(II) complexes that can be used as ECL labels are described in WO 2003 / 002974. Iridium(III) complexes that can be used as ECL labels are also described in WO 2012 / 107419, WO 2012 / 107420, and WO 2014 / 019709 and WO 2014 / 019708. Preferably, the iridium(III) complex is Ir 3+ and two substituted or unsubstituted 6-phenylphenanthridine ligands, optionally modified pyridine-2-carboxylic acid or 2-(1H-pyrazol-3-yl)pyridine. In particular, to allow for further conjugation, the 2-(1H-pyrazol-3-yl)pyridine is modified with a reactive unit, for example, the 2-(1H-pyrazol-3-yl)pyridine ligand is substituted with a 3-alkylcarboxylic acid group, which can be activated by an NHS group for conjugation.

[0035] Additional metal complexes suitable as labels for imaging and therapeutic purposes are known in the art (see, e.g., WO 2017 / 153574).

[0036] Radiolabeling employs radioactive isotopes (radionuclides), such as H, C, C, F, P, S, Cu, Gn, Y, Zr, TC, In, I, I, I, I, Xe, Lu, At, or Bi.

[0037] "Fluorophores" include rare earth chelates (europium chelates), fluorescein-type labels including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine-type labels including TAMRA, Lissamine, Texas Red; dansyl; cyanines; coumarins, phycoerythrin; and analogs thereof. Fluorescent labels can be attached to the azadibenzocyclooctyne derivatives of the present invention using methods known to those skilled in the art, preferably via amide bond formation. Fluorescent and non-fluorescent dyes, including Alexa, Atto, and DY dyes, and labeling reagents are commercially available from, for example, Invitrogen / Molecular Probes (Eugene, OR, USA), ThermoFisher Scientific (Waltham, MA, USA), Sigma Aldrich, Atto-Tec GmbH (Siegen), Dyomics GmbH (Jena), and Pierce Biotechnology, Inc. (Rockford, IL). For example, "fluorescence quenchers," such as black hole quenchers (BHQ), are known to those of skill in the art. "Dyes," such as dabsyl or azo dyes like dabsyl, are also known to those of skill in the art.

[0038] A "hapten" is an organic molecule having a molecular weight of 100 to 2000 daltons. In one embodiment, the hapten has a molecular weight of 100 to 1000 daltons. Typically, organic molecules of such molecular weight are not immunogenic or have relatively low immunogenicity. A hapten can be made immunogenic by conjugation to a carrier molecule, and anti-hapten antibodies can be generated according to standard procedures. In one embodiment, the hapten can be selected from the group consisting of sterols, bile acids, sex hormones, corticoids, cardenolides, cardenolide-glycosides, bufadienolides, steroid sapogenins, and steroid alkaloids, cardenolides, cardenolide-glycosides, and vitamins. Representative examples of these substance classes are digoxigenin, digitoxigenin, gitoxigenin, strophanthidin, digoxin, digitoxin, strophanthin fluorescein, biotin, and dinitrophenyl.

[0039] "Tyramine" is 4-(2-aminoethyl)phenol. When coupled to a label via the corresponding amide, it is used as a reagent for tyramide signal amplification by activation with horseradish peroxidase (HRP), e.g., antibody-HRP conjugates (see, e.g., Perkin-Elmer, ThermoFisher).

[0040] An "oligopeptide" is a peptide containing between 2 and 9 amino acid residues. A "polypeptide" is a peptide containing at least 10 amino acid residues. In some embodiments, the peptide contains at least 10 amino acid residues, or at least 20 amino acid residues. In some embodiments, the peptide contains 1000 or fewer amino acid residues, e.g., 500 or fewer amino acid residues, e.g., 100 or fewer amino acid residues. In some embodiments, the polypeptide is an enzyme or an antibody.

[0041] An "oligonucleotide" contains anywhere from two to nine covalently linked nucleotide monomers. A "polynucleotide" contains at least 10 covalently linked nucleotide monomers. In some embodiments, a polynucleotide contains 1000 or fewer nucleotide monomers. Oligonucleotides and / or polynucleotides are either single-stranded or double-stranded. The terms oligonucleotide or polynucleotide should be interpreted broadly and include DNA and RNA, as well as analogs and modifications thereof. "Analogs" can include, for example, substituted nucleotides with substitutions for the standard bases adenine, guanine, cytosine, thymine, and uracil. Examples of such nucleosides containing substituted nucleobases include 5-substituted pyrimidines such as 5-methyl-dC, aminoallyl-dU or -dC, 5-(aminoethyl-3-acrylimido)-dU, 5-propynyl-dU or -dC, and 5-halogenated dU or dC; N-substituted pyrimidines such as N4-ethyl-dC; N-substituted purines such as N6-ethyl-dA and N2-ethyl-dG; 8-substituted purines such as 8-[(6-amino-hex-1-yl)-amino]-dG or -dA, 8-halogenated dA or dG, and 8-alkyl-dG or -dA; and 2-substituted dA such as 2-amino-dA. "Analog" can include nucleotide or nucleoside analogs. That is, naturally occurring nucleobases can be replaced by using nucleobase analogs such as 5-nitroindole-d-riboside; 3-nitropyrrole-d-riboside, deoxyinosine (dI), deoxyxanthosine (dX); 7-deaza-dG, -dA, -dI, or -dX; 7-deaza-8-aza-dG, -dA, -dI, or -dX; 8-aza-dA, -dG, -dI, or -dX; d-formycin; pseudo-dU; pseudo-iso-dC; 4-thio-dT; 6-thio-dG; 2-thio-dT; iso-dG; 5-methyl-iso-dC; N-linked 8-aza-7-deaza-dA; 5,6-dihydro-5-aza-dC; and etheno-dA or pyrrolo-dC. As will be apparent to those skilled in the art, in the case of a double strand, the nucleobases in the complementary strand must be selected so that duplex formation is specific.For example, if 5-methyl-iso-dC is used in one strand (e.g., (a)), iso-dG must be in the complementary strand (e.g., (a')). In "analogs," the oligo- / polynucleotide backbone may be modified to contain substituted sugar residues, sugar analogs, modifications of the internucleoside phosphate moiety, and / or may be PNA. Oligonucleotides may contain nucleotides with substituted deoxyriboses, such as 2'-methoxy, 2'-fluoro, 2'-methylseleno, 2'-allyloxy, 4'-methyl-dN (where N is a nucleobase, e.g., A, G, C, T, or U).

[0042] In some preferred embodiments, R 6 is an oligonucleotide or polynucleotide, preferably a single-stranded DNA (ssDNA) having in the range of 4 to 12 nucleotides, more preferably in which all nucleotides are non-natural nucleotides, i.e., including nucleotide analogs or nucleoside analogs.

[0043] In some preferred embodiments, R 6 is an oligonucleotide or polynucleotide, preferably an LNA gapmer ("LNA" stands for locked nucleic acid, a "gapmer" is a short DNA antisense oligonucleotide structure with RNA-like segments on either side of the sequence; see PH Hagedorn et al., Drug Discovery Today 2018, 23(1), 101-114).

[0044] In some preferred embodiments, R 6 is an oligonucleotide or polynucleotide, preferably a beta-L-LNA single strand ("beta-L-LNA" refers to the L-configuration stereoisomer of LNA, see WO 2019 / 243391, WO 2020 / 245377).

[0045] Sugar analogs include, for example, xylose; 2',4'-bridged ribose, such as (2'-O,4'-C-methylene)-bridged ribose (oligomer known as LNA) or (2'-O,4'-C-ethylene)-bridged ribose (oligomer known as ENA); L-ribose, Ld-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (oligomer known as ANA); tricyclic ribose analogs in which the C3' and C5' atoms are linked by an ethylene bridge fused to a cyclopropane ring (oligomer known as tricycloDNA); glycerol (oligomer known as GNA); glucopyranose (oligomer known as homoDNA); carbaribose (containing cyclopentane instead of tetrahydrofuran subunits); and hydroxymethyl-morpholine (oligomer known as morpholinoDNA).

[0046] Many modifications, including modified internucleoside phosphate moieties, are also known to not interfere with hybridization properties, and such backbone modifications can be combined with substituted nucleotides or nucleotide analogs. Examples are phosphorothioate, phosphorodithioate, phosphoramidate, and methylphosphonate oligonucleotides.

[0047] PNAs (which have a backbone that does not contain phosphate and d-ribose) can also be used as DNA analogs.

[0048] The "solid phase" is typically glass or a polymer, with the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. As will be appreciated by those skilled in the art, the solid phase may contain aldehyde functional groups by its nature or may be chemically modified to introduce aldehyde groups. As will be further appreciated, the solid phase may be coated with any of polypeptides, carbohydrates, nucleotides, and nucleic acids. In some embodiments, the solid phase is coated with streptavidin. The solid phase may be in the form of a microplate tube, bead, or disk. In one embodiment, the solid phase is glass or a paramagnetic bead based on any of the aforementioned polymers.

[0049] A "carbohydrate" is a biological molecule composed of carbon (C), hydrogen (H), and oxygen (O) atoms, usually in a hydrogen-to-oxygen atomic ratio of 2:1 (as in water). In other words, the carbohydrate has the empirical formula C v (H2O) w where v is usually the same as w. Some exceptions exist (v is different from w); for example, deoxyribose, the sugar component of DNA, has the empirical formula C5H10O4. Carbohydrates are technically hydrates of carbon; structurally, it is more accurate to consider them as polyhydroxy aldehydes and ketones.

[0050] The term carbohydrate is most common in biochemistry and is a synonym for "saccharides," a group of molecules that includes sugars, starches, and cellulose. In one embodiment, the carbohydrate is selected from sugars, starches, and cellulose.

[0051] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity.

[0052] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with experimental, diagnostic, or therapeutic uses of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibodies will be prepared by at least one purification step.

[0053] "Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0054] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0055] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular toxicity.

[0056] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0057] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed., WB Saunders, Co. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0058] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment, as defined below. These terms specifically refer to an antibody having a heavy chain that includes an Fc region.

