Sulfonamide linker, conjugate thereof and preparation method

Abstract

The present invention relates to a compound comprising an α-end and an ω-end, wherein the compound comprises a reactive group Q at the α-end 1 and contains a target molecule at the ω-end, the Q 1 Can bind to the functional groups F present on biomolecules 1 The compound further comprises a group of formula (1) or a salt thereof; the compound may also be referred to as a linker-conjugate. The present invention also relates to a method for preparing a bioconjugate, the method comprising reacting a reactive group Q of the linker-conjugate of the present invention 1 Functional groups F of biomolecules 1 The invention also relates to a bioconjugate obtainable by the method of the invention. In a preferred embodiment, the invention relates to a method for preparing a bioconjugate by cycloaddition, such as a (4+2)-cycloaddition (e.g., a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g., a 1,3-dipolar cycloaddition). #imgabs0#

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201580066180.6, filed on October 5, 2015, and with the invention name “Sulfonamide linker, its conjugate and preparation method”. Technical Field

[0002] The present invention relates to the field of bioconjugation. The present invention relates to sulfonamide linkers and conjugates thereof, and to methods for preparing them. More specifically, the present invention relates to linkers comprising acylsulfonamide groups and / or carbamoylsulfonamide groups, and to conjugates comprising said linkers. The present invention also relates to methods for preparing bioconjugates comprising linkers, wherein the linkers comprise acylsulfonamide groups and / or carbamoylsulfonamide groups. Background Art

[0003] Bioconjugation is a method of connecting two or more molecules, at least one of which is a biomolecule. The biomolecule may also be referred to as a "biomolecule of interest", and the other molecules may also be referred to as a "target molecule" or "molecule of interest". Typically, the biomolecule of interest (BOI) will consist of a protein (or peptide), a polysaccharide, a nucleic acid (or an oligonucleotide), a lipid, a hormone, or a natural drug (or a fragment or combination thereof). Other molecules of interest (MOI) may also be biomolecules, thereby forming homo- or heterodimers (or higher oligomers), or other molecules may have specific features imparted to the biomolecule of interest by the conjugation process. For example, the regulation of protein structure and function by covalent modification using chemical probes for detection and / or separation has been developed as a powerful tool in proteome-based research and biomedical applications. Fluorescent labeling or affinity labeling of proteins is key to studying the transport of proteins in their native habitat. Vaccines based on protein-carbohydrate conjugates have been highlighted in the fight against HIV, cancer, malaria, and pathogens, and carbohydrates immobilized on microarrays help to elucidate the glycome. Synthetic DNA and RNA oligonucleotides (ON) need to be introduced with suitable functional groups for diagnostic and therapeutic applications, such as microarray technology, antisense and gene silencing therapies, nanotechnology and various materials science applications. For example, the attachment of cell-penetrating ligands is the most commonly used strategy to deal with the low ON internalization rates encountered during oligonucleotide-based therapies (antisense, siRNA). Similarly, the preparation of oligonucleotide-based microarrays requires the selective immobilization of ONs on suitable solid surfaces (e.g., glass).

[0004] There are numerous examples of chemical reactions suitable for covalently linking two (or more) molecular structures. However, labeling of biomolecules highly restricts the applicable reaction conditions (solvents, concentrations, temperatures), while the required chemically selective labeling limits the choice of reactive groups. Obviously, biological systems usually react best in an aqueous environment (flourish), which means that reagents used for bioconjugation should be suitable for application in aqueous systems. In general, two strategic concepts can be recognized in the field of bioconjugation technology: (a) conjugation based on functional groups already present in the target biomolecule, such as thiol, amine, alcohol or hydroxyphenol units; or (b) a two-stage approach, which involves the engineering insertion of one (or more) unique reactive groups into the BOI before the actual conjugation process.

[0005] The first approach generally involves reactive amino acid side chains in proteins (e.g., cysteine, lysine, serine, and tyrosine), or functional groups in polysaccharides (e.g., amines, aldehydes), or functional groups in nucleic acids (e.g., purine or pyrimidine functional groups or alcohols). As summarized in, inter alia, GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition. 2013 (incorporated by reference), in recent years, a large number of reactive functional groups have become available for chemoselective targeting of one of these functional groups (e.g., maleimide, haloacetamide, activated ester, activated carbonate, sulfonyl halide, activated thiol derivatives, olefins, alkynes, allenamide, etc.), each of which requires its own specific conjugation conditions (pH, concentration, chemical dosage, light, etc.). Most notably, cysteine-maleimide conjugation has stood out for protein conjugation due to its high reaction rate and chemoselectivity. However, when no cysteine ​​is available for conjugation, as in many proteins and certainly in other biomolecules, other approaches are generally required, each with its own disadvantages.

[0006] A well-established and widely applicable solution for bioconjugation involves a two-stage approach. Although more laborious, two-stage conjugation of engineered functional groups generally produces higher selectivity (site specificity) than conjugation on natural functional groups. In addition, complete stability can be achieved by appropriate selection of constructs, which may be an important disadvantage of one-stage conjugation on natural functional groups, especially for cysteine-maleimide conjugation. Typical examples of functional groups that can be added to BOI include (strained) alkynes, (strained) alkenes, norbornenes, tetrazines, azides, phosphines, nitrile oxides, nitrones, nitrile imines, diazo compounds, carbonyl compounds, (O-alkyl) hydroxylamines and hydrazines, which can be achieved by chemical or molecular biological methods. It is known that each of the above functional groups has at least one reaction partner, involving complete mutual reactivity in many cases. For example, cyclooctyne reacts selectively and exclusively with 1,3-dipoles, strained alkenes and tetrazines, and phosphines and azides to produce completely stable covalent bonds. However, some of the above functional groups have the disadvantage of being highly lipophilic, which may compromise the conjugation efficiency, especially when combined with lipophilic molecules of interest (see below).

[0007] The final linking unit between the biomolecule and the other molecule of interest should also be preferably fully compatible with an aqueous environment in terms of solubility, stability and biocompatibility. For example, highly lipophilic linkers may lead to aggregation (during or after conjugation), which can significantly increase reaction time and / or reduce conjugation yield, especially when the MOI also has hydrophobic properties. Similarly, highly lipophilic linker-MOI combinations may lead to non-specific binding to the surface or specific hydrophobic patches of the same or other biomolecules. If the linker is sensitive to hydrolysis or other water-induced cleavage reactions, the components comprising the initial bioconjugate are separated by diffusion. For example, when β-hydroxycarbonyl or γ-dicarbonyl compounds can lead to retro-aldol reactions or retro-Michael reactions, respectively, certain ester moieties are not suitable due to saponification. Finally, the linker should be inert toward the functional groups present in the bioconjugate or any other functional groups that may be encountered during the use of the bioconjugate, which specifically excludes the use of linkers featuring, for example, ketone or aldehyde moieties (which can lead to imine formation), α,β-unsaturated carbonyl compounds (Michael addition), thioesters or other active esters (amide bond formation).

[0008] Compounds consisting of linear oligomers of ethylene glycol (so-called PEG (polyethylene glycol) joints) are currently particularly popular in the process of biomolecule conjugation. PEG joints are highly water-soluble, non-toxic, non-antigenic, and result in negligible or no aggregation. For this reason, many types of linear, bifunctional PEG joints are commercially available from different sources, which can be selectively modified at either end using the target (bio) molecule. PEG joints are products of the polymerization process of ethylene oxide, and are therefore usually obtained as a random mixture of chain lengths, which can be partially decomposed into PEG constructs with an average weight distribution concentrated at about 1, 2, 4 kDa or more (up to 60 kDa). It is also known to have a uniform, discrete PEG (dPEG) with a molecular weight of up to 4 kDa, and its branched form is up to 15 kDa. Interestingly, the PEG unit itself imparts specific characteristics to biomolecules. Specifically, protein PEGylation can lead to prolonged residence time in the body, reduced degradation of metabolic enzymes, and reduced or eliminated protein immunogenicity. Several PEGylated proteins have been approved by the FDA and are currently on the market.

[0009] Due to its high polarity, PEG linkers are perfectly suitable for bioconjugation of small and / or water-soluble parts under aqueous conditions. However, in the case of conjugation of hydrophobic, water-insoluble molecules of interest, the polarity of the PEG unit may not be sufficient to offset the hydrophobicity, resulting in a significant reduction in reaction rate, decreased yield and aggregation problems. In this case, relatively long PEG linkers and / or large amounts of organic cosolvents may be required to dissolve the reagents. For example, in the field of antibody-drug conjugates, it is key to controllably connect different amounts of toxic payloads to monoclonal antibodies, where the payload is typically selected from auristatin E or F, maytansinoid, duocarmycin, calicheamicin or pyrrolobenzodiazepine (PBD), and there are many others in progress. Except for auristatin F, all toxic payloads have poor water solubility, which requires organic cosolvents to achieve successful conjugation, such as 25% dimethylacetamide (DMA) or 50% propylene glycol (PG). In the case of hydrophobic payloads, despite the use of the above-mentioned cosolvents, large stoichiometric amounts of reagents may be required during conjugation, while efficiency and yield may be significantly reduced due to aggregation (either in processing or after product isolation), as described, for example, by Senter et al. in Nat. Biotechn. 2014, 24, 1256-1263 (incorporated by reference). The use of long PEG spacers (12 units or more) may partially improve solubility and / or conjugation efficiency, but it has been shown that long PEG spacers may lead to more rapid in vivo clearance, thereby negatively affecting the pharmacokinetic properties of the ADC.

[0010] From the above, it can be seen that short polar spacers are needed that can quickly and efficiently conjugate hydrophobic moieties. Obviously, the latter belongs to even higher-level conjugation reactions in which hydrophobic reactive moieties such as strained alkynes, alkenes, and phosphines are applied (see above).

[0011] Linkers are known in the art and are disclosed in, for example, WO 2008 / 070291 (incorporated by reference). WO 2008 / 070291 discloses a linker for coupling a targeting agent to an anchoring component. The linker comprises a hydrophilic region represented by polyethylene glycol (PEG) and an extension portion lacking a chiral center, which is coupled to the targeting agent.

[0012] WO 01 / 88535, incorporated by reference, discloses a surface linker system for bioconjugation, in particular a linker system with a novel hydrophilic spacer group. The hydrophilic atom or group used in the linker system is selected from O, NH, C=O (keto group), OC=O (ester group) and CR 3 R 4 , where R 3 and R 4 Independently selected from H, OH, C 1 -C 4 Alkoxy and C 1 -C 4 Acyloxy.

[0013] WO 2014 / 100762 - incorporated by reference - describes compounds containing a hydrophilic self-immolative linker that can be cleaved under appropriate conditions and incorporates a hydrophilic group to provide a compound with better solubility. The compound comprises a drug moiety, a targeting moiety capable of targeting a selected cell population, and a linker comprising an acyl unit, an optional spacer unit for providing a distance between the drug moiety and the targeting moiety, a peptide linker cleavable under appropriate conditions, a hydrophilic self-immolative linker, and an optional second self-immolative spacer or a cyclized self-immolative linker. The hydrophilic self-immolative linker is, for example, a benzyloxycarbonyl group. Summary of the invention

[0014] The present invention relates to compounds comprising an α-terminus and an ω-terminus (also referred to as linker-conjugates), said compounds comprising a reactive group Q at the α-terminus 1 and contains the target molecule at the ω-end, the Q 1 Can bind to the functional groups F on biomolecules 1 Reaction, the compound further comprises a group of formula (1) or a salt thereof:

[0015]

[0016] in:

[0017] a is 0 or 1; and

[0018] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1-C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer;

[0019] And wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound.

[0020] More specifically, the present invention relates to a linker-conjugate of formula (4a) or (4b), or a salt thereof

[0021]

[0022] in:

[0023] a is independently 0 or 1;

[0024] b is independently 0 or 1;

[0025] c is 0 or 1;

[0026] d is 0 or 1;

[0027] e is 0 or 1;

[0028] f is an integer ranging from 1 to 150;

[0029] g is 0 or 1;

[0030] i is 0 or 1;

[0031] D is the target molecule;

[0032] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0033] Sp 1 For the interval part;

[0034] Sp 2 For the interval part;

[0035] Sp 3For the interval part;

[0036] Sp 4 For the interval part;

[0037] Z 1 is a connecting group, which connects Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 )connect;

[0038] Z 2 is a linking group, which connects D or Sp 4 With Sp 1 、N(R 1 ), O or C(O) connection; and

[0039] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl; or

[0040] R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 )g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above.

[0041] The present invention also relates to a method for preparing a bioconjugate, the method comprising: 1 Functional groups F of biomolecules 1 The step of reacting, wherein the linker-conjugate is a compound comprising an α-terminus and an ω-terminus, and the compound comprises a reactive group Q at the α-terminus 1 and contains the target molecule at the ω-end, the Q 1 Can bind to the functional groups F on biomolecules 1 Reaction, the compound further comprises a group of formula (1) or a salt thereof:

[0042]

[0043] Where a and R 1 As defined above, and wherein the group of formula (1) or a salt thereof is located between said α-end and said ω-end of the compound.

[0044] More specifically, the present invention also relates to a method for preparing a bioconjugate, said method comprising reacting a reactive group Q of a linker-conjugate of formula (4a) or (4b) as defined above 1 Functional groups F of biomolecules 1 In a preferred embodiment, the present invention relates to a method for preparing a bioconjugate by a cycloaddition, such as a (4+2)-cycloaddition (e.g., Diels-Alder reaction) or a (3+2)-cycloaddition (e.g., 1,3-dipolar cycloaddition), preferably a 1,3-dipolar cycloaddition, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q 1 is or comprises an alkynyl group (eg, a cycloalkynyl group) and F 1 is an azido group. In another preferred embodiment, the present invention relates to a method for preparing a bioconjugate, wherein the target molecule is hydrophobic (i.e. sparingly soluble in water), most preferably wherein the water solubility of the target molecule in water (20° C. and 100 kPa) is at most 0.1% (w / w). In a particularly preferred embodiment, the present invention relates to a method for preparing a bioconjugate by cycloaddition, wherein the cycloaddition is preferably a 1,3-dipolar cycloaddition, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q 1 is or contains an alkynyl group and F1 is an azido group, and wherein the target molecule is hydrophobic, most preferably wherein the water solubility of the target molecule in water (20° C. and 100 kPa) is at most 0.1% (w / w).

[0045] The present invention also relates to a bioconjugate obtainable by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Describes the general concept of biomolecule conjugation: a molecule containing one or more functional groups F 1 The target biomolecule (BOI) is covalently linked to the reactive group Q via a specific linker (excess) 1 In the bioconjugation process, F 1 and Q 1 A bioconjugate comprising a covalent bond between a BOI and an MOI is formed by a chemical reaction. The BOI may be, for example, a peptide / protein, a polysaccharide or a nucleic acid.

[0047] Figure 2 Several structures of derivatives of the UDP sugar of galactosamine are shown, which may be modified, for example, with 3-mercaptopropionyl (11a), azidoacetyl (11b), or azidodifluoroacetyl (11c).

[0048] Figure 3 Schematic representation of how any of the UDP-sugars 11a-c can be attached to a glycoprotein comprising a GlcNAc moiety 12 (e.g., a monoclonal antibody in which the glycan is endonucleoside trimmed) under the action of a galactosyltransferase mutant or a GalNAc-transferase, thereby generating β-glycosidic 1-4 bonds between the GalNAc derivative and the GlcNAc (compounds 13a-c, respectively).

[0049] Figure 4 It is shown how the modified antibodies 13a-c can be bioconjugated via nucleophilic addition with maleimide (for 13a, generating thioether conjugate 14) or via strain-promoted cycloaddition using cyclooctyne reagents (for 13b or 13c, generating triazoles 15 or 16, respectively).

[0050] Figure 5 The structures of several compounds are shown in which the N-maleimido reactive group Q is connected via a linker unit. 1 Attached to the pyrene group (D).

[0051] Figure 6 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q1 (also called a BCN group) is attached to the benzylamine (D).

[0052] Figure 7 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called a BCN group) or a dibenzoazocyclooctyne attached to pyrene.

[0053] Figure 8 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called a BCN group) attached to maytansin.

[0054] Fig. 9 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called BCN group) is attached to the Val-Cit-PABA-duocarmycin construct.

[0055] Fig.10 The structures of several compounds of the present invention are shown, wherein the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also known as the BCN group) conjugated to Val-Cit-PABA-Ahx-Maytansine.

[0056] Fig.11 HPLC retention times of compounds 19-23 and 30-38 using 0.1% TFA or in buffer pH 7.4 are shown.

[0057] Fig.12a The BCN-pyrene derivative conjugated via a sulfonamide linker (compound 26) or via a short PEG linker (compounds 24 or 25) is shown to be coupled to the trastuzumab antibody-N 3 (Compound 13b) conjugation efficiency.

[0058] Figure 12b The DIBAC-pyrene derivative conjugated via a sulfonamide linker (compound 29) or via a short PEG linker (compounds 27 or 28) is shown to be coupled to trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0059] Fig.13aBCN-maytansine derivatives conjugated via a sulfonamide linker (compound 33) or BCN-maytansine derivatives conjugated via a short PEG linker (compounds 30-32) are shown to be coupled to trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0060] Fig.13b BCN-maytansine derivatives conjugated via a sulfonamide linker (compound 33) or via a short PEG linker (compounds 30-32, where compounds 30 and 33 overlap) are shown with trastuzumab-F 2 -Conjugation efficiency of GalNAz (compound 13c).

[0061] Fig.14a The BCN-duocarmycin derivative conjugated via a sulfonamide linker (compound 35) or via a short PEG linker (compound 34) is shown with trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0062] Fig.14b BCN-duocarmycin derivatives conjugated via a sulfonamide linker (compound 35) or BCN-duocarmycin derivatives conjugated via a short PEG linker (compound 34) are shown with trastuzumab-F 2 -Conjugation efficiency of GalNAz (compound 13c).

[0063] Fig.15 A general synthetic scheme is shown for the conversion of alcohol (40) to carbamoylsulfonamide derivatives (42) by sequential treatment with chlorosulfonyl isocyanate (CSI) and amines. Carbamoylsulfonamide 42 can be converted to acylsulfonamide (44), which, if the starting alcohol is tert-butyl alcohol, affords sulfonamide 43 after removal of the tert-butyl protection with an acid, which can be acylated to afford 44.

[0064] Fig.16 It shows that Q 1 Synthesis of several linker-conjugates wherein D is N-maleimido and D is pyrene. Compound 18 is a compound of the present invention, while compound 17 is a comparative example.

[0065] Fig.17 The synthesis of several linker-conjugates is shown, in which Q 1 is bicyclo[6.1.0]non-4-yn-9-yl] (also known as a BCN group) and D is a benzyl group.

[0066] Fig.18 The synthesis of a linker-conjugate with two sulfonamide groups in the linker is shown, wherein Q 1is bicyclo[6.1.0]non-4-yn-9-yl] (also known as a BCN group) and D is a benzyl group.

[0067] Fig.19 The synthesis of several linker-conjugates is shown, in which Q 1 is bicyclo[6.1.0]non-4-yn-9-yl] (also known as a BCN group) and D is pyrenyl.

[0068] Fig. 20 The synthesis of several linker-conjugates is shown, in which Q 1 is heterocycloalkynyl (DIBAC) and D is pyrenyl.

[0069] Fig.21 The synthesis of a linker-conjugate comprising two target molecules D is shown, wherein Q 1 is bicyclo[6.1.0]non-4-yn-9-yl] (also known as the BCN group) and D is a cytotoxin (maytansine).

[0070] Fig. 22 A representative set of functional groups (F 1 ), which is in contact with the reactive group Q 1 After the reaction, the linker group Z is generated 3 . Linking group Z 3 It can also be F 2 and Q 2 The functional group F 1 Artificially introduced (engineered) into any chosen position in a biomolecule.

[0071] Fig.23 The extent of aggregation of bioconjugates 36-38 of the invention is shown.

[0072] Fig.24 Shown is the progression of aggregation over a 2-week period for a bioconjugate of the invention having a sulfonamide linker (36 conjugated to 13a) and a comparative bioconjugate having a PEG linker (30 conjugated to 13a), as well as trastuzumab. DETAILED DESCRIPTION

[0073] definition

[0074] The verb "to comprise" and its conjugated forms as used in this description and claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0075] Furthermore, unless the context clearly requires that there is one and only one element, referring to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present. Thus, the indefinite article "a" or "an" generally means "at least one".

[0076] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of the compounds may exist. Unless otherwise stated, the description of any compound in this specification and claims is intended to include all diastereomers and mixtures thereof. In addition, unless otherwise stated, the description of any compound in this specification and claims is intended to include individual enantiomers, as well as any mixtures, racemates or other forms of enantiomers. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to the specific enantiomer.

[0077] The compound can exist in different tautomeric forms. Unless otherwise indicated, the compound of the present invention is intended to include all tautomeric forms. When the structure of a compound is described as a specific tautomer, it should be understood that the invention of the present application is not limited to the specific tautomer.

[0078] The compounds disclosed in this specification and claims may also exist as exo and endo diastereomers. Unless otherwise indicated, the description of any compound in this specification and claims is intended to include a single exo and a single endo diastereomer of the compound, and mixtures thereof. When the structure of a compound is described as a specific endo or exo diastereomer, it is understood that the invention of the present application is not limited to the specific endo or exo diastereomer.

[0079] In addition, the compounds disclosed in this specification and claims may also exist as cis and trans isomers. Unless otherwise stated, the description of any compound in this specification and claims is intended to include a single cis isomer and a single trans isomer of the compound, and mixtures thereof. For example, when the structure of a compound is described as a cis isomer, it is understood that the corresponding trans isomer or a mixture of cis and trans isomers is not excluded from the invention of the present application. When the structure of a compound is described as a specific cis or trans isomer, it is understood that the invention of the present application is not limited to the specific cis or trans isomer.

[0080] Unsubstituted alkyl groups have the general formula C n H 2n+1and may be straight or branched. Optionally, the alkyl group is substituted with one or more substituents further specified herein. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, tert-butyl, 1-hexyl, 1-dodecyl, etc.

[0081] Cycloalkyl is a cyclic alkyl group. Unsubstituted cycloalkyl groups contain at least 3 carbon atoms and have the general formula C n H 2n-1 Optionally, the cycloalkyl group is substituted with one or more substituents as further specified herein. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0082] The alkenyl group contains one or more carbon-carbon double bonds and may be straight chain or branched. An unsubstituted alkenyl group containing one C—C double bond has the general formula C n H 2n-1 An unsubstituted alkenyl group containing two CC double bonds has the general formula C n H 2n-3 . The alkenyl group may contain a terminal CC double bond and / or an internal CC double bond. A terminal alkenyl group is an alkenyl group in which the CC double bond is located at the terminal position of the carbon chain. The alkenyl group may also contain two or more CC double bonds. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, tert-butenyl, 1,3-butadienyl, 1,3-pentadienyl, etc. Unless otherwise stated, the alkenyl group may be optionally substituted with one or more independently selected substituents as defined below. Unless otherwise stated, the alkenyl group may be optionally interrupted by one or more heteroatoms selected from O, N and S.

[0083] Alkynyl groups contain one or more carbon-carbon triple bonds and may be straight or branched. Unsubstituted alkynyl groups containing one C-C triple bond have the general formula C n H 2n-3 . Alkynyl groups may contain terminal C-C triple bonds and / or internal C-C triple bonds. A terminal alkynyl group is an alkynyl group in which the C-C triple bond is located at the terminal position of the carbon chain. Alkynyl groups may also contain two or more C-C triple bonds. Unless otherwise stated, alkynyl groups may be optionally substituted with one or more independently selected substituents as defined below. Examples of alkynyl groups include ethynyl, propynyl, isopropynyl, tert-butynyl, etc. Unless otherwise stated, alkynyl groups may be optionally interrupted by one or more heteroatoms selected from O, N and S.

[0084] Aryl contains 6 to 12 carbon atoms and may include a monocyclic or bicyclic structure. Optionally, the aryl may be substituted with one or more substituents as further specified herein. Examples of aryl are phenyl and naphthyl.

[0085] Arylalkyl and alkylaryl contain at least 7 carbon atoms and can include monocyclic and bicyclic structures. Optionally, the arylalkyl and alkylaryl can be substituted with one or more substituents further specified herein. Arylalkyl is, for example, benzyl. Alkylaryl is, for example, 4-tert-butylphenyl.

[0086] The heteroaryl group contains at least two carbon atoms (i.e., at least C 2 ) and one or more heteroatoms N, O, P or S. The heteroaryl group may have a monocyclic or bicyclic structure. Optionally, the heteroaryl group may be substituted with one or more substituents further specified herein. Examples of suitable heteroaryl groups include pyridyl, quinolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.

[0087] Heteroarylalkyl and alkylheteroaryl groups contain at least 3 carbon atoms (i.e., at least C 3 ), and may include monocyclic and bicyclic structures. Optionally, the heteroaryl group may be substituted with one or more substituents as further specified herein.

[0088] In the case where aryl is represented as (hetero)aryl, the representation is intended to include both aryl and heteroaryl. Similarly, alkyl (hetero)aryl is intended to include both alkyl aryl and alkyl heteroaryl, and (hetero)arylalkyl is intended to include both arylalkyl and heteroarylalkyl. Thus, C 2 -C 24 (Hetero)aryl is understood to include C 2 -C 24 Heteroaryl and C 6 -C 24 Similarly, C 3 -C 24 Alkyl (hetero) aryl means a group including C 7 -C 24 Alkyl aryl and C 3 -C 24 Alkyl heteroaryl, C 3 -C 24 (Hetero)arylalkyl refers to a group including C 7 -C 24 Arylalkyl and C 3 -C 24 Heteroarylalkyl.

[0089] Cycloalkynyl is a cyclic alkynyl group. Unsubstituted cycloalkynyl groups containing one triple bond have the general formula C n H 2n-5 Optionally, the cycloalkynyl group is substituted with one or more substituents as further specified herein. Examples of cycloalkynyl groups include cyclooctynyl.

[0090] Heterocycloalkynyl is a cycloalkynyl group interrupted by heteroatoms selected from oxygen, nitrogen and sulfur. Optionally, the heterocycloalkynyl group is substituted by one or more substituents as further specified herein. An example of a heterocycloalkynyl group is azacyclooctynyl.

[0091] (Hetero)aryl includes aryl and heteroaryl. Alkyl (hetero)aryl includes alkyl aryl and alkyl heteroaryl. (Hetero)arylalkyl includes arylalkyl and heteroarylalkyl. (Hetero)alkynyl includes alkynyl and heteroalkynyl. (Hetero)cycloalkynyl includes cycloalkynyl and heterocycloalkynyl.

[0092] (Hetero)cycloalkyne compounds are defined herein as compounds comprising a (hetero)cycloalkyne group.

[0093] Several compounds disclosed in the present specification and claims may be described as fused (hetero)cycloalkyne compounds, i.e., (hetero)cycloalkyne compounds in which a second ring structure is fused (i.e., annulated) to the (hetero)cycloalkyne group. For example, in a fused (hetero)cyclooctyne compound, a cycloalkyl group (e.g., cyclopropyl) or an aromatic group (e.g., benzene) may be annulated to the (hetero)cyclooctyne group. The triple bond of the (hetero)cyclooctyne group in the fused (hetero)cyclooctyne compound may be located in any of three possible positions, i.e., position 2, 3, or 4 of the cyclooctyne moiety (numbering according to "IUPAC Nomenclature of Organic Chemistry", item A31.2). The description of any fused (hetero)cyclooctyne compound in the present specification and claims is meant to include all three individual positional isomers of the cyclooctyne moiety.

[0094] Unless otherwise indicated, alkyl, cycloalkyl, alkenyl, alkynyl, (hetero)aryl, (hetero)arylalkyl, alkyl(hetero)aryl, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, (hetero)arylene, alkyl(hetero)arylene, (hetero)arylalkylene, (hetero)arylalkenylene, (hetero)arylalkynylene, alkenyl, alkoxy, alkenyloxy, (hetero)aryloxy, alkynyloxy and cycloalkyloxy may be substituted by one or more substituents independently selected from the following: 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, C 3 -C 12 Cycloalkyl, C 5 -C 12 Cycloalkenyl, C 8 -C 12 Cycloalkynyl, C 1 -C 12 Alkoxy, C 2 -C 12 Alkenyloxy, C 2 -C12 Alkynyloxy, C 3 -C 12 Cycloalkoxy, halogen, amino, oxo and silyl, wherein the silyl group can be represented by the formula (R 20 ) 3 Si-, where R 20 Independently selected from C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, C 3 -C 12 Cycloalkyl, C 1 -C 12 Alkoxy, C 2 -C 12 Alkenyloxy, C 2 -C 12 Alkynyloxy and C 3 -C 12 Cycloalkoxy, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, and the alkyl, alkoxy, cycloalkyl and cycloalkoxy are optionally interrupted by one or more heteroatoms selected from O, N and S.

[0095] As used herein, the general term "sugar" refers to monosaccharides, such as glucose (Glc), galactose (Gal), mannose (Man), and fucose (Fuc). As used herein, the term "sugar derivative" refers to a derivative of a monosaccharide, i.e., a monosaccharide that includes substituents and / or functional groups. Examples of sugar derivatives include amino sugars and sugar acids, such as glucosamine (GlcNH 2 ), galactosamine (GalNH 2 ), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) also known as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA).

[0096] The term "nucleotide" is used herein in its usual scientific sense. The term "nucleotide" refers to a molecule consisting of a nucleobase, a pentose (ribose or 2-deoxyribose) and one, two or three phosphate groups. Without a phosphate group, the nucleobase and the sugar constitute a nucleoside. Therefore, nucleotides are also referred to as nucleoside monophosphates, nucleoside diphosphates or nucleoside triphosphates. The nucleobase can be adenine, guanine, cytosine, uracil or thymine. Examples of nucleotides include uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

[0097] The term "protein" is used herein in its usual scientific sense. Polypeptides comprising about 10 or more amino acids are considered proteins herein. Proteins may comprise both natural and non-natural amino acids.

[0098] The term "glycoprotein" is used herein in its usual scientific sense and refers to a protein comprising one or more monosaccharide or oligosaccharide chains ("glycans") covalently bonded to the protein. The glycan may be attached to a hydroxyl group of the protein (O-linked glycans), such as to a hydroxyl group of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline; or to an amide functional group of the protein (N-glycoproteins), such as asparagine or arginine; or to a carbon of the protein (C-glycoproteins), such as tryptophan. A glycoprotein may comprise more than one glycan, may comprise a combination of one or more monosaccharide and one or more oligosaccharide glycans, and may comprise a combination of N-linked, O-linked, and C-linked glycans. It is estimated that more than 50% of all proteins have some form of glycosylation and are therefore identified as glycoproteins. Examples of glycoproteins include PSMA (prostate-specific membrane antigen), CAL (candida antartica lipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins, and antibodies.

