Peptide coupling using thiocycloheptine derivatives

Abstract

This invention relates to a novel compound of the following form, optionally coupled to a peptide or protein molecule, and to a method for synthesizing the same. The invention also relates to the use of the novel compound in coupling reactions with linkers and other molecules. Furthermore, the invention relates to the use of the novel compound in a click reaction, specifically a bio-orthogonal strain-promoting cycloaddition (copper-free). JPEG2026521256000050.jpg51166

Description

[Technical Field]

[0001] This invention relates to peptide or protein molecules. More particularly, it relates to compounds and methods for coupling peptide or protein molecules to other molecules, such as drugs, drug delivery systems, targeting and imaging ligands, especially using copper-free click chemistry. [Background technology]

[0002] Chemical methods for site-directed functionalization of peptides and proteins are useful for a variety of applications in synthesis, research, and biomedicine. Examples include functionalization of peptides and proteins with affinity tags to facilitate separation, purification, and characterization; imaging labels for detection; and several molecules such as polymers, ligands, and small molecules to create fusion proteins for the purpose of modulating either biological activity, pharmacokinetic properties, or targeting properties.

[0003] Several methods have been developed for the functionalization of peptides and proteins that enable site-specific coupling while maintaining the structural integrity of the protein or peptide. These methods include modification of cysteine ​​residues obtained by the reaction of thiol groups with electrophiles, introduction of non-native amino acids by specific reactivity in proteins, and enzymatic coupling and modification of the N-terminus of proteins and peptides, many of which require specific N-terminal amino acids.

[0004] Non-patent document 1 describes site-directed modification of the N-terminus of proteins using 2-pyridine carboxyaldehydes (2PCA). 2PCA-biotin reagents or 2PCA-PEG reagents bind to multiple protein substrates, and the resulting biotinylated proteins can be captured by streptavidin resin. Meanwhile, PEGylation of therapeutic proteins plays a role in improving in vivo properties.

[0005] Non-patent document 2 describes the use of 2PCA (6-(azidomethyl)-2-pyridinecarboxaldehyde; 6-AM-2PCA) with azide functional groups for site-specific modification of antibodies. The azide functional group of 6-AM-2PCA is coupled to dibenzylcyclooctyne (DBCO) via click chemistry.

[0006] However, these methods have drawbacks, such as limiting the range of protein coupling partners or requiring cumbersome additional steps. For example, the method in Non-Patent Document 2 requires the presence of an alkyne moiety in DBCO, the coupling partner in Non-Patent Document 2. However, incorporating such alkyne moieties may be complex in practice and may not be compatible with, or difficult to integrate with, many coupling partners of interest. Peptides are an example. DBCO and BCN, alkyne-containing click chemistry partners, are inherently unstable under acidic peptide synthesis reaction conditions due to their amide and cyclopropane moieties, respectively (Non-Patent Documents 3, 4, 5, and 6), and as a result, they cannot be coupled to peptides during solid-phase synthesis. Therefore, it is impossible to selectively couple two peptides using the method in Non-Patent Document 2. Furthermore, the yield of the final compound in Non-Patent Document 2 is relatively low (<50% to 79%). Another drawback of the method described in Non-Patent Document 2 is that DBCO is a hydrophobic and relatively large reagent, which can negatively affect the solubility of peptides / proteins, potentially leading to aggregation and, consequently, a decrease in subsequent conjugation efficiency.

[0007] Non-patent document 1 requires N-hydroxysuccinimide (NHS) chemistry to introduce chelating agents, fluorescent labels, or other small molecules such as biotin into the 2PCA molecule before N-terminal functionalization. Coupling of these molecules to 2PCA is not possible after N-terminal functionalization of the peptide / protein by the 2PCA moiety. Furthermore, non-patent document 1 showed variability in the percentage of achievable modifications per protein, ranging from 43-95%.

[0008] Therefore, there is still a need to increase the diversity of protein and peptide functionalization, particularly novel, multipurpose, specific, and controllable functionalization, which is widely applicable to a wide range of peptides and proteins and coupling partners, does not affect their functional groups, and is cost-effective due to high functionalization and (almost) quantitative conjugation efficiency and thus the use of economical materials. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] MacDonald et al., 2015 [Non-Patent Document 2] Li et al., 2018 [Non-Patent Document 3] Chigrinova et al., 2013 [Non-Patent Document 4] Erickson et al., 2021 [Non-Patent Document 5] Janson et al., 2020 [Non-Patent Document 6] La-Venia et al., 2021 [Overview of the project] [Problems that the invention aims to solve]

[0010] One object of the present invention is to expand the diversity and selectivity of functionalization of peptidic and proteinaceous molecules by other molecules, including peptides and proteins. Another object of the present invention is to provide compounds that enable such expansion. The compounds and methods of the present invention enable the specific modification of substantially any peptidic or proteinaceous molecule at its N-terminus, followed by a site-specific strain-enhanced azide-alkyne cycloaddition (SPAAC) reaction for coupling to substantially any molecule, including another peptide protein, that is non-toxic in biological systems. [Means for solving the problem]

[0011] In view of the above, the present invention provides a compound of formula (I). [ka] (wherein n and m are independently 0, 1, or 2 (where n+m is 2), R 1 -R 8 This includes hydrogen, halogens, hydroxyl groups, oxo groups, (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups, (C2-6) alkynyl groups, (C3-12) cycloalkyl groups, (C3-12) heterocycloalkyl groups, (C6-12) aryl groups, (C6-12) heteroaryl groups, (C7-12) alkyl(hetero)aryl groups, and (C7-12)(hetero)arylalkyl groups (provided that these include (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups, (C2-6)alkynyl groups, (C3-12)cycloalkyl groups, (C3-12)heterocycloalkyl groups, (C6-12)aryl groups, (C6-12)heteroaryl groups, (C7-12)alkyl(hetero)aryl groups, and (C7-12)(hetero)arylalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of halogens, oxo groups, amino groups, hydroxyl groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups), or R 1 and R 7 、R 1 and R 8 、 R 2 and R 7 、 R 2 and R 8 、 R 3 and R 5 、 R 3 and R 6 、 R 4 and R 5 、 R 4 and R 6 form a cycloalkyl group, a cyclo(hetero)aryl group, a cycloalkyl(hetero)aryl group, or a cyclo(hetero)arylalkyl group (provided that these cycloalkyl groups, cyclo(hetero)aryl groups, cycloalkyl(hetero)aryl groups, or cyclo(hetero)arylalkyl groups are optionally substituted by one or more substituents independently selected from the group consisting of a halogen atom, an oxo group, an amino group, a hydroxy group, a (C1-4)alkyl group, and a (C1-4)alkoxy group), and L is a linking group.)

[0012] In a further aspect, the present invention provides a compound comprising a compound of formula (I) according to the present invention which is coupled to a peptidic or proteinaceous molecule, particularly coupled to the N-terminus of a peptidic or proteinaceous molecule. In particular, the compound comprising a compound of formula (I) according to the present invention is specifically coupled to the N-terminus of a peptidic or proteinaceous molecule.

[0013] In a further embodiment, the present invention provides a compound comprising a compound of formula (I), (II), or (III) according to the present invention, wherein the alkyne group of thiocycloheptine of formula (I), (II), or (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, and more preferably comprises an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound.

[0014] In a further embodiment, the present invention provides the use of compounds of formula (I), (II), or (III) according to the present invention in a bioorthoth copper free-click reaction. These reactions result in the formation of a bioconjugate.

[0015] In further embodiments, the present invention provides a use of compounds of formula (I), (II), or (III) according to the present invention for bioconjugation, particularly for coupling molecules to peptide or proteinaceous molecules, wherein the molecule is selected from the group consisting of drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates).

[0016] In a further embodiment, the present invention provides a method for coupling a molecule to a peptide- or protein-like molecule, comprising reacting the molecule with a compound of formula (I), formula (II), or formula (III) according to the present invention, wherein the molecule comprises a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, and the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, wherein the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, more preferably an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound. [Brief explanation of the drawing]

[0017] [Figure 1] HPLC trace of the reaction between TMTHSI-2PCA and octreotide. [Figure 2] HPLC trace of the reaction between TMTHSI-2PCA and LTX. [Figure 3] TP10-2PCA-glycol-TMTHSI (right peak) exhibiting a peak shift, and TP10-2PCA-glycol-TMTHSI reacted with azide ethanol via a click reaction (left peak indicated by the arrow). [Figure 4] The reaction of Evasin-3 with (16) (top) and the chromatogram of the reaction mixture after 26 hours (bottom). [Figure 5] HPLC trace of Evasin-3-TMTHSI(20). [Figure 6] Reaction of Evasin-3 TMTHSI (20) with CDP-azide (21) (top) and trace of the purified product with inserted mass from the MS spectrum of the deconvoluted peak (bottom). [Figure 7] Time-dependent decrease in the radiochemical purity of B2. Gray dashed line: control experiment with His / Suc buffer. Black solid line: human serum: experiment with a 1:1 His / Suc buffer solution at 4°C. N=2. [Figure 8] Graph showing the decrease in C1 percentage over time. The gray dashed line shows the stability of C1 at pH 7, and the black solid line shows the stability of C1 at pH 2. N=2. [Modes for carrying out the invention]

[0018] In this specification, "includes" and its conjugations are used in a non-restrictive sense, meaning that the matter following the word is encompassed, but matters not specifically mentioned are not excluded. Furthermore, the verb "consist of" may be replaced with "substantially constitutes" meaning that the compounds or auxiliary compounds specified herein may contain (one or more) additional components in addition to those specifically identified.