[0059] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0060] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0061] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0062] Fab fragments contain heavy and light chain variable domains, and also contain a light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0063] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, for example, Plueckthun, In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994) pp. 269-315.

[0064] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, generating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in detail, for example, in EP 0404097; WO 1993 / 01161; Hudson, PJ et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., PNAS USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, PJ et al., Nat. Med. 9 (2003) 129-134).

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified, for example, to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, or create a multispecific antibody, and that an antibody comprising a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0066] A "lipid" is a substance, preferably natural, that is completely or at least largely insoluble in water (hydrophobic), but is highly soluble in hydrophobic (or lipophilic) solvents such as hexane due to its low polarity. The lipid is preferably selected from the group consisting of fatty acids, triglycerides (fats and fatty oils), waxes, phospholipids, sphingolipids, lipopolysaccharides, and isoprenoids. In some embodiments, the isoprenoid is a steroid. A "steroid" is a derivative of a hydrocarbon sterane.

[0067] An "active pharmaceutical ingredient" includes any pharmaceutically active chemical or biological compound, and any pharmaceutically acceptable salts thereof, and any mixtures thereof, that provides some pharmacological effect and is used to treat or prevent a pathological condition or disease. Preferably, particularly in the context of antibody-drug-conjugates (ADCs), the active pharmaceutical ingredient is a toxin or cytotoxin, such as amanitin or maytansine.

[0068] The meaning of the terms polyethylene glycol chain, polypropylene glycol chain and mixed polyethylene / polypropylene glycol chain will be clear to those skilled in the art. Preferably, each of these chain types has an average molecular weight in the range of 100 to 10,000 Da.

[0069] According to one embodiment, the conjugate has formula (IIa), (IIb) or (IIc): [ka] [ka] [ka] (Wherein L, n and R 6 has the same meaning as above, and Z 1 is a -C(=O)-O- group or a -C(=O)-NH- group.

[0070] According to one embodiment, the conjugate has formula (IIa-1), (IIa-2), (IIb-1), (IIb-2), (IIc-1) or (IIc-2): [ka] [ka] [ka] [ka] [ka] [ka] (Wherein, R in (IIa-1), (IIa-2), (IIb-1), (IIb-2), (IIc-1) or (IIc-2) 6 has the same meaning as above.

[0071] Third Aspect—Method of Modifying a Target Molecule In a third aspect, the present invention relates to a method for modifying a target molecule, comprising reacting a conjugate according to the second aspect with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group.

[0072] The reaction is carried out by strain-promoted cycloaddition of the cycloalkyne derivative of the present invention with, for example, azides (SPAAC). The reaction of cyclooctynes ​​with 1,3-(hetero)dienes is also known as the (hetero)Diels-Alder reaction. These reactions are also called metal-free click reactions. The alkoxy-substituted core increases the rate of cycloaddition compared to commercially available derivatives. The use of the complex according to the second aspect for the reaction with the target molecule avoids the need for a copper catalyst for the cycloaddition; i.e., the reaction is preferably carried out in the absence of a copper catalyst, more preferably in the absence of a catalyst.

[0073] With respect to the conjugate, all definitions apply as set out above in the section relating to the second aspect.

[0074] According to one embodiment of the method for modifying a target molecule, the 1,3-dipole group is selected from the group consisting of azide, nitrone, diazoalkane, diazoacetamide and nitrile oxide, preferably azide.

[0075] According to one embodiment of the method for modifying a target molecule, the 1,3-diene group is selected from the group consisting of 1,3-butadiene, 1,3-cyclopentadiene, 1,3-cyclohexadiene, furan and pyrrole.

[0076] According to one embodiment of the method for modifying a target molecule, the 1,3-heterodiene group is selected from the group consisting of tetrazine, 1-oxa-1,3-butadiene, 1-aza-1,3-butadiene, 2-aza-1,3-butadiene and 3-aza-1,3-butadiene.

[0077] According to one embodiment of the method for modification, the target molecule is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide; preferably a polypeptide, more preferably an antibody, more preferably a modified antibody with a 1,3-dipole group, more preferably a modified antibody with an azide group.

[0078] The terms fluorophore, fluorescence quencher, dye, hapten, tyramine, polyethylene glycol chain, polypropylene glycol chain, mixed polyethylene / polypropylene glycol chain, metal complex, radioisotope, steroid, active pharmaceutical ingredient, carbohydrate, solid phase, amino acid, oligopeptide, polypeptide, nucleotide, oligonucleotide and polynucleotide have the meanings explained above.

[0079] In some embodiments, R of the conjugate of formula (II) 6is selected from the group consisting of fluorophores, fluorescence quenchers, dyes, haptens, tyramines, metal complexes, active pharmaceutical compounds (drugs), solid phases, oligonucleotides, polynucleotides, lipids, polypeptides, in particular enzymes or antibodies, oligopeptides, polypeptides and polyethylene glycol, and preferably the target molecule is selected from the group consisting of antibodies, oligonucleotides and polynucleotides. With regard to oligo- and polynucleotides, it is preferred in some embodiments that these are antisense oligonucleotides such as LNA gapmers or L-LNA single strands. Thus, the complex according to the second aspect and the target molecule (herein also abbreviated as "target molecule complex" (hereinafter 第5 Conjugates obtained or obtainable from the reaction with an antibody-oligonucleotide conjugate (see embodiment (2)) may include, but are not limited to, antibody-metal complex conjugates, antibody-drug conjugates, antibody-oligonucleotide conjugates, antibody-solid-phase conjugates, antibody-fluorophore conjugates, antibody-fluorescence quencher conjugates, antibody-hapten conjugates, antibody-enzyme conjugates, antibody-antibody-antibody-antibody conjugates, antibody-polyethylene glycol conjugates, oligonucleotide-oligo- or polypeptide conjugates, oligonucleotide-lipid conjugates, oligonucleotide-solid-phase conjugates, tyramide conjugates, etc. Examples of antibody-oligonucleotide conjugates include, but are not limited to, antibodies modified with antisense oligonucleotides such as LNA gapmers (PH Hagedorn et al., Drug Discovery Today 2018, 23(1), 101-114) or L-LNA single strands (WO 2019 / 243391, WO 2020 / 245377). Antibody-oligonucleotide conjugates and their use for targeted delivery are described, for example, in WO 2020 / 247738. The antibody is as defined above and is more preferably selected from IgG and Fab fragments.

[0080] Fourth Aspect—Use of the Conjugate A fourth aspect of the invention relates to the use of a conjugate according to the second aspect for the bioorthogonal labelling and / or modification of a target molecule.

[0081] All definitions given above in the section relating to the second aspect also apply with respect to the uses described herein. With respect to the choice of target molecule or bioorthogonal label and / or modification, all definitions apply as given above in the section relating to the third aspect.

[0082] Fifth embodiment - modified target molecule In a fifth aspect, the present invention relates to a modified target molecule comprising the reaction product of a conjugate according to the second aspect, and to a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group obtained or obtainable from the method of the third aspect.

[0083] All definitions given above in the section relating to the second aspect also apply with respect to the uses described herein. With respect to the choice of target molecule or bioorthogonal label and / or modification, all definitions apply as given above in the section relating to the third aspect.

[0084] The reaction product of the conjugate according to the second embodiment with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group, also abbreviated as "target molecule conjugate", allows for a reduction of the background signal conjugate compared to the reaction product of a reference DBCO conjugate according to the state of the art. Without being bound by theory, it is hypothesized that this reduction of the background signal may be due to hydrophilization (by sulfonation or hydroxylation) of otherwise hydrophobic moieties.

[0085] Stability studies revealed higher recovery of ECL signal for the target molecule conjugates of the present invention compared to the state-of-the-art reference DBCO conjugate.

[0086] Sixth Aspect—Kit A sixth aspect of the invention comprises a modified target molecule according to the fifth aspect as a detection reagent, wherein the target molecule is preferably an antibody and R 6 is preferably a metal complex and is a suitable capture reagent.

[0087] All definitions given above in the section relating to the second aspect also apply with respect to the uses described herein. With respect to the choice of target molecule or bioorthogonal label and / or modification, all definitions apply as given above in the section relating to the third aspect.

[0088] As indicated above, the target molecule is preferably an antibody, preferably as defined above in the second related aspect of the third embodiment, more preferably selected from IgG and Fab fragments. As mentioned above, the metal complex is preferably a ruthenium(II)-based complex or an iridium(III)-based complex as described above in the section relating to the third embodiment. A solid phase is also preferably part of the kit, said solid phase being as defined above in the section relating to the third embodiment, preferably having a streptavidin coating.

[0089] The present invention is further exemplified by the following embodiments and combinations of embodiments shown by each dependent and subsequent reference. In particular, in each instance where a range of embodiments is mentioned, it should be noted that in the context of a term such as "the process of any one of embodiments 1 to 4," all embodiments within this range are meant to be expressly disclosed to those skilled in the art, that is, the wording of this term should be understood by those skilled in the art as being synonymous with "the process of any one of embodiments 1, 2, 3, and 4."

[0090] 1. Formula (I) [ka] (In the formula, R 1 , R 2 is, independently, -[(CH2) a CR x R y ] b R za group (wherein a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R z represents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - Based on: -R z (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - Not a group, c is 0; and - -[CR r R s ] d -R t a group wherein d is an integer selected from the range of 1 to 10, and R r represents a hydrogen atom, a hydroxyl group, and -[CR'(OH)] e -H groups, and R s is a hydrogen atom or -[CR”(OH)] f -H group, and R t represents a hydrogen atom, a C1-C5 alkyl group, and -[CR"'(OH)] g -H groups (each R', R", and R"' is independently a hydrogen atom or a -[CH(OH)] h -H groups, and each of d, e, f, g and h is R tis a hydrogen atom or a C1-C5 alkyl group, R r , R s are independently integers selected from the range of 1 to 10, provided that at least one of them is not a hydrogen atom; R 3 , R 4 are independently selected from the group consisting of a hydrogen atom, a C1-C3-alkyl group, a halogen atom, and an —O—C1-C3-alkyl group; R 5 is a carboxyl group, an activated carboxyl group and -NHR 5a R is selected from the group consisting of 5a is a hydrogen atom or a C1-C5 alkyl group; L comprises a chain of covalently bonded atoms (linker) forming the backbone and having a length in the range of 1 to 100 atoms; n is R 5 is either 0 or 1 if it is a carboxyl group or an activated carboxyl group, and R 5 Ga-NHR 5a group, it is 1.) or a salt thereof.