[0099] The term "glycan" is used herein with its common scientific meaning, and refers to a monosaccharide or oligosaccharide chain connected to a protein. Therefore, the term glycan refers to the carbohydrate portion of a glycoprotein. The glycan is connected to a protein by the C-1 carbon of a sugar, and the glycan may not contain other substitutions (monosaccharides) or may be further substituted (oligosaccharides) at one or more hydroxyl groups. Naturally occurring glycans generally contain 1 to about 10 sugar moieties. However, when a longer sugar chain is connected to a protein, the sugar chain is also considered to be a glycan herein. The glycan in a glycoprotein may be a monosaccharide. Typically, the monosaccharide glycan of a glycoprotein is composed of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently connected to a protein. A glycan may also be an oligosaccharide. The oligosaccharide chain of a glycoprotein may be linear or branched. In oligosaccharides, the sugar directly connected to a protein is called a core sugar. In oligosaccharides, the sugar that is not directly connected to a protein and is connected to at least two other sugars is called an endosugar. In oligosaccharides, sugars that are not directly connected to proteins but are connected to a single other sugar, that is, sugars that do not carry other sugar substituents on one or more of its other hydroxyl groups, are called terminal sugars. For the avoidance of doubt, multiple terminal sugars may be present in the oligosaccharides of glycoproteins, but there is only one core sugar. Polysaccharides may be O-linked polysaccharides, N-linked polysaccharides, or C-linked polysaccharides. In O-linked polysaccharides, monosaccharides or oligosaccharides are bonded to the O atom in the amino acids of proteins, usually via the hydroxyl groups of serine (Ser) or threonine (Thr). In N-linked polysaccharides, monosaccharides or oligosaccharides are bonded to proteins via the N atom in the amino acids of proteins (usually via the amide nitrogen on the side chains of asparagine (Asn) or arginine (Arg). In C-linked polysaccharides, monosaccharides or oligosaccharides are bonded to the C atom in the amino acids of proteins, usually bonded to the C atom of tryptophan (Trp).

[0100] The term "antibody" is used herein in its usual scientific sense. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Antibodies are an example of glycoproteins. Herein, the term antibody is used in its broadest sense, and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain antibodies and single-chain antibodies. Herein, the term "antibody" is also meant to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term "antibody" is meant to include whole antibodies, and antibody fragments (e.g., antibody Fab fragments, F(ab') from cleaved antibodies) 2, Fv fragment or Fc fragment; scFv-Fc fragment; miniantibody; diabody or scFv). In addition, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments and genetically engineered antibodies can be obtained by methods known in the art. Typical examples of antibodies include, among others, abciximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibrituximab tiuxetan, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and brentuximab.

[0101] A linker is defined herein as a moiety that connects two or more elements of a compound. For example, in a bioconjugate, the biomolecule and the target molecule are covalently linked to each other via a linker; in a linker-conjugate, the reactive group Q 1 Covalently linked to the target molecule via a linker; in the linker-construct, the reactive group Q 1 Through the linker and reactive group Q 2 Covalent attachment. A linker may comprise one or more spacer moieties.

[0102] A spacer moiety is defined herein as a moiety that spaces (i.e. provides a distance between) two parts (or more parts) of a linker and covalently links them together. The linker may be part of a linker-construct, linker-conjugate or bioconjugate, for example, as defined below.

[0103] A linker-construct is defined herein as one in which the reactive group Q1 Through the linker and reactive group Q 2 A covalently linked compound. A linker-construct comprises a reactive group Q that is capable of reacting with a reactive group present on a biomolecule. 1 , and a reactive group Q capable of reacting with a reactive group present on the target molecule 2 .Q 1 and Q 2 can be identical or different. The linker-construct may also contain more than one reactive group Q 1 and / or more than one reactive group Q 2 The linker-construct may also be represented as Q 1 -Sp-Q 2 , where Q 1 is a reactive group F that can react with the reactive group F on the biomolecule 1 Reactive group, Q 2 is a reactive group F that can react with the target molecule 2 When the linker-construct contains more than one reactive group Q 1 and / or more than one reactive group Q 2 When the linker-construct can be represented as (Q 1 ) y -Sp-(Q 2 ) z , wherein y is an integer in the range of 1 to 10, and z is an integer in the range of 1 to 10. Preferably, y is 1, 2, 3 or 4, more preferably, y is 1 or 2, most preferably, y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably, z is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, even more preferably, z is 1 or 2, and most preferably, z is 1. More preferably, y is 1 or 2, preferably 1, and z is 1, 2, 3 or 4; even more preferably, y is 1 or 2, preferably 1, and z is 1, 2 or 3; even more preferably, y is 1 or 2, preferably 1, and z is 1 or 2; and most preferably, y is 1 and z is 1.

[0104] A linker-conjugate is defined herein as a conjugate in which a target molecule is linked to a reactive group Q via a linker. 1 The linker-conjugate can be formed by making the reactive group Q present on the linker-construct 2 The linker-conjugate comprises a reactive group Q that is capable of reacting with a reactive group present on a biomolecule. 1 The linker-conjugate may contain one or more spacer moieties. The linker-conjugate may contain more than one reactive group Q 1and / or more than one target molecule.

[0105] A bioconjugate is defined herein as a compound in which a biomolecule is covalently linked to a target molecule via a linker. A bioconjugate comprises one or more biomolecules and / or one or more target molecules. The linker may comprise one or more spacer moieties.

[0106] When a compound is defined herein as a compound comprising an α end and an ω end, the compound comprises two (or more) ends, the first end being referred to as the α end, and the second end being referred to as the ω end. The compound may comprise more than two ends, i.e., a third, fourth end, etc. may be present in the compound.

[0107] A biomolecule is defined herein as any molecule that can be isolated from nature, or any molecule composed of smaller molecular building blocks, which are building blocks of macromolecular structures derived from nature, in particular nucleic acids, proteins, polysaccharides and lipids. Examples of biomolecules include enzymes, (non-catalytic) proteins, polypeptides, peptides, amino acids, oligonucleotides, monosaccharides, oligosaccharides, polysaccharides, glycans, lipids and hormones.

[0108] A target molecule, also referred to as a molecule of interest (MOI), is defined herein as a molecular structure having a desired property that is imparted to a biomolecule upon conjugation.

[0109] The term "salt thereof" means a compound formed when an acidic proton (usually a proton of an acid) is usually replaced by a cation (e.g., a metal cation or an organic cation, etc.). If applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts that are not intended for administration to a patient. For example, in a salt of a compound, the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid serving as the anionic component of the salt.

[0110] The term "pharmaceutically acceptable" salt means a salt that is acceptable for administration to a patient (e.g., a mammal) (for a given dosage regimen, it is a salt containing a counterion with acceptable mammalian safety). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases, as well as from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound, which are derived from a variety of organic and inorganic counterions known in the art, and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functional group, the salt is an organic or inorganic acid salt, such as hydrochloride, hydrobromide, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc.

[0111] Linker-Conjugate

[0112] Sulfonamide linkers and conjugates of the sulfonamide linkers are disclosed herein. The term "sulfonamide linker" refers to a sulfonamide group (more specifically, an acylsulfonamide group [-C(O)-N(H)-S(O) 2 -N(R 1 )-] and / or carbamoylsulfonamide [-OC(O)-N(H)-S(O) 2 -N(R 1 )-]) connector.

[0113] The present invention relates to the use of the sulfonamide linker of the present invention in a method for preparing a bioconjugate. The present invention also relates to a method for preparing a bioconjugate, and to a bioconjugate obtainable by the method. The method for preparing a bioconjugate will be described in more detail below.

[0114] In a first aspect, the present invention relates to a compound comprising an α end and an ω end, wherein the compound comprises a functional group F present on a biological molecule at the α end. 1 Reactive group Q 1 and comprising a target molecule D at the ω end, the compound further comprising a group of formula (1) or a salt thereof;

[0115]

[0116] in:

[0117] a is 0 or 1; and

[0118] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer;

[0119] And wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound.

[0120] Such compounds are also referred to as linker-conjugates. Linker-conjugates are defined herein as compounds in which a target molecule is linked to a reactive group Q via a linker. 1 The compound comprises an α end and an ω end, in other words, the compound comprises a first end and a second end. The first end of the compound may also be referred to as the α end, and the second end may also be referred to as the ω end of the compound. The terms "α end" and "ω end" are known to those skilled in the art. Therefore, the present invention also relates to a compound comprising a first end and a second end, wherein the compound comprises a functional group F present on a biomolecule at the first end. 1 Reactive group Q 1 and comprising a target molecule D at the second end, the compound further comprising a group of formula (1) or a salt thereof, wherein the group of formula (1) or a salt thereof is located between the first end and the second end of the compound, and wherein the group of formula (1) is as defined above.

[0121] In the compound of the present invention, the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound. 1 covalently bonded to the α-end of the compound, and the target molecule D covalently bonded to the ω-end of the compound.

[0122] The compounds of the present invention may also be referred to as linker-conjugates. In the linker-conjugates of the present invention, the target molecule D is linked to the reactive group Q via a linker. 1 covalently linked, and the linker comprises a group of formula (1) as defined above or a salt thereof.

[0123] When the linker-conjugate of the present invention comprises a salt of a group of formula (1), the salt is preferably a pharmaceutically acceptable salt.

[0124] The linker-conjugate of the present invention may contain more than one target molecule D. Thus, the linker-conjugate contains more than one ω-end, such as a second (third, fourth, fifth, etc.) ω-end, which can be covalently linked to the target molecule. Similarly, the linker-conjugate may contain more than one reactive group Q 1 , i.e. the linker-conjugate may contain more than one α-terminus. When more than one reactive group Q is present1 When the group Q 1 They may be the same or different. When more than one target molecule D is present, the target molecules D may be the same or different.

[0125] Therefore, the linker-conjugate of the present invention can also be represented as (Q 1 ) y -Sp-(D) z , wherein y is an integer in the range of 1 to 10, and z is an integer in the range of 1 to 10.

[0126] Therefore, the present invention also relates to compounds of the following formula:

[0127] (Q 1 ) y -Sp-(D) z ,

[0128] in:

[0129] y is an integer ranging from 1 to 10;

[0130] z is an integer ranging from 1 to 10;

[0131] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0132] D is the target molecule;

[0133] Sp is a spacer moiety, wherein the spacer moiety is defined as a spacer moiety that allows the reactive group Q 1 A moiety that spaces the target molecule D (i.e., provides a certain distance between them) and covalently links them; and wherein the spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0134] Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2, and most preferably y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, even more preferably z is 1 or 2, and most preferably z is 1. More preferably, y is 1 or 2, preferably 1, and z is 1, 2, 3 or 4; even more preferably y is 1 or 2, preferably 1, and z is 1, 2 or 3; even more preferably y is 1 or 2, preferably 1, and z is 1 or 2; and most preferably y is 1 and z is 1. In a preferred embodiment, the linker-conjugate is of formula Q 1 -Sp-(D) 4 , Q 1 -Sp-(D) 3 , Q 1 -Sp-(D) 2 or Q1 -Sp-D.

[0135] The linker-conjugate of the present invention comprises a group of formula (1) or a salt thereof as defined above. In a preferred embodiment, the linker-conjugate of the present invention comprises a group of formula (1) or a salt thereof wherein a is 0. Thus, in this embodiment, the compound comprises a group of formula (2) or a salt thereof:

[0136]

[0137] Where R 1 As defined above.

[0138] In another preferred embodiment, the linker-conjugate of the invention comprises a group of formula (1) or a salt thereof, wherein a is 1. Thus, in this embodiment, the compound comprises a group of formula (3) or a salt thereof:

[0139]

[0140] Where R 1 As defined above.

[0141] In the groups of formulae (1), (2) and (3), R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer;

[0142] In a preferred embodiment, R 1 is hydrogen or C 1 -C 20 Alkyl, more preferably R 1 is hydrogen or C 1 -C 16 Alkyl, even more preferably R 1 is hydrogen or C 1 -C 10 wherein the alkyl group is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms, preferably O, are present in the 3 are independently selected from hydrogen and C 1 -C 4 In a preferred embodiment, R 1 In another preferred embodiment, R 1 C 1 -C 20 Alkyl, more preferably C 1 -C 16 Alkyl, even more preferably C 1 -C 10 alkyl, wherein the alkyl is optionally interrupted by one or more O atoms, and wherein the alkyl is optionally substituted by an -OH group (preferably a terminal -OH group). In this embodiment, it is also preferred that R 1 is a (poly)ethylene glycol chain containing a terminal -OH group. In another preferred embodiment, R 1 is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl, more preferably selected from the group consisting of hydrogen, methyl, ethyl, n-propyl and isopropyl, and even more preferably selected from the group consisting of hydrogen, methyl and ethyl. Even more preferably R 1 is hydrogen or methyl, and most preferably R 1 For hydrogen.

[0143] In another preferred embodiment, R 1 is a target molecule D. Optionally, the target molecule D is connected to N via one or more spacer moieties. The spacer moiety, if present, is defined as a moiety that spaces the target molecule D and N apart (i.e., provides a certain distance between them) and covalently connects them. The target molecule D and preferred embodiments thereof will be described in more detail below.

[0144] When the linker-conjugate of the present invention comprises two or more target molecules D, the target molecules D may be different from each other.

[0145] In a preferred embodiment of the linker-conjugate of the present invention, the target molecule is selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, a nanoparticle, a microparticle and a biomolecule.

[0146] The inventors have found that the use of the sulfonamide linker of the present invention improves the solubility of the linker-conjugate, thereby significantly improving the efficacy of conjugation. Using conventional linkers, effective conjugation is usually hindered by the relatively low solubility of the linker-conjugate in aqueous media, especially when using relatively water-insoluble or hydrophobic target molecules. In a particularly preferred embodiment, the target molecule in a non-conjugated form is hydrophobic, and its water solubility is usually at most 1% (w / w), preferably at most 0.1% (w / w), and most preferably at most 0.01% (w / w), as measured at 20°C and 100kPa. Even this water-insoluble target molecule can be effectively conjugated when it is functionalized with the sulfonamide linker of the present invention. In this article, "non-conjugated form" refers to a target molecule that is not functionalized with the linker of the present invention or conjugated thereto. This non-conjugated form of the target molecule is known to those skilled in the art.

[0147] In this context, the term "active substance" refers to a pharmacological or biological substance, i.e., a substance with biological and / or pharmaceutical activity, such as a drug, a prodrug, a diagnostic agent, a protein, a peptide, a polypeptide, a peptide tag, an amino acid, a polysaccharide, a lipid, a vitamin, a steroid, a nucleotide, a nucleoside, a polynucleotide, RNA or DNA. Examples of peptide tags include cell permeable peptides such as human lactoferrin or polyarginine. Examples of polysaccharides are oligomannoses. Examples of amino acids are lysine.

[0148] When the target molecule is an active substance, the active substance is preferably selected from drugs and prodrugs. More preferably, the active substance is selected from pharmaceutically active compounds, especially low molecular weight to medium molecular weight compounds (e.g., about 2000 to about 2500 Da, preferably about 300 to about 1750 Da). In other preferred embodiments, the active substance is selected from cytotoxins, antiviral agents, antibacterial agents, peptides and oligonucleotides. Examples of cytotoxins include colchicine, vinca alkaloids, anthracyclines, camptothecins, doxorubicin, daunorubicin, taxanes, calicheamycins, tubulysins, irinotecans, inhibitory peptides, amanitin, deBouganin, duocarminsins, maytansines, auristatins or pyrrolobenzodiazepines (PBD). In view of their poor water solubility, preferred active substances include vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysin, amanitin, duocarmycin, maytansine, auristatins and pyrrolobenzodiazepines, especially vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulysin, amanitin, maytansine and auristatins.

[0149] The term "reporter molecule" as used herein refers to a molecule whose presence is readily detected, such as a diagnostic agent, a dye, a fluorophore, a radioisotope label, a contrast agent, a magnetic resonance imaging agent, or a mass marker.

[0150] Various fluorophores, also called fluorescent probes, are known to those skilled in the art. Several fluorophores are described in more detail in, for example, GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition 2013, Chapter 10: "Fluorescent probes", pages 395-463 (incorporated by reference). Examples of fluorophores include Alexa Fluor (e.g., Alexa Fluor 555), cyanine dyes (e.g., Cy3 or Cy5) and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthamide derivatives, phycobiliprotein derivatives (e.g., allophycocyanin), chromomycin, lanthanide chelates, and all classes of quantum dot nanocrystals. In view of their poor water solubility, preferred fluorophores include cyanine dyes, coumarin derivatives, fluorescein and its derivatives, pyrene derivatives, naphthylimide derivatives, chromomycin, lanthanide chelates and quantum dot nanocrystals, especially coumarin derivatives, fluorescein, pyrene derivatives and chromomycin.

[0151] Examples of radioisotope labels include 99m Tc, 111 In, 114m In, 115 In, 18 F. 14 C. 64 Cu, 131 I. 125 I. 123 I. 212 Bi, 88 y、 90 Y. 67 Cu, 186 Rh, 188 Rh, 66 Ga, 67 Ga and 10 B, which is optionally linked via a chelating moiety, such as DTPA (diethylenetriaminepentaacetic anhydride), DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid), NOTA (1,4,7-triazacyclononane N,N′,N″-triacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N″′-tetraacetic acid), DTTA (N 1 -(p-isothiocyanatobenzyl)-diethylenetriamine-N 1 , N 2 , N 3 , N 3-tetraacetic acid), deferoxamine or DFA (N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxysuccinamide) or HYNIC (hydrazinonicotinamide). Isotope labeling techniques are known to those skilled in the art and are described in more detail in GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition. 2013, Chapter 18: “PEGylation and synthetic polymer modification”, pages 787-838 (incorporated by reference).

[0152] Polymers suitable for use as target molecules D in the compounds of the present invention are known to those skilled in the art, and several examples are described in more detail in, for example, GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition. 2013, Chapter 18: "PEGylation and synthetic polymer modification", pages 787-838 (incorporated by reference). When the target molecule D is a polymer, preferably the target molecule D is independently selected from poly (ethylene glycol) (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1, x-diaminoalkane polymers (wherein x is the number of carbon atoms in the alkane, preferably x is an integer in the range of 2 to 200, preferably 2 to 10), (poly) ethylene glycol diamines (e.g. 1, 8-diamino-3, 6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (e.g. dextran), poly (amino acids) (e.g. poly (L-lysine)) and poly (vinyl alcohol). In view of their poor water solubility, preferred polymers include 1,x-diaminoalkane polymers and poly(vinyl alcohol).

[0153] Solid surfaces suitable for use as target molecules D are known to those skilled in the art. Solid surfaces are, for example, functional surfaces (e.g., surfaces of nanomaterials, carbon nanotubes, fullerenes or viral capsids), metal surfaces (e.g., titanium, gold, silver, copper, nickel, tin, rhodium or zinc surfaces), metal alloy surfaces (wherein the alloy is, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin, uranium, zinc and / or zirconium), polymer surfaces (wherein the polymer is, for example, polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly(dimethylsiloxane) or polymethyl methacrylate, polyacrylamide), glass surfaces, siloxane surfaces, chromatographic support surfaces (wherein the chromatographic support is, for example, a silica support, an agarose support, a cellulose support or an alumina support), etc. When the target molecule D is a solid surface, preferably D is independently selected from a functional surface or a polymer surface.

[0154] Hydrogels are known to those skilled in the art. Hydrogels are water-swollen networks formed by crosslinking between polymer components. See, for example, A Shoffman, Adv. Drug Delivery Rev. 2012, 64, 18 (incorporated by reference). When the target molecule is a hydrogel, preferably the hydrogel is composed of poly(ethylene glycol) (PEG) as a polymer base.

[0155] Microparticles and nanoparticles suitable for use as target molecules D are known to those skilled in the art. For example, GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition. 2013, Chapter 14: "Microparticles and nanoparticles", pages 549-587 (incorporated by reference) describe a variety of suitable microparticles and nanoparticles. The micron or nanoparticles can be in any shape, such as a sphere, rod, tube, cube, triangle and pyramid. Preferably, the microparticles or nanoparticles are spherical. The chemical composition of the microparticles and nanoparticles can be changed. When the target molecule D is a microparticle or nanoparticle, the microparticle or nanoparticle is, for example, a polymeric microparticle or polymeric nanoparticle, a silica microparticle or silica nanoparticle, or a gold microparticle or gold nanoparticle. When the particles are polymeric microparticles or polymeric nanoparticles, the polymer is preferably polystyrene or a styrene copolymer (e.g., a copolymer of styrene and divinylbenzene, butadiene, an acrylate and / or vinyltoluene), polymethylmethacrylate (PMMA), polyvinyltoluene, poly(hydroxyethylmethacrylate) (pHEMA), or poly(ethylene glycol dimethacrylate / 2-hydroxyethylmethacrylate) [poly(EDGMA / HEMA)]. Optionally, the surface of the microparticle or nanoparticle is modified, for example, using a detergent, by graft polymerization of a secondary polymer, or by covalent attachment of another polymer or a spacer moiety.

[0156] The target molecule D may also be a biomolecule. Biomolecules and preferred embodiments thereof will be described in more detail below. When the target molecule D is a biomolecule, the biomolecule is preferably selected from proteins (including glycoproteins and antibodies), polypeptides, peptides, polysaccharides, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides.

[0157] The linker-conjugate of the present invention comprises a functional group F present on a biomolecule 1 Reactive group Q 1. Functional groups are known to those skilled in the art and can be defined as any molecular entity that can impart specific properties to a molecule containing it. For example, the functional group in a biomolecule can constitute an amino group, a thiol group, a carboxylic acid group, an alcohol group, a carbonyl group, a phosphate group or an aromatic group. The functional group in a biomolecule can be naturally occurring or placed in the biomolecule by a specific technique (e.g., (bio)chemistry or genetic techniques). The functional group placed in the biomolecule can be a naturally occurring functional group in nature, or can be a functional group prepared by chemical synthesis, such as an azide, a terminal alkyne or a phosphine moiety. In this article, the term "reactive group" can refer to a certain group including a functional group, or can refer to the functional group itself. For example, a cyclooctyne group is a reactive group comprising a functional group (i.e., a C-C triple bond). Similarly, an N-maleimido group is a reactive group comprising a C-C double bond as a functional group. However, a functional group (e.g., an azido functional group, a thiol functional group or an amino functional group) can also be referred to as a reactive group in this article.

[0158] The linker-conjugate may contain more than one reactive group Q 1 When the linker-conjugate comprises two or more reactive groups Q 1 When the reactive group Q 1 Preferably, the linker-conjugate may comprise a reactive group Q 1 .

[0159] Reactive groups Q present in the linker-conjugate 1 Can bind to the functional groups F in biomolecules 1 In other words, the reactive group Q 1 It needs to be compatible with the functional groups F present in the biomolecule 1 Complementary. As used herein, a reactive group is referred to as being "complementary" to a functional group when it selectively reacts with the functional group, optionally in the presence of other functional groups. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below.

[0160] In a preferred embodiment, the reactive group Q 1is selected from: an optionally substituted N-maleimido group, a halogenated N-alkylamido group, a sulfonyloxy N-alkylamido group, an ester group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkenyl group, an alkynyl group, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl] group, a cycloalkenyl group, a tetrazine group, an azido group, a phosphino group, a nitrile oxide group, a nitrone group, a nitrile imide group, a diazo group, a keto group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halogenated N-maleimido group, a 1,1-bis(sulfonylmethyl)-methylcarbonyl group or an elimination derivative thereof, a carbonyl halide group, a propadienylamide group, a 1,2-quinone group or a triazine group.

[0161] In a preferred embodiment, Q 1 is N-maleimide. 1 When it is N-maleimide, Q 1 It is preferably unsubstituted. 1 Preferred is formula (9a), as shown below.

[0162] In another preferred embodiment, Q 1 is a halogenated N-alkylamido group. 1 When it is a halogenated N-alkylamido group, Q is preferably 1 It is formula (9b), as shown below, wherein k is an integer in the range of 1 to 10, and R 4 is selected from -Cl, -Br and -I. Preferably k is 1, 2, 3 or 4, more preferably k is 1 or 2 and most preferably k is 1. Preferably, R 4 is -I or -Br. More preferably, k is 1 or 2 and R 4 is -I or -Br, most preferably k is 1 and R 4 is -I or Br.

[0163] In another preferred embodiment, Q 1 is a sulfonyloxy N-alkylamido group. 1 When Q is sulfonyloxy N-alkylamido, it is preferred 1 It is formula (9b), as shown below, wherein k is an integer in the range of 1 to 10, and R 4 is selected from -O-methylsulfonyl, -O-phenylsulfonyl and -O-toluenesulfonyl. Preferably k is 1, 2, 3 or 4, more preferably k is 1 or 2, even more preferably k is 1. Most preferably k is 1 and R 4 is selected from -O-methylsulfonyl, -O-phenylsulfonyl and -O-toluenesulfonyl.

[0164] In another preferred embodiment, Q 1 is an ester group. 1When it is an ester group, it is preferred that the ester group is an activated ester group. Activated ester groups are known to those skilled in the art. An activated ester group is defined herein as an ester group comprising a good leaving group, wherein the ester carbonyl is bonded to the good leaving group. Good leaving groups are known to those skilled in the art. Further preferably, the activated ester group is of formula (9c), as shown below, wherein R 5 Selected from -N-succinimidyl (NHS), -N-sulfo-succinimidyl (sulfo-NHS), -(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl (TFP).

[0165] In another preferred embodiment, Q 1 is a carbonate group. 1 When being carbonate group, preferred carbonate group is activated carbonate group.Activated carbonate group is known to those skilled in the art.Activated carbonate group is defined as the carbonate group that comprises good leaving group in this article, and wherein carbonate carbonyl is bonded with described good leaving group.Further preferably, described carbonate group is formula (9d), as shown below, wherein R 7 Selected from -N-succinimidyl, -N-sulfo-succinimidyl, -(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl.

[0166] In another preferred embodiment, Q 1 is a sulfonyl halide group of formula (9e) shown below, wherein X is selected from F, Cl, Br and I. Preferably X is Cl or Br, more preferably Cl.

[0167] In another preferred embodiment, Q 1 is a thiol group (9f), or a derivative or precursor of a thiol group. A thiol group may also be referred to as a mercapto group. 1 When the thiol group is a derivative or precursor, the thiol derivative is preferably a thiol group represented by the formula (9g), (9h) or (9zb) shown below, wherein R 8 is an optionally substituted C 1 -C 12 Alkyl or C 2 -C 12 (Hetero)aryl, V is O or S and R 16 is an optionally substituted C 1 -C 12 More preferably R 8 is an optionally substituted C 1 -C 6 Alkyl or C 2 -C 6 (hetero)aryl, and even more preferably R 8 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or phenyl. Even more preferably, R8 is methyl or phenyl, most preferably methyl. More preferably R 16 is an optionally substituted C 1 -C 6 Alkyl, even more preferably R 16 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl, most preferably methyl. 1 is a thiol derivative of formula (9g) or (9zb), and Q 1 Reactive groups F on biomolecules 1 When Q 1 When it is formula (9h), Q 1 -SC(O)OR 8 OR-SC(S)OR 8 , preferably SC(O)OR 8 , where R 8 and preferred embodiments thereof are as defined above.

[0168] In another preferred embodiment, Q 1 is an alkenyl group, wherein the alkenyl group is straight-chain or branched, and wherein the alkenyl group is optionally substituted. The alkenyl group may be a terminal or internal alkenyl group. The alkenyl group may contain more than one CC double bond, and if so, preferably contains two CC double bonds. When the alkenyl group is a dienyl group, it is further preferred that the two CC double bonds are separated by a CC single bond (i.e., the dienyl group is preferably a conjugated dienyl group). Preferably, the alkenyl group is C 2 -C 24 Alkenyl, more preferably C 2 -C 12 Alkenyl, even more preferably C 2 -C 6 Alkenyl. Further preferably, the alkenyl is a terminal alkenyl. More preferably, the alkenyl is of formula (9i) as shown below, wherein l is an integer in the range of 0 to 10, preferably an integer in the range of 0 to 6, and p is an integer in the range of 0 to 10, preferably 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1. More preferably, p is 0, 1, 2, 3 or 4, more preferably p is 0, 1 or 2 and most preferably p is 0 or 1. Particularly preferably, p is 0 and l is 0 or 1, or p is 1 and l is 0 or 1.

[0169] In another preferred embodiment, Q 1 is an alkynyl group, wherein the alkynyl group is straight chain or branched, and wherein the alkynyl group is optionally substituted. The alkynyl group may be a terminal or internal alkenyl group. Preferably, the alkynyl group is C 2 -C 24 Alkynyl, more preferably C 2 -C12 Alkynyl, even more preferably C 2 -C 6 Alkynyl. Further preferably, the alkynyl is a terminal alkynyl. More preferably, the alkynyl is of formula (9j) as shown below, wherein l is an integer in the range of 0 to 10, preferably an integer in the range of 0 to 6. More preferably, l is 0, 1, 2, 3 or 4, more preferably l is 0, 1 or 2 and most preferably l is 0 or 1.

[0170] In another preferred embodiment, Q 1 The cycloalkenyl group is optionally substituted. Preferably, the cycloalkenyl group is C 3 -C 24 Cycloalkenyl, more preferably C 3 -C 12 cycloalkenyl, and even more preferably C 3 -C 8 Cycloalkenyl. In a preferred embodiment, the cycloalkenyl is a trans-cycloalkenyl, more preferably a trans-cyclooctenyl (also referred to as a TCO group), and most preferably a trans-cyclooctenyl of formula (9zi) or (9zj) as shown below. In another preferred embodiment, the cycloalkenyl is a cyclopropenyl, wherein the cyclopropenyl is optionally substituted. In another preferred embodiment, the cycloalkenyl is a norbornenyl, an oxa-norbornenyl, a norbornadienyl or an oxa-norbornenyl, wherein the norbornenyl, an oxa-norbornenyl, a norbornadienyl or an oxa-norbornenyl is optionally substituted. In other preferred embodiments, the cycloalkenyl is a formula (9k), (9l), (9m) or (9zc) as shown below, wherein T is CH 2 or O, R 9 independently selected from hydrogen, straight or branched chain C 1 -C 12 Alkyl or C 4 -C 12 (hetero)aryl, and R 19 is selected from hydrogen and fluorinated hydrocarbons. 9 are independently hydrogen or C 1 -C 6 Alkyl, more preferably R 9 are independently hydrogen or C 1 -C 4 Even more preferably, R 9 R is independently hydrogen or methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. Even more preferably, R 9 In other preferred embodiments, R 19 Selected from hydrogen and -CF 3 , -C 2 F 5 , -C3 F 7 and -C 4 F 9 , more preferably hydrogen and -CF 3 In other preferred embodiments, the cycloalkenyl group is of formula (9k), wherein one R 9 is hydrogen and the other R 9 In another preferred embodiment, the cycloalkenyl group is of formula (91), wherein the two R 9 In these embodiments, it is further preferred that l is 0 or 1. In another preferred embodiment, the cycloalkenyl group is a norbornene group of formula (9m) (T is CH 2 ) or oxa-norbornene (T is O), or a norbornadienyl (T is CH 2 ) or oxa-norbornadienyl (T is O), wherein R 9 is hydrogen and R 19 is hydrogen or -CF 3 , preferably -CF 3 .

[0171] In another preferred embodiment, Q 1 is a (hetero)cycloalkynyl group. The (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctynyl group, i.e., a heterocyclooctynyl group or a cyclooctynyl group, wherein the (hetero)cyclooctynyl group is optionally substituted. In other preferred embodiments, the (hetero)cyclooctynyl group is of formula (9n), also referred to as a DIBO group, or of formula (9o), also referred to as a DIBAC group, or of formula (9p), also referred to as a BARAC group, or of formula (9zk), also referred to as a COMBO group, all of which are as follows, wherein U is O or NR 9 , and R 9 Preferred embodiments of are as defined above. The aromatic ring in (9n) is optionally O-sulfonylated at one or more positions, while the rings of (9o) and (9p) may be halogenated at one or more positions.