[0019] In this specification, the singular notation is used to refer to one of the objects expressed in the singular, or to more than one (i.e., at least one). For example, "element" means one element or more than one element.

[0020] When the words "and" or "about" are used with a number (such as "about 10" or "approximately 10"), it preferably means that the value may be a value within ±10% of that value.

[0021] The use of options (for example, "or") should be understood to mean any one of those options, both of them, or any combination thereof.

[0022] The compounds described herein and in the claims may contain one or more chiral centers, and different diastereomers and / or enantiomers of the compound may exist. Any description of a compound herein and in the claims is intended to encompass all of its diastereomers and mixtures thereof, unless otherwise specified. Furthermore, any description of a compound herein and in the claims is intended to encompass both of its individual enantiomers, as well as mixtures and racemates of such enantiomers, unless otherwise specified. If the structure of a compound is described as a specific enantiomer, it should be understood that the present invention is not limited to that specific enantiomer.

[0023] The compounds may arise in different tautomers. Unless otherwise specified, the compounds according to the present invention are intended to encompass all tautomers.

[0024] The compounds described herein and in the claims may also exist as exo and endo stereoisomers. Unless otherwise specified, any description of a compound herein and in the claims is intended to encompass both the individual exo and endo stereoisomers of the compound, as well as mixtures thereof.

[0025] The compounds described herein and in the claims may exist as cis and trans isomers. Unless otherwise noted, any description of a compound herein and in the claims is intended to encompass both the individual cis and trans isomers of the compound, as well as mixtures thereof. For example, if the structure of a compound is described as a cis isomer, it should be understood that the corresponding trans isomer or mixtures of cis and trans isomers are not excluded from the present invention.

[0026] In this specification, 'halogen' refers to fluorine, chlorine, bromine, or iodine. Preferred halogen atoms are fluorine and chlorine.

[0027] In this specification, the term "(Cx-y)alkyl" refers to a branched or unbranched alkyl group having xy carbon atoms. For example, (C1-6)alkyl means a branched or unbranched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, or butyl. Similarly, "(C1-2)alkyl" means an alkyl group having 1 or 2 carbon atoms. Preferred alkyl groups are methyl and ethyl.

[0028] In this specification, the term "(Cx-y)alkoxy" refers to an alkoxy group having xy carbon atoms, where the alkyl portion is as defined above. For example, the term "(C2-6)alkoxy" means an alkoxy group having 2-6 carbon atoms. Preferred alkoxy groups are methoxy and ethoxy.

[0029] In this specification, the term "(Cx-y) alkenyl" refers to a branched or unbranched alkenyl group having xy carbon atoms, i.e., having at least one double bond. For example, the term "(C2-6) alkylene" means a saturated alkylene group having 2-6 carbon atoms. Suitable examples of alkenyl groups, but not limited to these, include ethenyl, propenyl, isopropenyl, butenyl, and pentenyl. Unsubstituted alkenyl groups may include a cyclic moiety.

[0030] In this specification, the term "(Cx-y)alkynyl" refers to a branched or unbranched alkynyl group having xy carbon atoms, where triple bonds may be present at different positions of the group, such as ethynyl, propanyl, 1-butynyl, and 2-butynyl. For example, the term "(C2-6)alkynyl" refers to a branched or unbranched alkynyl group having 2-6 carbon atoms.

[0031] In this specification, '(Cx-y)cycloalkyl' refers to a cyclic alkyl group having xy carbon atoms, which may be monocyclic or polycyclic (e.g., fused bicyclic). For example, the term '(C3-6)cycloalkyl' refers to a cyclic alkyl group having 3-6 carbon atoms, i.e., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl (these are preferred cycloalkyl groups).

[0032] In this specification, “(Cx-y) heterocycloalkyl group” refers to a cyclic group having a cyclic skeleton containing at least one heteroatom, preferably O, S, or N, i.e., having xy carbon atoms + heteroatom. The group may be monocyclic or polycyclic (e.g., fused bicyclic). For example, the term “(C3-12) heterocycloalkyl group” refers to a cyclic alkyl group having 3-6 carbon atoms, such as pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran, and thiomorpholine.

[0033] In this specification, '(Cx-y)aryl group' refers to a cyclic aromatic group having xy carbon atoms, which may include monocyclic, bicyclic, and polycyclic structures. Optionally, the aryl group may be substituted with one or more further specified substituents. Preferred examples of aryl groups include phenyl, naphthyl, and anthracyl. The preferred aryl group is phenyl.

[0034] In this specification, a '(Cx-y) heteroaryl group' refers to a cyclic aromatic group having xy carbon atoms + a heteroatom, and may include monocyclic, bicyclic, and polycyclic structures. Optionally, the aryl group may be substituted with one or more further specified substituents. Preferred examples of heteroaryl groups include furan, pyridine, pyrazine, pyrrole, imidazole, pyrazole, oxazole, and thiophene.

[0035] In this specification, the term "(Cx-y)alkyl(hetero)aryl group" includes (Cx-y)alkylheteroaryl groups and (Cx-y)alkylaryl groups having xy carbon atoms (in the case of (Cx-y)alkylaryl) or having xy carbon atoms + heteroatoms (in the case of (Cx-y)alkylheteroaryl groups), in addition to alkyl and (hetero)aryl groups.

[0036] In this specification, '(Cx-y)(hetero)arylalkyl' includes (Cx-y)heteroarylalkyls and (Cx-y)arylalkyls having xy carbon atoms (in the case of (Cx-y)arylalkyls) or having xy carbon atoms + a heteroatom (in the case of (Cx-y)heteroarylalkyls), in conjunction with the (hetero)aryl group and alkyl group.

[0037] Regarding substituents, the term "optionally substituted" indicates that a group may be unsubstituted or substituted with one or more substituents of a specified number and type. The term "independently substituted" means that if a group is substituted with more than one substituent, these substituents may be the same or different from one another.

[0038] The inventors have surprisingly discovered that 2PCA can be introduced into thiocycloheptin derivatives to provide a wide range of bioconjugates, and can be used for site-specific functionalization of peptides and proteins at the N-terminus. Furthermore, it has been found that the introduction of 2PCA into thiocycloheptin derivatives is highly versatile and provides a site-specific modification tool applicable to the efficient coupling of coupling partners with the extremely diverse range of peptide compounds reported to date, from small molecules to large molecules, including peptides, proteins, and supramolecular assemblies such as nanoparticles. Relatively small and hydrophilic TMTHSI-like 2PCA derivatives can achieve higher conversion rates and subsequent binding of large (strept)avidin moieties, such as those used by MacDonald et al. (2015), than, for example, larger 2PCA-DBCO derivatives or 2PCA-BCN (bicyclo[6.1.0]non-4-in) derivatives or larger biotin derivatives. Furthermore, the synthesis of another click reagent, 2PCA-DBCO, is thought to yield a relatively hydrophobic derivative, which has adverse effects on solubility and, consequently, on the reactivity of peptides and proteins. Moreover, the resulting 2PCA-DBCO reagent may not be sufficiently stable for N-terminal introduction. Another click reagent, 2PCA-BCN, is also relatively hydrophobic and is thought to be less reactive than the reagent of the present invention. Furthermore, before N-terminal functionalization of peptides / proteins, it is necessary to introduce N-hydroxysuccinimide (NHS) chemistry, chelating agents, fluorescent labels, or other small molecules required, for example, as described in Non-Patent Document 1, into the 2PCA molecule. While the introduction of these molecules is not possible after N-terminal functionalization of proteins, the thiocycloheptin-2PCA compound of the present invention enables click reactions of any type of small or large compound after N-terminal functionalization of peptides / proteins. Another advantage of the thiocycloheptin compound is its tolerance to strongly acidic conditions, which opens up the possibility of introducing the compound under peptide synthesis conditions.

[0039] Since the introduction of azides in molecules or coupling partners is easier than, for example, the introduction of strained alkynes, the compounds and methods of the present invention enable a broader range of applications than those known to date.

[0040] Following the specific introduction of strained alkynes, the subsequent reaction between the functionalized peptide molecule and the azide-containing molecule achieves extremely high yields (>90%), and furthermore, this reaction proceeds much faster than known coupling reactions between functionalized proteins and their coupling partners.

[0041] As shown in the examples, the TMTHSI-like 2PCA derivatives of the present invention exhibit good biological and chemical stability. They remain stable even in the presence of human serum (see Example 2 and Figure 7). Furthermore, stability over time was maintained for at least 56 hours under acidic conditions (pH 2) and for a longer period at pH 7 (see Example 3 and Figure 8). Since the derivatives of the present invention are intended for use under physiological conditions, acid sensitivity is only important during the synthesis, workup, and formulation processes. Since the conjugation of TMTHSI-like 2PCA to biomolecules works best between pH 7 and 8, the TMTHSI-like 2PCA conjugate is considered to be exposed to an acidic environment only during workup and / or analysis. The amount of 2PCA conjugate in solution remains above 95% for at least 56 hours, so it will remain stable during the workup and analysis processes.