[0091] 2.R 3 , R 4 are independently a hydrogen atom or a methyl group, and preferably R 3 , R 4 and each is a hydrogen atom.

[0092] 3.R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 In each of the formulas, a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R zrepresents a hydrogen atom, a C1-C3 alkyl group, and (CH2) c SO3 - groups, c is either 0 or an integer ranging from 1 to 4, and R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - It is a base; -R z (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - 3. The azadibenzocyclooctyne derivative or salt thereof according to embodiment 1 or 2, wherein c is not a group and c is 0.

[0093] 4.R 1 , R 2 are respectively [(CH2) a CR x R y ] b R z group, and each R 1 , R 2 In each of the formulas, a is either 0 or an integer ranging from 1 to 4, b is either 0 or an integer ranging from 1 to 3, and R x , R y , R z is a hydrogen atom and (CH2) c SO3 - wherein c is either 0 or an integer ranging from 1 to 4; R x , R y , R z At least one of the following conditions is met (CH2): c SO3 - Based on: -Rz (CH2) c SO3 - group, and when c is 0, R x , R y are both (CH2) c SO3 - is not a group, c is 0, or - If a is 0, R x and R y are both (CH2) c SO3 - The azadibenzocyclooctyne derivative or a salt thereof according to any one of embodiments 1 to 3, wherein c is not a group and c is 0.

[0094] 5.R 1 , R 2 are the same, and both are -(CH2) a CR x R y ] b R z 5. The azadibenzocyclooctyne derivative or a salt thereof according to any one of embodiments 1 to 4, wherein:

[0095] 6.R 1 , R 2 are respectively -[CR r R s ] d -R t group, and each R 1 , R 2 Regarding, independently, R r represents a hydrogen atom, a hydroxyl group, and -[CH(OH)] e -H, and R s is a hydrogen atom or -[CH(OH)] f -H group, and R t represents a hydrogen atom, C1-C5 alkyl, and -[CH(OH)] g -H groups, wherein each of d, e, f, and g is selected from the group consisting of R t is a hydrogen atom or a C1-C5 alkyl group, R r , R sare independently an integer selected from the range of 1 to 10, provided that at least one of is not a hydrogen atom.

[0096] 7.R 1 , R 2 are identical, and are -[CH(OH)] d 7. The azadibenzocyclooctyne derivative or salt thereof according to any one of embodiments 1, 2, and 6, wherein d is a —H group, and d is an integer in the range of 1 to 10, preferably selected from the range of 1 to 5, and more preferably 2 or 3.

[0097] 8.R 5 is an activated carboxyl group, and R 5 The azadibenzocyclooctyne derivative or salt thereof according to any one of embodiments 1 to 7, wherein the activating group is selected from the group consisting of a 4-nitrophenyl group, a pentafluorophenyl group, and an N-succinimidyl group, and is preferably an N-succinimidyl group.

[0098] 9.L has the structure -(CH2) p -(X) m -(CH2) q - and p and q are independently integers selected from the range of 2 to 10; X is selected from the group consisting of -C(=Y)-NH-, -NH-C(=Y)-, -C(=Y)-O-, and -OC(=Y)-, where Y is an oxygen atom or a sulfur atom; m is 0 or 1 [when m is 0, X is absent, and (CH2) p , (CH2) q are directly connected via a single bond]. The azadibenzocyclooctyne derivative or a salt thereof according to any one of embodiments 1 to 8.

[0099] 10. The azadibenzocyclooctyne derivative or salt thereof according to embodiment 9, wherein m is 1, X is a —C(═O)—NH— group, and / or p and q are the same and both are integers selected from the range of 2 to 5, preferably 2 or 3.

[0100] 11. Formula (Ia), (Ib) or (Ic): [ka] [ka] [ka] (Wherein L, n and R 5 11. The azadibenzocyclooctyne derivative or salt thereof according to any one of embodiments 1 to 10, wherein

[0101] 12. Formula (Ia-1), (Ib-1) or (Ic-1): [ka] (In the formula, R 5 is as defined in any one of embodiments 1 to 9), the azadibenzocyclooctyne derivative or a salt thereof according to any one of embodiments 1 to 11.

[0102] 13. The azadibenzocyclooctyne derivative or a salt thereof according to any one of embodiments 1 to 12, having the formula (Ia-1) or (Ib-1), preferably (Ia-1). [ka] [ka]

[0103] 14.Formula (II) [ka] (In the formula, L, R 1 , R 2 , R 3 , R 4 and n is as defined in any one of embodiments 1 to 13 for the azadibenzocyclooctyne derivative of formula (I) or a salt thereof; R 6 is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide; preferably a metal complex; Z is -C(=O)-O-, C(=O)-NR 7 -, and -NR 7 -C(=Y)-, R 7 is a hydrogen atom or a C1 to C5 alkyl group, and Y is an oxygen atom or a sulfur atom, preferably an oxygen atom.

[0104] 15.R 6 is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a metal complex, a radioisotope, an active pharmaceutical ingredient (drug), a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide and a polynucleotide, preferably a metal complex, and a further linker, preferably selected from the group consisting of an alkyl chain, a polyethylene glycol chain, a polypropylene glycol chain and a mixed polyethylene / polypropylene glycol chain, between Z and R 6 15. A conjugate of formula (II) as defined in embodiment 14, wherein:

[0105] 16. Formula (IIa) or (IIb) or (IIc); [ka] [ka] [ka] (Wherein L, n and R 6 , is as defined in embodiment 14 or 15, and Z 1 is a -C(=O)-O- group or a -C(=O)-NH- group.

[0106] 17. Formula (IIa-1), (IIa-2), (IIb-1), (IIb-2), (IIc-1) or (IIc-2): [ka] [ka] [ka] [ka] [ka] [ka] (Wherein, R in (IIa-1), (IIa-2), (IIb-1), (IIb-2), (IIc-1) or (IIc-2) 6 is as defined in any one of embodiments 14 to 16.

[0107] 18. A method for modifying a target molecule, comprising reacting the conjugate according to any one of embodiments 14 to 17 with a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group.

[0108] 19. The method of embodiment 18, wherein the 1,3-dipole group is selected from the group consisting of azide, nitrone, diazoalkane, diazoacetamide and nitrile oxide, preferably azide.

[0109] 20. The method of embodiment 18, wherein the 1,3-diene group is selected from the group consisting of 1,3-butadiene, 1,3-cyclopentadiene, 1,3-cyclohexadiene, furan, and pyrrole.

[0110] 21. The method of embodiment 18, wherein the 1,3-heterodiene group is selected from the group consisting of tetrazine, 1-oxa-1,3-butadiene, 1-aza-1,3-butadiene, 2-aza-1,3-butadiene, and 3-aza-1,3-butadiene.

[0111] 22. The method of any one of embodiments 18 to 21, wherein the target molecule is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide; preferably a polypeptide, more preferably an antibody, more preferably a modified antibody with a 1,3-dipole group, more preferably a modified antibody with an azide group.

[0112] 23. Use of a conjugate according to any one of embodiments 14 to 17 for the bioorthogonal labeling and / or modification of target molecules.

[0113] 24. A modified target molecule comprising the reaction product of a conjugate according to any one of embodiments 14 to 17 and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group, obtained or obtainable by a method according to any one of embodiments 18 to 22.

[0114] 25. The modified target molecule according to embodiment 24 is included as a detection reagent, wherein the target molecule is preferably an antibody, and R 6 is preferably a metal complex and is a suitable capture reagent.

[0115] Example The following examples are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the invention.

[0116] Testing Procedure: [ka] Aniline 1: After stirring a solution of m-anisidine (2.48 ml, 22.0 mmol) and m-anisaldehyde (2.44 ml, 20 mmol) in MeOH (200 ml) at room temperature for 1.5 h, NaBH4 (2.26 g, 60.0 mmol) was added and the reaction was stirred at that temperature for an additional 1.5 h. Water (100 ml) was added and the mixture was extracted with EtOAc. The combined organic phases were washed with 1 M NaOH, water, and brine, dried over MgSO4, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (hexane: EtOAc = 5:1, R f =0.3) to give aniline 1 (4.47 g, 18.4 mmol, 92%) as a yellowish oil.

[0117] R f = 0.3 [hexane / EtOAc, 5:1]. 1 H NMR(400 MHz,CDCl3)δ=7.25(t,J=7.84 Hz,1H),7.07(t,J=8.09 Hz,1H),6.95(dd,J=7.53,0.63 Hz,1H),6.92(m,1H),6.81(m,1H),6.27(dddd,J=11.26,8.13,2.32,0.75 Hz,2H),6.19(t,J=2.26 Hz,1H),4.29(s,2H),4.05(brs,1H),3.79(s,3H),3.75(s,3H)ppm. 13C NMR (150 MHz, CDCl3): δ=180.8,159.9,149.5,141.0,130.0,129.6,119.7,113.0,112.7,106.0,102.7,98.9,55.2,55.1,48.3 ppm. MS(ESI):C 15 H 18 NO2 + Calculated value for: 244.1 [M+H] + Measured value: 244.4 [M+H] + . [ka] Acylamine 2: DIPEA (4.31 ml, 24.7 mmol) was added to a solution of aniline 1 (3.00 g, 12.3 mmol), monomethyl glutarate (2.01 ml, 16.7 mmol), and HATU (6.35 g, 16.7 mmol) in DMF (31 ml), and the reaction was stirred at room temperature for 3 days. The mixture was diluted with EtOAc, washed with 1 M HCl, water, and brine, dried over MgSO, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc:hexane = 1:1) to give acylamine 2 (4.42 g, 11.9 mmol, 97%) as a pale yellow oil.