[0172] In a particularly preferred embodiment, compound (4b) 1 The nitrogen atom to which the bond is attached is the nitrogen atom in the ring of the heterocycloalkynyl group (e.g., the nitrogen atom in 9o). In other words, in compound (4b), c, d, and g are 0, and R 1 and Q 1 Together with the nitrogen atom to which they are attached, they form a heterocycloalkynyl group, preferably a heterocyclooctynyl group, most preferably a heterocyclooctynyl group of formula (9o) or (9p). Here, the carbonyl part of (9o) is replaced by a sulfonyl group of a group of formula (1). Alternatively, with R 1 The nitrogen atom to which it is attached is the same atom as the atom designated as U in formula (9n). In other words, when Q1 In the case of formula (9n), U may be the right nitrogen atom of the group of formula (1), or U=NR 9 And R 9 is the remainder of the group of formula (1), and R 1 It is the cyclooctyne part.

[0173] In another preferred embodiment, Q 1 is an optionally substituted bicyclo[6.1.0]non-4-yn-9-yl] group, also known as a BCN group. Preferably, the bicyclo[6.1.0]non-4-yn-9-yl] group is of formula (9q) as shown below.

[0174] In another preferred embodiment, Q 1 is a conjugated (hetero)diene group capable of reacting in a Diels-Alder reaction. Preferred (hetero)diene groups include optionally substituted tetrazine groups, optionally substituted 1,2-quinone groups and optionally substituted triazine groups. More preferably, the tetrazine group is of formula (9r), as shown below, wherein R 9 Selected from hydrogen, linear or branched C 1 -C 12 Alkyl or C 4 -C 12 (Hetero)aryl. Preferably, R 9 For hydrogen, C 1 -C 6 Alkyl or C 4 -C 10 (Hetero)aryl, more preferably R 9 For hydrogen, C 1 -C 4 Alkyl or C 4 -C 6 (Hetero)aryl. Even more preferably, R 9 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or pyridyl. Even more preferably, R 9 is hydrogen, methyl or pyridyl. More preferably, the 1,2-quinone group is of formula (9zl) or (9zm). The triazine group may be any positional isomer. More preferably, the triazine group is 1,2,3-triazine group or 1,2,4-triazine group, which may be connected via any possible site, as shown in formula (9zn). As the triazine group, the 1,2,3-triazine is most preferred.

[0175] In another preferred embodiment, Q 1 It is an azido group of the formula (9s) shown below.

[0176] In another preferred embodiment, Q 1is an optionally substituted triarylphosphine group suitable for Staudingerligation reaction. Preferably, the phosphine group is of formula (9t) shown below, wherein R 10 is hydrogen or (thio) ester. 10 When it is a (thio)ester group, preferably R 10 for -C(O)-VR 11 , where V is O or S and R 11 C 1 -C 12 Preferably, R 11 C 1 -C 6 Alkyl, more preferably C 1 -C 4 Most preferably, R 11 It is methyl.

[0177] In another preferred embodiment, Q 1 It is an oxide nitrile group of the formula (9u) shown below.

[0178] In another preferred embodiment, Q 1 Preferably, the nitrone group is of formula (9v) shown below, wherein R 12 Selected from linear or branched C 1 -C 12 Alkyl and C 6 -C 12 Preferably, R 12 C 1 -C 6 Alkyl, more preferably R 12 C 1 -C 4 Even more preferably, R 12 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. Even more preferably, R 12 It is methyl.

[0179] In another preferred embodiment, Q 1 Preferably, the nitrile imine group is a formula (9w) or (9zd) as shown below, wherein R 13 Selected from linear or branched C 1 -C 12 Alkyl and C 6 -C 12 Preferably, R 13 C 1 -C 6 Alkyl, more preferably R 13 C 1 -C4 Even more preferably, R 13 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. Even more preferably R 13 It is methyl.

[0180] In another preferred embodiment, Q 1 Preferably, the diazo group is of the formula (9x) shown below, wherein R 14 is selected from hydrogen or a carbonyl derivative. More preferably, R 14 For hydrogen.

[0181] In another preferred embodiment, Q 1 More preferably, the keto group is of formula (9y) shown below, wherein R 15 Selected from linear or branched C 1 -C 12 Alkyl and C 6 -C 12 Preferably, R 15 C 1 -C 6 Alkyl, more preferably R 15 C 1 -C 4 Even more preferably R 15 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl. Even more preferably R 15 It is methyl.

[0182] In another preferred embodiment, Q 1 More preferably, the (O-alkyl)hydroxyamino group is of the formula (9z) shown below.

[0183] In another preferred embodiment, Q 1 Preferably, the hydrazino group is of the formula (9za) shown below.

[0184] In another preferred embodiment, Q 1 is a halogenated N-maleimide group or a sulfonylated N-maleimide group. 1 When it is a halogenated or sulfonylated N-maleimide group, Q 1 Preferably, the formula (9ze) shown below is: 6 independently selected from hydrogen, F, Cl, Br, I and -S(O) 2 R 18 , where R 18 Selected from optionally substituted C 1 -C 12 Alkyl and C 4 -C12 (Hetero)aryl, provided that at least one R 6 is not hydrogen. 6 When it is a halogen (i.e. when R 6 is F, Cl, Br or I), preferably R 6 is Br. When R 6 -S(O) 2 R 18 When R 18 C 1 -C 6 Alkyl or C 4 -C 6 (Hetero)aryl, preferably phenyl.

[0185] In another preferred embodiment, Q 1 is a carbonyl halide of formula (9zf) as shown below, wherein X is selected from F, Cl, Br and I. Preferably, X is Cl or Br, and most preferably, X is Cl.

[0186] In another preferred embodiment, Q 1 It is an allenamide group of the formula (9zg).

[0187] In another preferred embodiment, Q 1 is a 1,1-bis(sulfonylmethyl)methylcarbonyl group of formula (9zh), or an elimination derivative thereof, wherein R 18 Selected from optionally substituted C 1 -C 12 Alkyl and C 4 -C 12 (hetero)aryl. More preferably, R 18 is an optionally substituted C 1 -C 6 Alkyl or C 4 -C 6 (Hetero)aryl, and most preferably phenyl.

[0188]

[0189]

[0190]

[0191] Among them k, l, X, T, U, V, R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 As defined above.

[0192] In a preferred embodiment of the conjugation method of the invention as described below, the conjugation is achieved by a cycloaddition such as a Diels-Alder reaction or a 1,3-dipolar cycloaddition, preferably a 1,3-dipolar cycloaddition. According to this embodiment, the reactive group Q 1 (and F on biomolecules 1 ) is selected from a group reactive in a cycloaddition reaction. Here, the reactive group Q 1 and F 1 are complementary, ie they are capable of reacting with each other in a cycloaddition reaction.

[0193] For the Diels-Alder reaction, F 1 and Q 1 One of them is a diene, and F 1 and Q 1 As will be appreciated by those skilled in the art, in the context of the Diels-Alder reaction, the term "diene" refers to 1,3-(hetero)dienes and includes conjugated dienes (R 2 C=CR-CR=CR2), imine (such as R 2 C=CR-N=CR 2 or R 2 C=CR-CR=NR、R 2 C=NN=CR 2 ) and a carbonyl group (e.g., R2C=CR-CR=O or O=CR-CR=O). Hetero-Diels-Alder reactions using N-containing dienes and O-containing dienes are known in the art. Any diene known in the art to be suitable for Diels-Alder reactions can be used as the reactive group Q 1 or F 1 Preferred dienes include tetrazines as described above, 1,2-quinones as described above, and triazines as described above. Although any dienophile known in the art to be suitable for Diels-Alder reactions may be used as the reactive group Q 1 or F 1 , but the dienophile is preferably an alkene or alkyne group as described above, most preferably an alkyne group. For conjugation via a Diels-Alder reaction, F is preferred. 1 is a diene and Q 1 Here, when Q 1 When it is a diene, F 1 is a dienophile; when Q1 When F is a dienophile, 1 Most preferably, Q 1 is a dienophile, preferably Q 1 is or contains an alkynyl group, and F 1 is a diene, preferably a tetrazine, 1,2-quinone or triazine group.

[0194] For the 1,3-dipolar cycloaddition, F 1 and Q 1 One of them is a 1,3-dipole, and F 1 and Q 1 Any 1,3-dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q 1 or F 1 Preferred 1,3-dipole groups include azido, nitrone, nitrile oxide, nitrile imine and diazo groups. Although any dipolephile known in the art to be suitable for 1,3-dipolar cycloaddition may be used as the reactive group Q 1 or F 1 , but the dipolarophile is preferably an alkene or alkyne group, most preferably an alkyne group. For conjugation via 1,3-dipolar cycloaddition, F is preferred 1 It is a 1,3-dipole and Q 1 Here, when Q 1 When it is a 1,3-dipole, F 1 It is a dipole-loving body; when Q 1 When it is a dipole-phile, F 1 is a 1,3-dipole. Most preferably, Q 1 For dipole philes, Q 1 is or contains an alkynyl group, and F 1 It is a 1,3-dipole, preferably an azido group.

[0195] Therefore, in a preferred embodiment, Q 1 is selected from the group consisting of a dipolephile and a dienophile. 1 In a particularly preferred embodiment, Q 1 It contains an alkynyl group, preferably selected from the alkynyl group as described above, the cycloalkenyl group as described above, the (hetero)cycloalkynyl group as described above, and the bicyclo[6.1.0]non-4-yn-9-yl] group, more preferably Q 1 is selected from (9j), (9n), (9o), (9p), (9q) and (9zk) as defined above and described below, more preferably selected from (9n), (9o), (9p), (9q) and (9zk). Most preferably, Q 1[6.1.0] non-4-yn-9-yl] group, preferably a group of formula (9q). These groups are known to be highly efficient in conjugation with azide-functionalized biomolecules as described herein, and when the sulfonamide linker of the present invention is used in such linker-conjugates and bioconjugates, any aggregation is advantageously minimized. In particular, for such hydrophobic reactive groups Q 1 , and for the conjugated bioconjugates, the sulfonamide linker of the present invention results in significantly reduced aggregation.

[0196] As described above, in the compounds of the present invention, Q 1 Can react with the reactive groups F on biomolecules 1 Reaction. Reactive group Q 1 The complementary reactive group F 1 It is known to those skilled in the art and is described in more detail below. 1 and Q 1 Some representative examples of the reactions between Fig. 22 middle.

[0197] As described above, in the linker-conjugate of the present invention, the target molecule D and the reactive group Q 1 Covalently linked to the linker. The covalent linking of the target molecule D to the linker can be achieved, for example, by a functional group F present on the target molecule. 2 With the reactive group Q present on the linker 2 Suitable organic reactions for connecting the target molecule D to the linker are known to those skilled in the art, and the reactive group Q 2 Complementary functional group F 2 Therefore, D can be connected to the linker via the linker group Z.

[0198] The term "linking group" as used herein refers to a structural element that connects one part of a compound to another part of the same compound. As will be appreciated by those skilled in the art, the nature of the linking group depends on the type of organic reaction employed to achieve the connection between the parts of the compound. For example, when the carboxyl group of RC(O)-OH is linked to H 2 When the amino group of N-R' is reacted to form RC(O)-N(H)-R', R is linked to R' via a linking group Z, and Z may be represented by the group -C(O)-N(H)-.

[0199] Reactive Group Q 1 Can be connected to the linker in a similar manner. 1 The spacer portion can be connected via a linker group Z.

[0200] A large number of reactions are known in the art for the attachment of target molecules to linkers, as well as for the reactive group Q 1 Linkage to Linkers. Thus, a variety of linker groups Z may be present in the linker-conjugates of the invention.

[0201] Therefore, the present invention also relates to compounds of the following formula:

[0202] (Q 1 ) y -(Z w )-Sp-(Z x )-(D) z ,

[0203] in:

[0204] y is an integer ranging from 1 to 10;

[0205] z is an integer ranging from 1 to 10;

[0206] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0207] D is the target molecule;

[0208] Sp is a spacer moiety, wherein the spacer moiety is defined as a spacer moiety that allows the reactive group Q 1 a moiety that is spaced from the target molecule D (i.e., provides a certain distance between them) and covalently links them;

[0209] Z w To make the reactive group Q 1 a linking group connected to the spacer moiety;

[0210] Z x A linking group for connecting the target molecule D to the spacer; and

[0211] The spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0212] In a preferred embodiment, a is 0 in the radical of formula (1). In another preferred embodiment, a is 1 in the radical of formula (1).

[0213] Preferred embodiments of y and z are as described above (Q 1 ) y -Sp-(D) z Further preferably, the compound is of formula Q 1 -(Z w )-Sp-(Z x )-(D) 4, Q 1 -(Z w )-Sp-(Z x )-(D) 3 , Q 1 -(Z w )-Sp-(Z x )-(D) 2 or Q 1 -(Z w )-Sp-(Z x )-D, more preferably Q 1 -(Z w )-Sp-(Z x )-(D) 2 or Q 1 -(Z w )-Sp-(Z x )-D and most preferably Q 1 -(Z w )-Sp-(Z x )-D, where Z w and Z x As defined above.

[0214] Preferably, Z w and Z x Independently selected from -O-, -S-, -NR 2 -, -N=N-, -C(O)-, -C(O)-NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 、-NR 2 -C(O)-、-NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -、-S(O)-、-S(O) 2 -、-OS(O) 2 -、-OS(O) 2 -O-, -OS(O) 2 -NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR 2 -、-NR 2 -OC(O)-、-NR2 -O-C(O)-O-、-NR 2 -O-C(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2 -C(S)-NR 2 -、-NR 2 -O-C(S)-、-NR 2 -O-C(S)-O-、-NR 2 -O-C(S)-NR 2 -、-O-C(S)-、-O-C(S)-O-、-O-C(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -、-S-S(O) 2 -、-S-S(O) 2 -O-、-S-S(O) 2 -NR 2 -、-NR 2 -O-S(O)-、-NR 2 -O-S(O)-O-、-NR 2 -O-S(O)-NR 2 -、-NR 2 -O-S(O) 2 -、-NR 2 -O-S(O) 2 -O-、-NR 2 -O-S(O) 2 -NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O) 2 -O-、-O-NR 2 -S(O) 2 -NR 2 -、-O-NR 2 -S(O) 2 -、-O-P(O)(R 2 ) 2 -、-S-P(O)(R 2 ) 2 -、-NR2 -P(O)(R 2 ) 2 -, and combinations of two or more thereof, wherein R 2 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0215] D and Q 1 Preferred embodiments are as defined above.

[0216] More specifically, the present invention relates to compounds of formula (4a) or (4b), or salts thereof:

[0217]

[0218]

[0219] in:

[0220] a is independently 0 or 1;

[0221] b is independently 0 or 1;

[0222] c is 0 or 1;

[0223] d is 0 or 1;

[0224] e is 0 or 1;

[0225] f is an integer ranging from 1 to 150;

[0226] g is 0 or 1;

[0227] i is 0 or 1;

[0228] D is the target molecule;

[0229] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0230] Sp 1 For the interval part;

[0231] Sp 2 For the interval part;

[0232] Sp 3 For the interval part;

[0233] Sp4 For the interval part;

[0234] Z 1 is a connecting group, which connects Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 )connect;

[0235] Z 2 is a linking group, which connects D or Sp 4 With Sp 1 、N(R 1 ), O or C(O) connection; and

[0236] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 1 -C 24 (Hetero)aryl, C 1 -C 24 Alkyl (hetero) aryl and C 1 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl; or

[0237] R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above.

[0238] The compound of formula (4a) or (4b) or a salt thereof may also be referred to as a linker-conjugate.

[0239] In a preferred embodiment, a is 1 in the compound of formula (4a) or (4b). In another preferred embodiment, a is 0 in the compound of formula (4a) or (4b).

[0240] As mentioned above, Z 1 To Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 ) connected to the linking group, and Z 2 To D or Sp 4 With Sp 1 、N(R 1 As described in more detail above, the term "linking group" refers to a structural element that connects one part of a compound to another part of the same compound.

[0241] In the compound of formula (4a), the linking group Z 1 - when present (ie when d is 1) - Q of the compound of formula (4a) 1 (optionally via a spacer portion Sp 3 ) and an O-atom or a C(O) group (optionally separated by a spacer portion Sp 2 ) connection. More specifically, when Z 1 exists (i.e. d is 1), and when Sp 3 and Sp 2 When it does not exist (i.e. g is 0 and c is 0), Z 1 The Q of the linker-conjugate of formula (4a) 1 is connected to an O atom (a is 1) or a C(O) group (a is 0). 1 Exists (that is, when d is 1), Sp 3 exists (i.e. g is 1) and Sp 2 When it does not exist (i.e. c is 0), Z 1 The spacer portion Sp of the linker-conjugate of formula (4a) 3 is connected to an O atom (a is 1) or a C(O) group (a is 0). 1exists (i.e. d is 1), and when Sp 3 and Sp 2 When exists (i.e. g is 1 and c is 1), Z 1 The spacer portion Sp of the linker-conjugate of formula (4a) 3 With the interval part Sp 2 Connect. When Z 1 Exists (that is, when d is 1), Sp 3 does not exist (i.e. g is 0) and Sp 2 When exists (i.e. c is 1), Z 1 The Q of the linker-conjugate of formula (4a) 1 With the interval part Sp 2 connect.

[0242] In the compound of formula (4b), the linking group Z 1 - when present (ie when d is 1) - Q in the linker-conjugate of formula (4b) 1 (optionally via a spacer portion Sp 3 ) and N(R 1 ) group (optionally separated by a spacer portion Sp 2 ) connection. More specifically, when Z 1 exists (i.e. when d is 1), and when Sp 3 and Sp 2 When it does not exist (i.e. g is 0 and c is 0), Z 1 The Q of the linker-conjugate of formula (4b) 1 With N(R 1 ) group. 1 Exists (that is, when d is 1), Sp 3 exists (i.e. g is 1) and Sp 2 When it does not exist (i.e. c is 0), Z 1 The spacer portion Sp of the linker-conjugate of formula (4b) 3 With N(R 1 ) group. 1 exists (i.e. when d is 1), and when Sp 3 and Sp 2 When exists (i.e. g is 1 and c is 1), Z 1 The spacer portion Sp of the linker-conjugate of formula (4b) 3 With the interval part Sp 2 Connect. When Z 1 Exists (that is, when d is 1), Sp 3 does not exist (i.e. g is 0) and Sp 2 When exists (i.e. c is 1), Z 1 The Q of the linker-conjugate of formula (4b)1 With the interval part Sp 2 connect.

[0243] In the compound of formula (4a), when c, d and g are all 0, then Q in the linker-conjugate of formula (4a) 1 Directly bonded to an O atom (when a is 1) or to a C(O) group (when a is 0).

[0244] In the compound of formula (4b), when c, d and g are all 0, then Q in the linker-conjugate of formula (4b) 1 With N(R 1 ) group is directly connected to the N atom of the group.

[0245] In the compound of formula (4a), the linking group Z 2 - when present (ie when e is 1) - D of the linker-conjugate of formula (4a) (optionally separated by a spacer moiety Sp 4 ) and N(R 1 ) group (optionally separated by a spacer portion Sp 1 ) connection. More specifically, when Z 2 exists (i.e. e is 1), and when Sp 1 and Sp 4 When it does not exist (i.e. b is 0 and i is 0), Z 2 The D of the linker-conjugate of formula (4a) is connected to N (R 1 ) group. 2 Exists (that is, when e is 1), Sp 4 exists (i.e., i is 1) and Sp 1 When it does not exist (i.e. b is 0), Z 2 The spacer portion Sp of the linker-conjugate of formula (4a) 4 With N(R 1 ) group. 2 exists (i.e. when e is 1), and when Sp 1 and Sp 4 When exists (i.e. b is 1 and i is 1), Z 2 The spacer portion Sp of the linker-conjugate of formula (4a) 1 With the interval part Sp 4 Connect. When Z 2 Exists (that is, when e is 1), Sp 4 does not exist (i.e., i is 0) and Sp 1 When exists (i.e. b is 1), Z 2 The D of the linker-conjugate of formula (4a) is connected to the spacer portion Sp 1 connect.

[0246] In the compound of formula (4a), when b, e and i are all 0, then D and N (R 1 ) group is directly connected to the N atom of the group.

[0247] In the compound of formula (4b), when b, e and i are all 0, then D in the linker-conjugate of formula (4b) is directly connected to the O atom (when a is 1) or to the C(O) group (when a is 0).

[0248] As will be appreciated by those skilled in the art, the nature of the linking group will depend on the type of organic reaction employed to achieve the linkage between the particular moieties of the compound. A wide variety of organic reactions can be used to link the reactive group Q 1 The target molecule is connected to the spacer part and is used to connect the target molecule to the spacer part. Therefore, there are a variety of linking groups Z 1 and Z 2 .

[0249] In a preferred embodiment of the linker-conjugates of (4a) and (4b), Z 1 and Z 2 Independently selected from -O-, -S-, -SS-, -NR 2 -, -N=N-, -C(O)-, -C(O)-NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 、-NR 2 -C(O)-、-NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -、-S(O)-、-S(O) 2 -、-OS(O) 2 -、-OS(O) 2 -O-, -OS(O) 2 -NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR 2 -、-NR 2 -OC(O)-、-NR 2 -OC(O)-O-、-NR 2 -OC(O)-NR2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2 -C(S)-NR 2 -、-NR 2 -O-C(S)-、-NR 2 -O-C(S)-O-、-NR 2 -O-C(S)-NR 2 -、-O-C(S)-、-O-C(S)-O-、-O-C(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -、-S-S(O) 2 -、-S-S(O) 2 -O-、-S-S(O) 2 -NR 2 -、-NR 2 -O-S(O)-、-NR 2 -O-S(O)-O-、-NR 2 -O-S(O)-NR 2 -、-NR 2 -O-S(O) 2 -、-NR 2 -O-S(O) 2 -O-、-NR 2 -O-S(O) 2 -NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O) 2 -O-、-O-NR 2 -S(O) 2 -NR 2 -、-O-NR 2 -S(O) 2 -、-O-P(O)(R 2 ) 2 -、-S-P(O)(R 2 ) 2 -、-NR 2 -P(O)(R 2 ) 2-, and combinations of two or more thereof, wherein R 2 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0250] As described above, in the compound of formula (4a) or (4b), Sp 1 、Sp 2 、Sp 3 and Sp 4 For the interval part. Sp 1 、Sp 2 、Sp 3 and Sp 4 Can be independently present or absent (b, c, g and i are independently 0 or 1). 1 (if present), may be different from Sp 2 (if present), may be different from Sp 3 and / or Sp 4 (if present).

[0251] Spacer moieties are known to those skilled in the art. Examples of suitable spacer moieties include (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), polyethylene glycol chains or polyethylene oxide chains, polypropylene glycol chains or polypropylene oxide chains, and 1,x-diaminoalkanes (wherein x is the number of carbon atoms in the alkane).

[0252] Another type of suitable spacer includes a cleavable spacer, or a cleavable linker. The cleavable linker is well known in the art. For example, Shabat et al., Soft Matter 2012, 6, 1073 - incorporated herein by reference - discloses a cleavable linker that is included in a biological trigger (e.g., enzymatic cleavage or oxidation event) to release a self-sacrificing part. Examples of some suitable cleavable linkers are disulfide linkers that are cleaved during reduction, peptide linkers that are cleaved under the specific recognition of proteases (e.g., cathepsins, plasmins, or metalloproteinases), or glycosidic linkers that are cleaved under the specific recognition of glycosidases (e.g., glucuronidase), or nitroaromatic compounds that are reduced in oxygen-deficient, anoxic regions. Here, suitable cleavable spacers also include spacers that include specific, cleavable amino acid sequences. Examples include, for example, spacers that include a Val-Cit (valine-citrulline) portion. Bioconjugates comprising cleavable linkers, such as Val-Cit linkers, especially Val-Cit-PABC, aggregate to a large extent due to their limited water solubility. For such bioconjugates, the inclusion of the sulfonamide linker of the present invention is particularly beneficial. Furthermore, conjugation reactions with linker conjugates comprising cleavable linkers are hampered by the limited water solubility of the linker-conjugate. Therefore, linker-conjugates comprising cleavable linkers, such as Val-Cit linkers, especially Val-Cit-PABC, and the sulfonamide linker of the present invention are superior to linker-conjugates comprising such cleavable linkers but lacking such sulfonamide linkers conjugated to biomolecules.

[0253] Thus, in a preferred embodiment of the linker-conjugates of formula (4a) and (4b), the spacer moiety Sp 1 、Sp 2 、Sp 3 and / or Sp 4 - if present - comprises an amino acid sequence. Spacer moieties comprising an amino acid sequence are known to those skilled in the art and may also be referred to as peptide linkers. Examples include spacer moieties comprising a Val-Cit moiety (e.g. Val-Cit-PABC, Val-Cit-PAB, Fmoc-Val-Cit-PAB, etc.). Preferably, a Val-Cit-PABC moiety is used in the linker-conjugate.

[0254] In a preferred embodiment of the linker-conjugates of formula (4a) and (4b), the spacer moiety Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C200 Alkylene, C 2 -C 200 Alkenylene, C 2 -C 200 Alkynylidene, C 3 -C 200 Cycloalkylene, C 5 -C 200 Cycloalkenylene, C 8 -C 200 Cycloalkynylene, C 7 -C 200 Alkyl arylene, C 7 -C 200 Arylalkylene, C 8 -C 200 Arylene and C 9 -C 200 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl are optionally substituted. When said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are interrupted by one or more heteroatoms as defined above, preferably said groups are interrupted by one or more O atoms, and / or by one or more SS groups.

[0255] More preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 100 Alkylene, C 2 -C 100 Alkenylene, C 2 -C 100 Alkynylidene, C 3 -C 100 Cycloalkylene, C 5 -C 100 Cycloalkenylene, C 8 -C100 Cycloalkynylene, C 7 -C 100 Alkyl arylene, C 7 -C 100 Arylalkylene, C 8 -C 100 Arylene and C 9 -C 100 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0256] Even more preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 50 Alkylene, C 2 -C 50 Alkenylene, C 2 -C 50 Alkynylidene, C 3 -C 50 Cycloalkylene, C 5 -C 50 Cycloalkenylene, C 8 -C 50 Cycloalkynylene, C 7 -C 50 Alkyl arylene, C 7 -C 50 Arylalkylene, C 8 -C 50 Arylene and C 9 -C 50 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0257] Even more preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, C 2 -C 20 Alkenylene, C 2 -C 20 Alkynylidene, C 3 -C 20 Cycloalkylene, C 5 -C 20 Cycloalkenylene, C 8 -C 20 Cycloalkynylene, C 7 -C 20 Alkyl arylene, C 7 -C 20 Arylalkylene, C 8 -C 20 Arylene and C 9 -C 20 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0258] In these preferred embodiments, it is further preferred that the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are unsubstituted and optionally selected from O, S and NR3 One or more heteroatoms, preferably O, are present in the 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, preferably hydrogen or methyl.

[0259] Most preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 wherein the alkylene radical is unsubstituted and is optionally selected from O, S and NR. 3 One or more heteroatoms, preferably O and / or SS, are present in the 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, preferably hydrogen or methyl.

[0260] Therefore, the preferred spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 Includes-(CH 2 ) n -、-(CH 2 CH 2 ) n -、-(CH 2 CH 2 O) n -、-(OCH 2 CH 2 ) n -、-(CH 2 CH 2 O) n CH 2 CH 2 -、-CH 2 CH 2 (OCH2 CH 2 ) n -、-(CH 2 CH 2 CH 2 O) n -、-(OCH 2 CH 2 CH 2 ) n -、-(CH 2 CH 2 CH 2 O) n CH 2 CH 2 CH 2 -and-CH 2 CH 2 CH 2 (OCH 2 CH 2 CH 2 ) n -, wherein n is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, even more preferably 1, 2, 3 or 4.

[0261] Due to Sp 1 、Sp 2 、Sp 3 and Sp 4 are chosen independently, so Sp 1 (if present), may be different from Sp 2 (if present), may be different from Sp 3 and / or Sp 4 (if present).

[0262] The reactive group Q is described in more detail above. 1 In the linker-conjugates of formula (4a) and (4b), preferably the reactive group Q 1is selected from the group consisting of an optionally substituted N-maleimido group, a halogenated N-alkylamido group, a sulfonyloxy N-alkylamido group, an ester group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkenyl group, an alkynyl group, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl] group, a cycloalkenyl group, a tetrazine group, an azido group, a phosphino group, an oxynitrile group, a nitrone group, a nitrile imide group, a diazo group, a keto group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halogenated N-maleimido group, a carbonyl halide group, an allenamide group, and a 1,1-bis(sulfonylmethyl)-methylcarbonyl group or an elimination derivative thereof. In other preferred embodiments, Q 1 are formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), 9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), where (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) and preferred embodiments thereof are as defined above. In a preferred embodiment, Q 1 is of formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn). In an even more preferred embodiment, Q 1 is of formula (9a), (9n), (9o), (9q), (9p), (9t), (9zh) or (9s), and in a particularly preferred embodiment, Q 1 is formula (9a), (9q), (9n), (9o), (9p), (9t) or (9zh) and preferred embodiments thereof, as defined above.

[0263] In the linker-conjugates of formula (4a) and (4b), the target molecule D and preferred embodiments of the target molecule D are as defined above. In a preferred embodiment of the linker-conjugates of formula (4a) and (4b), D is selected from active substances, reporter molecules, polymers, solid surfaces, hydrogels, nanoparticles, microparticles and biomolecules. Active substances, reporter molecules, polymers, hydrogels, solid surfaces, nanoparticles and microparticles and biomolecules, and preferred embodiments thereof have been described in more detail above.

[0264] As mentioned above, R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1, b, c, d, e, g and i are as defined above.

[0265] In a preferred embodiment, R 1 is hydrogen or C 1 -C 20 Alkyl, more preferably R 1 is hydrogen or C 1 -C 16 Alkyl, even more preferably R 1 is hydrogen or C 1 -C 10 wherein the alkyl group is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms, preferably O, are present in the 3 are independently selected from hydrogen and C 1 -C 4 In other preferred embodiments, R 1 In another preferred embodiment, R 1 C 1 -C 20 Alkyl, more preferably C 1 -C 16 Alkyl, even more preferably C 1 -C 10 alkyl, wherein the alkyl is optionally interrupted by one or more O atoms, and wherein the alkyl is optionally substituted by an -OH group, preferably a terminal -OH group. In this embodiment, it is further preferred that R 1 is a polyethylene glycol chain containing a terminal -OH group. In another preferred embodiment, R 1 C 1 -C 12 Alkyl, more preferably C 1 -C 6 Alkyl, even more preferably C 1 -C 4 alkyl, and even more preferably R 1 Selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl.

[0266] In another preferred embodiment, R 1 The target molecule D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 )g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above. When R 1 D or -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D], it is further preferred that the linker-conjugate is of formula (4a). In this embodiment, the linker-conjugate (4a) comprises two target molecules D, which may be the same or different. 1 For-[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D], in -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] in Sp 1 , b, Z 2 、e、Sp 4 , i and D can be combined with N(R 1 ) of the other N atom connected to -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] in Sp 1 , b, Z 2 、e、Sp 4 , i and D are the same or different. In a preferred embodiment, in N(R 1 ) on the N atom of the two -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] are the same.