[0042] Finally, the functional properties of peptides or proteins are not impaired when functionalized according to the present invention.

[0043] In a first aspect, in view of the above, the present invention provides a compound of formula (I). [ka] (wherein n and m are independently 0, 1, or 2 (where n+m is 2), R 1 -R 8This includes hydrogen, halogens, hydroxyl groups, oxo groups, (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups, (C2-6) alkynyl groups, (C3-12) cycloalkyl groups, (C3-12) heterocycloalkyl groups, (C6-12) aryl groups, (C6-12) heteroaryl groups, (C7-12) alkyl(hetero)aryl groups, and (C7-12)(hetero)arylalkyl groups (however, these (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups) (The (C2-6) alkynyl group, (C3-12) cycloalkyl group, (C3-12) heterocycloalkyl group, (C6-12) aryl group, (C6-12) heteroaryl group, (C7-12) alkyl(hetero)aryl group, and (C7-12)(hetero)arylalkyl group are optionally substituted with one or more substituents independently selected from the group consisting of halogens, oxo groups, amino groups, hydroxyl groups, (C1-4) alkyl groups, and (C1-4) alkoxy groups), or R 1 and R 7 、 R 1 and R 8 、 R 2 and R 7 、 R 2 and R 8 、 R 3 and R 5 、 R 3 and R 6 、 R 4 and R 5 、 R 4 and R 6L forms a cycloalkyl group, a cyclo(hetero)aryl group, a cycloalkyl(hetero)aryl group, or a cyclo(hetero)arylalkyl group (wherein these cycloalkyl groups, cyclo(hetero)aryl groups, cycloalkyl(hetero)aryl groups, or cyclo(hetero)arylalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo groups, amino groups, hydroxyl groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups), and L is a linking group.

[0044] The thiocycloheptine derivative shown below is described in International Publication No. 2020 / 013696. [ka] Here, this compound is used with various linkers and reactive groups for the coupling of two different coupling partners.

[0045] In formulas (I), (II), and (III), m and n are independently 0, 1, or 2 (where n+m is 2). That is, both m and n are 1, or m is 2 and n is 0, or m is 0 and n is 2. It is preferable that m is 1 and n is 1. Therefore, the preferred compound of formula (I) is the compound of formula (IA). [ka]

[0046] The preferred compound of formula (II) is the compound of formula (IIA). [ka]

[0047] The preferred compound of formula (III) is the compound of formula (IIIA). [ka]

[0048] In equations (I), (II), and (III), R 1 -R 8 Preferably, R is independently selected from the group consisting of hydrogen, halogens, hydroxyl groups, oxo groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups (these (C1-4)alkyl and (C1-4)alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of halogens, hydroxyl groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups). More preferably, 1 -R 8 R is independently selected from the group consisting of hydrogen, halogens, and C1-C4 alkyl groups, and is preferably methyl or ethyl. More preferably, 1 -R 8 R is independently selected from the group consisting of hydrogen, methyl, and ethyl, more preferably from the group consisting of hydrogen and methyl. In one particularly preferred embodiment, R 1 -R 4 R is a methyl group, 5 -R 8 is hydrogen. Furthermore, it is preferable that both m and n are 1. Therefore, preferred compounds of formula (I) are compounds of formula (1B). [ka]

[0049] The preferred compound of formula (II) is the compound of formula (IIB). [ka]

[0050] The preferred compound of formula (III) is the compound of formula (IIIB). [ka]

[0051] In formulas (I), (II), and (III), L is a linking group. In this specification, 'linking group' and 'linker' refer to chemical groups that are interchangeable and have the functional properties to connect different parts of a molecule or compound, typically two parts. In this disclosure, the linking group connects the thiocycloheptine moiety to the 2-pyridinecarboxaldehyde moiety (2PCA). A wide range of linkers can be used, and the linker may be selected based on other criteria, for example, related to the end use of the compound. Suitable linking groups are well known in the art and can be appropriately selected by those skilled in the art.

[0052] In one preferred embodiment, L is a degradable linker. Herein, the term “degradable linker” refers to a linker that can be cleaved over time and / or under specific conditions, such as physiological conditions. The linker is preferably degradable under physiological conditions, and more preferably hydrolyzable under physiological conditions or by enzymatic activity. Suitable degradable linkers are selected from linkers comprising esters, orthoesters, amides, carbonates, carbamates, anhydrides, ketals, acetals, and hydrazones. Another example of a suitable degradable linker is a linker comprising a valine-citrulline (VCit) dipeptide linker or a glutamate-valine-citrulline tripeptide linker. These linkers are commonly used as enzymatically cleaved linkers.

[0053] In one preferred embodiment, the linking group L is linear or branched, C1-C 24 Alkylene group, C2-C 24 Alkenylene group, C2-C 24 Alkynylene group, C3-C 24 Cycloalkylene group, C5-C 24 Cycloalkenylene group, C5-C 24 Cycloalkylene group, C7-C 24 Alkyl (hetero)alylene group, C7-C 24 (hetero)arylalkylene group, C5-C 24 (hetero)arylalkenylene group, C9-C 24(hetero)arylalkylylene group (however, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkyl(hetero)alylene group, (hetero)arylalkylene group, (hetero)arylalkenylene group, and (hetero)arylalkylylene group may optionally be C1-C 12 Alkyl alkyl group, C2-C 12 Alkenyl group, C2-C 12 Alkynyl group, C3-C 12 Cycloalkyl groups, C5-C 12 Cycloalkenyl group, C5-C 12 Cycloalkynyl group, C8-C 12 Alkoxy group, C2-C 12 Alkenyloxy group, C2-C 12 Alkynyloxy group, C3-C 12 It contains, or is selected from, one or more groups independently selected from the group consisting of cycloalkyloxy groups, halogens, amino groups, and oxo groups.

[0054] In another preferred embodiment, L is a linear or branched carbon atom chain with a chain length of 1 to 50 atoms, where the chain length is determined by the number of atoms in the longest linear chain, which may contain one or more heteroatoms and / or one or more saturated or unsaturated cyclic or heterocyclic moieties. Therefore, L is preferably at least a CH2 group. For example, in the following compound, the number of atoms in the longest linear chain of linking group L is 6. [ka] Preferably, the chain has a chain length of 1-40 atoms in the longest linear atomic chain, more preferably 1-30 atoms, more preferably 1-25 atoms, more preferably 1-20 atoms, and more preferably 1-15 atoms. The longest linear atomic chain may contain one or more heteroatoms such as O, N, S, and P. Preferably, the longest linear atomic chain may contain one or more N and / or O atoms, and more preferably one or more O atoms and one or more N atoms.

[0055] In a further preferred embodiment, a linear or branched carbon atom chain having a chain length of 1-50 atoms, preferably 1-40, 1-30, 1-25, 1-20, or 1-15 atoms, contains, in the longest molecular chain, one or more moieties independently selected from the group consisting of -S(O)2-, -S-, -SS-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, (C6-12)heteroaryl, and combinations thereof, preferably 1-10. In one preferred embodiment, the linear or branched carbon atom chain contains 1, 2, or 3 such moieties.

[0056] In further preferred embodiments, the linear or branched carbon atom chain comprises a (C3-12) heterocycloalkyl, preferably a (C3-8) heterocycloalkyl, more preferably a (C5-6) heterocycloalkyl, and most preferably a C6-heterocycloalkyl. Preferred examples of (C3-12) heterocycloalkyls are pyrrolidine, pyrazolidine, piperidine, piperazine, tetrahydrothiophene, thiane, dithiane, tetrahydrofuran, and thiomorpholine. Most preferred is piperazine.

[0057] Furthermore, preferably, chains with a length of 1-50 atoms, preferably 1-40, 1-30, 1-25, 1-20, or 1-15 atoms, are not branched, that is, they are linear except for one or more part atoms or heteroatoms that are not included in the longest linear atomic chain, such as -S(O)2-, -S-, -SS-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl, (C3-12)heterocycloalkyl, (C6-12)aryl, and (C6-12)heteroaryl.

[0058] In preferred embodiments, L comprises a piperazine or polyethylene glycol (PEG) portion.

[0059] In a particularly preferred embodiment, L is a linear or branched carbon atom chain that includes piperazine, and more preferably the portion described below. [ka] However, the following part [ka] Includes. In a particular preferred embodiment, the linking group L is structure [ka] (Here, -C(O)- is bonded to the nitrogen atom of thiocycloheptine).

[0060] In another preferred embodiment, the linking group L comprises or is a polyethylene glycol (PEG) moiety, preferably -(O-CH2-CH2) n - (wherein n is up to 13, and more preferably n is an integer from 2 to 10) including or including. In one preferred embodiment, the linking group L includes piperazine and PEG moieties, preferably the following moieties [ka] and the PEG portion, preferably the PEG2 portion (-(O-CH2-CH2) n - (where n=2)) is included. In another preferred embodiment, the linking group L has the following structure. [ka] (However, -C(O)- is bonded to the nitrogen atom of thiocycloheptine.)