[0118] R f = 0.4 [hexane / EtOAc, 1:1].

[0119] 1 H NMR(400 MHz,CDCl3)δ=7.19(m,2H),6.82(dd,J=8.34,2.2 Hz,1H),6.75(m,3H),6.55(brd,J=7.53 Hz,1H),6.48(m,1H),4.81(s,2H),3.74(s,3H),3.70(s,3H),3.59(s,3H),2.30(t,J=7.34 Hz,2H),2.15(t,J=7.22 Hz,2H),1.92(q,J=7.22 Hz,2H)ppm. 13C NMR(150 MHz, CDCl3):δ=173.6,171.9,160.3,159.6,143.3,139.1,130.2,129.3,12 1.1,120.6,114.1,113.5,113.0,55.3,55.2,52.8,51.4,33.24,33.16,20.7 ppm. MS(ESI):C 21 H 26 No. 5 + Calculated value for: 372.2 [M+H] + Measured value: 372.4 [M+H] + . [ka] Cyclooctene 3: Tetrachlorocyclopropene (0.80 mL, 6.55 mmol) was added dropwise to a suspension of AlCl (3.17 g, 23.8 mmol) in CHCl (50 mL), and the reaction was stirred at room temperature for 15 minutes. The solution was cooled to -78 °C, and a solution of acylamine 2 (2.21 g, 5.95 mmol) in CHCl was added slowly. After warming to room temperature overnight, water (45 mL) was added, and the reaction was stirred at room temperature for 30 minutes. The mixture was extracted with CHCl, dried over MgSO, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc / MeOH, 100:1, R f =0.4) to give cyclooctene 3 (1.13 g, 2.67 mmol, 45%) as a yellow oil.

[0120] R f = 0.4 [EtOAc / MeOH, 100:1].

[0121] 1H NMR(400 MHz,CDCl3)δ=8.02(d,J=8.83 Hz,1H),7.90(d,J=8.51 Hz,1H),7.26,(d,J=2.52 Hz,1H),7.06(dd,J=8.51,2.52 Hz,1H),6.96(dd,J=8.35,2.68 Hz,1H),6.89(d,J=2.52 Hz,1H),5.18(d,J=14.5 hz,1H),4.10(d,J=14.2 Hz,1H),3.93(s,3H),3.92(s,3H),3.56(s,3H),2.33(m,1H),2.14(m,1H),2.00(m,1H),1.93(m,1H),1.75(m,2H)ppm. 13 C NMR(150 MHz, CDCl3):δ=173.2,172.6,163.1,162.7,152.5,146.0,143.2,141.6,139.0,135.8,1 35.2,118.2,115.5,115.3,114.9,113.9,113.7,56.1,55.9,55.6,51.5,33.5,32.6,20.6 ppm. MS(ESI):C 24 H 24 No. 6 + Calculated value for: 422.2 [M+H] + Actual value: 422.4 [M+H] + .

[0122] [ka] Amine 4: A solution of 4-aminobutyric acid (5.00 g, 48.5 mmol) in SOCl (35 mL, 485 mmol) was stirred at room temperature for 2 h and concentrated under reduced pressure. NaHCO (8.95 g, 107 mmol) and t-BuOH (105 mL) were added, and the resulting suspension was stirred at room temperature overnight. All volatiles were removed under reduced pressure, and the residue was partitioned between EtOAc and 1 M NaOH. The organic phase was washed with water and brine, dried over MgSO, and concentrated under reduced pressure to give amine 4 (1.50 g, 9.39 mmol, 19%) as a light brown oil.

[0123] 1 H NMR(400 MHz,CDCl3)δ=2.72(t,J=7.03 Hz,2H),2.27(t,J=7.47 Hz,2H),1.73(q,J=7.22 Hz,2H),1.45(s,3H),1.26(brs,2H)ppm. 13 C NMR (150 MHz, CDCl3): δ=172.9,80.2,41.6,33.0,29.1,28.1 ppm. [ka] Amide 5: BBr3 (1.0 M in CHCl2, 26.6 mL, 26.6 mmol) was slowly added to a solution of cyclooctane 3 (1.12 g, 2.66 mmol) in CHCl2 (128 mL), and the solution was stirred at −78 °C for 1 h and at room temperature for 48 h. The reaction was quenched with water, basified with 4 M NaOH, and washed with CHCl2. (Two equal batches were combined at this stage.) The aqueous layer was acidified with concentrated HCl, and the resulting precipitate was collected. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. The combined solids were dissolved in MeOH (13 mL), THF (13 mL), and 1 M NaOH (21 mL) and stirred at room temperature for 3 h. The reaction mixture was acidified with concentrated HCl, and the resulting precipitate was collected. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over MgSO4, filtered, and concentrated under reduced pressure. The combined solid (1.06 g) and amine 4 (852 mg, 5.35 mmol) were dissolved in DMF (287 mL). HATU (2.03 g, 5.35 mmol) and DIPEA (3.73 mL, 21.4 mmol) were added sequentially, and the resulting mixture was stirred at room temperature for 20 hours and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (6% MeOH in CHCl2) to give amide 5 (1.14 g, 2.18 mmol, 41% over three steps) as a light brown solid.

[0124] R f = 0.3 [MeOH / CH2Cl2, 6:94].

[0125] 1 H NMR(400 MHz,d4-MeOH)δ=7.91(d,J=8.51 Hz,1H),7.77(d,J=8.2 Hz,1H),7.14(d,J=2.21 Hz,1H),7.01(dd,J=8.51,2.21 Hz,1H),6.93(d,J=2.21 Hz,1H),6.87(dd,J=8.35,2.36 Hz,1H),5.10(d,J=14.82 Hz,1H),4.21(d,J=14.5 Hz,1H),3.06(m,2H),2.31(m,1H),2.18(t,J=7.41 Hz,2H),1.93(m,3H),1.65(m,4H),1.43(s,9H)ppm. 13 C NMR(150 MHz, CDCl3):δ=175.3,175.0,174.4,164.1,163.3,154.6,148.0,144.4,142.5,138.9,137.0,136 .4,120.9,117.8,117.4,116.5,115.1,114.5,81.7,57.2,39.7,35.9,35.0,33.8,28.5,26.0,22.8 ppm. MS(ESI):C 29 H 33 N2O7 + Calculated value for: 521.2 [M+H] + Measured value: 521.3 [M+H] + . [ka] Acetonide 6: DEAD (40% in PhMe, 1.75 mL, 3.08 mmol) was added dropwise to a stirred solution of amide 5 (400 mg, 0.768 mmol), PPh3 (808 mg, 3.08 mmol), and (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (0.38 mL, 3.08 mmol) in THF (20 mL). The reaction was stirred at room temperature for 20 h and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (30-60% acetone in CHCl) to give acetonide 6 (478 mg, 0.638 mmol, 83%) as an off-white solid.

[0126] R f =0.5[アセトン / CH2Cl2,1:1].

[0127] 1 H NMR (400 MHz, CDCl3) δ=7.98(d,J=8.71 Hz,1H),7.87(d,J=8.31 ​​Hz,1H),7.25(m,1H),7.06(d,J=8.71 Hz,1H),6.96(m,2H),5.96(brs,1H),5.12(d,J=14.25 Hz,1H),4.50(m,2H),4.14(m,6H),4.05(d,J=15.04 Hz,1H),3.92(m,2H),3.16(m,2H),2.33(m,1H),2.20(t,Hz=7.12 Hz,2H),1.99(m,1H),1.91(m,2H),1.70(m,4H),1.45(s,6H),1.40(m,15H)ppm. 13 C NMR(150 MHz, CDCl3): δ=172.8,172.7,172.4,162.1,161.6,152.4,145.8,143.3,141.6,139.3,135.8,135.2,118.8,115.8,115.2,114.7,114.2, 110.1,109.9,80.6,79.77,73.74,73.70,69.4,69.0,66.6,66.5,56. 1,39.1,34.8,33.5,32.9,30.9,29.3,28.1,26.8,25.32,25.27,24.6 ppm. MS(ESI):C 41 H 53 N2O 11 + について Calculated value:749.4[M+H]+ Measured value: 749.4[M+H]+.

[0128]

change

[0129] 1 H NMR(400 MHz,d4-MeOH)δ=7.29(d,J=8.39 Hz,1H),7.27.(d,J=2.29 Hz,1H),7.12(m,2H),7.03(dd,J=8.56,2.48 Hz,1H),6.90(dd,J=8.39,2.29 Hz,1H),5.04(d,J=13.73 Hz,1H),4.13(m,2H),4.04(m,2H),3.99(m,2H),3.68(m,5H),3.09(t,J=7.06 Hz,2H),2.31(m,1H),2.25(t,J=7.44 Hz,2H),1.95(m,3H),1.68(m,4H)ppm. 13 C NMR(150 MHz,d4-MeOH):δ=177.0,175.4,175.1,160.7,160.3,153.8,151.1,128.5,127.3,120.51,120.45,117.7,116.9 ,116.2,115.9,115.0,114.7,107.9,71.9,71.8,71.4,70.7,64.3,64.2,57.0,39.8,36.1,35.2,32.4,25.9,22.9 ppm. MS(ESI):C 30 H 37 N2O 10 + Calculated value for: 585.2 [M+H] + Actual value: 585.4 [M+H] + . [ka] NHS-ester 8: DIPEA (31 μL, 0.176 mmol) was added to a solution of alkyne 7 (43 mg, 0.074 mmol) and TSTU (44 mg, 0.147 mmol) in DMF (2 mL), and the solution was stirred at room temperature for 2 h. The reaction was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase HPLC chromatography (YMC-Triart C18, 32–48% MeCN in HO, 0.1% TFA, 30 min) to afford NHS-ester 8 (31 mg, 0.045 mmol, 61%) as a white solid.