[0267] When R 1 For-[(Sp 2 ) c -(Z1 ) d -(Sp 3 ) g -Q 1 ], it is further preferred that the linker-conjugate is of formula (4b). In this embodiment, the linker-conjugate (4b) comprises two target molecules Q 1 , which may be the same or different. 1 For-[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], in -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] in Sp 2 ,c,Z 1 , d, Sp 3 , g and D can be combined with N(R 1 ) of the other N atom connected to -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ] in Sp 1 , b, Z 2 、e、Sp 4 , i and Q 1 In a preferred embodiment, in N(R 1 ) on the N atom of the two -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ] are the same.

[0268] In the linker-conjugates of formula (4a) and (4b), f is an integer in the range of 1 to 150. Thus, the linker-conjugate may contain more than one group of formula (1), the groups of formula (1) being as defined above. When more than one group of formula (1) is present, i.e. when f is 2 or greater, then a, b, Sp 1 and R 1 In other words, when f is 2 or greater, each a is independently 0 or 1, each b is independently 0 or 1, and each Sp1 can be the same or different, and each R 1 In a preferred embodiment, f is an integer in the range of 1 to 100, preferably in the range of 1 to 50, more preferably in the range of 1 to 25, and even more preferably in the range of 1 to 15. In this embodiment, more preferably, f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably f is 1, 2, 3, 4, 5 or 6, even more preferably f is 1, 2, 3 or 4, and most preferably f is 1. In another preferred embodiment, f is an integer in the range of 2 to 150, preferably in the range of 2 to 100, more preferably in the range of 2 to 50, more preferably in the range of 2 to 25, and even more preferably in the range of 2 to 15. In this embodiment, more preferably, f is 2, 3, 4, 5, 6, 7, 8, 9 or 10, even more preferably f is 2, 3, 4, 5, 6, 7 or 8, even more preferably f is 2, 3, 4, 5 or 6, even more preferably f is 2, 3 or 4, and most preferably f is 2.

[0269] As mentioned above, in a preferred embodiment, a in the compound of formula (4a) or (4b) is 0. Therefore, the present invention also relates to a compound of formula (6a) or (6b), or a salt thereof:

[0270]

[0271] Among them a, b, c, d, e, f, g, i, D, Q 1 、Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 and R 1 , and preferred embodiments thereof, as defined in (4a) and (4b) above.

[0272] As mentioned above, in another preferred embodiment, in the compound of formula (4a) or (4b), a is 1. Therefore, the present invention also relates to a compound of formula (7a) or (7b), or a salt thereof:

[0273]

[0274] Among them a, b, c, d, e, f, g, i, D, Q 1 、Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 and R1 , and preferred embodiments thereof, as defined in (4a) and (4b) above.

[0275] When Sp in the linker-conjugate of formula (4a) 4 When it does not exist (i.e. when i is 0), the target molecule D and Z 2 Connect (when e is 1), and Sp 1 Connect (when e is 0 and b is 1) or with N (R 1 ) connection (when e is 0 and b is 0). When in the linker-conjugate of formula (4b) Sp 4 When it does not exist (i.e. when i is 0), the target molecule D and Z 2 Connect (when e is 1), and Sp 1 The present invention also relates to a linker-conjugate of formula (4c) or (4d), or a salt thereof:

[0276]

[0277]

[0278] in:

[0279] a, b, c, d, e, f, g, D, Q 1 、Sp 1 、Sp 2 、Sp 3 , Z 1 , Z 2 and R 1 , and preferred embodiments thereof, as defined in (4a) and (4b) above.

[0280] In a preferred embodiment, a is 0 in the linker-conjugate of formula (4c) or (4d). In another preferred embodiment, a is 1 in the linker-conjugate of formula (4c) or (4d).

[0281] In a specific embodiment of the linker-conjugate of the invention, in particular in the linker-conjugate of (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b), Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, and Q 1 are formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v) , (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg)(9zh), (9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), where (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) and preferred embodiments thereof are as defined above. In a preferred embodiment, Q 1 is of formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn). In even further preferred embodiments, Q 1 is of formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and in a particularly preferred embodiment, Q 1 is formula (9a), (9q), (9n), (9p), (9t), (9zh) or (9o) and preferred embodiments thereof, as defined above.

[0282] A linker is defined herein as a moiety that connects two or more elements of a compound.

[0283] Thus, in the linker-conjugate of formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b) as defined above, the linker as defined above may be represented by formula (8a) and (8b), respectively:

[0284]

[0285] As will be appreciated by those skilled in the art, the preferred embodiments of the spacer moieties (8a) and (8b) may depend, for example, on the reactive groups Q in the linker-conjugation. 1 and the nature of D, the synthetic method for preparing the linker-conjugate (e.g., complementary functional groups F on the target molecule 2 properties of the bioconjugate prepared using the linker-conjugate (e.g., complementary functional groups F on the biomolecule 1 nature of the

[0286] When Q 1 For example, the above-defined formula (9n), (9 x ), (9p), (9q) or (9zk) is cyclooctyne, preferably Sp 3 exists (g is 1).

[0287] When, for example, the linker-conjugate is linked to a reactive group Q 2 — a cyclooctynyl group of formula (9n), (9o), (9p), (9q) or (9zk) — and an azido functional group F 2 When prepared by reaction, Sp 4 exists (i is 1).

[0288] In addition, Sp 1 、Sp 2 、Sp 3 and Sp 4 At least one of b, c, g and i is present, that is, at least one of b, c, g and i is not 0. In another preferred embodiment, Sp 1 and Sp 4 At least one of Sp 2 and Sp 3 At least one of them exists.

[0289] When f is 2 or greater, Sp 1 exists (b is 1).

[0290] These preferred embodiments of linker moieties (8a) and (8b) also apply to the linker moieties in the bioconjugates of the present invention described in more detail below.

[0291] Sp 1 、Sp 2 、Sp 3 and Sp 4 Preferred embodiments are as defined above.

[0292] Linker-Construct

[0293] A linker-construct is defined herein as one in which the reactive group Q 1 Through the linker and reactive group Q 2 A covalently linked compound. A linker-construct comprises a reactive group F present on a biomolecule. 1 Reactive group Q 1 , and the reactive group F present on the target molecule 2 Reactive group Q 2 .Q 1 and Q 2 Can be the same or different. A linker-construct may contain more than one reactive group Q 1 and / or more than one reactive group Q 2 When there is more than one reactive group Q 1 When the group Q 1 may be the same or different, and when more than one reactive group Q is present 2 When the group Q 2 Can be the same or different.

[0294] The present invention also relates to a compound, more specifically to a linker-construct, wherein the compound comprises an α end and an ω end, wherein the α end of the compound comprises a functional group F present on a biological molecule. 1 Reactive functional group Q 1 , and a functional group F present on the target molecule at the ω end 2 Reactive functional group Q 2 , the compound further comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above, and wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound.

[0295] Reactive Group Q 1 covalently bonded to the α-terminus of the compound, and the reactive group Q 2 Covalently bonded to the ω-end of the compound.

[0296] The compound of the present invention may also be referred to as a linker-construct. In the linker-construct of the present invention, the reactive group Q 2 Through the linker and reactive group Q 1 covalently linked, and the linker comprises a group of formula (1) as defined above or a salt thereof. When the linker-construct of the present invention comprises a salt of a group of formula (1), the salt is preferably a pharmaceutically acceptable salt.

[0297] The linker-constructs of the invention may contain more than one reactive group Q 2Thus, the linker may comprise, for example, a third (fourth, fifth, etc.) terminus, which may be referred to as a psi, chi, phi, etc. terminus, comprising a reactive group Q 2 Similarly, the linker-conjugate may contain more than one reactive group Q 1 .

[0298] Therefore, the linker-conjugate of the present invention can also be represented as (Q 1 ) y -Sp-(Q 2 ) z , wherein y is an integer in the range of 1 to 10, and z is an integer in the range of 1 to 10.

[0299] Therefore, the present invention also relates to linker-constructs of the following formula:

[0300] (Q 1 ) y -Sp-(Q 2 ) z ,

[0301] in:

[0302] y is an integer ranging from 1 to 10;

[0303] z is an integer ranging from 1 to 10;

[0304] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0305] Q 2 is the functional group F that can bind to the target molecule 2 Reactive groups for reactions;

[0306] Sp is a spacer moiety, wherein the spacer moiety is defined as a spacer moiety that allows the reactive group Q 1 and reactive group Q 2 a moiety that spaces them apart (i.e. provides a certain distance between them) and covalently links them; and

[0307] The spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0308] Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2, and most preferably y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, even more preferably z is 1 or 2, and most preferably z is 1. More preferably, y is 1 or 2, preferably 1, and z is 1, 2, 3 or 4; even more preferably y is 1 or 2, preferably 1, and z is 1, 2 or 3; even more preferably y is 1 or 2, preferably 1, and z is 1 or 2; and most preferably y is 1 and z is 1. In a preferred embodiment, the linker-construct is of formula Q 1 -Sp-(Q 2 ) 4 , Q 1 -Sp-(Q 2 ) 3 , Q 1 -Sp-(Q 2 ) 2 or Q 1 -Sp-Q 2 .

[0309] The linker-construct of the present invention comprises a group of formula (1) or a salt thereof as defined above. In a preferred embodiment, the linker-construct of the present invention comprises a group of formula (1) or a salt thereof wherein a is 0. Therefore, in this embodiment, the linker-construct comprises a group of formula (2) or a salt thereof:

[0310]

[0311] Where R 1 As defined above.

[0312] In another preferred embodiment, the linker-construct of the present invention comprises a group of formula (1) or a salt thereof, wherein a is 1. Thus, in this embodiment, the linker-construct comprises a group of formula (3) or a salt thereof:

[0313]

[0314] Where R 1 As defined above.

[0315] In the linker-construct of the present invention, R 1 and R 1 Preferred embodiments of are as defined above. In addition, the reactive group Q 1 and the spacer moiety Sp, and their embodiments, are as defined above for the linker-conjugate of the present invention.

[0316] More specifically, the present invention relates to compounds of the following formula:

[0317] (Q1 ) y -(Z w )-Sp-(Z x )-(Q 2 ) z ,

[0318] in:

[0319] y is an integer ranging from 1 to 10;

[0320] z is an integer ranging from 1 to 10;

[0321] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0322] Q 2 is capable of binding to the functional group F on the biomolecule 2 Reactive groups for reactions;

[0323] Sp is a spacer moiety, wherein the spacer moiety is defined as a spacer moiety that allows the reactive group Q 1 and Q 2 a moiety that spaces them apart (i.e., provides a certain distance between them) and covalently links them;

[0324] Z w To convert the reactive group Q 1 a linking group connected to the spacer portion;

[0325] Z x To convert the reactive group Q 2 a linking group connected to the spacer portion; and

[0326] The spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0327] In a preferred embodiment, a in the group of formula (1) is 0. In another preferred embodiment, a in the group of formula (1) is 1.

[0328] Preferred embodiments of y and z are as described above (Q 1 ) y -Sp-(Q 2 ) z It is further preferred that the compound is of formula Q 1 -(Z w )-Sp-(Z x )-(Q 2 ) 4 , Q 1 -(Z w )-Sp-(Zx )-(Q 2 ) 3 , Q 1 -(Z w )-Sp-(Z x )-(Q 2 ) 2 or Q 1 -(Z w )-Sp-(Z x )-Q 2 , more preferably Q 1 -(Z w )-Sp-(Z x )-(Q 2 ) 2 or Q 1 -(Z w )-Sp-(Z x )-Q 2 , and most preferably Q 1 -(Z w )-Sp-(Z x )-Q 2 , where Z w and Z x As defined above.

[0329] In the linker compound of the present invention, Z w and Z x Preferably, they are independently selected from -O-, -S-, -NR 2 -, -N=N-, -C(O)-, -C(O)-NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 、-NR 2 -C(O)-、-NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -、-S(O)-、-S(O) 2 -、-OS(O) 2 -、-OS(O) 2 -O-, -OS(O) 2 -NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR2 -、-NR 2 -O-C(O)-、-NR 2 -O-C(O)-O-、-NR 2 -O-C(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-、-O-NR 2 -C(S)-NR 2 -、-NR 2 -O-C(S)-、-NR 2 -O-C(S)-O-、-NR 2 -O-C(S)-NR 2 -、-O-C(S)-、-O-C(S)-O-、-O-C(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-、-NR 2 -C(S)-NR 2 -、-S-S(O) 2 -、-S-S(O) 2 -O-、-S-S(O) 2 -NR 2 -、-NR 2 -O-S(O)-、-NR 2 -O-S(O)-O-、-NR 2 -O-S(O)-NR 2 -、-NR 2 -O-S(O) 2 -、-NR 2 -O-S(O) 2 -O-、-NR 2 -O-S(O) 2 -NR 2 -、-O-NR 2 -S(O)-、-O-NR 2 -S(O)-O-、-O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O) 2 -O-、-O-NR 2 -S(O) 2 -NR 2 -、-O-NR 2 -S(O) 2 -、-O-P(O)(R 2 ) 2-、-SP(O)(R 2 ) 2 -、-NR 2 -P(O)(R 2 ) 2 -, and combinations of two or more thereof, wherein R 2 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0330] Q 1 Preferred embodiments are as defined above.

[0331] In the linker-construct of the present invention, Q 2 is the functional group F that can bind to the target molecule 2 Reactive groups that can react with such functional groups F 2 Reactive group Q 2 In a preferred embodiment, Q 2 is a reactive group selected from the group consisting of an optionally substituted N-maleimido group, a halogenated N-alkylamido group, a sulfonyloxy N-alkylamido group, an ester group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkenyl group, an alkynyl group, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl] group, a cycloalkenyl group, a tetrazine group, an azido group, a phosphino group, an oxynitrile group, a nitrone group, a nitrile imide group, a diazo group, a keto group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halogenated N-maleimido group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or an elimination derivative thereof, a carbonyl halide group and an allenamide group, -[C(R 17 ) 2 C(R 17 ) 2 O] q -R 17 (where q is in the range of 1 to 200), -CN, -NCV, -VCN, -VR 17 、-N(R 17 ) 2 、- + N(R 17 ) 3 、-C(V)N(R 17 ) 2 ,-C(R 17 ) 2 VR 17、-C(V)R 17 、-C(V)VR 17 、-S(O)R 17 、-S(O) 2 R 17 、-S(O)OR 17 、-S(O) 2 OR 17 、-S(O)N(R 17 ) 2 、-S(O) 2 N(R 17 ) 2 、-OS(O)R 17 、-OS(O) 2 R 17 、-OS(O)OR 17 、-OS(O) 2 OR 17 、-P(O)(R 17 )(OR 17 )、-P(O)(OR 17 ) 2 、-OP(O)(OR 17 ) 2 、-Si(R 17 ) 3 、-VC(V)R 17 、-VC(V)VR 17 、-VC(V)N(R 17 ) 2 、-N(R 17 )C(V)R 17 、-N(R 17 )C(V)VR 17 and -N(R 17 )C(V)N(R 17 ) 2 , where V is O or S, and where R 17 are independently selected from hydrogen, halogen, C 1 -C 24 Alkyl, C 6 -C 24 (Hetero)aryl, C 7 -C 24 Alkyl (hetero) aryl and C 7 -C 24 (Hetero)arylalkyl.

[0332] In other preferred embodiments, Q 2are formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v) , (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), where (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) and preferred embodiments thereof are as defined above. In this embodiment, further preferred is Q 2 It is the formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn), more preferably the formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and even more preferably Q 2 is formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferred embodiments thereof, as defined above. Most preferably, Q 2 is formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferred embodiments thereof, as defined above.

[0333] In another other preferred embodiment, Q 2 Selected from -[C(R 17 ) 2 C(R 17 ) 2 O] q -R 17 , where q is in the range of 1 to 200, -CN, -NCV, -VCN, -VR 17 、-N(R 17 ) 2 、- + N(R17 ) 3 、-C(V)N(R 17 ) 2 ,-C(R 17 ) 2 VR 17 、-C(V)R 17 、-C(V)VR 17 、-S(O)R 17 、-S(O) 2 R 17 、-S(O)OR 17 、-S(O) 2 OR 17 、-S(O)N(R 17 ) 2 、-S(O) 2 N(R 17 ) 2 、-OS(O)R 17 、-OS(O) 2 R 17 、-OS(O)OR 17 、-OS(O) 2 OR 17 、-P(O)(R 17 )(OR 17 )、-P(O)(OR 17 ) 2 、-OP(O)(OR 17 ) 2 、-Si(R 17 ) 3 、-VC(V)R 17 、-VC(V)VR 17 、-VC(V)N(R 17 ) 2 、-N(R 17 )C(V)R 17 、-N(R 17 )C(V)VR 17 and -N(R 17 )C(V)N(R 17 ) 2 , where V and R 17 As defined above. In this embodiment, further preferably Q 2 Select from -OR 17 , SR 17 、-N(R 17 ) 2 、- + N(R 17 ) 3 、-C(O)N(R 17 )2 ,-C(R 17 ) 2 OR 17 、-C(O)R 17 、-C(O)OR 17 、-S(O)R 17 、-S(O) 2 R 17 、-S(O)OR 17 、-S(O) 2 OR 17 、-S(O)N(R 17 ) 2 、-S(O) 2 N(R 17 ) 2 、-OS(O)R 17 、-OS(O) 2 R 17 、-OS(O)OR 17 、-OS(O) 2 OR 17 、-P(O)(R 17 )(OR 17 )、-P(O)(OR 17 ) 2 、-OP(O)(OR 17 ) 2 、-Si(R 17 ) 3 、-OC(O)R 17 、-OC(O)OR 17 、-OC(O)N(R 17 ) 2 、-N(R 17 )C(O)R 17 、-N(R 17 )C(O)OR 17 and -N(R 17 )C(O)N(R 17 ) 2 , where R 17 As defined above.

[0334] In a specific embodiment of the linker-construct of the invention, Q 1are formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w) ), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), where (9a), (9b), (9c ), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) and preferred embodiments thereof, as defined above; and a spacer portion Sp is selected from a linear or branched C 1 -C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl; wherein the spacer portion Sp is interrupted by one or more groups of formula (1) or a salt thereof; and wherein (1) is as defined above.

[0335] In a preferred embodiment, the spacer portion Sp is spaced by one or more groups of formula (2) or salts thereof, wherein (2) is as defined above. In another preferred embodiment, the spacer portion Sp is spaced by one or more groups of formula (3) or salts thereof, wherein (3) is as defined above.

[0336] In other preferred embodiments, Q 1 is formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9zk), (9r), (9zl), (9zm) or (9zn). In a further preferred embodiment, Q 1is of formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and in a particularly preferred embodiment, Q 1 is formula (9a), (9p), (9q), (9n), (9t), (9zh), (9zk) or (9o), and preferred embodiments thereof, as defined above. Most preferably Q 1 is formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferred embodiments thereof, as defined above.

[0337] Methods for preparing linker-conjugates

[0338] The present invention also relates to a method for preparing the linker-conjugate of the present invention. Specifically, the present invention relates to a method for preparing the linker-conjugate of the present invention, the method comprising: 2 Functional group F of the target molecule 2 The step of reacting, wherein the linker-construct is a compound comprising an α-terminus and an ω-terminus, and the compound comprises a functional group Q at the α-terminus 1 and contains a functional group Q at the ω-terminus 2 , the Q 1 Can bind to the functional groups F on biomolecules 1 reaction, and the Q 2 Capable of binding to the functional group F present on the target molecule 2 The compound further comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above, and wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound.

[0339] Including Q 1 , Q 2 The linker-construct and preferred embodiments of the target molecule D are described in detail above.

[0340] In a preferred embodiment of the method for preparing a linker-construct, the linker-construct is as defined above (Q 1 ) y -Sp-(Q 2 ) z In other preferred embodiments of the method for preparing a linker-construct, the linker-construct is as defined above (Q 1 ) y -(Z w )-Sp-(Z x )-(Q 2 ) z .

[0341] The present invention also relates to the use of the sulfonamide linker-construct of the present invention in a bioconjugation process. The linker-construct of the present invention and its preferred embodiments have been described in detail above. The present invention particularly relates to formula (Q 1 ) y -Sp-(Q 2 ) z The invention relates to the use of a linker-construct in a bioconjugation process, and relates to formula (Q 1 ) y -(Z w )-Sp-(Z x )-(Q 2 ) z Use of linker-constructs in bioconjugation processes.

[0342] The present invention also relates to the use of the linker-conjugate of the present invention in a bioconjugation process. The linker-conjugate of the present invention and its preferred embodiments are described in detail above. The present invention particularly relates to the use of the linker-conjugate of formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b) in a bioconjugation process.

[0343] Bioconjugates

[0344] Bioconjugates are defined herein as compounds in which a biomolecule is covalently linked to a target molecule via a linker. The bioconjugate comprises one or more biomolecules and / or one or more target molecules. The linker may comprise one or more spacer moieties. The bioconjugates of the present invention are conveniently prepared by the method for preparing the bioconjugates of the present invention, wherein the biomolecule comprising the reactive group Q is covalently linked to a target molecule via a linker. 1 The linker-conjugate is conjugated to a reactive group F 1 In this conjugation reaction, the reactive group Q 1 and F 1 react with each other to form a linker moiety, which connects the linker conjugate and the biomolecule. Therefore, all preferred embodiments of the linker-conjugate and the biomolecule described herein are equally applicable to the bioconjugate of the present invention, except that all described Q 1 and F 1 In addition, the bioconjugate of the present invention contains Q as defined above 1 and F 1 reaction product.

[0345] The present invention also relates to a compound comprising an α-terminus and an ω-terminus, the compound comprising a biomolecule at the α-terminus and a target molecule at the ω-terminus, and the compound further comprising a group of formula (1) or a salt thereof:

[0346]

[0347] in:

[0348] a is 0 or 1; and

[0349] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or

[0350] R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer; and wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the compound.

[0351] In a preferred embodiment, the compound further comprises a moiety obtainable by a cycloaddition reaction (preferably a 1,3-dipolar cycloaddition reaction, most preferably a 1,2,3-triazole ring), which is located between the α-terminus and the group of formula (1). Therefore, when viewed from the α-terminus to the ω-terminus, the compound described herein comprises a biomolecule, a moiety obtainable by a cycloaddition reaction, a group of formula (1), and a target molecule.

[0352] In other preferred embodiments, the target molecule is hydrophobic as defined above.

[0353] The compound of the present invention may also be referred to as a bioconjugate. When the bioconjugate of the present invention comprises a salt of the group of formula (1), the salt is preferably a pharmaceutically acceptable salt.

[0354] The biomolecule is covalently linked to the α-terminus of the bioconjugate of the invention, and the target molecule is covalently linked to the ω-terminus of the bioconjugate of the invention.

[0355] The conjugates of the present invention may comprise more than one target molecule. Similarly, the bioconjugates may comprise more than one biomolecule. Biomolecule B and target molecule D, and preferred embodiments thereof, have been described in more detail above. As described in more detail above, preferred embodiments of D in the bioconjugates of the present invention correspond to preferred embodiments of D in the linker-constructs of the present invention. As described in more detail above, preferred embodiments of linkers (8a) or (8b) in the bioconjugates of the present invention correspond to preferred embodiments of linkers in the linker-conjugates of the present invention.

[0356] In the bioconjugates of the present invention, biomolecule B is preferably selected from proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides. More preferably, biomolecule B is selected from proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes. Most preferably, biomolecule B is selected from proteins (including glycoproteins and antibodies), polypeptides, peptides and glycans.

[0357] The bioconjugate of the present invention may also be defined as a bioconjugate in which a biomolecule is conjugated to a target molecule via a spacer moiety, wherein the spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0358] The bioconjugate of the present invention can also be represented by (B) y -Sp-(D) z , wherein y is an integer in the range of 1 to 10, and z is an integer in the range of 1 to 10.

[0359] Therefore, the present invention also relates to bioconjugates of the following formula:

[0360] (B) y -Sp-(D) z ,

[0361] in:

[0362] y is an integer ranging from 1 to 10;

[0363] z is an integer ranging from 1 to 10;

[0364] B is a biomolecule;

[0365] D is the target molecule;

[0366] Sp is a spacer moiety, wherein a spacer moiety is defined as a moiety that spaces the biomolecule B and the target molecule D (i.e., provides a certain distance between them) and covalently links them; and

[0367] The spacer moiety comprises a group of formula (1) or a salt thereof, wherein the group of formula (1) is as defined above.

[0368] In a preferred embodiment, the spacer moiety further comprises a moiety obtainable by a cycloaddition reaction (preferably a 1,3-dipolar cycloaddition reaction, most preferably a 1,2,3-triazole ring), which is located between B and the group of formula (1).

[0369] Preferably, y is 1, 2, 3 or 4, more preferably y is 1 or 2, and most preferably y is 1. Preferably, z is 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4, even more preferably z is 1, 2 or 3, even more preferably z is 1 or 2, and most preferably z is 1. More preferably, y is 1 or 2, preferably 1, and z is 1, 2, 3 or 4; even more preferably y is 1 or 2, preferably 1, and z is 1, 2 or 3; even more preferably y is 1 or 2, preferably 1, and z is 1 or 2; and most preferably y is 1 and z is 1. In a preferred embodiment, the bioconjugate is of the formula B-Sp-(D) 4 、B-Sp-(D) 3 、B-Sp-(D) 2 or B-Sp-D.

[0370] As described above, the bioconjugate of the present invention comprises a group of formula (1) or a salt thereof as defined above. In a preferred embodiment, the bioconjugate comprises a group of formula (1) or a salt thereof wherein a is 0. Thus, in this embodiment, the bioconjugate comprises a group of formula (2) or a salt thereof, wherein (2) is as defined above.

[0371] In another preferred embodiment, the bioconjugate comprises a group of formula (1) or a salt thereof, wherein a is 1. Thus, in this embodiment, the bioconjugate comprises a group of formula (3) or a salt thereof, wherein (3) is as defined above.

[0372] In the bioconjugates of the present invention, R 1 , the interval part Sp, and R 1 Preferred embodiments of and Sp are as defined above for the linker-conjugates of the invention.

[0373] In a preferred embodiment, the bioconjugate is of formula (5a) or (5b), or a salt thereof:

[0374]

[0375] Among them, a, b, c, d, e, f, g, h, i, D, Sp 1 、Sp 2 、Sp 3 , Z 1 , Z 2 , Z 3 and R 1 , and preferred embodiments thereof are as defined above in the linker-conjugates of (4a) and (4b); and

[0376] h is 0 or 1;

[0377] Z 3 is a linking group that connects B to Sp 3 , Z 1 、Sp 2 , O or C(O) connection;

[0378] B is a biomolecule.

[0379] Preferably, h is 1.

[0380] Preferred embodiments of biomolecule B are as defined above.

[0381] When the bioconjugate of the present invention is a salt of (5a) or (5b), the salt is preferably a pharmaceutically acceptable salt.

[0382] Z 3 As mentioned above, the term "linking group" herein refers to a structural element that connects one part of a compound to another part of the same compound. Typically, bioconjugates are linked by reactive groups Q present in the linker-conjugate. 1 With the functional group F present in the biomolecule 1 As will be appreciated by those skilled in the art, the linking group Z 3 The nature of Z depends on the type of organic reaction used to establish the connection between the biomolecule and the linker-conjugate. 3 The nature of the linker-conjugate depends on the reactive group Q 1 The properties of the functional group F in the biomolecule 1 Since there are a large number of different chemical reactions that can be used to establish the connection between the biomolecule and the linker-conjugate, Z 3 There are many possibilities.

[0383] Fig. 22 It shows that when Q is included 1 The linker-conjugate comprises a complementary functional group F 1 When conjugated with biomolecules, F 1 and Q 1Several examples of suitable combinations of, and the presence of linker groups Z in the bioconjugates 3 Several examples of .

[0384] When F 1 When it is a thiol group, for example, the complementary group Q 1 Includes N-maleimide and alkenyl groups, and the corresponding linking group Z 3 like Fig. 22 As shown. 1 When it is a thiol group, the complementary group Q 1 Also included are allenamide groups.

[0385] When F 1 When it is, for example, an amino group, the complementary group Q 1 Including keto group, activated ester group and azido group, and the corresponding linking group Z 3 like Fig. 22 shown.

[0386] When F 1 When it is, for example, a keto group, the complementary group Q 1 including (O-alkyl)hydroxylamino and hydrazine groups, and the corresponding linking group Z 3 like Fig. 22 shown.

[0387] When F 1 When it is, for example, an alkynyl group, the complementary group Q 1 includes an azido group, and the corresponding linking group Z 3 like Fig. 22 shown.

[0388] When F 1 When it is, for example, an azido group, the complementary group Q 1 Including alkynyl, and the corresponding linking group Z 3 like Fig. 22 shown.

[0389] When F 1 When it is, for example, cyclopropenyl, trans-cyclooctenyl or cyclooctynyl, the complementary group Q 1 Including tetrazine group, and the corresponding linking group Z 3 like Fig. 22 In these specific cases, Z 3 It is only an intermediate structure and will discharge N 2 , thereby generating a dihydropyridazine (from the reaction with the alkenyl) or a pyridazine (from the reaction with the alkyne).

[0390] F 1 and Q 1 Other suitable combinations of and the resulting linking group Z 3The properties of are known to those skilled in the art and are described, for example, in GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition. 2013 (ISBN: 978-0-12-382239-0) (especially Chapter 3, pages 229-258) (incorporated by reference). A list of complementary reactive groups suitable for bioconjugation processes is disclosed in Table 3.1 on pages 230-232 of Chapter 3 of GT Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition. 2013 (ISBN: 978-0-12-382239-0), and the contents of the table are expressly incorporated herein by reference.

[0391] In the bioconjugates of (5a) and (5b), preferably Z 3 、Sp 3 , Z 1 and Sp 2 At least one of h, g, d and c is present, that is, at least one of h, g, d and c is not 0. It is also preferred that Sp 1 , Z 2 and Sp 4 At least one of b, e and i is present, that is, at least one of b, e and i is not 0. More preferably, Z 3 、Sp 3 , Z 1 and Sp 2 At least one of them exists, and Sp 1 , Z 2 and Sp 4 At least one of b, e and i is present, that is, preferably at least one of h, g, d and c is not 0.

[0392] Methods of preparing bioconjugates

[0393] The present invention also relates to a method for preparing a bioconjugate, the method comprising reacting a reactive group Q of a linker-conjugate of the present invention 1 Functional groups F of biomolecules 1 Steps of the reaction. The linker-conjugate of the present invention and its preferred embodiments have been described in more detail above.

[0394] Figure 1 shows the general concept of biomolecule conjugation: a molecule containing one or more functional groups F 1 The target biomolecule (BOI) is bound to (excess) a specific linker and a reactive group Q 1 The target molecule D (also called molecule of interest or MOI) is incubated with the covalently linked target molecule D (also called molecule of interest or MOI).1 and Q 1 The chemical reaction between the BOI and the MOI forms a bioconjugate comprising a covalent bond between the BOI and the MOI. The BOI may be, for example, a peptide / protein, a polysaccharide or a nucleic acid. In the method of the present invention, the linker is a sulfonamide linker.