[0061] In a preferred embodiment, the compound of formula (I) is [ka] and [ka] Selected from.

[0062] The present invention also provides methods for preparing compounds of formula (I), (IA), and (IB). Compound (I) can be prepared by methods known in the art. The method for preparing the compounds of the following formulas is: [ka] As described in detail in International Publication No. 2020 / 013696, which is incorporated herein by reference, suitable methods for preparing the compounds of formulas (I), (IA), and (IB) are described in the examples herein.

[0063] For example, to briefly explain, 1-imino-3,3,6,6-tetramethyl-4,5-didehydro-2,3,6,7-tetrahydro-1H-1λ6-thiepine 1-oxide (TMTHSI) was derivatized with its succinimidylcarbamate using a solution of DSC in ACN to obtain TMTHSI-OSu. The tosylated form of piperazine-2PCA was reacted with TMTHSI-OSu in a 1:1 ACN:DCM mixture using DIPEA as the base. The product was purified on silica, triturated from Et2O, filtered, washed with pentane, and air-dried.

[0064] Compounds of formula (I), in which L is an ethylene glycol spacer, can be synthesized, for example, by a seven-step reaction starting with an ethylene glycol derivative and pyridinebismethylene alcohol, as shown in Scheme 4 of the examples herein.

[0065] As described above, the compounds of formulas (I), (IA), and (IB) can be appropriately coupled to peptide-like or protein-like molecules, preferably peptides or proteins. Therefore, compounds comprising the compounds of formulas (I), (IA), and (IB) that are coupled to peptide-like or protein-like molecules, preferably peptides or proteins, and particularly to the N-terminus of peptide-like or protein-like molecules, are also provided. In particular, the compounds comprising the compound of formula (I) according to the present invention are specifically coupled to the N-terminus of peptide-like or protein-like molecules.

[0066] In particular, compounds of formula (II) or (III) are provided herein. [ka] [ka]

[0067] In equations (II) and (III), m, n, R 1 -R 8 , and L are defined above.

[0068] In preferred embodiments, the compound is a compound of formula (IIA) or (IIIA), [ka] [ka] More preferably, the compound is of formula (IIB) or (IIIB). [ka] [ka]

[0069] In formulas (II), (IIA), (IIB), (III), (IIIA), and (IIIB), X is a peptide or protein molecule. Hereinafter, “peptidic molecule” and “protein molecule” refer to molecules containing at least one amino acid residue, preferably more than one amino acid residue. Typically, the term “peptidic molecule” is used for molecules containing relatively short amino acid chains, e.g., up to about 50 amino acids, while the term “protein molecule” is used for molecules containing longer amino acid chains, e.g., from about 50 amino acids. In preferred embodiments, X is a peptide or protein.

[0070] Before coupling to the compounds of formula (I), (IA), or (IB), the peptidic or proteinaceous molecule contains at least two amino acid residues linked to each other via peptide bonds, i.e., the peptidic or proteinaceous molecule contains at least one peptide. The N-terminal amino acid of this peptidic or proteinaceous molecule reacts with 2PCA to form a cyclic structure, in particular a five-membered ring in compounds (II), (IIA), and (IIB) or a six-membered ring in compounds (III), (IIIA), and (IIIB). In the compounds of formula (II), (IIA), (IIB), (III), (IIIA), and (IIIB), X is a peptidic or proteinaceous molecule without an N-terminal amino acid that reacts with 2PCA.

[0071] Any peptide-like or protein-like molecule described herein preferably comprises one or more peptides and proteins, and optionally comprises one or more non-amino acid moieties and cleavable sites.

[0072] In one embodiment, a peptidic or proteinaceous molecule contains or is a peptide or protein. Non-limiting examples include therapeutic peptides and proteins, including antibodies, antigens and ligands, imaging and targeting ligands, and any other bioactive peptides or proteins. The term “bioactive peptide or protein” refers to a peptide or protein that exerts activity when administered to a subject. This activity may be any activity, including, but is not limited to, therapeutic or prophylactic activity, binding activity, diagnostic activity, or targeting activity. In one preferred embodiment, a peptidic or proteinaceous molecule is a peptide or protein, meaning that it consists of amino acid residues as a whole.

[0073] In another embodiment, the peptide-like or protein-like molecule comprises one or more non-amino acid moieties. In principle, any molecule can be coupled to at least one peptide to form a peptide-like or protein-like molecule as defined herein. Non-limiting examples of non-amino acid moieties include oligonucleotides, peptide mimetic substances, targeting and imaging labels, sugars, and any other bioactive molecules. These non-amino acids are bonded to at least one amino acid via a secondary amine.

[0074] Therefore, in preferred embodiments, the peptidic or proteinaceous molecule is a peptide, a protein, or a molecule coupled to at least a dipeptide, preferably selected from the group consisting of oligonucleotides, peptide mimetic substances, targeting and imaging labels, sugars, and any other bioactive molecules. Herein, at least a dipeptide means a chain of at least two amino acid residues linked to each other via peptide bonds.

[0075] In one embodiment, a peptidic or proteinaceous molecule contains a cleavable moiety or linker between at least one peptide coupled to a compound of formula (I) of the present invention and the peptide, protein, or other molecule of interest. Such a cleavable moiety or linker results in the release of the peptide, protein, or other molecule of interest under appropriate conditions. The cleavable moiety or linker is preferably gradable under physiological conditions and more preferably hydrolyzable under physiological conditions or by enzymatic activity. Suitable cleavable moieties or linkers are selected from the group consisting of esters, orthoesters, amides, carbonates, carbamates, anhydrides, ketals, acetals, and hydrazones. Another example of a suitable cleavable moiety or linker is a linker comprising a valine-citrulline (VCit) dipeptide linker or a glutamic acid-valine-citrulline tripeptide linker.

[0076] The peptide-like or protein-like molecule may contain any number of amino acids and any non-amino acid moieties, since its ability to be coupled to the compounds according to the present invention is not limited by the size of the peptide-like or protein-like molecule or the properties and size of one or more non-amino acid moieties. For example, the molecule may contain peptides of 2 to 50, 2 to 100, 2 to 500, or 2 to 1000 amino acids. The peptide-like or protein-like molecule may contain a single amino acid chain. Alternatively, the peptide-like or protein-like molecule may contain two or more amino acid chains linked by non-peptide bonds.

[0077] At least two amino acid residues linked to each other via a peptide bond within a peptidic or proteinaceous molecule prior to being coupled to a compound of formula (I), (IA), or (IB) must not have a free N-terminus. The free N-terminus can be utilized for coupling to the 2PCA moiety of the compounds of the invention of formula (I), (IA), or (IB). The N-terminal amino acid of the peptidic or proteinaceous molecule prior to being coupled to a compound of formula (I), (IA), or (IB) can be any amino acid, either a natural amino acid or a non-natural amino acid, and can be either an L-amino acid or a D-amino acid. However, it is preferred that the amino acid is an α-amino acid or a β-amino acid. Coupling of a peptidic or proteinaceous molecule containing an N-terminal α-amino acid to a compound of formula (I), (IA), and (IB) results in a compound of formula (II). Coupling of a peptidic or proteinaceous molecule containing an N-terminal β-amino acid to a compound of formula (I), (IA), and (IB) results in a compound of formula (III).

[0078] Furthermore, the second amino acid (and thus the first amino acid in X) in any peptidic or proteinaceous molecule coupled to a compound of formula (I), (IA), or (IB) of the invention is not proline, and more preferably is neither proline nor another amino acid containing a tertiary amine.

[0079] In formula (II), (IIA), and (IIB), R 9 is an amino acid side chain. In formula (III), (IIIA), and (IIIB), one of R 9 and R 10 is an amino acid side chain and the other of R 9 and R 10 is hydrogen, or both of R 9 and R 10 are amino acid side chains. The term "amino acid side chain" is well known in the art and refers to the substituent that characterizes the relevant amino acid. This term refers to the substituent attached to the α-carbon of the amino acid. R 9 and / or R10 The side chain can be any amino acid, and may be either a natural or unnatural α-amino acid.

[0080] The coupling of peptidic or proteinaceous molecules to compounds of formula (I), (IA), or (IB) can be achieved by methods well known in the art. Suitable methods for such coupling are described in the examples herein. Briefly, (I), (1A), or (1B) is dissolved in pH 7.4 phosphate buffer containing 10% ACN. A 10 mM solution of this is added to a peptidic or proteinaceous molecule dissolved at a concentration of 0.1 mM in pH 7.4 phosphate buffer. This is reacted overnight at room temperature. The reaction mixture was purified by dialysis on a PD Sephadex column G10 or G25 membrane with a molecular weight cutoff of 1 kDA, depending on the size of the peptide molecule. In all cases, 20 mM phosphate buffer at pH 7.4 was used as the eluent. Optionally, any excess thiocycloheptin-2PCA reagent can be removed by other known techniques, for example, by incubation with hydroxylamine-containing beads that capture the aldehyde of the reagent by oxime formation.