[0130] 1 H NMR(400 MHz,d4-MeOH)δ=7.30(d,J=8.53 Hz,1H),7.27(d,J=2.38 Hz,1H),7.13(m,2H),7.03(dd,J=8.53,2.51 Hz,1H),6.90(dd,J=8.41,2.51 Hz,1H),5.04(d,J=13.93 Hz,1H),4.11(m,2H),4.00(m,4H),3.70(d,J=9.16 Hz,1H),3.66(m,4H),3.16(t,J=,6.84 Hz,2H),2.80(s,4H),2.60(t,J=7.4 Hz,2H),2.27(m,1H),1.93(m,3H),1.79(q,J=7.12 Hz,2H),1.65(m,2H)ppm. 13 C NMR(150 MHz,d4-MeOH):δ=174.0,173.5,170.4,168.5,159.1,158.7,155.9,152.2,149.5,127.3,127.0,125.7,119.5, 118.9,116.1,115.3,114.6,114.3,70.3,70.2,69.5,62.7,62.6,55.4,37.8,34.5,33.6,27.7,25.1,24.0,21.3 ppm. MS(ESI):C 34 H 40 N3O12 + Calculated value for: 682.3 [M+H]+ Found value: 682.4 [M+H]+. [ka] Sulfonic acid 9: A suspension of amide 5 (100 mg, 0.192 mmol), K2CO3 (159 mg, 1.15 mmol), and sodium 2-bromoethanesulfonate (243 mg, 1.15 mmol) in MeCN (2.8 mL) was stirred at 80 °C for 5 days. The reaction mixture was purified by reverse-phase HPLC chromatography (YMC-Triart C18, 23–39% MeCN in HO, 0.1% TFA, over 30 min) to give sulfonic acid 9 (104 mg, 0.131 mmol, 68%) as a white solid.

[0131] 1 H NMR(400 MHz,d4-MeOH)δ=7.92(d,J=8.51 Hz,1H),7.76(d,J=8.51 Hz,1H),7.26(d,J=1.89 Hz,1H),7.19(d,J=2.21 Hz,1H),7.14(dd,J=8.51,2.21 Hz,1H),6.98(dd,J=8.51,1.89 Hz,1H),5.10(d,J=14.5 Hz,1H),4.43(m,4H),4.13(d,J=14.5 Hz,1H),3.24(m,5H),3.10(t,J=6.94 Hz,2H),2.38(m,1H),2.21(t,J=7.25 Hz,1H),2.12(m,1H),2.07(m,1H),1.84(m,1H),1.62(m,4H),1.33(s,5H),1.33(s,5.5H)1.13(s,2H9,1.09(s,0.5H)ppm(mixture of rotamers). 13C NMR (150 MHz, d4-MeOH): δ = 177.0, 176.9, 174.8, 174.1, 173.7, 163.9, 163.2, 154.3, 147.4, 144.0, 143.0, 139.6, 136.6, 136.1, 119.9, 117.0, 116.3, 115.6, 115.2, 81.5, 65.7, 65.2, 56.9, 51.5, 51.3, 40.7, 40.6, 36.7, 34.63, 34.55, 33.2, 31.7, 31.0, 28.2, 27.1, 24.9, 24.8, 22.43, 22.39 ppm (mixture of rotamers). MS(ESI):C 33 H 41 N2O 13 S2 + Calculated value for: 737.2 [M+H]+ Found value: 737.4 [M+H]+. [ka] Carboxylic acid 10: A solution of sulfonic acid 9 (84 mg, 0.105 mmol) in CHCl (0.83 mL), iPrSiH (0.08 mL), water (0.08 mL), and TFA (2.5 mL) was stirred at room temperature for 2 h and concentrated under reduced pressure. The residue was coevaporated twice with acetone and MeCN. The resulting residue was dissolved in MeOH (6.7 mL) and DIPEA (0.40 mL), irradiated (360 nm) for 1.5 h, and concentrated under reduced pressure. The resulting residue was purified by reverse-phase HPLC chromatography (YMC-Triart C18, 22–38% MeCN in HO, 0.1% TFA, over 30 min) to give carboxylic acid 10 (41 mg, 0.045 mmol, 43% over two steps) as a white solid.

[0132] 1H NMR(400 MHz,d4-MeOH)δ=7.29(d,J=8.53 Hz,1H),7.25(d,J=2.26 Hz,1H),7.15(d,J=2.38 Hz,1H),7.12(d,J=8.41 Hz,1H)),7.03(dd,J=8.60,2.45 Hz,1H),6.90(dd,J=8.53,2.38 Hz,1H),5.04(d,J=14.05 Hz,1H),4.42(m,5H),3.69(spt,J=6.61 Hz,4H),3.29(m,4H),3.18(m,6H),2.30(m,4H),2.07(m,1H),1.90(m 1H),1.73(m,4H),1.34(m,30H)ppm. 13 C NMR(150 MHz,d4-MeOH):δ=174.9,173.7,173.1,158.6,158.2,152.1,149.6,127.0,125.8,119.1,116.3,115.4,114.7, 114.5,113.2,113.0,106.4,64.0,63.6,55.4,50.4,50.1,42.4,38.8,34.0,33.5,30.5,23.8,21.3,17.3,15.9 ppm. MS(ESI):C 28 H 33 N2O 12 S2 + Calculated value for: 653.1 [M+H] + Actual value: 653.1 [M+H] + . [ka] NHS-ester 11: DIPEA (0.17 mL, 0.998 mmol) was added to a solution of carboxylic acid 10 (87 mg, 0.095 mmol) and TSTU (80 mg, 0.266 mmol) in DMF (3 mL). The reaction was stirred at room temperature for 2 h and concentrated under reduced pressure. The resulting residue was purified by reverse-phase HPLC chromatography (YMC-Triart C18, 24-40% MeCN, 0.1% TFA in HO over 30 min) to give NHS-ester 10 (39 mg, 0.045 mmol, 41) as a white solid.

[0133] 1 H NMR(400 MHz,d4-MeOH)δ=7.31(d,J=8.53 Hz,1H),7.27(d,J=2.51 Hz,1H),7.17(d,J=2.51 Hz,1H),7.14(d,J=8.41 Hz,1H),7.05(dd,J=8.53,2.51 Hz,1H),6.92(dd,J=8.47,2.45 Hz,1H),5.06(d,J=13.93 Hz,1H),4.44(m,5H),3.71(m,4H),3.31(m,4H),3.21(m,6H),2.83(s,4H),2.68(m,1H),2.63(t,J=7.34 Hz,1H),2.34(m,2H),2.26(q,J=7.49 Hz,1H),2.08(m,1H),1.90(m,4H),1.36(m,30H)ppm. MS(ESI):C 32 H 36 N3O 14 S2 + Calculated value for: 750.2 [M+H] + Actual value: 750.4 [M+H] + . [ka] Acylamine 12: DIPEA (3.85 ml, 11.1 mmol) was added to a solution of aniline 1 (5.66 g, 11.1 mmol), monomethyl succinate (1.98 g, 14.9 mmol), and HATU (5.66 g, 14.9 mmol) in DMF (28 ml), and the reaction was stirred at room temperature for 3 days. The mixture was diluted with EtOAc, washed with 1 M HCl, water, and brine, dried over MgSO4, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (1:1 EtOAc:hexane, R f =0.5)) to give acylamine 12 (3.85 g, 10.8 mmol, 98%) as a pale yellow oil.

[0134] R f = 0.5 [hexane / EtOAc, 1:1].

[0135] 1 H NMR(400 MHz,CDCl3)δ=7.22(t,J=8.09 Hz,1H),7.16(dd,J=9.06 Hz,7.28 Hz,1H),6.83(dd,J=8.28,2.13 Hz,1H),6.76(m,3H),6.64(d,J=7.65 Hz,1H),6.56(m,1H),4.86(s,2H),3.75(s,1H),3.71(s,1H),3.66(s,1H),2.63(m,2H),2.39(m,2H)ppm. 13 C NMR(150 MHz, CDCl3):δ=173.5,171.2,160.3,159.6,143.2,139.1,130.2,129.3,121.0,120.6,114.1,113.9,113.1,55.3,55.2,53.0,51.7,29.3 ppm. MS(ESI):C 20 H 24 No. 5 + Calculated value for: 358.2 [M+H] + Actual value: 358.4 [M+H] + .

[0136] [ka] Cyclooctene 13: Tetrachlorocyclopropene (0.57 mL, 5.72 mmol) was added dropwise to a suspension of AlCl (2.74 g, 20.7 mmol) in CHCl (45 mL), and the reaction was stirred at room temperature for 15 minutes. The solution was cooled to -78 °C, and a solution of acylamine 12 (1.85 g, 5.16 mmol) in CHCl (30 mL) was added slowly. After warming to room temperature overnight, water (39 mL) was added, and the reaction was stirred at room temperature for 30 minutes. The mixture was extracted with CHCl, dried over MgSO, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc / MeOH, 100:1, R f =0.4) to give cyclooctene 13 (1.60 g, 3.93 mmol, 38%) as a yellowish oil.

[0137] R f =0.4[EtOAc / MeOH,100:1].

[0138] 1 H NMR (400 MHz, CDCl3) δ=8.00(d,J=8.66 Hz,1H),7.90(d,J=8.53 Hz,1H),7.25(d,J=2.51 Hz,1H),7.18(d,J=2.51 Hz,1H),7.06(dd,J=8.66,2.51 Hz,1H),6.95(dd,J=8.47,2.57 Hz,1H),5.21(d,J=14.4 Hz,1H),4.12(d,J=14.4 Hz,1H),3.94(s,3H),3.91(s,3H),3.59(s,3H),2.76(m,1H),2.66(m,1H),2.36(m,1H),1.94(m,1H)ppm. MS(ESI):C 24 H 24 NO6 + について Calculated value: 408.1[M+H] + Actual measurement value:408.3[M+H] + .

change

[0139] MS(ESI):C 21 H 18 No. 6 + Calculated value for: 380.1 [M+H] + Actual value: 380.3 [M+H] + . [ka] Acetonide 15: DEAD (40% in PhMe, 94 μL, 0.207 mmol) was added dropwise to a stirred solution of phenol 14 (26 mg, 0.069 mmol), PPh3 (54 mg, 0.207 mmol), and (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (26 μL, 0.207 mmol) in THF (1.5 mL). The reaction was stirred at room temperature for 20 h and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (10-30% acetone in CHCl, R f =0.3 (20% acetone in CH2Cl2)) to give acetonide 15 (33 mg, 0.054 mmol, 79%) as an off-white solid.