[0395] The present invention also relates to a method for preparing a bioconjugate, the method comprising: 1 Functional groups F of biomolecules 1 The step of reacting, wherein the linker-conjugate is a compound comprising an α-terminus and an ω-terminus, and the compound comprises a reactive group Q at the α-terminus 1 and contains the target molecule at the ω-end, the Q 1 Can bind to the functional groups F present on biomolecules 1 Reaction, the compound further comprises a group of formula (1) or a salt thereof:

[0396]

[0397] in:

[0398] a is 0 or 1; and

[0399] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer;

[0400] And wherein the group of formula (1) or a salt thereof is located between the α-end and the ω-end of the linker conjugate.

[0401] In a preferred embodiment, the present invention relates to a method for preparing a bioconjugate by a cycloaddition, such as a (4+2)-cycloaddition (e.g., a Diels-Alder reaction) or a (3+2)-cycloaddition (e.g., a 1,3-dipolar cycloaddition). Preferably, the conjugation is a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is a reverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, a 1,3-dipolar cycloaddition is used, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q 1 is or contains an alkynyl group and F 1 is an azido group. Cycloadditions (e.g., Diels-Alder reactions and 1,3-dipolar cycloadditions) are known in the art and the skilled person knows how to perform them. In other preferred embodiments, the present invention relates to a method for preparing a bioconjugate, wherein the target molecule is hydrophobic (i.e., sparingly soluble in water), most preferably wherein the solubility of the target molecule in water is at most 0.1% (w / w) (20° C. and 100 kPa). In a particularly preferred embodiment, the present invention relates to a method for preparing a bioconjugate by cycloaddition, wherein the cycloaddition is preferably a 1,3-dipolar cycloaddition, more preferably an alkyne-azide cycloaddition, and most preferably wherein Q 1 is or contains an alkynyl group and F 1 is an azido group, and wherein the target molecule is hydrophobic, most preferably wherein the water solubility of the target molecule in water is at most 0.1% (w / w) (20° C. and 100 kPa).

[0402] In the method of the present invention, Q 1 With F 1 Reaction to form a covalent link between the biomolecule and the linker moiety. The complementary reactive group Q 1 and functional group F 1 Described in more detail in the context.

[0403] In a preferred embodiment of the method of the invention, a in the group of formula (1) is 0. Thus, in this embodiment, the linker-conjugate comprises a group of formula (2), as defined above. In another preferred embodiment of the method of the invention, a in the group of formula (1) is 1. Thus, in this embodiment, the linker-conjugate comprises a group of formula (3), as defined above.

[0404] Biomolecules have been described in more detail above. Preferably, in the method of the present invention, the biomolecule is selected from proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides. More preferably, biomolecule B is selected from proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes. Most preferably, biomolecule B is selected from proteins (including glycoproteins and antibodies), polypeptides, peptides and glycans.

[0405] The target molecule has been described in more detail above. In a preferred embodiment of the method of the present invention, the target molecule is selected from active substances, reporter molecules, polymers, solid surfaces, hydrogels, nanoparticles, microparticles and biomolecules. Active substances, reporter molecules, polymers, solid surfaces, hydrogels, nanoparticles and microparticles have been described in detail above, as have their preferred embodiments. In view of the significantly increased water solubility of the linker-conjugate when the sulfonamide linker of the present invention is used, a preferred embodiment of the method for preparing a bioconjugate uses a hydrophobic target molecule. The water solubility of the hydrophobic target molecule in its non-conjugated form, measured at 20° C. and 100 kPa, is typically at most 1% (w / w), preferably at most 0.1% (w / w), most preferably at most 0.01% (w / w).

[0406] In the method of the present invention, preferably the reactive group Q 1 is selected from an optionally substituted N-maleimido group, a halogenated N-alkylamido group, a sulfonyloxy N-alkylamido group, an ester group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkenyl group, an alkynyl group, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl] group, a cycloalkenyl group, a tetrazine group, an azido group, a phosphino group, a nitrile oxide group, a nitrone group, a nitrile imide group, a diazo group, a keto group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halogenated N-maleimido group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or an elimination derivative thereof, a carbonyl halide group and an allenamide group.

[0407] In other preferred embodiments, Q 1are formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9 w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm) or (9zn), where (9a), (9b), ( (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk), (9zl), (9zm), (9zn) and preferred embodiments thereof are as defined above for the linker-conjugate of the present invention. More preferably, Q 1 is formula (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9zh), (9r), (9zl), (9zm) or (9zn). Even more preferably, Q 1 is of formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and most preferably, Q 1 is formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o), and preferred embodiments thereof, as defined above.

[0408] In a particularly preferred embodiment, Q 1 It contains an alkynyl group, preferably selected from the alkynyl group as described above, the cycloalkenyl group as described above, the (hetero)cycloalkynyl group as described above and the bicyclo[6.1.0]non-4-yn-9-yl] group, more preferably Q 1 is selected from (9j), (9n), (9o), (9p), (9q) and (9zk) as defined above. Most preferably, Q 1 is a bicyclo[6.1.0]non-4-yn-9-yl] group, preferably a group of formula (9q).

[0409] In other preferred embodiments of the method of the present invention, the linker-conjugate is of formula (4a) or (4b), or a salt thereof:

[0410]

[0411] in:

[0412] a is independently 0 or 1;

[0413] b is independently 0 or 1;

[0414] c is 0 or 1;

[0415] d is 0 or 1;

[0416] e is 0 or 1;

[0417] f is an integer ranging from 1 to 150;

[0418] g is 0 or 1;

[0419] i is 0 or 1;

[0420] D is the target molecule;

[0421] Q 1 is capable of binding to the functional group F on the biomolecule 1 Reactive groups for reactions;

[0422] Sp 1 For the interval part;

[0423] Sp 2 For the interval part;

[0424] Sp 3 For the interval part;

[0425] Sp 4 For the interval part;

[0426] Z 1 is a connecting group, which connects Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 )connect;

[0427] Z 2 is a linking group, which connects D or Sp 4 With Sp 1 、N(R 1 ), O or C(O) connection; and

[0428] R 1 Selected from hydrogen, C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl, the C 1 -C 24 Alkyl, C 3 -C 24 Cycloalkyl, C 2 -C 24 (Hetero)aryl, C 3 -C 24 Alkyl (hetero) aryl and C 3 -C 24 (Hetero)arylalkyl is optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 Alkyl; or

[0429] R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above.

[0430] Sp 1 、Sp 2 、Sp 3 and Sp 4 is independently a spacer portion, in other words, Sp 1 、Sp 2 、Sp 3 and Sp 4 Can be different from each other. 1 、Sp 2 、Sp 3 and Sp 4May be present or absent (b, c, g and i are independently 0 or 1). However, preferably Sp 1 、Sp 2 、Sp 3 and Sp 4 At least one of b, c, g and i is present, that is, preferably at least one of b, c, g and i is not 0.

[0431] If present, preferably Sp 1 、Sp 2 、Sp 3 and Sp 4 independently selected from linear or branched C 1 -C 200 Alkylene, C 2 -C 200 Alkenylene, C 2 -C 200 Alkynylidene, C 3 -C 200 Cycloalkylene, C 5 -C 200 Cycloalkenylene, C 8 -C 200 Cycloalkynylene, C 7 -C 200 Alkyl arylene, C 7 -C 200 Arylalkylene, C 8 -C 200 Arylene and C 9 -C 200 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, said alkyl, alkenyl, alkynyl and cycloalkyl are optionally substituted. When said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are interrupted by one or more heteroatoms as defined above, preferably said groups are interrupted by one or more O atoms, and / or by one or more SS groups.

[0432] More preferably, the spacer portion Sp 1 、Sp2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 100 Alkylene, C 2 -C 100 Alkenylene, C 2 -C 100 Alkynylidene, C 3 -C 100 Cycloalkylene, C 5 -C 100 Cycloalkenylene, C 8 -C 100 Cycloalkynylene, C 7 -C 100 Alkyl arylene, C 7 -C 100 Arylalkylene, C 8 -C 100 Arylene and C 9 -C 100 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0433] Even more preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 50 Alkylene, C 2 -C 50 Alkenylene, C 2 -C 50 Alkynylidene, C 3 -C 50 Cycloalkylene, C 5 -C 50 Cycloalkenylene, C 8 -C 50 Cycloalkynylene, C7 -C 50 Alkyl arylene, C 7 -C 50 Arylalkylene, C 8 -C 50 Arylene and C 9 -C 50 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0434] Even more preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, C 2 -C 20 Alkenylene, C 2 -C 20 Alkynylidene, C 3 -C 20 Cycloalkylene, C 5 -C 20 Cycloalkenylene, C 8 -C 20 Cycloalkynylene, C 7 -C 20 Alkyl arylene, C 7 -C 20 Arylalkylene, C 8 -C 20 Arylene and C 9 -C 20 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1-C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0435] In these preferred embodiments, it is further preferred that the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are unsubstituted and optionally selected from O, S and NR 3 One or more heteroatoms, preferably O, are present in the 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, preferably hydrogen or methyl.

[0436] Most preferably, the spacer portion Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 wherein the alkylene radical is unsubstituted and is optionally selected from O, S and NR. 3 One or more heteroatoms, preferably O and / or SS, are present in the 3 are independently selected from hydrogen and C 1 -C 4 Alkyl, preferably hydrogen or methyl.

[0437] Particularly preferred spacer parts Sp 1 、Sp 2 、Sp 3 and Sp 4 Includes-(CH 2 ) n -、-(CH2 CH 2 ) n -、-(CH 2 CH 2 O) n -、-(OCH 2 CH 2 ) n -、-(CH 2 CH 2 O) n CH 2 CH 2 -、-CH 2 CH 2 (OCH 2 CH 2 ) n -、-(CH 2 CH 2 CH 2 O) n -、-(OCH 2 CH 2 CH 2 ) n -、-(CH 2 CH 2 CH 2 O) n CH 2 CH 2 CH 2 -and-CH 2 CH 2 CH 2 (OCH 2 CH 2 CH 2 ) n -, wherein n is an integer in the range of 1 to 50, preferably in the range of 1 to 40, more preferably in the range of 1 to 30, even more preferably in the range of 1 to 20, and even more preferably in the range of 1 to 15. More preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1, 2, 3, 4, 5, 6, 7 or 8, even more preferably 1, 2, 3, 4, 5 or 6, even more preferably 1, 2, 3 or 4.

[0438] In another preferred embodiment of the method of the present invention, in the linker-conjugate of formula (4a) or (4b), the spacer portion Sp 1 、Sp 2 、Sp 3 and / or Sp 4- if present - comprises an amino acid sequence. Spacer moieties comprising an amino acid sequence are known to those skilled in the art and may also be referred to as peptide linkers. Examples include spacer moieties comprising a Val-Cit moiety (e.g., Val-Cit-PABC, Val-Cit-PAB, Fmoc-Val-Cit-PAB, etc.).

[0439] As mentioned above, Z 1 and Z 2 In a preferred embodiment of the method of the present invention, Z 1 and Z 2 Independently selected from -O-, -S-, -NR 2 -, -N=N-, -C(O)-, -C(O)NR 2 -, -OC(O)-, -OC(O)-O-, -OC(O)-NR 2 、-NR 2 -C(O)-、-NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -SC(O)-, -SC(O)-O-, -SC(O)-NR 2 -、-S(O)-、-S(O) 2 -、-OS(O) 2 -、-OS(O) 2 -O-, -OS(O) 2 -NR 2 -, -OS(O)-, -OS(O)-O-, -OS(O)-NR 2 -、-O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR 2 -、-NR 2 -OC(O)-、-NR 2 -OC(O)-O-、-NR 2 -OC(O)-NR 2 -、-O-NR 2 -C(S)-、-O-NR 2 -C(S)-O-, -O-NR 2 -C(S)-NR 2 -、-NR 2 -OC(S)-、-NR 2 -OC(S)-O-、-NR 2 -OC(S)-NR 2-, -OC(S)-, -OC(S)-O-, -OC(S)-NR 2 -、-NR 2 -C(S)-、-NR 2 -C(S)-O-, -NR 2 -C(S)-NR 2 -、-SS(O) 2 -、-SS(O) 2 -O-, -SS(O) 2 -NR 2 -、-NR 2 -OS(O)-、-NR 2 -OS(O)-O-、-NR 2 -OS(O)-NR 2 -、-NR 2 -OS(O) 2 -、-NR 2 -OS(O) 2 -O-, -NR 2 -OS(O) 2 -NR 2 -、-O-NR 2 -S(O)-, -O-NR 2 -S(O)-O-, -O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O) 2 -O-, -O-NR 2 -S(O) 2 -NR 2 -、-O-NR 2 -S(O) 2 -、-OP(O)(R 2 ) 2 -、-SP(O)(R 2 ) 2 -、-NR 2 -P(O)(R 2 ) 2 -, and combinations of two or more thereof, wherein R 2 are independently selected from hydrogen, C 1 -C 24 Alkyl, C 2 -C 24 Alkenyl, C 2 -C 24 Alkynyl and C 3 -C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0440] In a particularly preferred embodiment of the present invention, Sp 1 、Sp 2 、Sp 3 and Sp 4 - if present - independently selected from linear or branched C 1 -C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C 1 -C 4 alkyl, and wherein Q 1 It is the formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o):

[0441]

[0442] in:

[0443] R 10 is hydrogen or a (thio)ester group; and

[0444] R 18 Selected from optionally substituted C 1 -C 12 Alkyl and C 4 -C 12 (Hetero)aryl.

[0445] As described above, in the method of preparing the bioconjugate, the reactive group Q present in the linker-conjugate 1 With the functional group F present in biological molecules 1 In the methods of the present invention, more than one functional group may be present in the biomolecule. When two or more functional groups are present, the groups may be the same or different. Similarly, more than one reactive group may be present in the linker-conjugate. When two or more reactive groups are present, the groups may be the same or different. In a preferred embodiment of the methods of the present invention, the linker-conjugate comprises one reactive group Q 1, and one or more target molecules D which may be the same or different. The linker-conjugate comprises, for example, 1, 2, 3, 4, 5 or 6, preferably 1, 2, 3 or 4, more preferably 1, 2 or 3, even more preferably 1 or 2 target molecules D. In a particularly preferred embodiment, the linker-conjugate comprises 1 target molecule D. In another particularly preferred embodiment, the linker-conjugate comprises 2 target molecules D, which may be the same or different. In another preferred embodiment, the biomolecule comprises two or more functional groups F, which may be the same or different, and the two or more functional groups react with the complementary reactive groups Q of the linker-conjugate. For example, a biomolecule comprising two functional groups F (i.e., F 1 and F 2 ) can be combined with two functional groups Q 1 The bioconjugate is reacted with a linker-conjugate (which may be the same or different) to form a bioconjugate.

[0446] Functional group F in biomolecules 1 Examples of include amino, thiol, carboxylic acid, alcohol, carbonyl, phosphate or aromatic groups. The functional group in the biomolecule may be naturally occurring or placed in the biomolecule by a specific technique (e.g. (bio)chemical or genetic techniques). The functional group placed in the biomolecule may be a functional group naturally occurring in nature, or may be a functional group prepared by chemical synthesis, such as an azide, a terminal alkyne, a cyclopropene moiety or a phosphine moiety. In view of the preferred mode of conjugation by cycloaddition, F is preferably 1 is a group capable of reacting in a cycloaddition, such as a diene, a dienophile, a 1,3-dipole or a dipolephile, preferably F 1 is selected from a 1,3-dipole (usually an azido group, a nitrone group, an oxynitrile group, an nitrile imine group or a diazo group) or a dipolephile (usually an alkene group or an alkynyl group). 1 When it is a dipole-phile, F 1 It is a 1,3-dipole; when Q 1 When it is a 1,3-dipole, F 1 is a dipole-loving body; or when Q 1 When F is a dienophile, 1 For diene; when Q 1 When it is a diene, F 1 Most preferably, F 1 It is a 1,3-dipole, preferably F 1 is or contains an azido group.

[0447] Figure 2 Several examples of functional groups placed in biomolecules are shown. Figure 2Several structures of derivatives of the UDP sugar of galactosamine are shown, which may be modified, for example, with mercaptopropionyl (11a), azidoacetyl (11b) or azidodifluoroacetyl (11c).

[0448] Figure 3 Schematic representation of how an arbitrary UDP-sugar 11a-c can be attached to a glycoprotein containing a GlcNAc moiety 12 (e.g., a monoclonal antibody in which the glycan is trimmed by an endonucleoside enzyme) under the action of a galactosyltransferase mutant or a GalNAc-transferase, thereby generating a B-glycosidic 1-4 linkage between the GalNAc derivative and GlcNAc (compounds 13a-c, respectively).

[0449] Naturally occurring functional group F 1 Preferred examples of include thiol and amino groups. Preferred examples of functional groups prepared by chemical synthesis for incorporation into biomolecules include keto, terminal alkynyl, azido, cyclo(hetero)alkynyl, cyclopropenyl, or tetrazine.

[0450] As mentioned above, the complementary reactive group Q 1 and functional group F 1 is known to those skilled in the art, and Q 1 and F 1 Several suitable combinations of Fig. 22 The complementary group Q 1 and F 1 A list of is disclosed in Table 3.1 on pages 230-232 of Chapter 3 of GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition. 2013 (ISBN: 978-0-12-382239-0), and the contents of this table are expressly incorporated herein by reference.

[0451] Figure 4 Embodiments of the method of the present invention are described in . Figure 4 It is shown how the modified antibodies 13a-c can be bioconjugated via nucleophilic addition with maleimide (for 13a, generating thioether conjugate 14) or under strain-promoted cycloaddition conditions using cyclooctyne reagents (for 13b or 13c, generating triazoles 15 or 16, respectively).

[0452] The present invention also relates to a bioconjugate obtainable by the process of the present invention for the preparation of a bioconjugate.

[0453] An advantage of the methods of the invention for preparing bioconjugates and the linker-conjugates and sulfonamide linkers of the invention is that the conjugation efficiency is increased when a sulfonamide linker is used instead of a conventional polyethylene glycol (PEG) spacer. For example, as demonstrated in Example 58, a competition experiment involving incubation of trastuzumab containing a single engineered free cysteine ​​with a stoichiometric mixture of maleimide 17 (comprising a comparative linker) and 18 (comprising a sulfonamide linker of the invention) demonstrated that the conjugation efficiency of maleimide 18 containing a sulfonamide was higher than that of 17.

[0454] Figure 5 The structures of linker-conjugates 17 and 18 are shown, where Q 1 is N-maleimido and D is pyrene. Compound 18 is a compound of the present invention, and Compound 17 is a comparative example. Fig.16 The syntheses of 17 and 18 are shown.

[0455] same, Figure 6-9 19-35 with trastuzumab-N 3 The conjugation of sulfonamide-containing linker conjugates showed that the azide-cyclooctyne cycloaddition-based conjugation process was always faster, and in most cases significantly faster, than that of traditional PEG-containing linker conjugates (see Tables 1 and 2 and Figures 12-14).

[0456] The conjugates of 20, 21, 23, 26, 29, 33 and 35 with trastuzumab-N3 are the present invention, and the conjugates of 19, 22, 24, 25, 27, 28, 30, 31, 32 and 34 are comparative examples. In addition, in several instances, conjugation using PEG-based constructs did not achieve complete conversion even after extended incubation times, e.g. Fig.12a , 12b , 13b and 14a.

[0457] Table 1

[0458]

[0459] *Based on remaining starting trastuzumab-N 3 .

[0460] nd = not determined.

[0461] Table 2

[0462]

[0463] Fig.12aThe BCN-pyrene derivative conjugated via a sulfonamide linker (compound 26) or via a short PEG linker (compounds 24 or 25) is shown to be coupled to the trastuzumab antibody-N 3 (Compound 13b) conjugation efficiency.

[0464] Figure 12b The DIBAC-pyrene derivative conjugated via a sulfonamide linker (compound 29) or via a short PEG linker (compounds 27 or 28) is shown to be coupled to trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0465] Fig.13a BCN-maytansine derivatives conjugated via a sulfonamide linker (compound 33) or BCN-maytansine derivatives conjugated via a short PEG linker (compounds 30-32) are shown to be coupled to trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0466] Fig.13b BCN-maytansine derivatives conjugated via a sulfonamide linker (compound 33) or via a short PEG linker (compounds 30-32, where compounds 30 and 33 overlap) are shown with trastuzumab-F 2 -Conjugation efficiency of GalNAz (compound 13c).

[0467] Fig.14a The BCN-duocarmycin derivative conjugated via a sulfonamide linker (compound 35) or via a short PEG linker (compound 34) is shown with trastuzumab-N 3 (Compound 13b) conjugation efficiency.

[0468] Fig.14b BCN-duocarmycin derivatives conjugated via a sulfonamide linker (compound 35) or BCN-duocarmycin derivatives conjugated via a short PEG linker (compound 34) are shown with trastuzumab-F 2 -Conjugation efficiency of GalNAz (compound 13c).

[0469] Another advantage of sulfonamide groups (especially acylsulfonamide or carbamoylsulfonamide groups) is their high polarity, which has a positive effect on the solubility of the linker containing such groups before, during and after conjugation, as well as on the construct as a whole. Fig.11The increased polarity of the sulfonamide spacer can be clearly seen in Table 3 (as shown graphically in Figure 3), and the retention times of compounds 19-23 and 30-38 in RP-HPLC are summarized in Table 3. In view of this increased polarity, conjugation using linker-conjugates containing the sulfonamide linker of the present invention is particularly suitable for conjugating hydrophobic target molecules to biomolecules.

[0470] Fig.11 Shown are the HPLC retention times of compounds 19-23 and 30-38 using 0.1% TFA or in buffer pH 7.4.

[0471] Figure 6 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called BCN group) is attached to benzylamine (D). Compounds 20, 21 and 23 are of the present invention, while compounds 19 and 22 are comparative examples. Fig.17 The synthesis of 19-22 is shown (see also Examples 21-28) and Fig.18 The synthesis of 23 is shown (see also Examples 29-31).

[0472] Figure 7 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called BCN group) or dibenzazepine octyne reactive group Q 1 (also called DIBAC group or DBCO group) attached to pyrene. Compounds 26 and 29 are of the present invention, while compounds 24, 25, 27 and 28 are comparative examples. Fig.19 The synthetic routes to compounds 24-26 are shown, and Fig. 20 Synthetic routes to compounds 27-29 are shown.

[0473] Figure 8 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called BCN group) is attached to maytansine. Compounds 33 and 29 are of the present invention, while compounds 30, 31 and 32 are comparative examples.

[0474] Fig. 9 The structures of several compounds are shown in which the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also called BCN group) is attached to the Val-Cit-PABA-duocarmycin construct. Compound 35 is of the present invention, while compound 34 is a comparative example.

[0475] Fig.10 The structures of compounds 36-38 of the present invention are shown, wherein the bicyclo[6.1.0]non-4-yn-9-yl] reactive group Q 1 (also known as the BCN group) conjugated to Val-Cit-PABA-Ahx-Maytansine.

[0476] The synthesis of compounds 30-35 is described in Examples 43-39, and the synthetic routes of compounds 36-38 are described in Fig.21 This is shown graphically in FIG. 1 and described in Examples 50-55.

[0477] Table 3. Retention times of compounds 19-23 and 30-38 measured by RP-HPLC

[0478]

[0479] The lower values ​​of compounds 20, 21 and 23 relative to compounds 19 and 22 at pH 7.4 (no effect was visible at low pH) clearly reflect the polarity of the sulfonamide spacer compared to the standard amide linker construct (compound 19), as more polar compounds show reduced retention times. Methylated compound 22, although also a sulfonamide in the strict sense, does not show enhanced polarity due to the lack of an acid proton on the acylated nitrogen. The fact that no significant difference in retention times was observed using 0.1% TFA (no deprotonation occurs at this pH) also suggests that the acid proton has a key role in defining polarity. The bissulfonamide compound 22 finally shows that additional sulfonamide units in a single linker (i.e., when f is 2 or greater in the compounds and methods of the present invention) further enhance polarity. The latter observation also facilitates the smooth conjugation of BCN-constructs with two lipophilic toxins (maytansine) to azido-mAbs, as in bissulfonamide compounds 38 and 39.

[0480] The high polarity of sulfonamides also has a positive impact in the context of imparting a hydrophobic moiety to a target molecule, which is known to require large amounts of organic co-solvents during conjugation and / or reduce aggregation of bioconjugates. High levels of co-solvents (up to 50% DMA, polypropylene glycol, or DMSO) can induce protein denaturation during conjugation and / or may require special equipment during manufacturing. Examples of stability of bioconjugates include highly polar bis-sulfonamide-containing compound 38, which once conjugated to trastuzumab showed zero to negligible aggregation upon long-term storage, which is actually also applicable to mono-sulfonamide 37.

[0481] In view of this reduced tendency to aggregate, the sulfonamide linkers of the present invention are particularly advantageous for use in preparing bioconjugates in which the reaction product of the conjugation reaction (i.e., the reactive group Q 1 and F 1 The reaction of ( ) provides a linking moiety that is slightly soluble in water. In a particularly preferred embodiment, the conjugation is accomplished by cycloaddition, preferably 1,3-dipolar cycloaddition, more preferably alkyne-azide cycloaddition. As shown in the examples, the use of the sulfonamide linker of the present invention advantageously reduces any aggregation herein, which greatly improves the stability of the product by reducing the tendency to aggregate. Thus, in the formation of the bioconjugate, by the target molecule and the reactive group Q in the linker-conjugate 1 The use of the sulfonamide linker of the present invention in the spacer between the two effectively solves the aggregation problem associated with the hydrophobic linking part in the bioconjugate.

[0482] Another advantage of the sulfonamide linker of the present invention and its use in bioconjugation processes is that it is easy to synthesize and has high yields. Fig.15 Schematically shown, the synthesis of the carbamoylsulfonamide spacer involves the reaction of the primary alcohol 40 with the commercially available reagent CSI (chlorosulfonyl isocyanate) to give the intermediate sulfonyl chloride 41, which reacts with an amine without workup to give the stable carbamoylsulfonamide 42. 1 When R is tert-butyl, the latter compound can be easily converted to the acylsulfonamide, which, after acid treatment, affords the primary sulfonamide product 43. 1 =benzyl, deprotection can be performed using hydrogenation. The primary sulfonamide 43 can be conveniently acylated with an activated ester to give the acyl sulfonamide 44 using conventional procedures.

[0483] The ease of synthesis of the sulfonamide linkers of the present invention, as well as their excellent performance in bioconjugation processes, are also clearly seen in the synthesis and use of compounds 23 and 38, both of which are bissulfonamide constructs (compounds 23 and 38) that are easily generated by repeating the events summarized above, i.e., treatment of an alcohol with CSI followed by reaction with an amino alcohol to generate an alcohol that can again undergo the sequence of events described to give the bissulfonamide. Further repetition of the sequence generates tri-, tetra-, and higher sulfonamide oligomers, depending on the number of repeats.

[0484] In particular, long PEG spacers (e.g., PEG 24 The fact that the sulfonamide linker in the final conjugate is short may have an additional advantage in the sense that ) may have a negative impact on the pharmacokinetics of the final protein conjugate, as is known for antibody-drug conjugates, especially those with high drug load.

[0485] Example

[0486] Example 1. Synthesis of α-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate

[0487] α-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate was prepared from D-galactosamine according to the method described for D-glucosamine in Linhardt et al., J. Org. Chem. 2012, 77, 1449-1456 (incorporated by reference).

[0488] 1 H-NMR (300 MHz, CD 3 OD): δ (ppm) 5.69 (dd, J = 7.2, 3.3 Hz, 1H), 5.43-5.42 (m, 1H), 5.35 (dd, J = 11.1, 3.3 Hz, 1H), 4.53 (t, J = 7.2 Hz, 1H), 4.21-4.13 (m, 1H), 4.07-4.00 (m, 1H), 3.82 (dt, J = 10.8, 2.7 Hz, 1H), 2.12 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H). LRMS (ESI-) m / z calculated value C 12 H 17 N 3 O 11 P(MH + )=410.06; Actual value 410.00.

[0489] Example 2. Synthesis of α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose

[0490] α-2-Azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose 1-phosphate prepared as in Example 1 was linked to UMP according to Baisch et al. Bioorg. Med. Chem., 1997, 5, 383-391.

[0491] Therefore, with DOWEX 50Wx8 (H + form) treated and dissolved in H 2 O (15mL) of D-uridine-5'-monophosphate disodium salt (1.49g, 4.05mmol) was added dropwise to the filtrate while vigorously stirring at room temperature. After stirring for another 30 minutes, the reaction mixture was lyophilized and filtered under vacuum. 2 O5 Dry again for 5h.

[0492] In an argon atmosphere, the resulting tributylammonium uridine-5′-monophosphate was dissolved in dry DMF (25 mL). Carbonyl diimidazole (1.38 g, 8.51 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. Then, dry MeOH (180 μL) was added and stirred for 15 min to remove excess carbonyl diimidazole. The remaining MeOH was removed under high vacuum for 15 min. The resulting compound (2.0 g, 4.86 mmol) was dissolved in dry DMF (25 mL) and added dropwise to the reaction mixture. The reactants were stirred at room temperature for 2 days before vacuum concentration. The consumption of the imidazole-UMP intermediate was monitored by MS. Gradient flash column chromatography (7:2:1→5:2:1 EtOAc:MeOH:H 2 O) to give α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose (1.08 g, 1.51 mmol, 37%).

[0493] 1 H-NMR (300 MHz, D 2 (O): δ (ppm) 7.96 (d, J = 8.0 Hz, 1H), 5.98-5.94 (m, 2H), 5.81-5.79 (m, 1H), 5.70 (dd, J = 7.1, 3.3 Hz, 1H), 5.49 (dd, J = 15.2, 2.6 Hz, 1H), 5.30 (ddd, J = 18.5, 11.0, 3.2 Hz, 2H), 4.57 (q, J = 6.0 Hz, 2H), 4.35-4.16 (m, 9H), 4.07-3.95 (m, 2H), 2.17 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H). LRMS (ESI-) m / z calculated value C 21 H 29 N 5 O 19 P 2 (MH + )=716.09; measured value 716.3.

[0494] Example 3: Synthesis of α-UDP-2-azido-2-deoxy-D-galactose

[0495] According to Kiso et al., Glycoconj. J., 2006, 23, 565, α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose prepared as in Example 2 was deacetylated.

[0496] Therefore, α-UDP-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose (222 mg, 0.309 mmol) was dissolved in H 2 O (2.5 mL), add Et 3 N (2.5 mL) and MeOH (6 mL). The reaction mixture was stirred for 3 h and then concentrated in vacuo to give crude α-UDP-2-azido-2-deoxy-D-galactose. 1 H-NMR (300 MHz, D 2 O): δ (ppm) 7.99 (d, J = 8.2 Hz, 1H), 6.02-5.98 (m, 2H), 5.73 (dd, J = 7.4, 3.4 Hz, 1H), 4.42-4.37 (m, 2H), 4.30-4.18 (m, 4H), 4.14-4.04 (m, 2H), 3.80-3.70 (m, 2H), 3.65-3.58 (m, 1H). LRMS (ESI-) m / z calculated value C 15 H 23 N 5 O 16 P 2 (MH + )=590.05; measured value 590.20.