[0081] The alkyne group of the compounds of the present invention is reactive and can be functionalized, for example, using cycloaddition reactions, particularly to provide bioconjugates. The term "bioponjugate" refers to the coupling of two molecules, at least one of which is a biological molecule, particularly a peptide or protein. In one embodiment, the alkyne group can be reacted with a compound containing a thiol, a 1,3-dipole, or a 1,3-(hetero)diene. In some embodiments, the compound containing a thiol, a 1,3-dipole, or a 1,3-(hetero)diene is an azide-containing compound, a nitrone-containing compound, or a nitrile oxide-containing compound. Most preferably, the azide-containing compound is used. In preferred embodiments, the azide-containing compound can be coupled using a copper-free click reaction. This coupling results in a triazole-type structure. Therefore, the compounds of the present invention are suitable for coupling peptide or protein molecules to other molecules. Compounds comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene can be coupled to the thiocycloheptin compound of the present invention either before or after the coupling of a peptidic or proteinaceous molecule to the thiocycloheptin compound of the present invention.

[0082] Compounds are provided that include a compound according to any of the present invention, wherein the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene. Preferably, the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene comprises an azide, a nitrone, or a nitrile oxide, and more preferably an azide, where the azide-alkyne coupling results in the formation of a triazole compound.

[0083] Functionalization can be carried out with any compound, for example, drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates), particularly those containing thiols, 1,3-dipoles, or 1,3-(hetero)dienes, preferably those containing azides, nitrones, or nitrile oxides, more preferably those containing azides.

[0084] Methods for introducing azides into azides-free compounds are well known in this art, and those skilled in the art can select appropriate reactions. Suitable methods for introducing azides into primary amines are described, for example, in Presser and Bertozzi (2005) and Meng et al. (2019).

[0085] Therefore, in preferred embodiments, the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene comprises one or more drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates).

[0086] The molecule may be bonded to the compound of formula (I), (II), or (III) via a cleavable moiety or linker. In one embodiment, a compound comprising a thiol, 1,3-dipole, or 1,3-(hetero)diene contains a cleavable moiety or linker between the thiol, 1,3-dipole, or 1,3-(hetero)diene coupled to the compound of formula (I), (II), or (III) of the present invention, and a peptide, protein, or other molecule. Such a cleavable moiety or linker results in the release of the molecule under appropriate conditions. The cleavable moiety or linker can be any of the cleavable moieties or linkers described above. The moiety or linker is preferably cleavable under physiological conditions, and more preferably hydrolyzable under physiological conditions or by enzymatic activity. Suitable cleavable moieties or linkers are selected from the group consisting of esters, orthoesters, amides, carbonates, carbamates, anhydrides, ketals, acetals, and hydrazones. Another example of a suitable cleavable portion or linker is a linker containing a valine-citrulline (VCit) dipeptide linker or a glutamate-valine-citrulline tripeptide linker.

[0087] The present invention further provides the use of any compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) in a bioorthogonal copper free-click reaction. Copper free-click chemistry is a bioorthogonal reaction, and since cytotoxic copper catalysts are eliminated, the reaction can be carried out without toxicity to biological systems such as cells and tissues.

[0088] Also provided is the use of any compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) for coupling a molecule to a peptide or protein molecule, wherein the molecule is preferably selected from the group consisting of drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates).

[0089] Also provided is a method for coupling a molecule to a peptide- or protein-like molecule, comprising reacting the molecule with a compound of any of the formulas (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB), wherein the molecule comprises a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, and the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, wherein the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, more preferably an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound.

[0090] In some embodiments, the molecule coupled to the compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) of the present invention is a drug delivery carrier, in particular, a nanoparticle. That is, the compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) of the present invention is coupled to a nanoparticle via the alkyl group of thiocycloheptine of the compound. Drug delivery systems are increasingly used in the pharmaceutical field. The use of these drug delivery systems, such as nanoparticles or liposomes, is rapidly developing in the pharmaceutical field. In the pharmaceutical field, these systems are used to reduce drug toxicity and side effects and improve delivery, and imaging ligand support is added for efficacy monitoring.

[0091] Nanoparticles, typically having a diameter of <100 nm, are manufactured from a wide range of materials and have been anticipated to have medical applications including drug delivery, both in vitro and in vivo diagnostics, nutritional supplements, and the manufacture of improved biocompatible materials. Nanoparticles can be customized for specific applications, and a wide range of materials are available. Many pharmaceutically interesting nanoparticles are based on (bio)polymer materials, which can take various forms. The materials may be of biological origin, such as phospholipids, lipids, lactic acid, dextran, and chitosan, or they may have more "chemical" properties, such as various (co)polymers. These nanoparticles are typically functionalized by (covalently) linking, coupling, or binding active ingredients (drugs, ligands, imaging ligands, etc.). Many of these nanoparticle functionalizations can be achieved by a wide range of chemistry, such as copper-free click chemistry, using the thiocycloheptine derivatives of the present invention.

[0092] In some embodiments, the nanoparticles are self-assembling polymer micelles, preferably derived from temperature-sensitive block copolymers. Particularly preferred are copolymers based on PEG-b-poly(N-hydroxyalkylmethacrylamide-oligoacetate) having partially methacrylated oligolactic acid units, but other (meth)acrylamide esters can also be used to construct temperature-sensitive blocks, for example, esters of HPMAm (hydroxypropyl methacrylamide) and HEMAm (hydroxyethyl methacrylamide), and optionally (oligo)lactic acid esters, as well as N-(meth)acryloyl amino acid esters. Furthermore, preferred temperature-sensitive block copolymers are derived from monomers having functional groups that may be modified with derivatized and non-derivatized methacrylate groups, such as HPMAm-lactic acid polymers. That is, this modification involves the incorporation of linker moieties.

[0093] Other functional temperature-sensitive (co)polymers that can be used include copolymers of hydrophobically modified poly(N-hydroxyalkyl)(meth)acrylamide, N-isopropylacrylamide (NIPAAm) with monomers having reactive functional groups (e.g., acidic acrylamide or other parts such as N-acryloxysuccinimide), or homologous copolymers of poly(alkyl) 2-oxazalines. Further preferred temperature-sensitive groups are obtained based on NIPAAm and / or alkyl-2-oxazolines, and these monomers may be reacted with monomers having reactive functional groups such as (meth)acrylamide or (meth)acrylate having a hydroxy, carboxy, amine, or succinimide group.

[0094] Suitable temperature-sensitive polymers are described in U.S. Patent No. 7,425,581 and European Patent Application Publication No. 1776400, which are incorporated herein by reference. Furthermore, they are also described in International Publication No. 2010 / 033022 and International Publication No. 2013 / 002636, which are incorporated herein by reference.

[0095] International Publication No. 2012 / 039602, incorporated herein by reference, describes drug-polymer matrix particles using these polymers. Furthermore, International Publication No. 2012 / 039602 describes biodegradable linker molecules that may be used in such known polymer matrix particles.

[0096] Typically, nanoparticles based on temperature-sensitive block copolymers as outlined above can be coupled to the compounds of the present invention using azide-alkyne copper-free coupling. Examples thereof are described in International Publication No. 2017 / 086794, which is incorporated herein by reference.

[0097] Therefore, in some preferred embodiments of the present invention, nanoparticles are prepared in which the compound of the present invention is coupled to azide-containing nanoparticles. In some embodiments, the nanoparticles are self-assembling polymer micelles, preferably derived from a temperature-sensitive block copolymer.

[0098] In one embodiment, the molecule coupled to the compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) of the present invention is an antibody. That is, the compound of formula (I), (IA), (IB), (II), (IIA), (IIB), (III), (IIIA), or (IIIB) of the present invention is coupled to an azide-containing antibody via the alkyl group of thiocycloheptin of the compound. Antibody-drug conjugates (ADCs) are increasingly being developed in the pharmaceutical field. Antibody-drug conjugates combine the specificity and target-directivity of monoclonal antibodies (mABs) with, for example, the potency of cytotoxic molecules. Such ADCs have been developed, for example, for the treatment of cancer, where they target and kill tumor cells while preserving non-tumor cells. Therefore, an example of an antibody-drug conjugate according to the present invention is a bioconjugate of a cytotoxic peptide functionalized with the thiocycloheptin-2PCA compound of the present invention. Any cytotoxic peptide can be functionalized with the thiocycloheptin-2PCA compound of the present invention and then coupled to an antibody to form an ADC.

[0099] For the sake of clarity and conciseness, features may be described herein as part of the same or distinct aspects or embodiments of the invention. Those skilled in the art will understand that embodiments having all or some combinations of the features described herein as part of the same or distinct embodiments may fall within the scope of the invention.

[0100] The present invention will be described in more detail by the following non-limiting examples.

[0101] (Examples) (Example 1) We investigated whether the 2-pyridinecarboxaldehyde (2PCA) reagent can be combined with the TMTHSI-click handle that we recently published (Weterings et al. 2020).