[0140] R f =0.3 [acetone / CH2Cl2, 1:4].

[0141] 1 H NMR(400 MHz,CDCl3)δ=7.99(d,J=8.66 Hz,1H),7.89(d,J=8.41 Hz,1H),7.25(t,J=2.64 Hz,1H),7.21(t,J=2.26 Hz,1H),7.07(m,1H),8.96(dt,J=8.47,2.60 Hz,1H),5.17(d,J=14.3 Hz,1H),4.50(m,2H),4.18(m,7H),3.93(m,2H),3.58(s,3H),2.68(m,2H),2.35(m,1H),1.92(m,1H),1.47(s,6H),1.41(s,6H)ppm. MS(ESI):C 33 H 38 NO 10 + Calculated value for: 608.2 [M+H]+ Found value: 608.3 [M+H]+. [ka] Tetraol 16: 1M HCl (0.1 ml) was slowly added to a solution of acetonide 15 (11 mg, 0.018 mmol) in MeOH (0.2 ml), and the reaction was stirred at room temperature for 2 h. The mixture was directly subjected to flash column chromatography (10% MeOH in CHCl) to give tetraol 16 (9.7 mg, 0.018 mmol, 100%) as a colorless oil.

[0142] R f = 0.2 [methanol / CH2Cl2, 1:9].

[0143] 1H NMR(400 MHz,d4-MeOH)δ=8.04(d,J=8.66 Hz,1H),7.88(d,J=8.53 Hz,1H),7.34(m,2H),7.24(d,J=8.60,2.45 Hz,1H),7.08(dd,J=8.53,2.51 Hz,1H),5.17(d,J=14.6 Hz,1H),4.28(d,J=14.3 Hz,1H),4.20(m,4H),4.02(m,2H),3.70(m,4H),3.46(s,3H).2.65(m,1H),2.42(m,2H),2.03(m,1H)ppm. MS(ESI):C 27 H 30 NO 10 + Calculated value for: 528.2 [M+H]+ Found value: 528.4 [M+H]+. [ka] Alkyne 17: A solution of tetraol 16 (9.7 mg, 0.018 mmol) in MeOH (1.5 mL) was irradiated (360 nm) for 30 min. The solution was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (10% MeOH in CHCl, R f =0.2) to give alkyne 17 (4.3 mg, 8.6 μmol, 48%) as a colorless oil.

[0144] R f = 0.2 [methanol / CH2Cl2, 1:9].

[0145] 1H NMR(400 MHz,d4-MeOH)δ=7.31(d,J=8.53 Hz,1H),7.26(m,2H),7.12(d,J=8.41 Hz,1H),7.05(dd,J=8.6,2.57 Hz,1H),6.90(dd,J=8.47,2.57 Hz,1H)5.014(d,J=14.1 Hz,1H).,4.06(m,6H),3.73(d,J=13.8 Hz,1H),3.67(m,4H),3.53(s,3H),2.74(m,1H),2.47(m,1H),2.38(m,1H),2.01(m,1H)ppm. MS(ESI):C 33 H 38 NO 10 + Calculated value for: 500.2 [M+H]+ Measured value: 500.4 [M+H]+. [ka] Carboxylic acid 18: 1M NaOH (100 μL) was added to a solution of alkyne 17 (15.8 mg, 0.032 mmol) in THF (1 mL) and MeOH (1 mL), and the reaction was stirred at room temperature overnight. The reaction was concentrated under reduced pressure, and the resulting residue was purified by reverse-phase HPLC chromatography (Chromolith RP18e, C 18 , 18-34% MeCN, 0.1% TFA in HO, over 30 min) to give carboxylic acid 18 (3.2 mg, 6.6 μmol, 21%) as a white solid.

[0146] 1 H NMR(400 MHz,d4-MeOH)δ=7.30(m,2H),7.26(d,J=2.38 Hz,1H),7.12(d,J=8.41 Hz,1H),7.04(dd,J=8.53,2.51 Hz,1H),6.90(dd,J=8.41,2.51 Hz,1H),5.05(d,J=14.1 Hz,1H),4.07(m,6H),3.73(d,J=13.9 Hz,1H),3.68(m,4H),2.76(m,1H),2.50(m,1H),2.31(m,1H),1.96(m,1H)ppm. MS(ESI):C25 H 28 No. 9 + Calculated value for: 486.2 [M+H]+ Found value: 486.4 [M+H]+. [ka] tert-Butyl ester 19: BBr3 (1.0 M in CHCl2, 39 mL, 39 mmol) was slowly added to a solution of cyclooctane 13 (1.60 g, 3.93 mmol) in CHCl2 (190 mL), and the solution was stirred at −78 °C for 1 h and at room temperature for 48 h. The reaction was quenched with water, basified with 4 M NaOH, and washed with CHCl2. The aqueous layer was acidified with concentrated HCl, and the resulting precipitate was collected. The aqueous phase was extracted with EtOAc, and the organic phases were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The solids were combined, dissolved in MeOH (10 mL), THF (10 mL), and 1 M NaOH (16 mL), and stirred at room temperature for 3 h. The reaction mixture was acidified with concentrated HCl, and the resulting precipitate was collected. The aqueous phase was extracted with EtOAc, and the organic phases were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The combined solid (702 mg) and glycine tert-butyl ester hydrochloride (322 mg, 1.92 mmol) were dissolved in DMF (22 mL). HATU (728 mg, 1.92 mmol) and DIPEA (1.32 mL, 7.63 mmol) were added sequentially, and the resulting mixture was stirred at room temperature for 20 hours and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (4-6% MeOH in CHCl, R f =0.4, 6% MeOH in CH2Cl2) to afford the tert-butyl ester 19 (333 mg, 0.696 mmol, 18% over three steps) as a brownish solid.

[0147] R f =0.4 [methanol / CH2Cl2, 6:94]. 1H NMR(400 MHz,d4-MeOH)δ=7.90(d,J=8.53 Hz,1H),7.75(d,J=8.28 Hz,1H),7,12(m,1H),7.02(m,2H),6.85(dd,J=8.34,2.32 Hz,1H),5.08(d,J=14.6 Hz,1h),4.20(d,J=14.4 Hz,1H),3.68(s,2H),2.71(m,1H),2.41(m,1H),2.24(m,1H),2.05(m,1H),1.42(s,9H)ppm. MS(ESI):C 26 H 27 N2O7 + Calculated value for: 479.2 [M+H] + Actual value: 479.0 [M+H] + . [ka] Acetonide 20: DEAD (40% in PhMe, 0.48 ml, 0.836 mmol) was added dropwise to a solution of tert-butyl ester 19 (100 mg, 0.209 mmol), PPh3 (219 mg, 0.836 mmol), and (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (103 μL, 0.836 mmol) in THF (5.5 ml). The reaction was stirred overnight at room temperature, after which the solvent was removed under reduced pressure and the residue purified by column chromatography (30–50% acetone in CHCl, R f =0.5 (40% acetone in CH2Cl2) to give acetonide 20 (128 mg, 0.181 mmol, 87%) as a white solid.

[0148] R f =0.5 [acetone / CH2Cl2, 2:3].

[0149] 1H NMR(400 MHz,CDCl3)δ=7.95(d,J=8.28 Hz,1H),7.86(d,J=8.41 Hz,1H),7.24(m,2H),7.05(dt,J=8.66,2.89 Hz,1H),6.94(dt,J=8.53,2.32 Hz,1H),6.05(brt,J=4.77 Hz,1H),5.15(d,J=14.4 Hz,1H),4.49(m,2H),4.12(m,7H),3.90(m,2H),3.83(d,J=5-14 Hz,2H),2.85(m,1H),2,60(m,1H),2.16(m,1H),1.89(m,1H),1.45(s,6H),1.44(s,9H),1.39(s,6H)ppm. MS(ESI):C 38 H 47 N2O 11 + Calculated value for: 707.3 [M+H] + Actual value: 707.6 [M+H] + .

[0150] [ka] Carboxylic acid 21: TFA (0.5 mL) was added to a mixture of acetonide 20 (64 mg, 0.091 mmol), iPrSiH (0.1 mL), water (0.1 mL), and CHCl (1 mL). The reaction was stirred at room temperature for 6 h, diluted with CHCl, and extracted with water. The combined aqueous phases were lyophilized to give carboxylic acid 21 (48.5 mg, 0.085 mmol, 93%).

[0151] 1H NMR(400 MHz,d4-MeOH)δ=8.00(d,J=8.66 Hz,1H),7.84(d,J=8.53 Hz,1H),7.36(t,J=2.13 Hz,1H),7.31(d,J=2.51 Hz,1H),7.20(dd,J=8.60,2.32 Hz,1H),7.04(dd,J=8.47,2.45 Hz,1H),5.15(d,J=14.56 Hz,1H),4.18(m,5H),4.02(m,2H),3.77(s,2H),3.70(m,4H),2.73(m,1H),2.44(m,1H),2.26(m,1H),2.01(m,1H)ppm. 13 C NMR(150 MHz,d4-MeOH):δ=174.8,174.3,173.1,163.2,162.3,153.2,146.2,142.6,141.6,138.3,135.2,134.6 ,118.5,115.6,115.2,115.0,114.2,113.9,70.2,70.1,69.9,69.4,62.6,62.5,55.7,40.3,30.0,29.4 ppm. MS(ESI):C 35 H 45 N4O 12 + Calculated value for: 571.2 [M+H] + Actual value: 571.4 [M+H] + .