[0497] Example 4. α-UDP-D-galactosamine (UDP-GalNH 2 )

[0498] Dissolve in 1:1H 2 Lindlar catalyst (50 mg) was added to a solution of α-UDP-2-azido-2-deoxy-D-galactose prepared in Example 3 in a mixture of O-MeOH (4 mL). The reaction was stirred for 5 h under a hydrogen atmosphere and then filtered through celite. 2 O (10 mL) and the filtrate was concentrated under vacuum to obtain α-UDP-D-galactosamine (UDP-GalNH 2 ) (169 mg, 0.286 mmol, 92% yield for two steps). 1 H-NMR (300 MHz, D 2 O): δ (ppm) 7.93 (d, J = 8.1 Hz, 1H), 5.99-5.90 (m, 2H), 5.76-5.69 (m, 1H), 4.39-4.34 (m, 2H), 4.31-4.17 (m, 5H), 4.05-4.01 (m, 1H), 3.94-3.86 (m, 1H), 3.82-3.70 (m, 3H), 3.30-3.16 (m, 1H). LRMS (ESI-) m / z calculated value C15 H 25 N 3 O 16 P 2 (MH + )=564.06; measured value 564.10.

[0499] Example 5. Synthesis of UDP-GalNProSAc

[0500] UDP-α-D-galactosamine (44 mg, 0.078 mmol) was dissolved in 0.1 M NaHCO 3 (1 mL). 3-AcS-propionic acid succinimidyl ester (38 mg, 0.156 mmol) and DMF (1 mL) were added, and the reaction was stirred at room temperature overnight and then concentrated under reduced pressure. Gradient flash column chromatography (7:2:1→5:2:1 EtOAc:MeOH:H 2 O) UDP-GalNProSAc (6 mg, 0.009 mmol, 11%) + UDP-GalNAc impurity was obtained.

[0501] LRMS (ESI-) calcd. 20 H 31 N 3 O 18 P 2 S(M-)694.08 Measured value 694.1.

[0502] 1 H-NMR (300 MHz, D 2 O): δ (ppm) 7.79 (m, 1H), 5.83-5.80 (m, 2H), 5.48-5.45 (m, 1H), 4.28-4.05 (m, 6H), 3.8 8-3.85 (m, 2H), 3.63-3.55 (m, 3H), 3.17-3.16 (m, 2H), 2.60-2.55 (m, 2H) 2.50 (s, 3H).

[0503] Example 6. Synthesis of UDP-GalNProSH (11a)

[0504] UDP-GalNProSAc (3 mg, 0.005 mmol) was dissolved in degassed 1 M NaOH (1 mL), stirred for 1.5 h, and then concentrated under vacuum. This product was used crude in glycosylation experiments.

[0505] 1 H-NMR (400 MHz, D 2O): δ (ppm) 7.86 (d, J=8.0Hz, 1H), 5.88-5.86 (m, 2H), 5.48-5.45 (m, 1H), 4.20-4.05 ( m, 8H), 3.95-3.85 (m, 1H), 3.68-3.65 (m, 2H), 2.69-2.67 (m, 2H), 2.60-2.55 (m, 2H).

[0506] Example 7. Synthesis of ethyl 2-azido-2,2-difluoroacetate

[0507] To a solution of ethyl 2-bromo-2,2-difluoroacetate (950 mg, 4.68 mmol) dissolved in dry DMSO (5 mL) was added sodium azide (365 mg, 5.62 mmol). After stirring overnight at room temperature, the reaction mixture was poured into water (150 mL). The layers were separated, dichloromethane was added to the organic layer, and the layer was washed with Na 2 SO 4 After filtration, the solvent was removed under reduced pressure (300 mbar) at 35°C to give crude ethyl 2-azido-2,2-difluoroacetate (250 mg, 1.51 mmol, 32%).

[0508] 1 H-NMR (300 MHz, CDCl 3 ): δ (ppm) 4.41 (q, J=7.2hz, 2H), 1.38 (t, J=6.9Hz, 3H).

[0509] Example 8. Synthesis of α-2-amino-3,4,6-tri-O-acetyl-D-galactose 1-phosphate

[0510] To a solution of α-2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-galactose-1-phosphate (105 mg, 0.255 mmol) prepared as in Example 1 in MeOH (3 mL) was added Pd / C (20 mg). The reaction was stirred under a hydrogen atmosphere for 2 h and filtered through celite. The filter was rinsed with MeOH (10 mL) and the filtrate was dried under vacuum to give the free amine (94 mg, 0.244 mmol, 96%).

[0511] 1 H-NMR (300 MHz, D 2O): δ (ppm) 5.87-5.76 (m, 1H), 5.44 (br. s, 1H), 5.30-5.20 (m, 1H), 4.55 (t, J = 6.3 Hz, 1H), 4.28-4.00 (m, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H). LRMS (ESI-) m / z calculated value C 12 H 19 NO 11 P(MH + )=384.07; Actual value 384.10.

[0512] Example 9. Synthesis of α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose 1-phosphate

[0513] To a solution of α-2-amino-3,4,6-tri-O-acetyl-D-galactose 1-phosphate (94 mg, 0.244) prepared as in Example 8 in dry DMF (3 mL) was added ethyl 2-azido-2,2-difluoroacetate (48 mg, 0.293 mmol) and Et 3 N (68 μL, 0.488 mmol). The reaction was stirred for 6 h and then concentrated under vacuum to give the crude product. Gradient flash column chromatography (100:0→50:50 EtOAc:MeOH) gave α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose 1-phosphate (63 mg, 0.125 mmol, 51%).

[0514] Example 10. Synthesis of UDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose

[0515] α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-glucose 1-phosphate prepared as in Example 9 was coupled to UMP according to Baisch et al. Bioorg. Med. Chem., 1997, 5, 383-391 (incorporated by reference).

[0516] Therefore, with DOWEX 50Wx8 (H + form) treated and dissolved in H 2O (1mL) of D-uridine-5'-monophosphate disodium salt (98mg, 0.266mmol) was added dropwise to the filtrate while vigorously stirring at room temperature. After stirring for another 30 minutes, the reaction mixture was lyophilized and concentrated under vacuum with P 2 O 5 Dry again for 5h. In an argon atmosphere, the resulting tributylammonium uridine-5′-monophosphate was dissolved in dry DMF (15mL). Carbonyldiimidazole (35mg, 0.219mmol) was added and the reaction mixture was stirred at room temperature for 30min. Then, dry MeOH (4.63μL) was added and stirred for 15min to remove excess carbonyldiimidazole. The remaining MeOH was removed under high vacuum (15min). Subsequently, N-methylimidazole.HCl (61mg, 0.52mmol) was added to the reaction mixture, and the resulting compound (63mg, 0.125mmol) was dissolved in dry DMF (15mL) and added dropwise to the reaction mixture. The reaction was stirred at room temperature overnight before the mixture was concentrated under reduced pressure. The consumption of imidazole-UMP intermediates was monitored by MS analysis. Gradient flash column chromatography (7:2:1→5:2:1 EtOAc:MeOH:H 2 O) to give UDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose.

[0517] 1 H-NMR (300 MHz, D 2 O): δ (ppm) 7.87 (d, J=8.1Hz, 1H), 5.913-5.85 (m, 2H), 5.67 (dd, J=6.6, 2.7Hz, 1H), 5.56-5.50 (m, 1H), 5.47-5 .43(m, 1H), 5.31-5.25(m, 2H), 4.61-4.43(m, 2H), 4.31-4.05(m, 5H), 2.16(s, 3H), 2.02(s, 3H), 1.94(s, 3H).

[0518] LRMS (ESI-) m / z calculated for C 23 H 29 F 2 N 6 O 20 P 2 (MH + )=809.09; measured value 809.1.

[0519] Example 11. Synthesis of α-UDP-2-(2′-azido-2′, 2′-difluoroacetamido)-2-deoxy-D-galactose (UDP-F2-GalNAz, 11c)

[0520] Deacetylation of UDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose was performed according to Kiso et al., Glycoconj. J., 2006, 23, 565, incorporated by reference.

[0521] Therefore, UDP-α-(2′-azido-2′,2′-difluoroacetamido)-3,4,6-tri-O-acetyl-D-galactose prepared as in Example 10 was dissolved in H 2 To a 5% ethanol solution (1 mL) was added triethylamine (1 mL) and MeOH (2.4 mL). The reaction mixture was stirred for 2 h and then concentrated in vacuo. Gradient flash column chromatography (7:2:1→5:2:1 EtOAc:MeOH:H 2 O) to give α-UDP-2-(2′-azido-2′,2′-difluoroacetamido)-2-deoxy-D-galactose (11c).

[0522] 1 H-NMR (300 MHz, D 2 O): δ (ppm) 7.86 (d, J = 8.1 Hz, 1H), 5.91-5.85 (m, 2H), 5.54 (dd, J = 6.6, 3.6 Hz, 1H), 4.31-3.95 (m, 9H), 3.74-3.62 (m, 2H). LRMS (ESI-) m / z calculated value C 17 H 23 F 2 N 6 O 17 P 2 (MH + )=683.06; measured value 683.10.

[0523] Trimming of trastuzumab with endoS to produce 12

[0524] Mass spectrometry analysis of monoclonal antibodies

[0525] Will be at 37 ℃, by total volume about 70 μ L of 50 μ g (modified) IgG, 1M Tris-HCl pH 8.0, 1mM EDTA and 30mM DTT solution incubated for 20 minutes, to reduce disulfide bridges, thereby allow analysis of light chain and heavy chain. If present, the azide functional group is also reduced to amine under these conditions. The reduced sample is washed 3 times with milliQ using Amicon Ultra-0.5, Ultracel-10 membrane (Millipore), and then concentrated to 10 μ M (modified) IgG. By electrospray ionization time of flight (ESI-TOF) on JEOL AccuTOF, reduced IgG is analyzed. Deconvoluted spectra are obtained using Magtran software.

[0526] Example 12. Preparation of trimmed trastuzumab 12

[0527] Glycan trimming of trastuzumab was performed using endoS from Streptococcus pyogenes (commercially available from Genovis, Lund, Sweden). Therefore, trastuzumab (10 mg / mL) was incubated with endoS (40 U / mL) in 25 mM Tris pH 8.0 at 37°C for approximately 16 hours. Deglycosylated IgG was concentrated and lysed using Amicon Ultra-0.5, Ultracel-10 membranes (Millipore) with 10 mM MnCl 2 and washed with 25 mM Tris-HCl pH 8.0.

[0528] The sample was subjected to MS analysis, and after deconvolution of the peaks, the mass spectrum showed one light chain peak and two heavy chain peaks. The two peaks of the heavy chain belonged to a major product (49496Da, 90% of the total heavy chain) (generated by core GlcNAc (Fuc) substituted trastuzumab), and a minor product (49351Da, ± 10% of the total heavy chain) (generated by deglycosylated trastuzumab).

[0529] Transient expression of trastuzumab mutants and carbonyltransferases in CHO

[0530] Proteins (enzymes and trastuzumab mutants) were transiently expressed in CHO K1 cells using Evitria (Zurich, Switzerland) in a 20-25 mL format.

[0531] GalT double mutant identified by SEQ ID NO: 1 (Y289L, C342T)

[0532] RDLRRLPQLVGVHPPLQGSSHGAAAIGQPSGELRLRGVAPPPPLQNSSKPRSRAPSNLDAYSHPGPGPGPGSNLTSAPVPSTTTRSLTACPEESPLLVGPMLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDKFGFSLPYVQLFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKTRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS

[0533] CeGalNAcT[30-383] identified by SEQ ID NO: 2

[0534] KIPSLYENLTIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKTNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYKMIKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCF

[0535] CeGalNAcT(30-383)-His identified by SEQ ID NO: 3 6

[0536] KIPSLYENITIGSSTLIADVDAMEAVLGNTASTSDDLLDTWNSTFSPISEVNQTSFMEDIRPILFPDNQTLQFCNQTPPHLVGPIRVFLDEPDFKTLEKIYPDTHAGGHGMPKDCVARHRVAIIVPYRDREAHLRIMLHNLHSLLAKQQLDYAIFIVEQVANQTFNRGKLMNVGYDVASRLYPWQCFIFHDVDLLPEDDRNLYTCPIQPRHMSVAIDKFNYKLPYSAIFGGISALTKDHLKKINGFSNDFWGWGGEDDDLATRTSMAGLKVSRYPTQIARYKMIKHSTEATNPVNKCRYKIMGQTKRRWTRDGLSNLKYKLVNLELKPLYTRAVVDLLEKDCRRELRRDFPTCFHHHHHH

[0537] Trastuzumab (heavy chain N300C) identified by SEQ ID NO: 4

[0538] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0539] Trastuzumab (light chain) identified by SEQ ID NO: 5

[0540] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0541] Example 13-1. Purification of CeGalNAcT

[0542] The purification scheme was based on cation exchange and subsequent size exclusion chromatography on a SP column (GE Healthcare).

[0543] In conventional purification experiments, the supernatant containing the expressed CeGalNAcT produced CHO was dialyzed with 20mM Tris buffer, pH 7.5. The supernatant (usually 25mL) was filtered through a 0.45μM-pore diameter filter and then purified through a cation exchange column (SP column, 5mL, GE Healthcare), which was balanced with 20mM Tris buffer, pH7.5 before use. Purification was performed on an AKTA Prime column chromatography system equipped with an external fraction collector. The sample was loaded from system pump A. Unbound protein was eluted from the column by washing the column with 10 column volumes (CV) of 20mM Tris buffer, pH 7.5. The retained protein was eluted with elution buffer (20mM Tris, 1NaCl, pH7.5; 10mL). The collected fractions were analyzed by SDS-PAGE on polyacrylamide gel (12%), and the fractions containing the target protein were combined and concentrated to a volume of 0.5 mL using spin filtration. The protein was then purified on a preparative Superdex size exclusion column on an AKTA purification system (UNICORN v6.3). This purification step identified and separated the dimer fraction and the monomer fraction of the target protein. The two fractions were analyzed by SDS-PAGE and stored at -80°C before further use.

[0544] Example 13-2. CeGalNAcT-His 6 Purification

[0545] In conventional purification experiments, CHO supernatant was filtered through a 0.45 μM-pore diameter filter and applied to a Ni-NTA column (GE Healthcare, 5 mL), which was balanced with buffer A (20 mM Tris buffer, 20 mM imidazole, 500 mM NaCl, pH 7.5) before use. Before filtration, imidazole was added to the CHO supernatant to a final concentration of 20 mM to minimize nonspecific binding to the column. The column was first washed with buffer A (50 mL). The retained protein was eluted with buffer B (20 mMTris, 500 mM NaCl, 250 mM imidazole, pH 7.5, 10 mL). The fractions were analyzed on polyacrylamide gel (12%) by SDS-PAGE, and the fractions containing the purified target protein were combined and exchanged with 20 mM Tris (pH 7.5) by dialysis performed overnight at 4 ° C. The purified protein was stored at -80 °C before further use. Note: For both monomeric and dimeric CeGalNAcT-His 6 For identification of the species, additional SEC purification was performed (as described above).

[0546] 11a-c are transferred to 12 to prepare 13a-c

[0547] Example 14. Trastuzumab (GalNProSH) 2 Preparation of 13a

[0548] Deglycosylated trastuzumab (10 mg / mL) was mixed with UDP-galactose derivative 11a (1.3 mM) and β(1,4)-Gal-T1 (Y289L, C342T) (2 mg / mL) in 10 mM MnCl at 30 °C. 2 and 50 mM Tris-HCl pH 6.0 for 16 h.

[0549] Subsequently, functionalized trastuzumab was incubated with protein A agarose (40 μL / mg IgG) at room temperature for 1 hour. Protein A agarose was washed 3 times with TBS (pH 6.0) and IgG was eluted with 100 mM glycine-HCl pH 2.5. The eluted IgG was neutralized with 1 M Tris-HCl pH 7.0, concentrated, and washed with 50 mM Tris-HCl pH 6.0 to a concentration of 15-20 mg / mL using Amicon Ultra-0.5, Ultracel-10 membrane (Millipore). Spectral analysis after digestion with Fabricator and subsequent washing with MiliQ using Amicon Ultra-0.5, Ultracel-10 membrane (Millipore) showed that two products were formed, a major product (24387 Da) containing introduced GalNProSH and a minor product (25037 Da) containing introduced GalNProSH+UDPGalNProSH as a disulfide. The ratio of the products was about 60:40.

[0550] Transglycosylation of UDP-galactose derivatives using Gal-T1 (Y289L, C342T), general protocol

[0551] Enzymatic introduction of UDP-galactose derivatives to deglycosylated trastuzumab was performed using a double mutant of bovine β(1,4)-galactosyltransferase [β(1,4)-Gal-T1(Y289L, C342T)]. Deglycosylated trastuzumab (10 mg / mL) was incubated with appropriate UDP-galactose derivatives (0.4 mM) and Gal-T double mutant (1 mg / mL) at 30°C in 10 mM MnCl 2 and incubated in 25 mM Tris-HCl pH 8.0 for 16 h.

[0552] Subsequently, the functionalized trastuzumab was incubated with protein A agarose (40 μL / mg IgG) at 4°C for 2 hours. The protein A agarose was washed 3 times with PBS and IgG was eluted with 100 mM glycine-HCl pH 2.7. The eluted IgG was neutralized with 1 M Tris-HCl pH 8.0, concentrated, and washed with PBS to a concentration of 15-20 mg / mL using Amicon Ultra-0.5, Ultracel-10 membrane (Millipore).

[0553] Transglycosylation of UDP-GalNAc derivatives using CeGalNAcT (general protocol)

[0554] Enzymatic introduction of GalNAc derivatives to IgG was performed using CeGalNAc-transferase. Deglycosylated IgG (prepared as described above, 10 mg / mL) was mixed with a modified UDP-GalNAc derivative (e.g., an azide-modified sugar-UDP derivative) (0.4 mM) and CeGalNAc-T (1 mg / mL) at 30°C in 10 mM MnCl 2 The IgG was incubated in 25 mM Tris-HCl pH 8.0 for 16 hours. Functionalized IgG (e.g., azido-functionalized IgG) was incubated with protein A agarose (40 μL / mg IgG) for 2 hours at 4°C. The protein A agarose was washed 3 times with PBS and the IgG was eluted with 100 mM glycine-HCl pH 2.7. The eluted IgG was neutralized with 1 M Tris-HCl pH 8.0, concentrated, and washed with PBS to a concentration of 15-20 mg / mL using an Amicon Ultra-0.5, Ultracel-10 membrane (Millipore).

[0555] Example 15. Preparation of trastuzumab (GalNAz) using GalT double mutant 2 13b

[0556] Glycosylation transfer protocol for trastuzumab using UDP-N-azidoacetylgalactosamine (UDP-GalNAz) and Gal-T double mutants. After protein A affinity purification, mass analysis showed the formation of a major product (49713 Da, 90% of the total heavy chain), which resulted from the transfer of GalNAz to core GlcNAc (Fuc) substituted trastuzumab, and a minor product (49566 Da, ±10% of the total heavy chain), which resulted from the transfer of GalNAz to core GlcNAc substituted trastuzumab.

[0557] This is an example of an azide-modified glycoprotein of formula (13b).

[0558] Example 16-1. Preparation of trastuzumab (GalNAz) using CeGalNAc-T 2 13b

[0559] Glycosylation transfer protocol was performed on trimmed trastuzumab using UDP-N-azidoacetylgalactosamine (UDP-GalNAz) and CeGalNAc-T. After protein A affinity purification, a small amount of sample was reduced with DTT and then subjected to MS analysis, indicating the formation of a major product (49713 Da, 90% of the total heavy chain) resulting from GalNAz transfer to core GlcNAc (Fuc) substituted trastuzumab, and a minor product (49566 Da, ±10% of the total heavy chain) resulting from GalNAz transfer to core GlcNAc substituted trastuzumab.

[0560] Example 16-2. Preparation of trastuzumab (F) using CeGalNAc-T 2 -GalNAz) 2 13c

[0561] UDP-N-azidodifluoroacetylgalactosamine (UDP-F 2 -GalNAz, 13c) and CeGalNAc-T or CeGalNAcT-His 6 Glycosylation transfer protocol was performed on trimmed trastuzumab. After protein A affinity purification, a small amount of sample was reduced with DTT and then subjected to MS analysis, which showed the formation of a major heavy chain product (49865 Da, approximately 90% of the total heavy chain), which consisted of F 2 -GalNAz was transferred to core GlcNAc(Fuc)-substituted trastuzumab that had been reacted with DTT during sample preparation.

[0562] This is an example of an azide-modified glycoprotein of formula (13c).

[0563] Synthesis of 17-68

[0564] Example 17. Synthesis of 1-pyrenecarboxylic acid OSu ester (45)

[0565] To a solution of 1-pyrenecarboxylic acid (65 mg, 0.24 mmol) in DCM / DMF (2 mL each) was added N-hydroxysuccinimide (34 mg, 0.29 mmol) and EDC.HCl (70 mg, 0.36 mmol). The reaction was stirred for 2 h, then diluted with DCM (10 mL) and washed with aqueous citric acid (10%, 5 mL) and saturated NaHCO 3 (3x5mL), washed with Na 2 SO 4 Dry, filter and concentrate in vacuo to afford crude 45.

[0566] Example 18. Synthesis of (46)

[0567] Compound 45 (480 mg, 1.38 mmol) was dissolved in DCM (15 mL), and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamoyl ester (348 mg, 1.4 mmol) and Et 3 N (286 μL, 2.1 mmol). The reaction mixture was stirred overnight and quenched with water (15 mL). The organic layer was washed with water (1×15 mL) and saturated NaHCO 3 The solution was washed with Na 2 SO 4 Dry, filter and concentrate in vacuo. Purification by gradient flash column chromatography (DCM→DCM:MeOH 95:5) gave the product 46 (460 mg, 0.97 mmol, 70%).

[0568] Example 19. Synthesis of (17)

[0569] The Boc-protected pyreneamine 46 (460 mg, 0.97 mmol) was dissolved in methanol (10 mL) and acetyl chloride (140 μL, 1.9 mmol) was added. After 1 h and 3 h, additional acetyl chloride (2x140 μL, 1.9 mmol) was added. After stirring for 4 h, the mixture was concentrated under reduced pressure. Subsequently, the crude product (100 mg, 0.24 mmol) was dissolved in DCM (3 mL) and 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyric acid 2,5-dioxopyrrolidin-1-yl ester (50 mg, 0.17 mmol) and Et 3 N (73 μL, 0.52 mmol). After stirring overnight, the solution was quenched with water (3 mL), washed with water (2 x 3 mL), and washed with Na 2 SO 4 Dry, filter and concentrate. Purification by gradient flash column chromatography (DCM→DCM:MeOH 95:5) gave product 17 (69 mg, 0.13 mmol, 75%). 1 H NMR (400 MHz, CDCl 3)δ (ppm) 8.48 (d, 1H, J = 9.2Hz), 8.12 (d, 2H, J = 7.6Hz), 8.04-7.93 (m, 6H), 6.59 (bs, 1H), 6.38 (s, 2H), 5.99 (bs, 1H), 3.76-3.71 (m, 4H), 3.62-3.60 (m, 2H), 3.54-3.52 (m, 2H), 3.37 (t, J=5.2Hz, 2H), 3.23-3.17 (m, 4H), 1.82 (t, J=6.8Hz, 2H), 1.63 (q, J=7.2Hz, 2H).

[0570] Example 20-1. Synthesis of maleimide alcohol derivative (47)

[0571] Dissolve in saturated NaHCO 3 To a cold (0°C) solution of 5-aminopentan-1-ol (100 mg, 0.97 μmol) in aqueous solution (16 mL) was added N-methoxycarbonylmaleimide (150 mg, 0.64 μmol). The mixture was stirred for 1.5 h and extracted with DCM (2×20 mL). The combined organic layers were dried (Na 2 O 4 3 O 4 ... 2 SO 4 ) and concentrated to give the product 47 (137 mg, 0.75 mmol, 75%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 6.69 (s, 2H), 3.70-3.67 (m, 1H), 3.67-3.60 (m, 2H), 3.53 (t, J=7.1Hz, 2H), 1.68-1.54 (m, 4H), 1-42-1.31 (m, 2H).

[0572] Example 20-2. Synthesis of maleimide-pyrene derivative (18)

[0573] To a solution of alcohol 47 (7.7 mg, 42 μmol) in DCM (1 mL) was added chlorosulfonyl isocyanate (CSI, 3.9 μL, 5.9 mg, 42 μmol). After the solution was stirred for 15 min, Et 3 N (18 μL, 13 mg, 126 μmol) and 1-pyrenemethylamine.HCl (49, 11 mg, 42 μmol) and Et 3 N (18 μL, 13 mg, 126 μmol) solution. After 80 min, saturated NH 4 Cl aqueous solution (20 mL) and DCM (20 mL). After separation, the organic phase was dried (Na 2 SO 4) and concentrated. After gradient flash column chromatography (DCM→2% MeOH in DCM), the product 18 (7.4 mg, 14.2 μmol, 34%) was obtained as a light yellow solid. 1 HNMR (400 MHz, CDCl 3 / CD 3 OD) δ (ppm) 8.32-8.26 (m, 1H), 8.20-7.90 (m, 8H), 6.61 (s, 2H), 4.90 (s, 2H), 3.6 5 (t, J=6.5Hz, 2H), 3.30 (t, J=7.2Hz, 2H). 1.40-1.20 (m, 4H), 1.08-0.97 (m, 2H).

[0574] Example 21. Synthesis of BCN-heptanoic acid (52)

[0575] To a solution of BCN-OSu derivative 51 in MeCN (5 mL) was added 0.1 M NaHCO 3 7-Aminoheptanoic acid 50 (145 mg, 1.0 mmol) in aqueous solution (30 mL) and MeCN (25 mL) was stirred for 4 h and partially concentrated. Saturated NH 4 Cl aqueous solution (30 mL) and after extraction with DCM (2 x 30 mL), the combined organics were dried (Na 2 SO 4 The product 52 was used directly in the step without further purification. 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 4.68 (bs, 1H), 4.14 (d, J = 7.9Hz, 2H), 3.17 (dd, J = 12.8, 6.3Hz, 2H), 2.35 (t, J=7.5Hz, 2H), 2.32-2.09 (m, 6H), 1.70-1.25 (m, 11H), 0.94 (t, J=9.7Hz, 2H).

[0576] Example 22. Synthesis of BCN-heptanoic acid benzylamide (19)

[0577] To a mixture of 51 (291 mg, 1.00 mmol) dissolved in DCM (25 mL) was added 7-aminoheptanoic acid 50 and Et 3 N (417 μl, 303 mg, 3.00 mmol) and DMF (10 mL). After DCM was evaporated (40 °C), the resulting mixture was stirred for 10 min and NaHCO was added 3 After the reaction mixture was stirred for another 3 h, it was poured into saturated NH4 Cl aqueous solution (50mL) and extracted with DCM (2×50mL). The combined organic phases were dried and concentrated. The residue was dissolved in DCM (25mL), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI.HCl, 249mg, 1.30mmol) and N-hydroxysuccinimide (150, 1.30mmol) were added, and the resulting mixture was stirred for 18h. After adding water (50mL), the layers were separated and the aqueous phase was extracted with DCM (2x25mL). The combined organic phases were washed with brine (50mL), dried (Na 2 SO 4 ) and concentrated. Column chromatography afforded the intermediate NHS ester derivative of 52 (233 mg, 0.56 mmol, 56%) as a colorless thick oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)4.69(s,1H),4.14(d,J=8.0Hz,2H),3.17(dd,J=13.6,6.9Hz,2H),2.91-2.77(m,4H),2.61(t,J=7.4Hz,2H),2.37-2.12(m,6H),1.83-1.69(m,2H),1.66-1.17(m,9H),1.01-0.90(m,2H). Subsequently, to a solution of the intermediate BCN-aminoheptanoic acid NHS ester (49 mg, 0.12 mmol) dissolved in DCM (12 mL), benzylamine (19 μL, 19 mg, 0.18 mmol) and Et 3 N (50 μL, 36 mg, 0.36 mmol). The mixture was stirred for 19 h, then DCM (10 mL) and saturated NH 4 Cl aqueous solution (20 mL). Dry (Na 2 SO 4 ) and the organic layer was concentrated. After gradient column chromatography (25%→50% EtOAc in heptane), compound 19 (31 mg, 0.076 mmol, 63%) was obtained as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 7.38-7.27 (m, 5H), 5.72 (bs, 1H), 4.64 (bs, 1H), 4.45 (d, J = 5.6Hz, 2H), 4.13 (d, J = 8.1H z, 2H), 3.16 (dd, J=12.8, 6.3Hz, 2H), 2.38-2.13 (m, 8H), 1.75-1.11 (m, 11H), 1.00-0.88 (m, 2H).

[0578] Example 23. Synthesis of tert-butyl N-benzylsulfamoylcarbamate (53)

[0579] In N 2 atmosphere, dissolved in Et 2 To a cooled solution (-78°C) of tert-butyl alcohol in DCM (20 mL), chlorosulfonyl isocyanate (CSI) was added and the mixture was allowed to reach room temperature. After 45 min, the mixture was concentrated and the resulting tert-butyl chlorosulfonylcarbamate was used in the next step without further purification (considered to be 68% pure). Therefore, to a solution of crude tert-butyl chlorosulfonylcarbamate (199 mg crude = 135 mg, 0.63 mmol) in DCM (10 mL), Et 3 N (263 μL, 191 mg, 1.89 mmol) and benzylamine (82 μL, 81 mg, 0.85 mmol). The mixture was stirred for 2 h, and saturated NH 4 Cl. DCM aqueous solution (10 mL) was used to quench and the layers were separated. The organic layer was dried (Na 2 SO 4 ) and concentrated. After gradient column chromatography (25%→50% EtOAc in heptane), Compound 53 (169 mg, 0.59 mmol) was obtained as a white solid. 1 HNMR (400 MHz, CDCl 3 )δ (ppm) 7.41-7.29 (m, 5H), 5.41-5.30 (m, 1H), 4.30-4.20 (m, 2H), 1.46 (s, 9H).

[0580] Example 24. Synthesis of N-benzylsulfonamide (54)

[0581] To a solution of tert-butyl N-benzylsulfamoylcarbamate 53 (108 mg, 0.38) in DCM (10 mL) was added trifluoroacetic acid (2 mL). The reaction mixture was stirred for 1.5 h and then poured into saturated NaHCO 3 Aqueous solution (50 mL) was added. Another 50 mL of saturated NaHCO 3 After the aqueous solution, the aqueous mixture was extracted with DCM (50 mL). The organic layer was dried (Na 2 SO 4 ) and concentrated to afford the product 54 (36 mg, 0.19 mmol, 50%) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)7.41-7.30(m, 5H), 4.32(d, J=6.1Hz, 2H).

[0582] Example 25. Synthesis of (20)

[0583] To a solution of 52 (44 mg, 0.136 mmol) in DCM (5 mL) were added EDCI.HCl (39 mg, 0.204 mmol), DMAP (2.9 mg, 0.024 mmol) and N-benzylsulfonamide 54 (13 mg, 0.068 mmol). After the reaction mixture was stirred at room temperature for 22 h, EtOAc (20 mL) and saturated NH 4 Cl aqueous solution (20 mL). After separation, the aqueous phase was extracted with EtOAc (20 mL). The combined organic phases were dried (Na 2 SO 4 After gradient column chromatography (25%→50% EtOAc in heptane), the product 20 was obtained as an inseparable mixture of the title compound and N-benzylsulfonamide 54 (mass ratio 3.2 / 1) (14.4 mg). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 7.38-7.27 (m, 5H), 5.99 (bs, 1H), 5.01 (t, J=6.2Hz, 1H), 4.20 (s, 2H), 4.85-4.70 (m, 2H), 4.13 (d, J=8.12Hz, 2H), 3.15 (q, J=6.50, 2H), 2.35-2.15 (m, 6H), 2.09 (t, J=7.4Hz, 2H), 1.65-0.75 (m, 13H).