[0102] The first click construct (3) that we evaluated was obtained in 50% yield by combining 2PCA (2) and TMTHSI via a piperazine linker in reaction with the TMTHSI-hydroxysuccinamide derivative (1) that we previously described (Scheme 1). [ka] (Scheme 1: Synthesis of TMTHSI-2PCA(3))

[0103] Octreotide (4), a peptide clinically used for the treatment of acromegaly, was used to test the novel TMTHSI-2PCA reagent (3) in this study. Octreotide is a cyclic peptide with several functional groups, including a lysine side chain NH2 and a disulfide bridge, making it a useful model for evaluating the potential N-specific reagent (3) in this study. After reaction with 20 equivalents of TMTHSI-2PCA (3), a conversion rate of >90% was achieved based on a decrease in the octreotide peak (Scheme 2), which was confirmed by LC-MS to be selectively functionalized at the N-terminus (Figure 1). Not only was the expected mass of modified octreotide observed, but evidence of cyclization arising from an N-terminal specific Edman degradation-like cyclization reaction was also evident from the presence of two diastereomers with the same mass that could be separated by LC-MS (Rutjes, 2015). [ka] (Scheme 2: Reaction of TMTHSI-2PCA(3) and octreotide(4))

[0104] Next, the oncolytic LTX-315 peptide (Sveinbjo / rnsson et al. 2017; Zhou et al. 2016) (6), which contains multiple (five) lysine residues, was exposed to TMTHSI-2PCA (3) (Scheme 3). Here again, an LTX peptide adduct (7) with only one TMTHSI-linker moiety was formed (Figure 2). As expected, no (stable) Schiff base adducts were formed on the side chains of the lysine residues. As mentioned above, N-terminal modification was also evident from the formation of a diastereomer mixture (Figure 2). [ka] (Scheme 3: Reaction of MTHSI-2PCA(3) with LTX(6) having multiple lysine side chains)

[0105] Therefore, the TMTHSI-2PCA(3) reagent is suitable for N-specific introduction of a TMTHSI-containing linker into multifunctional peptides.

[0106] For larger peptides and proteins, we envisioned TMTHSI derivatives with a longer distance between the N-terminal aldehyde and the TMTHSI moiety of (16) (Scheme 4). The longer distance formed by the ethylene glycol spacer provides a suitable distance between the reagent binding site and the strain-promoting azidoalkyne cycloaddition reaction in peptides or proteins of considerable size, and may also be favorable for the water solubility of the adduct. The TMTHSI-glycol-2PCA derivative (16) was obtained by a simple seven-step synthesis starting from the ethylene glycol derivative (8) and pyridinebismethylene alcohol (11) (Scheme 4). [ka] (Scheme 4: Synthesis of TMTHSI-glycol-2PCA(16))

[0107] This TMTHSI-glycol-2PCA derivative (16) was used in the N-terminal modification of the peptide Angiopep2 (17) (Scheme 5). Angiopep2 is applied as a conjugate for intracellular delivery of oligonucleotides and transport across the blood-brain barrier (Lei et al. 2022). Excess TMTHSI-glycol-2PCA can be removed by incubation with hydroxylamine-containing beads after the reaction is complete (Scheme 5). These beads capture the aldehyde of TMTHSI-glycol-2PCA by forming an oxime, which is shown in the workup of the reaction between peptodo TP10 (sequence) and hydroxylamine beads. [ka] (Scheme 5: Capture of excess TMTHSI-glycol-2PCA(16) using hydroxylamine beads)

[0108] At this point, we attempted to verify that the TMTHSI moiety was still intact and reactive. We performed this verification by adding azide ethanol to the workup reaction mixture of the TP10 peptide (AGYLLGKINLKALAALAKKIL, membrane transport peptide (Soomets et al. 2000; Zhang et al. 2019; Islam et al. 2014)), N-terminal modification, Scheme 6. This resulted in a complete shift of the diastereomerized peptide to a more polar region of the HPLC trace (Figure 3). [ka] (Scheme 6: Click reaction between TMTHSI-glycol-2PCA peptide construct and azideethanol)

[0109] Finally, we aimed to demonstrate that TMTHSI-glycol-2PCA has the ability to specifically react with the N-terminus of larger proteins. We selected this linker because N-terminal modification by Evasin-3 should enable binding for the purpose of modified vaccine administration (Figure 4, top).

[0110] Twelve equivalents of (16) were reacted with Evasin-3 for 26 hours. Under these conditions, complete conversion of Evasin-3 (19) was not observed (Figure 4, bottom), but separation by HPLC was possible, and Evasin-3-TMTHSI (20) was isolated by collecting the HPLC fraction. This yielded a pure product with a final yield of 40% (Figure 5).

[0111] Considering that 12 equivalents of (16) were used and purification was performed by preparative HPLC, a 40% yield of the pure product is very satisfactory and is expected to be significantly higher with the development of further processing steps.

[0112] For vaccine applications, Evasin-3-TMTHSI (20) was reacted with azide-functionalized CDP (21) in a click reaction (Figure 6). Two equivalents of Evasin-3-TMTHSI (20) were added dropwise to one equivalent of CDP-azide (21). After 3.5 hours of reaction, the reaction mixture was purified using HPLC, and the product peak was collected. Confirmation by MS revealed a pure Evasin-CDP conjugate (22). The overall yield of the conjugate was 60%.

[0113] (Example 2: Biological stability of TMTHSI-2PCA conjugate containing BSA in human serum) The objective of this embodiment is to determine the stability of the TMTHSI-2PCA BSA conjugate under physiological conditions. Bovine serum albumin (BSA) was used as a model protein. Conjugation with the 2PCA moiety is expected to occur. To track stability, DFO-N3 is bonded to the TMTHSI moiety via an alkyne functional group. 89 Zr markings are now possible. • Stability was confirmed by incubating the product in serum at 4°C, BSA-2PCA-DFO- 89 This was determined by monitoring the decrease in the radiochemical purity of Zr(B2). [Chemical]

[0114] (Synthesis of B1) The following were placed in an Eppendorf tube. · 439.5 μl of 0.1 M PBS (pH = 7.4) · 10.5 μl of TMTHSI-PEG2-2PCA (50 mM) · 50 μl of BSA (0.1 g / ml)

[0115] The reaction mixture was incubated on a thermoshaker at 37 °C for 2.5 days. The reaction mixture was purified using a PD10 cartridge column according to the following method. · Pipette 500 μl of BSA-modified solution into the column. · Pipette 1750 μl of 0.1 M PBS into the column. · Pipette 1750 μl of PBS into the column. Collect this fraction.

[0116] The fraction collected from the PD-10 column was further purified using the following 30 kDa spin filter method. · Fill three 30 kDa spin filters with 500 μl each and one 30 kDa spin filter with 250 μl, and centrifuge the solution at 10000 rcf for 3 minutes. · Wash the spin filter with 200 μl of 1.0 M PBS and centrifuge at 10000 rcf for 3 minutes. · Repeat the washing step a total of 3 times. · Invert the spin filter and place it in a new Eppendorf vial and centrifuge at 10000 rcf for 3 minutes. · Adjust the collected solution to 500 μl using PBS.

[0117] ( 89 Zr Pre-labeling) 20 MBq of 89 Zr (volume depends on the manufacturing date) was placed in an Eppendorf. 89Zr was prepared in 20 μl with oxalic acid (1.0 M, volume dependent on the volume of 89 Zr). Na2CO3 (2.0 M, 9 μl) was added to this solution, and then the volume was adjusted with 1.0 M HEPES buffer (131 μl). A solution of DFO-N3 (0.5 mM, 0.1 equivalent, volume dependent on the final concentration-modified BSA) was prepared. 89 The Zr solution was added to the resulting solution, which was then stirred in a thermos shaker at 20°C for 15 minutes.

[0118] (B2 synthesis) 89 350 μl of modified BSA solution was added to the Zr prelabeled solution. The resulting solution was incubated in a thermoshaker at 20°C for 1 hour. 89 Zr-labeled modified BSA was rebuffered in 240 mM histidine / scroll (His / Suc) buffer using a PD-10 column according to the following method. • Pipette 500 μl of the labeled solution into the column. • Pipette 1750 μl of 240 mM His / Suc buffer into the column. • Pipette 1500 μl of 240 mM His / Suc buffer into the column. Collect this fraction.

[0119] (Stability of B2 over time) The collected B2 fraction was filtered and placed in a sterile vial with 1.5 mL of human serum. B2 dissolved in His / Suc buffer was used as a control. A 100 μl aliquot was taken immediately after filtration as the measurement at t=0. The material was incubated at 4°C. Subsequently, 100 μl aliquots were taken at t=1, 24, 48, 72, 144, and 168 hours. A 3 μl aliquot was taken and diluted in 6 μl of 240 mM His / Suc buffer to prepare a sample for spin filtration analysis. Spin filtration analysis was performed twice for each sample according to the following method. Using a pipette, 3 μl of diluted spin-filtered aliquots were added to 100 μl of 240 mM His / Suc buffer containing 5% DMSO. The spin filter was centrifuged at 14,000 rcf for 7 minutes. The washing and centrifugation processes were performed a total of three times. After the final centrifugation step, the spin filter and Eppendorf vial are separated into different tubes, and the activity is measured using a Hidex fully automated gamma counter #425-601. The counting window is between 400 and 1100 keV, and the counting time is set to 1 minute. The entire gamma counting procedure takes approximately 15 minutes and is performed at room temperature.