[0152] [ka] Amide 22: DIPEA (29 μL, 0.168 mmol) was added to a solution of carboxylic acid 21 (48 mg, 0.084 mmol), N-Boc-ethylenediamine (27 mg, 0.168 mmol), and HATU (64 mg, 0.168 mmol) in DMF (1 ml). The reaction was stirred overnight at room temperature, after which the solvent was removed under reduced pressure and the residue was purified by reverse-phase HPLC chromatography (Chromolith RP18e, C 18The residue was purified by 14-30% MeCN, 0.1% TFA in HO over 30 min) to give amide 22 (30 mg, 0.042 mmol, 50%) as a white solid.

[0153] 1 H NMR(400 MHz,d4-MeOH)δ=8.02(d,J=8.53 Hz,1H),7.86(d,J=8.53,1H),7.34(m,2H),7.23(dd,J=8.66,2.38 Hz,1H),7.07(dd,J=8.53,2.51 Hz,1H),5.21(d,J=14.8 Hz,1H),4.19(m,5H),4.02(m,2H),3.82(d,J=16.8 Hz,1H),3.69(m,4H),3.60(d,J=16.8 Hz,1H),3.18(m,2H),2.83(m,1H),2.29(m,2H),2.03(m,1H),1.42(s,9H)ppm. MS(ESI):C 35 H 45 N4O 12 + Calculated value for: 713.3 [M+H] + Actual value: 713.6 [M+H] + . [ka] Amine 23: TFA (0.5 ml) was added to a mixture of amide 22 (30 mg, 0.042 mmol), iPrSiH (0.1 ml), water (0.1 ml), and CHCl (1 ml). The reaction was stirred at room temperature for 3 h, diluted with CHCl, and extracted with water. The combined aqueous phases were lyophilized to give the crude deprotected amine.

[0154] The residue was dissolved in MeOH (3 ml) and irradiated (360 nm) for 45 min. All volatiles were removed under reduced pressure and the resulting residue was purified by reverse phase HPLC chromatography (Chromolith RP18e, C 18, 15-31% MeCN, 0.1% TFA in H2O, over 30 min) to give amine 23 (18.7 mg, 0.027 mmol, 64% over two steps) as a white solid.

[0155] 1 H NMR(400 MHz,d4-MeOH)δ=7.32(d,J=8.66,1H),7.23(d,J=2.51 Hz,2H),7.14(d,J=8.53 Hz,1H),7.06(dd,J=8.60,2.57 Hz,1H),6.92(dd,J=8.47,2.32 Hz,1H),5.04(d,J=14.2 Hz,1H),4.06(m,6H),3.87(dd,J=17.1,1.69 Hz,1H),3.76(d,J=14.1 Hz,1H),3.68(m,5H),3.51(t,J=5.84 Hz,2H),3.07,m,2H),2.85(m,1H),2.29(m,2H),2.07(m,1H)ppm. 13 C NMR(150 MHz,d4-MeOH):δ=174.7,173.0,171.8,159.2,159.2,158.5,151.9,149.6,127.0,125.9,119.2,119.2,1 16.2,115.5,114.6,114.4,112.8,106.3,70.3,70.2,69.5,69.2,62.7,62.6,42.5,39.7,36.6,30.5,29.8 ppm. MS(ESI):C 29 H 37 N4O9 + Calculated value for: 585.3 [M+H] + Measured value: 585.5 [M+H] + . [ka] Ether 24: NaOH (30%, 42 mL) was slowly added to a mixture of NZ-ethanolamine (4.00 g, 20.5 mmol), tert-butyl bromoacetate (6.06 mL, 41.0 mmol), and BuNHSO (2.96 g, 8.72 mmol) in PhMe (84 mL), and the reaction was stirred at room temperature overnight. tert-Butyl bromoacetate (1.74 mL, 11.8 mmol) was added, and the reaction was stirred at room temperature for 6 hours. The layers were separated, and the organic phase was washed with 5% AcOH and water, dried over MgSO, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc:hexane = 1:4) to give ether 24 (1.35 g, 4.36 mmol, 21%) as a colorless oil.

[0156] 1 H NMR(400 MHz, CDCl3)δ=7.34(m,4H),5.44(brs,1H),5.10(s,2H),3.95(s,2H),3.61(m,2H),3.41(m,2H),1.46(s,9H)ppm. [ka] Amine 25: Pd / C (710 mg) was added to a solution of ether 24 (1.35 g, 4.36 mmol) in EtOAc (15 mL), and the reaction vessel was placed under an atmosphere of H. The reaction was stirred at room temperature for 3 h and then filtered through Celite. The solution was concentrated under reduced pressure to give amine 25 (670 mg, 3.82 mmol, 88%) as a colorless oil.

[0157] 1 H NMR(400 MHz, CDCl3)δ=3.99(s,2H),3.57(t,J=5.04 Hz,2H),2.91(t,J=5.20 Hz,2H),1.87(brs,2H),1.48(s,9H)ppm. [ka] Amide 27: DIPEA (48 μL, 0.178 mmol) was added to a solution of carboxylic acid 26 (50 mg, 0.137 mmol) and TSTU (54 mg, 0.178 mmol) in DMF (2 mL), and the reaction was stirred at room temperature for 2 h. Amine 25 (31 mg, 0.178 mmol) was added, and the reaction was stirred at room temperature for 4 h and concentrated under reduced pressure. The resulting residue was purified by reverse-phase HPLC chromatography (Chromolith RP18e, C 18 , 35-51% MeCN, 0.1% TFA in H2O, over 30 min) to give amide 27 (31.6 mg, 60 μmol, 44%) as a white solid.

[0158] 1 H NMR(400 MHz,d4-MeOH)δ=7.91(d,J=8.71 Hz,1H),7.76(d,J=8.31 ​​Hz,1H),7.13(d,J=2.37 Hz,1H),7.05(d,J=1.98 Hz,1H),7.01(dd,J=8.31,2.37 Hz,1H),6.86(dd,J=8.51,2.18 Hz,1H),5.09(d,J=14.6 Hz,1H),4.21(d,J=14.3 Hz,1H),3.95(s,2H),3.45(m,2H),3.23(m,2H),2.68(m,1H),2.34(m,1H),2.23(m,1H),2.06(m,1H),1.46(s,9H)ppm. MS(ESI):C 16 H 24 No. 5 + Calculated value for: 523.2 [M+H] + Actual value: 523.4 [M+H] + . [ka] Sulfonic acid 28: A suspension of amide 27 (100 mg, 0.191 mmol), KCO (132 mg, 0.955 mmol), and sodium 2-bromoethanesulfonate (202 mg, 0.955 mmol) in MeCN (2.0 mL) was stirred at 80 °C for 20 h. The reaction mixture was purified by reverse-phase HPLC chromatography (Chromolith RP18e, C18 , 22-38% MeCN, 0.1% TFA in HO, over 30 min) to give sulfonic acid 28 (46 mg, 0.058 mmol, 30%) as a white solid.

[0159] MS(ESI):C 32 H 39 N2O 14 S2 + Calculated value for: 739.2 [M+H]+ Measured value: 739.3 [M+H]+.

[0160] [ka] A solution of carboxylic acid 29::sulfonic acid 28 (45 mg, 0.061 mmol) in CHCl (2 mL), iPrSiH (0.2 mL), water (0.2 mL), and TFA (1 mL) was stirred at room temperature for 6 h, diluted with water, and lyophilized. The resulting residue was purified by reverse-phase HPLC chromatography (Chromolith RP18e, C 18 , C18, 16-32% MeCN, 0.1% TFA in HO, over 30 min) to give carboxylic acid 29 (15.2 mg, 0.021 mmol, 34%) as a white solid.

[0161] MS(ESI):C 28 H 31 N2O 14 S2 + Calculated value for: 683.1 [M+H] + Actual value: 683.3 [M+H] + .

[0162] [ka] Alkyne 30: A solution of carboxylic acid 29 (17 mg, 0.023 mmol) in MeOH (2 mL) and DIPEA (0.10 mL) was irradiated (360 nm) for 0.5 h and concentrated under reduced pressure. The resulting residue was purified by reverse-phase HPLC chromatography (YMC-Triart C18, 18–30% MeCN, 0.1% TFA in HO, over 30 min) to give carboxylic acid 30 (16.7 mg, 0.018 mmol, 80%) as a white solid.

[0163] MS(ESI):C 27 H 31 N2O 13 S2 + Calculated value for: 655.1 [M+H] + Actual value: 655.3 [M+H] + .

[0164] [ka] Ruthenium complex 33: DIPEA (28 μL, 0.158 mmol), NHS (5.0 mg, 43.5 μmol), and EDC HCl (15.2 mg, 79.0 μmol) were added sequentially to a solution of 10 (36 mg, 40 μmol) in DMF (1.0 mL), and the reaction was stirred at room temperature for 2 h. Amine 34 (45 mg, 40 μmol) was added, and the reaction was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the resulting residue was purified by HPLC chromatography (C18, 2–90% MeCN in HO, 0.05% TFA, over 90 min) to give ruthenium complex 33 (31 mg, 21.6 mmol, 55%) as a red solid.

[0165] MS(ESI):C 69 H 74 N 10 O 14 S2Ru + Calculated value for: 717.20 [M+2H] 2+ / 2 Measured value: 717.27 [M+2H] 2+ / 2.

[0166] [ka] [ka] Ruthenium complex 35: A solution of amide 36 (7.7 mg, 7.1 mmol), NHS-ester 8 (7.2 mg, 10.6 mmol), and DIPEA (2.5 μL, 14.4 μmol) in DMF (1.0 mL) was stirred at room temperature for 1 day. The solvent was removed under reduced pressure, and the resulting residue was purified by HPLC chromatography (C18, 0–100% MeCN, 0.1% TFA in HO, over 80 min) to give ruthenium complex 35 (3.7 mg, 2.2 mmol, 31%) as a red solid.

[0167] MS(ESI):C 71 H 80 N 10 O 12 Ru + Calculated value for: 683.26 [M+2H] 2+ / 2 Measured value: 683.39 [M+2H] 2+ / 2.