[0584] Example 26. Synthesis of (56)

[0585] To a solution of 51 (430 mg, 1.48 mmol) in DCM (20 mL) was added 5-nitropropene (4 mL) and Et 3 A solution of 5-aminopentan-1-ol 55 (152 mg, 1.47 mmol) in N (619 μL, 449 mg, 4.44 mmol) was added. The mixture was stirred at room temperature for 1.5 h, after which saturated NaHCO 3 After separation, the organic layer was dried (Na 2 SO 4 ) and concentrated. The residue was purified by gradient column chromatography (EtOAc / heptane 1 / 1→3 / 1). The product 56 (356 mg, 1.27 mmol, 81%) was obtained as a colorless viscous liquid. 1 H NMR (400 MHz, CDCl 3)δ (ppm) 4.68 (s, 1H), 4.14 (d, J = 8.0Hz, 2H), 3.65 (dd, J = 11.7, 6.3Hz, 2H), 3.19 ( dd, J=13.2, 6.7Hz, 2H), 2.35-2.15(m, 6H), 1.66-1.30(m, 7H), 1.02-0.88(m, 2H).

[0586] Example 27. Synthesis of (21)

[0587] To a solution of 56 (51 mg, 0.18 mmol) in DCM (10 mL) was added chlorosulfonyl isocyanate (16 μl, 25 mg, 0.18 mmol). After stirring the mixture for 40 min, Et 3 N (75 μl, 55 mg, 0.54 mmol) and benzylamine (19 μl, 19 mg, 0.18 mmol). The mixture was stirred for another 1.5 h and the mixture was heated to 40 ℃ by adding NH 4 It was quenched with Cl (saturated) aqueous solution. After separation, the aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried (Na 2 SO 4 ) and concentrated. The residue was purified by gradient column chromatography (20%→50% EtOAc in pentane) to give the product 21 (57 mg, 0.12 mmol, 67%) as a colorless viscous oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)7.41-7.28(m, 5H), 5.55(s, 1H), 4.75(s, 1H), 4.29-4.24(m, 2H), 4.20-4.08(m, 2H) , 3.19 (dd, J=13.4, 6.6Hz, 2H), 2.37-2.16 (m, 6H), 1.74-1.31 (m, 9H), 0.94 (t, J=9.7Hz, 2H).

[0588] Example 28. Synthesis of (22)

[0589] Under inert atmosphere, 21 (93 mg, 0.19 mmol) was dissolved in anhydrous THF (10 mL). PPh 3 (49 mg, 0.19 mmol) and MeOH (50 μL, 1.23 mmol), and the mixture was cooled to 0°C. A solution of DIAD (37 μL, 0.19 mmol) dissolved in anhydrous THF (5 mL) was slowly added dropwise and the mixture was allowed to reach room temperature, after which the reaction was stirred for 18 h and then concentrated. Gradient column chromatography (20→50% EtOAc in heptane) gave the product 22 as a colorless viscous oil.1 H NMR (400 MHz, CDCl 3 )δ (ppm) 7.39-7.26 (m, 5H), 5.94-5.84 (m, 1H), 4.80-4.64 (m, 1H), 4.19 (d, J=6.4Hz, 2H,), 4.13 (d, J=7.4H z, 2H), 4.08 (t, J=6.5Hz, 2H), 3.17 (q, 2H, J=6.5Hz), 3.12 (s, 3H), 2.35-2.14 (m, 6H), 1.80-1.65 (m, 13H).

[0590] Example 29. Synthesis of (58)

[0591] In N 2 To a solution of 57 (1.5 g, 10 mmol) in DCM (150 mL) was added CSI (0.87 mL, 1.4 g, 10 mmol), Et 3 N (2.8 mL, 2.0 g, 20 mmol) and 2-(2-aminoethoxy)ethanol (1.2 mL, 1.26 g, 12 mmol). The mixture was stirred for 10 min and NH 4 It was quenched with aqueous Cl (saturated, 150 mL). After separation, the aqueous layer was extracted with DCM (150 mL). The combined organic layers were dried (Na 2 SO 4 ) and concentrated. The residue was purified by column chromatography to obtain the product 58 (2.06 g, 5.72 mmol, 57%) as a light yellow viscous oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)6.0(bs, 1H), 4.28(d, J=8.2 Hz, 2H), 3.78-3.73(m, 2H), 3.66-3.61(m, 2H), 3.61-3.55(m, 2H), 3.34(t, J=4.9 Hz, 2H), 2.37-2.15(m, 6H), 1.64-1.48(m, 2H), 1.40(quartet, J=8.7 Hz, 1H), 1.05-0.92(m, 2H).

[0592] Example 30. Synthesis of (59)

[0593] Subsequently, to a solution of 58 (130 mg, 0.36 mmol) were added CSI (31 μL, 51 mg, 0.36 mmol), Et 3N (151 μL) and 2-(2-aminoethoxy)ethanol (36 μL, 38 mg, 0.36 mmol). After 15 min, water (20 mL) was added and after separation, the aqueous layer was acidified to pH 3 with 1M HCl aqueous solution and then extracted with DCM (20 mL). The DCM layer was dried and concentrated. After column chromatography, the product 59 (87 mg, 0.15 mmol, 42%) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)6.15-5.95(m, 2H), 4.40-4.32(m, 2H), 4.31(d, J=8.3Hz, 2H), 3.85-3.55(m, 10H), 3.45-3.25(m, 4H), 2.40-2.15(m, 6H), 1.65-1.47(m, 2H), 1.40(quartet, J=8.7Hz, 1H), 1.06-1.92(m, 2H).

[0594] Example 31. Synthesis of (23)

[0595] Subsequently, to a solution of 59 (63 mg, 0.11 mmol) in DCM (10 mL) were added p-nitrophenyl chloroformate (22 mg, 0.11 mmol) and Et 3 N (46 μL, 33 mg, 0.33 mmol). After 20 h, benzylamine (22 μL, 21.6 mg, 0.20 mmol) was added to the reaction mixture. The mixture was stirred for another 24 h, after which the mixture was concentrated and the residue was purified by gradient column chromatography (first column: 0 → 20% MeOH in DCM, second column: 0 → 8% MeOH in DCM). The product 23 (18 mg, 0.026 mmol, 23%) was obtained as a colorless film. 1 H NMR (400 MHz, CD 3 OD) δ (ppm) 7.38-7.18 (m, 5H), 4.31-4.22 (m, 6H), 4.22-4.16 (m, 2H), 3.70-3.63 (m, 4H), 3.63-3.54 (m, 4H), 3.34 (s, 1H), 3.24-3.15 (m, 4H), 2.30-2.10 (m, 6H), 1.68-1.52 (m, 2H), 1.42 (quartet, J = 8.7 Hz, 1H), 1.02-0.90 (m, 2H).

[0596] Example 32. BCN-dPEG 4 Synthesis of -C(O)OSu(60a)

[0597] Then, amino-dPEG was dissolved in anhydrous DMF (30 mL). 4 -acid (1.23 g, 4.23 mmol), 51 (1.02 g, 3.85 mmol) and triethylamine (1.60 mL, 11.53 mmol) were added. The reaction mixture was stirred at room temperature for 3 h, after which EDCI.HCl (0.884 g, 4.61 mmol) and NHS (88 mg, 0.77 mmol) were added. The resulting solution was stirred overnight at room temperature, and then 100 mL of NaHCO 3 (saturated) and 150 mL of EtOAc. The layers were separated and washed with saturated NaHCO 3 (90 mL) and H 2 O (75 mL) and the organic phase was dried (Na 2 SO 4 ), filtered, and concentrated in vacuo. Gradient column chromatography (MeCN→MeCN∶H 2 O30:1) gave the product 60a (800 mg, 1.48 mmol, 40%) as a colorless oil.

[0598] Example 33. BCN-dPEG 4 Synthesis of -pyrene (24)

[0599] To a solution of 60a (50 mg, 0.095 mmol) in DCM (10 mL) were added 49 (30 mg, 0.11 mmol) and Et 3 N (17 μL, 0.12 mmol). After stirring at room temperature overnight, the reaction mixture was concentrated under reduced pressure. It was then purified by flash column chromatography (DCM→DCM:MeOH 9:1) to give the product 24 (38 mg, 61%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.29-8.26 (m, 2H), 8.19-8.11 (m, 4H), 8.06-7.97 (m, 4H), 7.04 (br.s, 1H), 5.24 (br.s, 1H), 5.16 (d, 2H, J=4Hz), 4.06 (d, 2H, J=4H z), 3.76 (t, 2H, J = 5.6Hz), 3.50 (m, 2H), 3.39-3.22 (m, 14H), 2.56-2.53 (m, 2H), 2.27-2.13 (m, 7H), 1.29-1.25 (m, 2H), 0.87-0.83 (m, 2H).

[0600] Example 34. BCN-PEG 8 Synthesis of -C(O)OSu(60b)

[0601] Then, amino-dPEG was dissolved in anhydrous DMF (3 mL). 8 -acid (217 mg, 0.492 mmol) was added to a solution of 51 (143 mg, 0.492 mmol) and Et 3 N (204 μL, 1.47 mmol). The reaction mixture was stirred at room temperature for 3 h, after which EDCI.HCl (0.88 g, 4.61 mmol) and NHS (88 mg, 0.77 mmol) were added. The resulting solution was stirred overnight at room temperature, and then 50 mL of NaHCO 3 (saturated) and 50 mL of EtOAc. The layers were separated and washed with saturated NaHCO 3 (50 mL) and H 2 O (30 mL) to wash the organic phase. 2 SO 4 ), filtered, and concentrated in vacuo. Gradient column chromatography (MeCN→MeCN∶H 2 O30:1) gave the product 60b (212 mg, 0.30 mmol, 60%) as a colorless oil.

[0602] 1 HNMR (300 MHz, CDCl 3 ): δ (ppm) 4.13 (d, J = 8.1 Hz, 2H), 3.84 (t, J = 6.3 Hz, 2H), 3.68-3.59 (m, 28H), 3.54 (t, J = 5.1 Hz, 2H), 3.36 (q, J = 5.4 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.82 (s, 4H), 2.35-2.15 (m, 6H), 1.68-1.48 (m, 2H), 1.44-1.23 (m, 1H), 1.00-0.86 (m, 2H). LRMS (ESI+) m / z calculated value C 34 H 54 N 2 O 14 (M+Na + )=737.8; measured value 737.3.

[0603] Example 35. BCN-PEG 8 Synthesis of -pyrene (25)

[0604] To a solution of 60b (100 mg, 0.14 mmol) in DCM (15 mL) was added 1-aminomethylpyrene.HCl 49 (50 mg, 0.19 mmol) and Et 3N (47 μL, 0.25 mmol). After stirring for 3 h, the reaction mixture was concentrated under reduced pressure. Subsequent purification by flash column chromatography (DCM→DCM:MeOH 95:5) gave the product 25 (35 mg, 30%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.30-8.28 (m, 1H), 8.21-8.13 (m, 4H), 8.05-7.99 (m, 4H), 7.22 (bs, 1H), 5.16 (d, 2H, J=5.2Hz), 4.12 (d, 2H, J=8.0Hz), 3.76 (t, 2H, J=6.0Hz), 3. 65-3.49(m, 21H), 3.45-3.42(m, 2H), 3.36-3.32(m, 4H), 2.59-2.54(m, 4H), 2.28-2.20(m, 4H), 1.59-1.54(m, 2H), 1.38-1.25(m, 2H), 0.93-0.91(m, 2H).

[0605] Example 36. Synthesis of (61)

[0606] To a solution of 57 (0.15 g, 1.0 mmol) in DCM (15 mL) were added CSI (87 μL, 0.14 g, 1.0 mmol), Et 3 N (279 μL, 202 mg, 2.0 mmol) and H dissolved in DCM (1 mL) 2 N-PEG 3 -OH (251 mg, 1.3 mmol). After stirring for 2.5 h, NH 4 The reaction mixture was quenched with Cl (saturated, 20 mL) solution. After separation, the aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried (Na2O3) and purified by centrifugation. 2 SO 4 ) and concentrated. The residue was purified by gradient column chromatography (0→10% MeOH in DCM). The product 61 (254 mg, 0.57 mmol, 57%) was obtained as a colorless viscous oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)6.81(br.s,1H),4.26(d,J=8.2Hz,2H),3.80-3.70(m,4H),3.70-3.58(m,10H),3.36(t,J=4.7Hz,2H),2.36-2.16(m,6H),1.64-1.49(m,2H),1.40(quartet,J=8.7Hz,1H),1.04-0.92(m,2H).

[0607] Example 37. Synthesis of (62)

[0608] To a solution of 61 (242 mg, 0.54 mmol) in DCM (30 mL) was added p-nitrophenyl chloroformate (218 mg; 1.08 mmol) and Et 3 N (226 μL, 164 mg, 1.62 mmol). The mixture was stirred for 17 h and quenched with water (20 mL). After separation, the organic layer was dried (Na 2 SO 4 ) and concentrated. The residue was purified by gradient column chromatography (EtOAc / pentane 1 / 1→EtOAc) to give product 62 (259 mg, 0.38 mmol). LRMS (ESI) m / z calculated value C 33 H 42 N 5 O 16 S(M+NH 4 + )=796.23; measured value 796.52.

[0609] Example 38. Synthesis of BCN-sulfonamide-pyrene (26)

[0610] Compound 62 (40 mg, 0.11 mmol) was dissolved in DCM (10 mL), and 49 (34 mg, 0.13 mmol) and Et 3 N (30 μL, 0.21 mmol). The reaction mixture was stirred for 4 h, concentrated under reduced pressure, and purified on gradient flash column chromatography (DCM→DCM:MeOH 96:4). The fractions containing the product were washed with saturated NaHCO 3 (3x100mL), washed with Na 2 SO 4 Dry, filter and concentrate in vacuo to afford 26 (25 mg, 36%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.29-8.26 (m, 2H), 8.19-8.11 (m, 4H), 8.06-7.97 (m, 4H), 6.87 (d, 1H, J = 12Hz), 5.78 (br.s, 2H), 5.12 (d, 2H, J = 5.6Hz) , 4.31-4.26 (m, 2H), 3.97 (d, 2H, J=8.4Hz), 3.69-3.62 (m, 4H), 3.28 (m, 2H), 2.18-1.96 (m, 7H), 1.28 (m, 1H), 0.79-0.74 (m, 2H).

[0611] Example 39. DIBAC-PEG 4 Synthesis of -pyrene (27)

[0612] Compound 45 (75 mg, 0.22 mmol) was dissolved in DCM (3 mL), followed by the addition of amino-PEG 4 -formic acid (53 mg, 0.20 mmol) and Et 3 N (83 μL, 0.6 mmol). After stirring overnight, additional 45 (25 mg, 0.07 mmol) was added and the reaction mixture was stirred for another 1 h. After complete conversion (based on TLC analysis), N-hydroxysuccinimide (5 mg, 0.04 mmol) and EDC.HCl (70 mg, 0.36 mmol) were added and the reaction was stirred at room temperature overnight. To the reaction mixture, 64 (60 mg, 0.2 mmol) and Et 3 N (50 μL, 0.36 mmol), after 1 h, the reaction mixture was concentrated under reduced pressure. Purification by gradient flash column chromatography (DCM→DCM:MeOH 9:1) gave the product 27 (12 mg, 8%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.61 (d, 1H, J = 9.2Hz), 8.23-8.21 (m, 2H), 8.17-8.10 (m, 4H), 8.07-8.02 (m, 3H), 7.59 (d, 1H, J = 7.2Hz), 7.38-7.18 (m, 7H), 7.06 (b s, 1H), 6.45 (m, 1H), 5.00 (d, 1H, J=13.6), 3.86-3.79 (m, 3H), 3.70-3. 19(m, 14H), 2.42-2.38(m, 1H), 2.19-2.15(m, 2H), 1.89-1.85(m, 1H).

[0613] Example 40. DIBAC-PEG 8 Synthesis of -pyrene (28)

[0614] Compound 45 (24 mg, 0.07 mmol) was dissolved in DCM (1 mL), followed by the addition of amino-PEG 8 -formic acid (27 mg, 0.06 mmol) and Et 3N (14 μL, 0.10 mmol). After stirring for 2 h, additional 45 (10 mg, 0.03 mmol) was added and the reaction mixture was stirred further. NHS (10 mg, 0.08 mmol) and EDC.HCl (17 mg, 0.09 mmol) were then added and the reaction was stirred for 2 h. Subsequently, 64 (21 mg, 0.08 mmol) and Et 3 N (14 μL, 0.10 mmol), and after 4 h, the reaction was quenched by the addition of water (3 mL). The organic layer was washed with water (2 x 3 mL) and washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure. Purification by flash column chromatography (DCM→DCM:MeOH 9:1) gave the product (8 mg, 14%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.55 (d, 1H, J = 9.2Hz), 8.19-7.86 (m, 7H), 7.59 (d, 1H, J = 6.8Hz), 7.33-7.18 (m, 8H), 6.99 (m, 1H), 6.45 (m, 1H) , 5.03 (d, 1H, J=14.0), 3.78-3.74 (m, 3H), 3.65-3.29 (m, 29H), 2.43-2.37 (m, 1H) 2.25-2.21 (m, 2H), 1.91-1.84 (m, 1H).

[0615] Example 41. Synthesis of (65)

[0616] Compound 45 (50 mg, 0.14 mmol) was dissolved in DCM (3 mL), followed by the addition of 2-aminoethanol (10 μL, 0.16 mmol) and Et 3 N (30 μL, 0.22 mmol). After 2 h, additional 2-aminoethanol (10 μL, 0.16 mmol) was added and the reaction mixture was stirred overnight at room temperature. Subsequently, water (5 mL) was added, the organic layer was washed with water (3 x 5 mL), and Na 2 SO 4 Dry, filter and concentrate in vacuo. Purification by gradient flash column chromatography (DCM→DCM:MeOH 97:3) gave product 65 (32 mg, 79%). LRMS (ESI + )m / z calculated value C 19 H 16 NO 2 (M+H + )=290.12; measured value 290.30.

[0617] Example 42. Synthesis of DIBAC-sulfonamide-pyrene (29)

[0618] Compound 65 (26 mg, 0.09 mmol) was dissolved in DCM (2 mL), followed by the addition of CSI (8 μL, 0.09 mmol). After 5 min, Et 3 N (37 μL, 0.27 mmol) and 64 (26 mg, 0.09 mmol) were added and the reaction was stirred at room temperature for 2 h, after which NH 4 The reaction was quenched with aqueous Cl solution (saturated, 5 mL). The organic layer was washed with water (3 x 5 mL) and washed with Na 2 SO 4 Dry, filter and concentrate in vacuo. Purification by gradient flash column chromatography (DCM→DCM:MeOH 95:5) gave the product 29 (10 mg, 17%). 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.49 (d, 1H, J = 9.2Hz), 8.25-8.18 (m, 3H), 8.11-7.95 (m, 5H), 7. 38-7.00(m, 8H), 6.05(m, 1H), 5.96(m, 1H), 4.62-4.58(m, 1H).4.52-4.47 (m, 1H), 4.11-4.08 (m, 1H), 3.81-3.78 (m, 1H), 3.64-3.60 (m, 1H), 3.06 ( d, 1H, J = 14Hz), 2.90-2.87 (m, 2H), 2.17-2.09 (m, 1H), 1.62-1.56 (m, 2H).

[0619] Example 43-1. Synthesis of (30)

[0620] Dissolve BCN-PEG in 1 mL DMF 4 -C(O)OSu (60a, 7.1 mg, 0.013 mmol) and Et 3 A solution of N (9.1 μL, 6.6 mg, 65.5 μmol) was added to H-Ahx-woodenin.TFA (10 mg, 0.011 mmol). The reaction was stirred at room temperature for 20 h and then concentrated under reduced pressure. The residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2 Purification was carried out by HPLC-MS / MS (ESI / 400 μL). The product 30 (8.9 mg, 7.5 μmol, 68%) was obtained as a colorless liquid. + )m / z calculated value C 60 H 87 C1N5 O 16 (M + -H 2 O) = 1168.58; measured value 1168.87.

[0621] Example 43-2. ​​BCN-PEG 12 Synthesis of -C(O)OSu

[0622] To amino-dPEG dissolved in anhydrous DMF (1 mL) 12 -acid (43 mg, 0.069 mmol) was added to a solution of 51 (22 mg, 0.076 mmol) and triethylamine (24 μL, 0.174 mmol). The reaction mixture was stirred at room temperature for 5 h, after which EDCI.HCl (27 mg, 0.139 mmol) and NHS (8 mg, 0.076 mmol) were added. The resulting solution was stirred at room temperature overnight, and 10 mL of saturated NaHCO was poured into the mixture. 3 and 10 mL DCM. The layers were separated and washed with H 2 The organic phase was washed with 4% CO (2x10 mL). The combined aqueous layers were extracted with DCM (10 mL). The combined organic layers were dried (Na2O3) and 2 SO 4 ), filtered and concentrated in vacuo. Gradient flash column chromatography (MeCN→MeCN∶H 2 O20:1, 10:1) to obtain BCN-PEG 12 -C(O)OSu.

[0623] 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 4.14 (d, J = 8.0 Hz, 2H), 3.85 (t, J = 6.4 Hz, 2H), 3.69-3.60 (m, 44H), 3.56 (t, J = 5.2 Hz, 2H), 3.36 (q, J = 5.2 Hz, 2H), 2.91 (t, J = 6.4 Hz, 2H), 2.84 (s, 4H), 2.36-2.17 (m, 6H), 1.65-1.51 (m, 2H), 1.44-1.23 (, J = 8.4 Hz, 1H), 1.00-0.88 (m, 2H). LRMS (ESI+) m / z calculated value C 42 H 70 N 2 O 18 (M+H + )=892.0; measured value 891.6.

[0624] Example 44. Synthesis of (31)

[0625] Dissolve BCN-PEG in 1.1 mL DMF 12 -C(O)OSu (14 mg, 15.7 μmol) and Et 3 A solution of N (9.1 μL, 6.6 mg, 65.5 μmol) was added to H-Ahx-woodenin.TFA (10 mg, 0.011 mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.3 μL, 2.3 mg, 16 μmol) was added and the mixture was concentrated. The residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2 Purification was carried out by HPLC-MS / MS (ESI / 400 μL) and HPLC-MS / MS (ESI / 400 μL) to afford the product 31 (6.6 mg, 4.3 μmol, 39%) as a colorless film. + )m / z calculated value C 75 H 118 C1N 5 O 25 (M + -H 2 O)=1520.79; measured value 1520.96.

[0626] Example 45. BCN-PEG 24 Synthesis of -C(O)OSu

[0627] To amino-dPEG dissolved in anhydrous DMF (1 mL) 24 -acid (48 mg, 0.042 mmol) was added to a solution of 51 (14 mg, 0.048 mmol) and triethylamine (17 μL, 0.125 mmol). The reaction mixture was stirred at room temperature overnight, after which DCM (10 mL) and citric acid (10% aqueous solution, 5 mL) were added. The aqueous phase was extracted with DCM (15 mL) and the combined organic layers were dried (Na 2 O 4 3 O 4 4 4 4 0 0 4 ... 2 SO 4 ). After filtration, the solvent was removed in vacuo. The crude product was redissolved in anhydrous DMF (1 mL), and EDCI.HCl (17 mg, 0.088 mmol) and NHS (8 mg, 0.070 mmol) were added. The resulting mixture was stirred at room temperature overnight, after which another equivalent of EDCI.HCl (16 mg) and NHS (7 mg) were added. After 6 h, the reaction mixture was poured into 10 mL of saturated NaHCO 3 and 15 mL of EtOAc. The layers were separated and the organic phase was washed with saturated NaHCO 3 (10 mL) and H 2 O (10 mL). The organic phase was dried (Na 2 SO4 ), filtered and concentrated in vacuo. Gradient flash column chromatography (MeCN→MeCN∶H 2 O20:1, 10:1, 5:1) to obtain BCN-PEG in the form of a colorless oil 24 -C(O)OSu (32 mg, 0.022 mmol, 54%).

[0628] 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 4.08 (d, J = 8.0 Hz, 2H), 3.78 (t, J = 6.4 Hz, 2H), 3.63-3.54 (m, 92H), 3.49 (t, J = 5.2 Hz, 2H), 3.30 (q, J = 5.2 Hz, 2H), 2.84 (t, J = 6.4 Hz, 2H), 2.78 (s, 4H), 2.29-2.08 (m, 6H), 1.59-1.43 (m, 2H), 1.35-1.23 (m, 1H), 0.93-0.80 (m, 2H). LRMS (ESI+) m / z calculated value C 66 H 118 N 2 O 30 (M+H + )=1420.66; measured value 1420.0.

[0629] Example 46. Synthesis of (32)

[0630] Dissolve BCN-PEG in 0.78 mL DMF 24 -C(O)OSu (25 mg, 17.6 μmol) and Et 3 A solution of N (9.1 μL, 6.6 mg, 65.5 μmol) was added to H-Ahx-woodenin.TFA (10 mg, 0.011 mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.3 μL, 2.3 mg, 16 μmol) was added and the mixture was concentrated. The residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2 Purification was carried out by HPLC-MS / MS (ESI / 500 % yield) and purification by HPLC-MS / MS (ESI / 500 % yield). The product was obtained as a colorless film (11.4 mg, 5.5 μmol, 50%). + )m / z calculated value C 100 H 171 C1N 5 O 37 3+ (M+3H + ) / 3=690.05; Actual value: 690.05.

[0631] Example 47. Synthesis of (33)

[0632] A solution of H-Ahx-xyloglucinol.TFA (20 mg, 0.023 mmol) in DMF (2 mL) was added to a solution of 62 (14 mg, 0.023 mmol) and Et 3 N (9.5 μL, 6.9 mg, 0.068 mmol) and the resulting reaction mixture was stirred for 24 h. After silica gel column chromatography, the title compound 33 was obtained in quantitative yield (29 mg, +99%). Proof of identity was performed at the ADC stage.

[0633] Example 48. Synthesis of (34)

[0634] 60a (6.6 mg, 0.012 mmol) and Et 3 A solution of N (6.8 μL, 4.9 mg, 48.5 μmol) was added to H-Val-Cit-PABA-duocarmycin (10 mg, 0.0097 mmol). After 18 h, 2,2′-(ethylenedioxy)bis(ethylamine) (1.8 μL, 1.8 mg, 12 μmol) was added and the mixture was concentrated under reduced pressure. The residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2 Purification was carried out by HPLC-MS / MS (ESI / 400 μL) and HPLC-MS / MS (ESI / 400 μL) to afford the product 34 (7.5 mg, 5.1 μmol, 53%) as a colorless film. + )m / z calculated value C 71 H 94 C1N 10 O 21 (M+H + )=1457.63; measured value 1456.89.

[0635] Example 49. Synthesis of (35)

[0636] 62 (6.0 mg, 9.8 μmol) and Et 3 A solution of N (6.8 μL, 4.9 mg, 48.5 μmol) was added to H-Val-Cit-PABA-duocarmycin (10 mg, 0.0097 mmol). After 22 h, 2,2′-(ethylenedioxy)bis(ethylamine) (2.8 μL, 2.8 mg, 19 μmol) was added. After 1 h, the reaction mixture was concentrated under reduced pressure and the residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2Purification was carried out by evaporation of 1% MeCN (1% AcOH) in 2% O (1% AcOH). The product 35 (6.4 mg, 4.2 μmol. 44%) was obtained as a white solid. LRMS (ESI + )m / z calculated value C 69 H 92 C1N 12 O 22 S(M+H + )=1507.59; measured value 1508.00.

[0637] Example 50. Synthesis of (36)

[0638] To a solution of 58 (229 mg, 0.64 mmol) in DCM (20 mL) was added p-nitrophenyl chloroformate (128 mg, 0.64 mmol) and Et 3 N (268 μL, 194 mg, 1.92 mmol). The mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was purified by gradient column chromatography (20→70% EtOAc (1% AcOH) in heptane) to give the PNP carbonate derivative of 58 (206 mg, 0.39 mmol, 61%) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ (ppm) 8.31-8.26 (m, 2H), 7.45-7.40 (m, 2H), 5.56 (t, J=6.0Hz, 1H), 4.48-4.40 (m, 2H), 4.27 (d, J=8.2Hz, 2H), 3.81-3.75 (m, 2H) , 3.68 (t, J=5.0Hz, 2H), 3.38-3.30 (m, 2H), 2.36-2.14 (m, 6H), 1.61-1.45 (m, 2H), 1.38 (quintet, J=8.7Hz, 1H), 1.04-0.94 (m, 2H).

[0639] Subsequently, the PNP derivative of 58 (4.1 mg, 7.8 μmol) and Et 3 N (3.3 μL, 2.4 mg, 23.4 μmol), a solution of H-Val-Cit-PABA-Ahx-maytansine (10 mg, 8.6 μmol) dissolved in DMF (100 μL) was added. After 20 h, 2,2′-(ethylenedioxy)bis(ethylamine) (5.7 μL, 5.6 mg, 38 μmol) was added, and the mixture was concentrated under reduced pressure. The residue was purified by reverse phase (C18) HPLC column chromatography (30→90% in H 2Purification of 36 (2.2 mg, 1.4 μmol, 18%) by LRMS (ESI + )m / z calculated value C 73 H 103 C1N 11 O 21 S(M-18+H + )=1536.67; measured value 1537.08.

[0640] Example 51. Synthesis of (66)

[0641] To a stirred solution of 57 (500 mg, 3.33 mmol) in DCM (100 mL) was added CSI (290 μL, 471 mg, 3.33 mmol). After 20 min, Et 3 N (1.4 mL, 1.0 g, 10 mmol) and a solution of diethanolamine.HCl (571 mg, 4.0 mmol) in DMF (5 mL). After an additional 45 min, the reaction mixture was concentrated under reduced pressure, and the residue was purified by gradient column chromatography (0→15% MeOH in DCM). The product 66 (767 mg, 2.13 mmol, 64%) was obtained as a colorless viscous oil. 1 H NMR (400 MHz, CDCl 3 )δ(ppm)4.26(d, J=8.2Hz, 2H),3.87(t, J=4.9Hz, 4H),3.55(t, J=4.9Hz, 4H),2.37-2.16(m, 6H),1.65-1.45(m, 2H),1.39(quartet, J=8.6Hz, 1H),1.05-0.92(m, 2H)

[0642] Example 52. Synthesis of (67)

[0643] To a suspension of 66 (206 mg, 0.57 mmol) in DCM (20 mL) was added Et 3 N (318 μL, 231 mg, 2.28 mmol) and p-nitrophenyl chloroformate (230 mg, 1.14 mmoL, 2 equiv.). The reaction mixture was stirred at room temperature for 28 h and then concentrated. The residue was purified by gradient column chromatography (20→75% EtOAc in heptane) to give 67 (83 mg, 0.12 mmol, 21%) as a light yellow viscous oil. 1 H NMR (400 MHz, CDCl 3)δ(ppm)8.31-8.24(m, 4H), 7.43-7.34(m, 4H), 4.53(t, J=5.4 Hz, 2H), 4.22(d, J=4.2 Hz, 2H), 3.87(t, J=5.4 Hz, 4H), 2.35-2.15(m, 6H), 1.55-1-40(m, 2H), 1.35(quartet, J=8.8 Hz, 1H), 1.03-0.92(m, 2H).