[0120] The radiochemical purity of B2 was determined by examining the radiochemical purity of the tube containing the spin filter.

[0121] Monitoring of B2 stability according to the spin filtering method described above yielded the radiochemical purity results shown in Figure 7. While the control experiment (gray dashed line) was not monitored as long as human serum experiments (black solid line) were performed, the decrease in radiochemical purity showed a similar trend in both experiments. Furthermore, considering the standard deviation, the difference in radiochemical purity over time between the control and serum experiments does not appear to be significant. The results indicate that the stability of B2 is not affected by exposure to human serum. The observed decrease in radiochemical purity in DFO was due to the effects of human serum exposure. 89 Due to the instability of Zr, or 89 This can be caused by other decompositions triggered by irradiation from Zr.

[0122] (Example 3: Stability of TMTHSI-2PCA with model peptide under harsh acidic conditions) The purpose of this example was to evaluate the stability of the MTHSI-2PCA conjugate at acidic pH values. • Tuftosin was used as a model peptide. Since 2PCA exhibits good absorbance at 267 nm, it was not necessary to use a chromophore to label the tuftosin-CliCr construct (C1) via alkyne. Stability was determined by monitoring the time-dependent decrease in C1 (in the control experiment) and the generation of TMTHSI-PEG-2PCA. [ka]

[0123] Aqueous solutions with pH values ​​of 2 and 7 were prepared using oxalic acid (0.86 mM and 1.4 μM, respectively). C1 (2 mg, 1.89 μl) was dissolved in 200 μl of oxalic acid solution. Two 60 μl aliquots were prepared from this stock solution, with each aliquot containing 0.57 μmol of C1. LC-MS measurements were taken of each aliquot at 1, 5, 24, 32, 56, 144, and 168 hours. C1 decomposition was determined by integrating the peaks corresponding to TMTHSI-PEG-2PCA and C1 at 267 nm and taking the average value of each measurement.

[0124] The results shown in Figure 8 were obtained by monitoring the time-dependent stability of C1 under acidic conditions. After one week, 85.88% (0.49 μmol) and 80.73% (0.46 μmol) of C1 remained at pH 7 and 2, respectively. Figure 8 also showed that the decrease in the amount of C1 was faster at pH 2 (solid black line) compared to pH 7 (dashed gray line). However, since TMTHSI-2PCA is intended to be used under physiological conditions, acid sensitivity is only important during the synthesis, workup, and formulation processes. Since the conjugation of TMTHSI-2PCA to biomolecules works best between pH 7 and 8, it is assumed that the TMTHSI-2PCA conjugate is only exposed to an acidic environment during workup and / or analysis (Timmers et al. 2023). As shown in Figure 8, the amount of C1 in solution remains above 95% for at least 56 hours, which means that the 2PCA conjugate will remain stable during the workup and analysis processes.

[0125] (References) Chigrinova, M.; McKay, C. S.; Beaulieu, L.-P. B.; Udachin, K. A.; Beauchemin, A. M.; Pezacki, J. P. Rearrangements and addition reactions of biarylazacyclooctynones and the implications to copper-free click chemistry. Organic & Biomolecular Chemistry 2013, 11(21), 3436-3441 Erickson, P. W.; Fulcher, J. M.; Spaltenstein, P.; Kay, M. S. Traceless Click-assisted native chemical ligation enabled by protecting dibenzocyclooctyne from acid-mediated rearrangement with copper(I). Bioconjugate chemistry 2021, 32(10), 2233-2244 Islam, M. Z.; Ariyama, H.; Alam, J. M.; Yamazaki, M. Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating across Lipid Membrane and Its Induced Pores. Biochemistry 2014, 53(2), 386-396. Janson, N.; Kru¨ger, T.; Karsten, L.; Boschanski, M.; Dierks, T.; Mu¨ller, K. M.; Sewald, N. Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs. ChemBioChem 2020, 21(24), 3580-3593 La-Venia, A.; Dzijak, R.; Rampmaier, R.; Vrabel, M. An Optimized Protocol for the Synthesis of Peptides Containing trans‐Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes. Chemistry-A European Journal 2021, 27(54), 13632-13641 Lei, Y.; Chen, S.; Zeng, X.; Meng, Y.; Chang, C.; Zheng, G. Angiopep-2 and Cyclic RGD Dual-Targeting Ligand Modified Micelles across the Blood-Brain Barrier for Improved Anti-Tumor Activity. J. Appl. Polym. Sci. 2022, 139(24), 52358. Meng G, Guo T, Ma T, Zhang J, Shen Y, Sharpless KB, Dong J. Nature. 2019; 574(7776):86-89. doi: 10.1038 / s41586-019-1589-1. Prescher, J. A. and Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13- 21. Rutjes, F. P. J. T. How to Pick a Single Amine?Nat. Chem. Biol. 2015 115 2015, 11(5), 306-307. Soomets, U.; Lindgren, M.; Gallet, X.; Hallbrink, M.; Elmquist, A.; Balaspiri, L.; Zorko, M.; Pooga, M.; Brasseur, R.; Langel, U¨. Deletion Analogues of Transportan. Biochim. Biophys. Acta 2000, 1467(1), 165-176. Sveinbjo / rnsson, B.; Camilio, KA; Haug, BE; Rekdal, O / . LTX-315: A First-in-Class Oncolytic Peptide That Reprograms the Tumor Microenvironment. Future Med. Chem. 2017, 9(12), 1339-1344. M. Timmers, W. Peeters, NJ Hauwer, CJF Rijcken, T. Vermonden, I. Dijkgraaf, RMJ Liskamp, ​​Specific N-Terminal Attachment of TMTHSI Linkers to Native Peptides and Proteins for Strain-Promoted Azide Alkyne Cycloaddition, Chem. Commun., 2023, 59, 11397-11400, DOI: https: / / doi.org / 10.1039 / D3CC03397J Weterings, J.; Rijcken, C. J. F.; Veldhuis, H.; Meulemans, T.; Hadavi, D.; Timmers, M.; Honing, M.; Ippel, H.; Liskamp, R. M. J. TMTHSI, a Superior 7-Membered Ring Alkyne Containing Reagent for Strain-Promoted Azide-Alkyne Cycloaddition Reactions. Chem. Sci. 2020, 11(33), 9011-9016. Zhang, C.; Ren, W.; Liu, Q.; Tan, Z.; Li, J.; Tong, C. Transportan-Derived Cell-Penetrating Peptide Delivers SiRNA to Inhibit Replication of Influenza Virus in Vivo . Drug Des. Devel. Ther. 2019, 13, 1059-1068. Zhou, H.; Forveille, S.; Sauvat, A.; Yamazaki, T.; Senovilla, L.; Ma, Y.; Liu, P.; Yang, H.; Bezu, L.; Mu¨ller, K.; et al. The Oncolytic Peptide LTX-315 Triggers Immunogenic Cell Death. Cell Death Dis. 2016 73 2016, 7(3), e2134-e2134.

[0126] [Supplementary Note] [Supplementary Note 1] The compound of formula (I). [Chemical Structure] (I) (where n and m are independently 0, 1, or 2 (where n + m is 2), and R 1 -R 8This includes hydrogen, halogens, hydroxyl groups, oxo groups, (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups, (C2-6) alkynyl groups, (C3-12) cycloalkyl groups, (C3-12) heterocycloalkyl groups, (C6-12) aryl groups, (C6-12) heteroaryl groups, (C7-12) alkyl(hetero)aryl groups, and (C7-12)(hetero)arylalkyl groups (however, these (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups) (The (C2-6) alkynyl group, (C3-12) cycloalkyl group, (C3-12) heterocycloalkyl group, (C6-12) aryl group, (C6-12) heteroaryl group, (C7-12) alkyl(hetero)aryl group, and (C7-12)(hetero)arylalkyl group are optionally substituted with one or more substituents independently selected from the group consisting of halogens, oxo groups, amino groups, hydroxyl groups, (C1-4) alkyl groups, and (C1-4) alkoxy groups), or R 1 and R 7 、 R 1 and R 8 、 R 2 and R 7 、 R 2 and R 8 、 R 3 and R 5 、 R 3 and R 6 、 R 4 and R 5 、 R 4 and R 6L forms a cycloalkyl group, a cyclo(hetero)aryl group, a cycloalkyl(hetero)aryl group, or a cyclo(hetero)arylalkyl group (wherein these cycloalkyl groups, cyclo(hetero)aryl groups, cycloalkyl(hetero)aryl groups, or cyclo(hetero)arylalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo groups, amino groups, hydroxyl groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups), and L is a linking group.

[0127] [Note 2] The compound described in Appendix 1, wherein m is 1 and n is 1.

[0128] [Note 3] R 1 -R 8 is independently selected from the group consisting of hydrogen, halogens, hydroxyl groups, oxo groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups (these (C1-4)alkyl and (C1-4)alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of halogens, hydroxyl groups, (C1-4)alkyl groups, and (C1-4)alkoxy groups), preferably R 1 -R 4 R is a methyl group, 5 -R 8 The compound is hydrogen, as described in Appendix 1 or 2.