[0168] Solubility Experiments: [ka] Inventive compound 8 (16.9 mg, 0.0248 mmol), inventive compound 11 (10.4 mg, 0.0139 mmol), standard compound 31 (5.0 mg, 0.015 mmol), and standard compound 32 (4.2 mg, 0.0104 mmol) were successively mixed with water to reach theoretical concentrations of 515 mM, 16.5 mM, 12.4 mM, 6 mM, and 0.6 mM, respectively. After each addition, the mixture was sonicated for 10 seconds. If the solid compound was still visible to the naked eye, it was considered undissolved. If the solid compound was not visible, it was considered dissolved. The results of the visual inspection are listed in Table 1, with "Yes" or "No" indicating whether the compound was considered dissolved based on visual inspection.

[0169] [Table 1]

[0170] The compounds of the present invention have significantly higher hydrophilicity as indicated by their better solubility in water. - It is clear that those bearing the group are more hydrophilic than the compounds of the invention bearing hydroxyl groups.

[0171] Hydrophobicity as shown by HPLC: [ka] Compounds 7 and 10 of the present invention and standard compound 31 were analyzed by reversed phase (RP HPLC, YMC-Triart C 18 HPLC chromatograms were obtained using a 250-mL HPLC system (0-100% MeCN in HO, 0.1% TFA in 25 min) and their respective retention times are listed in Table 2. A later retention corresponds to a better interaction with the hydrophobic stationary phase, and therefore the compound can be considered more hydrophobic. Conversely, a faster retention corresponds to a higher hydrophilicity.

[0172] [Table 2]

[0173] The compounds of the present invention were found to be more hydrophilic, and SO3 - It was found that the compounds of the present invention having a group were more hydrophilic than the compounds of the present invention having a hydroxyl group.

[0174] Antibody conjugation A modified target molecule, herein MAB, comprising the reaction product of a conjugate of formula (II) with a target molecule. <tn-t>The chim-5D8-IgG antibody (anti-troponin T monoclonal IgG antibody prepared by Roche, Penzberg, Germany, Roche substance number 05074991001; the monoclonal antibody 5D8 is known in the art, for example, from Jaffe AS et al. Journal of the American College of Cardiology 58 (2011) 1819-1824) (containing a 1,3-dipole group, here an azide group) was prepared. These target molecule complexes are also referred to below as "conjugates."

[0175] MAB <tn-t>The chim-5D8-IgG antibody was treated with increasing excesses (5, 10, 15, 20-fold) of NHS-PEG5-DBCO (for the reference compound; entries 1-4) or NHS-PEG4-azide (for the compounds of the present invention; entries 5-12). Unconjugated excess label was removed by dialysis. These conjugates were further treated with a 3-fold excess (compared to the previously used NHS ester) of BPRu-(O2OC)3-azide (entries 1-4) or 33 (entries 5-8) or 35 (entries 9-12), respectively. After the reaction, the conjugates were resuspended in 50 mM K phosphate buffer. + Unconjugated excess label was separated by size exclusion chromatography on Superdex™ 200 Increase 10 / 300GL using 5% DMSO in phosphate, 150 mM KCl pH 7.4. Antibodies treated with 33 achieved higher incorporation of label compared to the reference compound (Table 3).

[0176] [Table 3]

[0177] All conjugates were evaluated in a model sandwich immunoassay (Elecsys Troponin T hs, Roche: 05092744 190) containing streptavidin-coated beads (Roche: 05092744) measured on an Elecsys e170 module using the ECL signal as readout. Measurements without analyte in buffer (Diluent Universal, Roche: 11732277 122) showed that 33 could reduce background by 71-281 fold and 35 by 2-8 fold. Using serum (Diluent MultiAssay, Roche: 03609987 190), the reduction in background signal increased to 56-1065 fold for 33 and 9-62 fold for 35 (Table 4).

[0178] The reduction in background signal of the 33 and 35 conjugates compared to that of the reference DBCO conjugate can be attributed to hydrophilization (by sulfonation or hydroxylation) of the hydrophobic moiety.

[0179] Measurements of calibrator 2 (id.05092752 190) yield signals within or slightly above background when a reference complex is used (see Cal2 / MA above). Using 33 and 35, the signal-to-background ratio was increased to 374 and 334, respectively (see Cal2 / MA above).

[0180] Stability studies showed higher recovery of ECL signals for conjugates with 33 and 35 compared to the reference compounds after 8 days of storage at 35°C.

[0181] [Table 4]

[0182] References - European Patent No. 3004062 - International Publication No. 2014 / 189370 - Debets et al., Chem.Commun.2010,46,97-99 - Org.Biomol.Chem.,2014,12,5031-5037 - U.S. Patent No. 8,912,322 - International Publication No. 2003 / 002974 - International Publication No. 2012 / 107419 - International Publication No. 2012 / 107420 - International Publication No. 2014 / 019709 - International Publication No. 2014 / 019708 - International Publication No. 2017 / 153574 - PH Hagedorn et al., Drug Discovery Today 2018, 23(1), 101-114 - International Publication No. 2019 / 243391 - International Publication No. 2020 / 245377 - Abbas et al., Cellular and Mol. Immunology, 4th ed., WB Saunders, Co. (2000) - Plueckthun, In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994) pp. 269-315 - European Patent Application Publication No. 0404097 - International Publication No. 1993 / 01161 - Hudson, PJ et al., Nat. Med. 9 (2003) 129-134 - Holliger, P. et al., PNAS USA 90 (1993) 6444-6448

Claims

1. Formula (I) 【Chemistry 1】 (In the formula, R １ , R ２ are the same and both are -[(CH ２ ) ａ CR ｘ R ｙ ] ｂ R ｚ a is a group, a is either 0 or an integer ranging from 1 to 4, and b is either 0 or 1; R ｘ , R ｙ , R ｚ represents a hydrogen atom, a C1-C3 alkyl group, and (CH ２ ) ｃ SO ３ － groups, c is either 0 or an integer ranging from 1 to 4, and R ｘ , R ｙ , R ｚ At least one of the following conditions is satisfied (CH ２ ) ｃ SO ３ － is a group: -R ｚ But (CH ２ ) ｃ SO ３ － group, and when c is 0, R ｘ , R ｙ are both (CH ２ ) ｃ SO ３ － is not a group, c is 0, or - when a is 0, R ｘ and R ｙ are both (CH ２ ) ｃ SO ３ － is not a group and c is 0; R ３ , R ４ are independently selected from the group consisting of a hydrogen atom, a C1-C3-alkyl group, a halogen atom, and an —O—C1-C3-alkyl group; R ５ represents a carboxyl group, an activated carboxyl group, and -NHR ５ａ groups, and R ５ａ is a hydrogen atom or a C1-C5 alkyl group; L comprises a chain of covalently bonded atoms (linker) forming the backbone and having a length in the range of 1 to 100 atoms; n is R ５ is either 0 or 1 when R is a carboxyl group or an activated carboxyl group; ５ Ga-NHR ５ａ group, the value is 1.) or a salt thereof.

2. R ３ , R ４ are independently a hydrogen atom or a methyl group, and preferably R ３ , R ４ and each represent a hydrogen atom.

3. Formula (Ia) or (Ib): 【Chemistry 2】 【Transformation 3】 (Wherein L, n and R ５ The azadibenzocyclooctyne derivative or salt thereof according to claim 1 or 2, wherein

4. Formula (Ia-1) or (Ib-1): 【Chemistry 4】 【Transformation 5】 (In the formula, R ５ The azadibenzocyclooctyne derivative or salt thereof according to claim 1, wherein

5. Formula (II) 【Transformation 6】 (In the formula, L, R １ , R ２ , R ３ , R ４ and n is as defined in claim 1 for the azadibenzocyclooctyne derivative of formula (I) or a salt thereof; R ６ is selected from the group consisting of a fluorophore, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide; preferably a metal complex; Z is -C(=O)-O-, C(=O)-NR ７ -, and -NR ７ -C(=Y)-; R ７ is a hydrogen atom or a C1-C5 alkyl group, and Y is an oxygen atom or a sulfur atom, preferably an oxygen atom.

6. R ６ is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a metal complex, a radioisotope, an active pharmaceutical ingredient (drug), a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide and a polynucleotide, preferably a metal complex, and a further linker is preferably selected from the group consisting of an alkyl chain, a polyethylene glycol chain, a polypropylene glycol chain and a mixed polyethylene / polypropylene glycol chain, between Z and R ６ 6. The complex of formula (II) according to claim 5, wherein:

7. Formula (IIa) or (IIb); 【Transformation 7】 【Transformation 8】 (Wherein L, n and R ６ is as defined in claim 5, and Z １ is a -C(=O)-O- group or a -C(=O)-NH- group.

8. Formula (IIa-1), (IIa-2), (IIb-1), or (IIb-2): 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 (Wherein, R in (IIa-1), (IIa-2), (IIb-1), or (IIb-2) ６ is as defined in claim 5.

9. A method for modifying a target molecule, comprising reacting the complex according to claim 5 with a target molecule containing a 1,3-dipole group or a 1,3-(hetero)diene group.

10. 10. The method of claim 9, wherein the target molecule is selected from the group consisting of a fluorophore, a fluorescence quencher, a dye, a hapten, a tyramine, a polyethylene glycol chain, a polypropylene glycol chain, a mixed polyethylene / polypropylene glycol chain, a metal complex, a radioisotope, an active pharmaceutical ingredient, a carbohydrate, a solid phase, a lipid, an amino acid, an oligopeptide, a polypeptide, a nucleotide, an oligonucleotide, and a polynucleotide, and is preferably a polypeptide, more preferably an antibody, more preferably a modified antibody having a 1,3-dipole group, more preferably a modified antibody having an azide group.

11. 7. Use of the conjugate according to claim 5 or 6 for the bioorthogonal labelling and / or modification of target molecules.

12. A modified target molecule comprising the reaction product of a conjugate according to claim 5 and a target molecule comprising a 1,3-dipole group or a 1,3-(hetero)diene group obtained or obtainable by the method according to claim 9.

13. A kit comprising a modified target molecule according to claim 12 as a detection reagent and a suitable capture reagent.

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