[0129] [Note 4] The compound according to any one of the appendices 1 to 3, wherein L is a linear or branched carbon chain with a chain length of 1 to 50 atoms (where the chain length is determined by the number of atoms in the longest linear chain of atoms, and the longest linear chain may contain one or more heteroatoms and / or one or more saturated or unsaturated cyclic or heterocyclic portions).

[0130] [Note 5] The compound as described in Appendix 4, wherein the linear or branched carbon atom chain comprises one or more portions independently selected from the group consisting of -S(O)2-, -S-, -SS-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6-12)heteroaryl group, and combinations thereof.

[0131] [Note 6] The part of the following formula [ka] This is the part of the following equation [ka] A compound containing any one of the compounds listed in Appendix 1 to 5.

[0132] [Note 7] A compound described in any one of the appendices 1 to 6 of formula (IA). [ka] (However, L is defined as specified in one of the appendices 1 through 6.)

[0133] [Note 8] A compound described in any one of the appendices 1 to 7, which is coupled to the N-terminus of a peptide or protein molecule.

[0134] [Note 9] A compound of formula (II) or formula (III) as described in Appendix 8. [ka] [ka] (However, m, n, R 1 -R 8, and L are as described in any one of the appendices 1 to 7, and in formula (II), R 9 R is an amino acid side chain, and in formula (III), 9 and R 10 One of them is an amino acid side chain, R 9 and R 10 The other is hydrogen, or R 9 and R 10 Both are amino acid side chains, and X is a peptide or protein molecule.

[0135] [Note 10] Compounds of formula (IIB) or formula (IIIB) as described in Appendix 9. [ka] [ka] (However, L is defined in one of the appendices 1 to 7, and X and R 9 (This is defined in Appendix 9.)

[0136] [Note 11] The alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, wherein the compound comprising the thiol, a 1,3-dipole, or a 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, and more preferably comprises an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound, as described in any one of Appendix 1 to 10.

[0137] [Note 12] The compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene is a compound according to Appendix 10, comprising one or more of the following: a drug, small molecule, antibody, protein, peptide, nucleic acid molecule (including oligonucleotides, antisense oligonucleotides, and mRNA), ligand, imaging label (including radiolabeling), targeting ligand, delivery agent, drug delivery carrier (such as nanoparticles), carrier compound, and solid support (such as a surface plasmon resonance (SPR) plate).

[0138] [Note 13] Use of any one of the compounds described in Appendix 1 to 10 in the bioorthogonal copper free-click reaction.

[0139] [Note 14] Use of any one of the compounds described in Appendix 1 to 10 for bioconjugation, particularly for coupling a molecule to a peptide or protein molecule, wherein the molecule is selected from the group consisting of drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates).

[0140] [Note 15] A method for coupling a molecule to a peptide- or protein-like molecule, comprising reacting the molecule with a compound described in any one of appendices 1 to 10, wherein the molecule comprises a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, and the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to the compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene, wherein the compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, more preferably an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound.

Claims

1. A compound of formula (I). 【Chemistry 1】 (I) (However, n and m are independently 0, 1, or 2 (where n + m is 2), and R １ -R ８ is selected independently from the group consisting of hydrogen, halogen, hydroxy group, oxo group, (C1-6) alkyl group, (C1-6) alkoxy group, (C2-6) alkenyl group, (C2-6) alkynyl group, (C3-12) cycloalkyl group, (C3-12) heterocycloalkyl group, (C6-12) aryl group, (C6-12) heteroaryl group, (C7-12) alkyl(hetero)aryl group, and (C7-12)(hetero)arylalkyl group (where these (C1-6) alkyl groups, (C1-6) alkoxy groups, (C2-6) alkenyl groups, (C2-6) alkynyl groups, (C3-12) cycloalkyl groups, (C3-12) heterocycloalkyl groups, (C6-12) aryl groups, (C6-12) heteroaryl groups, (C7-12) alkyl(hetero)aryl groups, and (C7-12)(hetero)arylalkyl groups are optionally substituted by one or more substituents independently selected from the group consisting of halogen, oxo group, amino group, hydroxy group, (C1-4) alkyl group, (C1-4) alkoxy group)), or R １ and R ７ 、 R １ and R ８ 、 R ２ and R ７ 、 R ２ and R ８ 、 R ３ and R ５ 、 R ３ and R ６ 、 R ４ and R ５ 、 R ４ and R ６ L forms a cycloalkyl group, a cyclo(hetero)aryl group, a cycloalkyl(hetero)aryl group, or a cyclo(hetero)arylalkyl group (wherein these cycloalkyl groups, cyclo(hetero)aryl groups, cycloalkyl(hetero)aryl groups, or cyclo(hetero)arylalkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of halogen atoms, oxo groups, amino groups, hydroxyl groups, (C1-4) alkyl groups, and (C1-4) alkoxy groups), and L is a linking group.

2. The compound according to claim 1, wherein m is 1 and n is 1.

3. R １ -R ８ is independently selected from the group consisting of hydrogen, halogens, hydroxyl groups, oxo groups, (C1-4) alkyl groups, and (C1-4) alkoxy groups (these (C1-4) alkyl groups and (C1-4) alkoxy groups are optionally substituted with one or more substituents independently selected from the group consisting of halogens, hydroxyl groups, (C1-4) alkyl groups, and (C1-4) alkoxy groups), preferably R １ -R ４ R is a methyl group, ５ -R ８ The compound according to claim 1 or 2, wherein is hydrogen.

4. The compound according to any one of claims 1 to 3, wherein L is a linear or branched carbon chain having a chain length of 1 to 50 atoms (where the chain length is determined by the number of atoms in the longest linear chain of atoms, and the longest linear chain may include one or more heteroatoms and / or one or more saturated or unsaturated cyclic or heterocyclic portions).

5. The linear or branched carbon atom chains are configured such that the longest linear chain contains -S(O) ２ The compound according to claim 4, comprising one or more parts independently selected from the group consisting of -, -S-, -S-S-, -C(O)NH-, -NHC(O)-, -C(O)-, -C(O)O-, -O-, -OC(O), (C3-12)cycloalkyl group, (C3-12)heterocycloalkyl group, (C6-12)aryl group, (C6-12)heteroaryl group, and combinations thereof.

6. The part of the following formula 【Chemistry 2】 This is the part of the following equation 【Transformation 3】 A compound according to any one of claims 1 to 5, comprising:

7. A compound according to any one of claims 1 to 6 of formula (IA). 【Chemistry 4】 (However, L is defined as in any one of claims 1 to 6.)

8. The compound according to any one of claims 1 to 7, which is coupled to the N-terminus of a peptide or protein molecule.

9. The compound according to claim 8, of formula (II) or formula (III). 【Transformation 5】 【Transformation 6】 (However, m, n, R １ -R ８ , and L are as described in any one of claims 1 to 7, and in formula (II), R ９ R is an amino acid side chain, and in formula (III), ９ and R １０ One of them is an amino acid side chain, R ９ and R １０ The other is hydrogen, or R ９ and R １０ Both are amino acid side chains, and X is a peptide or protein molecule.

10. The compound according to claim 9, of formula (IIB) or formula (IIIB). 【Transformation 7】 【Transformation 8】 (However, L is as defined in any one of claims 1 to 7, and X and R ９ (This is defined in claim 9.)

11. The compound according to any one of claims 1 to 10, wherein the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to a compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, the compound comprising a thiol, a 1,3-dipole, or a 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, and more preferably comprises an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound.

12. The compound according to claim 10, wherein the compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene comprises one or more of the following: a drug, a small molecule, an antibody, a protein, a peptide, a nucleic acid molecule (including oligonucleotides, antisense oligonucleotides, and mRNA), a ligand, an imaging label (including radiolabeling), a targeting ligand, a delivery agent, a drug delivery carrier (such as nanoparticles), a carrier compound, and a solid support (such as a surface plasmon resonance (SPR) plate).

13. Use of the compound according to any one of claims 1 to 10 in a bioorthogonal copper free-click reaction.

14. Use of a compound according to any one of claims 1 to 10 for bioconjugation, particularly for coupling a molecule to a peptide or protein molecule, wherein the molecule is selected from the group consisting of drugs, small molecules, antibodies, proteins, peptides, nucleic acid molecules (including oligonucleotides, antisense oligonucleotides, and mRNA), ligands, imaging labels (including radiolabels), targeting ligands, delivery agents, drug delivery carriers (such as nanoparticles), carrier compounds, and solid supports (such as surface plasmon resonance (SPR) plates).

15. A method for coupling a molecule to a peptide- or protein-like molecule, comprising reacting the molecule with a compound according to any one of claims 1 to 10, wherein the molecule comprises a thiol, a 1,3-dipole, or a 1,3-(hetero)diene, and the alkyne group of thiocycloheptine of formula (I), formula (II), or formula (III) is coupled to the compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene, wherein the compound comprising the thiol, 1,3-dipole, or 1,3-(hetero)diene preferably comprises an azide, a nitrone, or a nitrile oxide, more preferably an azide, wherein the azide-alkyne coupling results in the formation of a triazole compound.

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