[0644] Example 53. Synthesis of (37)

[0645] To 67 (2.7 mg, 3.9 μmol) and Et 3 N (2.7 μ L, 2.0 mg, 19.5 μ mol) solution, added H-Val-Cit-PABA-Ahx-maytansine (10 mg, 0.0086 mmol) dissolved in DMF (100 μ L). The mixture was allowed to react overnight at room temperature and then concentrated. A solution of 2,2′-(ethylenedioxy)bis(ethylamine) (2.8 μ L, 2.8 mg, 19 μ mol) dissolved in DMF (1 mL) was added. The reaction mixture was concentrated and the residue was purified by reverse phase (C18) HPLC column chromatography (30 → 90% in H 2 Purification of MeCN (1% AcOH) in DHO (1% AcOH) afforded compound 37 (4.8 mg, 1.7 μmol, 45%). LRMS (ESI + )m / z calculated value C 131 H 186 Cl 2 N 20 O 38 S(M+2H + ) / 2=1375.12; actual measured value 1375.51.

[0646] Example 54. Synthesis of (68)

[0647] To a stirred solution of 58 (47 mg, 0.13 mmol) in DCM (10 mL) was added CSI (11 μL, 18 mg, 0.13 mmol). After 30 min, Et 3 A solution of N (91 μL, 66 mg, 0.65 mmol) and diethanolamine (16 mg, 0.16 mmol) dissolved in DMF (0.5 mL) was prepared. After 30 minutes, p-nitrophenyl chloroformate (52 mg, 0.26 mmol) and Et 3N (54 μL, 39 mg, 0.39 mmol).After an additional 4.5 h, the reaction mixture was concentrated and the residue was purified by gradient column chromatography (33→66% EtOAc / heptane (1% AcOH)) to give 68 (88 mg, 0.098 mmol, 75%) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 8.28-8.23 (m, 4H), 7.42-7.35 (m, 4H), 4.52 (t, J = 5.4Hz, 4H), 4.30 (d, J = 8.3Hz, 2H), 4.27-4.22 (m, 2H), 3.86 (t, J = 5.3Hz, 4H), 3. 69-3.65 (m, 2H), 3.64-3.59 (m, 2H), 3.30-3.22 (m, 2H), 2.34-2.14 (m, 6H), 1.62-1.46 (m, 2H), 1.38 (quartet, J = 8.7Hz, 1H), 1.04-0.92 (m, 2H).

[0648] Example 55. Synthesis of (38)

[0649] To 68 (3.9 mg, 4.3 μmol) and Et 3 To a solution of N (3.0 μL, 2.2 mg, 21.5 μmol) was added a solution of H-Val-Cit-PABA-Ahx-maytansine (10 mg, 8.6 μmol) dissolved in DMF (100 μL). The mixture was reacted and concentrated. The residue was purified by reverse phase (C18) HPLC chromatography (30→90% in H 2 Purification of MeCN (1% AcOH) in D2O (1% AcOH) afforded the product 38 (3.9 mg, 1.32 μmol, 31%). LRMS (ESI + )m / z calculated value C 136 H 196 Cl 2 N 22 O 43 S 2 (M+2H + ) / 2=1480.13;Measured 1480.35.LRMS(ESI + )m / z calculated value C 85 H 119 C1N 14 O 31 S 2 (M+2H + ) / 2=965.36; actual measured value 965.54.

[0650] As a byproduct, the monosubstituted Ahx-maytansine derivative of 68 was isolated (not shown). LRMS (ESI + ) Calculated value C 85 H 119 C1N 14 O 31 S 2 2+ m / z 965.36; found 965.54.

[0651] Example 56. Synthesis of (39)

[0652] To the monosubstituted Ahx-maytansine derivative of 68 (isolated as a by-product in Example 55) was added Et 3 N (0.7 μL, 0.5 mg, 5 μmol) and 1.2 mg (1.1 μmol) of H-Val-Cit-PABA-MMAF. The mixture was allowed to react overnight, and then 2,2′-(ethylenedioxy)bis(ethylamine) (1 μL, 1.0 mg, 6.7 μmol) was added. After 15 min, the reaction mixture was concentrated. Reverse phase (C18) HPLC chromatography (30→90% in H 2 MeCN (1% AcOH) in D2O (1% AcOH) gave 1.0 mg of the desired product. LRMS (ESI + )m / z calculated value C 137 H 206 C1N 23 O 41 S 2 (M+2H + ) / 2=1464.69; measured value 1465.66.

[0653] Example 57. Determination of HPLC retention time

[0654] Samples of compounds 19-38 were injected into a HPLC system equipped with a Phenomenex Luna C18 (2) 5μ, 150×4.6 mm, Column, and eluted with a gradient of 10% MeCN (0.1% TFA) / 90% water (0.1% TFA) to 90% MeCN (0.1% TFA) / 10% water (0.1% TFA), or a gradient of 10% MeCN / 90% 10mM potassium phosphate buffer pH 7.4 to 90% MeCN / 10% 10mM potassium phosphate buffer pH 7.4. The retention times of these compounds obtained are shown in Table 3.

[0655] Time program:

[0656] 0-12min: 10% MeCN to 90% MeCN

[0657] 12-14min: 90% MeCN

[0658] 14-15min: 90% MeCN to 10% MeCN

[0659] 15-17min: 10% MeCN

[0660] Table 3: Retention time of compounds 19-23 and 30-38 on RP-HPLC

[0661]

[0662] Example 58. Competitive conjugation of 17 and 18 to trastuzumab (N300C)

[0663] Trastuzumab (N300C) (100 μM) was incubated in PBS containing 30 mM EDTA and 1 mM TCEP (10 equiv) for 2 h at room temperature. After buffer exchange with PBS, partially reduced trastuzumab (N300C) (66.7 μM) was incubated with 1.33 mM dehydroascorbic acid (20 equiv). Reoxidized trastuzumab (N300C) (66.7 μM) was incubated with 0.4 mM maleimide 17 (6 equiv) and 0.4 mM maleimide 18 (6 equiv). After incubation for 1 h at room temperature, the reaction was terminated by adding a 5-fold molar excess of N-acetylcysteine. The reaction products were washed with Fabricator TM (purchased from Genovis) digested and analyzed by MS-analysis (AccuTOF). About 45% of the Fc-fragment was conjugated to maleimide 18 (24297 Da, expected mass = 24299), and about 35% of the Fc-fragment was conjugated to maleimide 17 (24319 Da, expected mass = 24323). The remaining 20% ​​of the Fc-fragment consisted of various unconjugated forms (ranging from 23776 to 23874 Da).

[0664] Example 59. Conjugation of 19-39 to 13b or 13c

[0665] Trastuzumab (azide) as appropriate 2(8.8 μL, 0.2 mg, 22.7 mg / ml, dissolved in Tris buffer pH 7.5 10 mM), Tris buffer pH 7.5 10 mM (4 μL) and substrate (2 μL, dissolved in MiliQ 2mM solution + 5% DMA) were added. The reaction was incubated at room temperature and samples were collected (2 μL) at pre-set time points. These samples were incubated with DTT (2 μL 0.2M), diluted with MiliQ (30 μL), and subjected to MS analysis (AccuTof) to determine the conjugation efficiency (see Tables 1 and 2).

[0666] Example 60. Aggregation

[0667] Studies 36, 37, and 38 compared with trastuzumab (F 2 -GalNAz) 2 Aggregation tendency of the conjugate of (13c) (prepared according to Example 59). ADC was incubated at 37°C at a concentration of 1 mg / mL in PBS pH 7.4. Aggregation levels were analyzed after 0, 1 and 2 weeks using a Superdex200 PC 3.2 / 30 column (GE Healthcare). The results are shown in Fig.23 Aggregation remained below 1% from conjugate 36 with a drug-to-antibody ratio of 2 (DAR2) to conjugate 37 with a drug-to-antibody ratio of 4 (DAR4), indicating that the sulfonamide spacer has the potential to compensate for the increased lipophilicity conferred by doubling the number of payloads. No aggregation was observed at all for the bis-sulfonamide linker of 38. These results greatly improve conventional bioconjugates prepared by alkyne-azide cycloaddition ligation.

[0668] Example 61. Aggregation

[0669] During the 14-day period, monitor 30 and 36 with trastuzumab (F 2 -GalNAz) 2 The aggregation tendency of the conjugate of (13c) (prepared according to Example 59), as well as trastuzumab itself, was measured. The conjugate was stored at 40°C at pH 5 for 2 weeks, and the degree of aggregation was determined at 0, 2, 7, 10 and 14 days. The level of aggregation was analyzed using a Superdex200 PC 3.2 / 30 column (GE Healthcare). The results are shown in Fig.24 Compared to Example 61, conditions of increased stress (elevated T, lower pH) resulted in increased aggregation. However, compared to 30 with trastuzumab (F 2 -GalNAz) 2 Comparative bioconjugates of (13c) (using PEG 4 spacer), the conjugate of the present invention (36 and trastuzumab (F2 -GalNAz) 2 The aggregation of the conjugate of (13c) was significantly reduced. Notably, trastuzumab itself did not show any tendency to aggregate, given the lack of hydrophobic groups such as 1,2,3-triazole moieties fused to the cyclooctane and maytansine moieties. Sequence Listing <110> Sinafox Co., Ltd. <120> Sulfonamide linker, conjugate thereof and preparation method <130> P6051929PCT <150> 14187615.1 <151> 2014-10-03 <160> 5 <170> PatentIn Version 3.3 <210> 1 <211> 358 <212> PRT <213> Artificial <220> <223> GalT double mutant (Y289L, C342T) <400> 1 Arg Asp Leu Arg Arg Leu Pro Gln Leu Val Gly Val His Pro Pro Leu 1 5 10 15 Gln Gly Ser Ser His Gly Ala Ala Ala Ile Gly Gln Pro Ser Gly Glu 20 25 30 Leu Arg Leu Arg Gly Val Ala Pro Pro Pro Pro Leu Gln Asn Ser Ser 35 40 45 Lys Pro Arg Ser Arg Ala Pro Ser Asn Leu Asp Ala Tyr Ser His Pro 50 55 60 Gly Pro Gly Pro Gly Pro Gly Ser Asn Leu Thr Ser Ala Pro Val Pro 65 70 75 80 Ser Thr Thr Thr Arg Ser Leu Thr Ala Cys Pro Glu Glu Ser Pro Leu 85 90 95 Leu Val Gly Pro Met Leu Ile Glu Phe Asn Ile Pro Val Asp Leu Lys 100 105 110 Leu Val Glu Gln Gln Asn Pro Lys Val Lys Leu Gly Gly Arg Tyr Thr 115 120 125 Pro Met Asp Cys Ile Ser Pro His Lys Val Ala Ile Ile Ile Pro Phe 130 135 140 Arg Asn Arg Gln Glu His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro 145 150 155 160 Ile Leu Gln Arg Gln Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln 165 170 175 Ala Gly Glu Ser Met Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe 180 185 190 Lys Glu Ala Leu Lys Asp Tyr Asp Tyr Asn Cys Phe Val Phe Ser Asp 195 200 205 Val Asp Leu Ile Pro Met Asn Asp His Asn Thr Tyr Arg Cys Phe Ser 210 215 220 Gln Pro Arg His Ile Ser Val Ala Met Asp Lys Phe Gly Phe Ser Leu 225 230 235 240 Pro Tyr Val Gln Leu Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln 245 250 255 Phe Leu Ser Ile Asn Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly 260 265 270 Glu Asp Asp Asp Ile Tyr Asn Arg Leu Ala Phe Arg Gly Met Ser Val 275 280 285 Ser Arg Pro Asn Ala Val Ile Gly Lys Thr Arg Met Ile Arg His Ser 290 295 300 Arg Asp Lys Lys Asn Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala 305 310 315 320 His Thr Lys Glu Thr Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr 325 330 335 Met Val Leu Glu Val Gln Arg Tyr Pro Leu Tyr Thr Lys Ile Thr Val 340 345 350 Asp Ile Gly Thr Pro Ser 355 <210> 2 <211> 354 <212> PRT <213> Artificial <220> <223> CeGalNAcT [30-383] <400> 2 Lys Ile Pro Ser Leu Tyr Glu Asn Leu Thr Ile Gly Ser Ser Thr Leu 1 5 10 15 Ile Ala Asp Val Asp Ala Met Glu Ala Val Leu Gly Asn Thr Ala Ser 20 25 30 Thr Ser Asp Asp Leu Leu Asp Thr Trp Asn Ser Thr Phe Ser Pro Ile 35 40 45 Ser Glu Val Asn Gln Thr Ser Phe Met Glu Asp Ile Arg Pro Ile Leu 50 55 60 Phe Pro Asp Asn Gln Thr Leu Gln Phe Cys Asn Gln Thr Pro Pro His 65 70 75 80 Leu Val Gly Pro Ile Arg Val Phe Leu Asp Glu Pro Asp Phe Lys Thr 85 90 95 Leu Glu Lys Ile Tyr Pro Asp Thr His Ala Gly Gly His Gly Met Pro 100 105 110 Lys Asp Cys Val Ala Arg His Arg Val Ala Ile Ile Val Pro Tyr Arg 115 120 125 Asp Arg Glu Ala His Leu Arg Ile Met Leu His Asn Leu His Ser Leu 130 135 140 Leu Ala Lys Gln Gln Leu Asp Tyr Ala Ile Phe Ile Val Glu Gln Val 145 150 155 160 Ala Asn Gln Thr Phe Asn Arg Gly Lys Leu Met Asn Val Gly Tyr Asp 165 170 175 Val Ala Ser Arg Leu Tyr Pro Trp Gln Cys Phe Ile Phe His Asp Val 180 185 190 Asp Leu Leu Pro Glu Asp Asp Arg Asn Leu Tyr Thr Cys Pro Ile Gln 195 200 205 Pro Arg His Met Ser Val Ala Ile Asp Lys Phe Asn Tyr Lys Leu Pro 210 215 220 Tyr Ser Ala Ile Phe Gly Gly Ile Ser Ala Leu Thr Lys Asp His Leu 225 230 235 240 Lys Lys Ile Asn Gly Phe Ser Asn Asp Phe Trp Gly Trp Gly Gly Glu 245 250 255 Asp Asp Asp Leu Ala Thr Arg Thr Ser Met Ala Gly Leu Lys Val Ser 260 265 270 Arg Tyr Pro Thr Gln Ile Ala Arg Tyr Lys Met Ile Lys His Ser Thr 275 280 285 Glu Ala Thr Asn Pro Val Asn Lys Cys Arg Tyr Lys Ile Met Gly Gln 290 295 300 Thr Lys Arg Arg Trp Thr Arg Asp Gly Leu Ser Asn Leu Lys Tyr Lys 305 310 315 320 Leu Val Asn Leu Glu Leu Lys Pro Leu Tyr Thr Arg Ala Val Val Asp 325 330 335 Leu Leu Glu Lys Asp Cys Arg Arg Glu Leu Arg Arg Asp Phe Pro Thr 340 345 350 Cys Phe <210> 3 <211> 360 <212> PRT <213> Artificial <220> <223> CeGalNAcT(30 - 383)-His6) <400> 3 Lys Ile Pro Ser Leu Tyr Glu Asn Leu Thr Ile Gly Ser Ser Thr Leu 1 5 10 15 Ile Ala Asp Val Asp Ala Met Glu Ala Val Leu Gly Asn Thr Ala Ser 20 25 30 Thr Ser Asp Asp Leu Leu Asp Thr Trp Asn Ser Thr Phe Ser Pro Ile 35 40 45 Ser Glu Val Asn Gln Thr Ser Phe Met Glu Asp Ile Arg Pro Ile Leu 50 55 60 Phe Pro Asp Asn Gln Thr Leu Gln Phe Cys Asn Gln Thr Pro Pro His 65 70 75 80 Leu Val Gly Pro Ile Arg Val Phe Leu Asp Glu Pro Asp Phe Lys Thr 85 90 95 Leu Glu Lys Ile Tyr Pro Asp Thr His Ala Gly Gly His Gly Met Pro 100 105 110 Lys Asp Cys Val Ala Arg His Arg Val Ala Ile Ile Val Pro Tyr Arg 115 120 125 Asp Arg Glu Ala His Leu Arg Ile Met Leu His Asn Leu His Ser Leu 130 135 140 Leu Ala Lys Gln Gln Leu Asp Tyr Ala Ile Phe Ile Val Glu Gln Val 145 150 155 160 Ala Asn Gln Thr Phe Asn Arg Gly Lys Leu Met Asn Val Gly Tyr Asp 165 170 175 Val Ala Ser Arg Leu Tyr Pro Trp Gln Cys Phe Ile Phe His Asp Val 180 185 190 Asp Leu Leu Pro Glu Asp Asp Arg Asn Leu Tyr Thr Cys Pro Ile Gln 195 200 205 Pro Arg His Met Ser Val Ala Ile Asp Lys Phe Asn Tyr Lys Leu Pro 210 215 220 Tyr Ser Ala Ile Phe Gly Gly Ile Ser Ala Leu Thr Lys Asp His Leu 225 230 235 240 Lys Lys Ile Asn Gly Phe Ser Asn Asp Phe Trp Gly Trp Gly Gly Glu 245 250 255 Asp Asp Asp Leu Ala Thr Arg Thr Ser Met Ala Gly Leu Lys Val Ser 260 265 270 Arg Tyr Pro Thr Gln Ile Ala Arg Tyr Lys Met Ile Lys His Ser Thr 275 280 285 Glu Ala Thr Asn Pro Val Asn Lys Cys Arg Tyr Lys Ile Met Gly Gln 290 295 300 Thr Lys Arg Arg Trp Thr Arg Asp Gly Leu Ser Asn Leu Lys Tyr Lys 305 310 315 320 Leu Val Asn Leu Glu Leu Lys Pro Leu Tyr Thr Arg Ala Val Val Asp 325 330 335 Leu Leu Glu Lys Asp Cys Arg Arg Glu Leu Arg Arg Asp Phe Pro Thr 340 345 350 Cys Phe His His His His His His 355 360 <210> 4 <211> 449 <212> PRT <213> Artificial <220> <223> Trastuzumab (Heavy Chain N300C) <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 5 <211> 107 <212> PRT <213> Artificial <220> <223> Trastuzumab (light chain) <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

Claims

1. A method for preparing a bioconjugate, the method comprising: 1 Functional group F of glycoprotein (B) 1 The step of reacting, wherein the linker-conjugate is a compound comprising an a-terminus and an ω-terminus, and the compound comprises a reactive group Q at the a-terminus 1 and contains a target molecule (D) at the ω-terminus, the Q 1 Can bind to the functional group F on glycoprotein (B) 1 Reaction, wherein the target molecule is a cytotoxin, and the compound further comprises a group of formula (1) or a salt thereof: in: a is 0 or 1; and R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C1-C4 alkyl, or R 1 is a second target molecule D, wherein the second target molecule is optionally linked to N via a spacer; and wherein the group of formula (1) or a salt thereof is located between the a-terminal and the ω-terminal of the compound, The reaction is a 1,3-dipolar cycloaddition.

2. The method of claim 1, wherein In the group of formula (1), a is 0.

3. The method of claim 1, wherein In the group of formula (1), a is 1.

4. The method of any one of claims 1 to 3, wherein the cytotoxin is selected from the group consisting of colchicine, vinca alkaloids, anthracyclines, camptothecins, taxanes, calicheamicins, tubulysins, inhibitory peptides, amanitin, deBouganin, duocarmycin, maytansine, auristatins, and pyrrolobenzodiazepines (PBDs).

5. The method of claim 4, wherein the cytotoxin is selected from the group consisting of doxorubicin, daunorubicin and irinotecan.

6. The method of claim 1, wherein Q 1 and F 1 is selected from the group consisting of alkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9-yl], cycloalkenyl, tetrazine, azido, nitrile oxide, nitrone, nitrile imine, diazo, conjugated diene, conjugated heterodiene, 1,2-quinone and triazine, wherein when F 1 When Q is an azido group, an oxynitrile group, a nitrone group, an nitrile imine group, a diazo group, a conjugated diene group, a conjugated heterodiene group, a 1,2-quinone group, or a triazine group, 1 is an alkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9-yl] group or cycloalkenyl, and vice versa.

7. The method of claim 6, wherein Q 1 is alkenyl or alkynyl, and F 1 It is an azido group.

8. The method of claim 1, wherein the linker-conjugate is of formula (4a) or (4b), or a salt thereof in: a is independently 0 or 1; b is independently 0 or 1; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer ranging from 1 to 150; g is 0 or 1; i is 0 or 1; D is the target molecule; Q 1 is alkenyl or alkynyl; Sp 1 For the interval part; Sp 2 For the interval part; Sp 3 For the interval part; Sp 4 For the interval part; Z 1 is a connecting group, which connects Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 )connect; Z 2 is a linking group, which connects D or Sp 4 With Sp 1 、N(R 1 ), O or C(O) connection; and R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, and C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 independently selected from hydrogen and C1-C4 alkyl; or R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above.

9. The method of claim 7 or 8, wherein Q 1 It is a cycloalkenyl group, a cycloalkynyl group or a heterocycloalkynyl group.

10. The method of claim 9, wherein Q 1 is a bicyclo[6.1.0]non-4-yn-9-yl] group.

11. The method of claim 8, wherein Sp 1 、Sp 2 、Sp 3 and Sp 4 independently selected from linear or branched C1-C 200 Alkylene, C2-C 200 Alkenylene, C2-C 200 Alkynylidene, C3-C 200 Cycloalkylene, C5-C 200 Cycloalkenylene, C8-C 200 Cycloalkynylene, C7-C 200 Alkyl arylene, C7-C 200 Aryl alkylene, C8-C 200 Arylene and C9-C 200 arylalkynylene, the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene being optionally substituted and optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen, C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl and C3-C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

12. The method of claim 8 or 11, wherein Z 1 and Z 2 are independently selected from -O-, -S-, -NR 2 -, -N=N-, -C(O)-, -C(O)NR 2 -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-NR 2 -, -NR2-C(O)-, -NR 2 -C(O)-O-, -NR 2 -C(O)-NR 2 -, -S-C(O)-, -S-C(O)-O-, -S-C(O)-NR 2 -, -S(O)-, -S(O)2-, -O-S(O)2-, -O-S(O)2-O-, -O-S(O)2-NR 2 -, -O-S(O)-, -O-S(O)-O-, -O-S(O)-NR 2 -, -O-NR 2 -C(O)-, -O-NR 2 -C(O)-O-, -O-NR 2 -C(O)-NR 2 -, -NR 2 -O-C(O)-, -NR 2 -O-C(O)-O-, -NR 2 -O-C(O)-NR 2 -, -O-NR 2 -C(S)-, -O-NR 2 -C(S)-O-, -O-NR 2 -C(S)-NR 2 -, -NR 2 -O-C(S)-, -NR 2 -O-C(S)-O-, -NR 2 -O-C(S)-NR 2 -, -O-C(S)-, -O-C(S)-O-, -O-C(S)-NR 2 -, -NR 2 -C(S)-, -NR 2 -C(S)-O-, -NR 2 -C(S)-NR 2 -, -S-S(O)2-, -S-S(O)2-O-, -S-S(O)2-NR 2 -, -NR 2 -O-S(O)-, -NR 2 -O-S(O)-O-, -NR 2 -OS(O)-NR 2 -、-NR 2 -OS(O)2-、-NR 2 -OS(O)2-O-、-NR 2 -OS(O)2-NR 2 -、-O-NR 2 -S(O)-, -O-NR 2 -S(O)-O-, -O-NR 2 -S(O)-NR 2 -、-O-NR 2 -S(O)2-O-, -O-NR 2 -S(O)2-NR 2 -、-O-NR 2 -S(O)2-、-OP(O)(R 2 )2-、-SP(O)(R 2 )2-、-NR 2 -P(O)(R 2 )2- and combinations of two or more thereof, wherein R 2 independently selected from hydrogen, C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl and C3-C 24 Cycloalkyl, the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

13. The method of claim 8, wherein Sp 1 、Sp 2 、Sp 3 and Sp 4 , if present, independently selected from linear or branched C1-C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C1-C4 alkyl, and wherein Q 1 It is the formula (9a), (9q), (9n), (9o) or (9p): Where U is O or NR 9 , and R 9 is hydrogen, a straight or branched C1-C 12 Alkyl, C4-C 12 Aryl or C4-C 12 Heteroaryl.

14. A bioconjugate obtainable by the method of any one of claims 1 to 13.

15. A compound comprising an a-terminus and an ω-terminus, wherein the compound comprises a reactive group Q at the a-terminus 1 and contains a target molecule (D) at the ω-terminus, the Q 1 Can bind to the functional group F on glycoprotein 1 reaction, the target molecule (D) is selected from cytotoxins, and the compound further comprises a group of formula (1) or a salt thereof: in: a is 0 or 1; and R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C1-C4 alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer; wherein the group of formula (1) or a salt thereof is located between the a-end and the ω-end of the compound, wherein Q 1 It is a dipolarophile selected from an alkenyl group or an alkynyl group that reacts in a 1,3-dipolar cycloaddition reaction.

16. The compound of claim 15, wherein Q 1 is an optionally substituted cycloalkynyl or heterocycloalkynyl group.

17. The compound of claim 16, wherein Q 1 is a bicyclo[6.1.0]non-4-yn-9-yl] group.

18. The compound of claim 15, wherein the compound is of formula (4a) or (4b), or a salt thereof: Among them, a, b, c, d, e, f, g, i, D, Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 and R 1 As defined in claim 8.

19. The compound of claim 15, wherein Q 1 It is the formula (9a), (9q), (9n), (9o) or (9t): Where U is O or NR 9 , and R 9 is hydrogen, a straight or branched C1-C 12 Alkyl, C4-C 12 Aryl or C4-C 12 Heteroaryl.

20. The compound of claim 15, wherein Sp 1 、Sp 2 、Sp 3 and Sp 4 , if present, independently selected from linear or branched C1-C 20 Alkylene, which is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 Independently selected from hydrogen and C1-C4 alkyl.

21. A compound comprising an a-terminus and an ω-terminus, the compound comprising a glycoprotein (B) at the a-terminus and a target molecule at the ω-terminus, the target molecule being selected from cytotoxins, the compound further comprising a group of formula (1) or a salt thereof: in: a is 0 or 1; and R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 independently selected from hydrogen and C1-C4 alkyl; or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer; And wherein the group of formula (1) or a salt thereof is located between the a-terminal and the ω-terminal, and wherein the compound further comprises a portion obtained by a cycloaddition between the a-terminal and the group of formula (1), wherein the compound comprises a portion between the a-terminal and the group of formula (1) obtainable by a 1,3-dipolar cycloaddition.

22. The compound of claim 21, wherein the target molecule is selected from the group consisting of colchicine, vinca alkaloids, anthracyclines, camptothecins, taxanes, calicheamicins, tubulysins, inhibitory peptides, amanitin, deBouganin, duocarmycin, maytansine, auristatins, or pyrrolobenzodiazepines.

23. The compound of claim 22, wherein the target molecule is selected from the group consisting of the following cytotoxins: doxorubicin, daunorubicin, and irinotecan.

24. The compound of claim 21, which is a bioconjugate of the formula: (B)y-Sp-(D)z, in: y is an integer ranging from 1 to 10; z is an integer ranging from 1 to 10; B is glycoprotein; D is the target molecule; Sp is a spacer moiety which covalently links the glycoprotein B and the target molecule D, and comprises a group of formula (1) or a salt thereof.

25. The compound of claim 24, wherein y is 1, and z is 1, 2, 3 or 4.

26. The compound of claim 21, which is of formula (5a) or (5b), or a salt thereof: in: a is independently 0 or 1; b is independently 0 or 1; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer ranging from 1 to 150; g is 0 or 1; h is 0 or 1; i is 0 or 1; D is the target molecule; Sp 1 For the interval part; Sp 2 For the interval part; Sp 3 For the interval part; Sp 4 For the interval part; Z 1 is a connecting group, which connects Q 1 or Sp 3 With Sp 2 , O or C(O) or N(R 1 )connect; Z 2 is a linking group, which connects D or Sp 4 With Sp 1 、N(R 1 ), O or C(O) connection; Z 3 is a linking group that connects B to Sp 3 , Z 1 、Sp 2 , O or C(O) connection; R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C1-C4 alkyl; or R 1 D, -[(Sp 1 ) b -(Z 2 ) e -(Sp 4 ) i -D] or -[(Sp 2 ) c -(Z 1 ) d -(Sp 3 ) g -Q 1 ], where Sp 1 、Sp 2 、Sp 3 、Sp 4 , Z 1 , Z 2 , D, Q 1 , b, c, d, e, g and i are as defined above; and B is glycoprotein.

27. The compound of claim 26, wherein Z 3 Selected from (10a) – (10h): The connecting group (10h) can emit N2.

28. The compound of claim 21, wherein the moiety obtainable by a 1,3-dipolar cycloaddition reaction is a 1,2,3-triazole ring, which moiety is located between the a-terminus and the group of formula (1).

29. The compound of claim 21, wherein glycoprotein B is an antibody.

30. The compound of claim 29, wherein the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, and an antibody that specifically binds to a cancer antigen.

31. The compound of claim 22, wherein the target molecule is camptothecin.

32. The compound of claim 22, wherein the target molecule is auristatin.

33. The compound of claim 22, wherein the target molecule is maytansine.

34. The compound of claim 22, wherein the target molecule is calicheamicin.

35. A method for preparing a compound according to any one of claims 22 to 34, the method comprising reacting the reactive group Q of the linker-conjugate 1 Functional group F of glycoprotein (B) 1 The step of reacting, wherein the linker-conjugate is a compound comprising an a-terminus and an ω-terminus, and the compound comprises a reactive group Q at the a-terminus 1 and contains the target molecule at the ω-end, the Q 1 Can bind to the functional group F on glycoprotein (B) 1 Reaction, the compound further comprises a group of formula (1) or a salt thereof: in: a is 0 or 1; and R 1 Selected from hydrogen, C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl, the C1–C 24 Alkyl, C3–C 24 Cycloalkyl, C2–C 24 Aryl, C2–C 24 Heteroaryl, C3–C 24 Alkylaryl, C3–C 24 Alkyl heteroaryl, C3–C 24 Arylalkyl and C3–C 24 Heteroarylalkyl is optionally substituted and is optionally selected from O, S and NR 3 One or more heteroatoms are separated, wherein R 3 are independently selected from hydrogen and C1-C4 alkyl, or R 1 is a target molecule D, wherein the target molecule is optionally linked to N via a spacer; and The group of formula (1) or a salt thereof is located between the a-end and the ω-end of the compound, and wherein the target molecule is selected from colchicine, vinca alkaloids, anthracycline antibiotics, camptothecins, taxanes, calicheamicin, tubulysin, inhibitory peptides, amanitin, deBouganin, duocarmycin, maytansine, auristatin or pyrrolobenzodiazepine, and wherein the reaction is a 1,3-dipolar cycloaddition.

36. The method of claim 35, wherein the target molecule is selected from the group consisting of doxorubicin, daunorubicin, and irinotecan.

37. The method of claim 35, wherein Q 1 and F 1 is selected from the group consisting of alkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9-yl], cycloalkenyl, tetrazine, azido, nitrile oxide, nitrone, nitrile imine, diazo, conjugated diene, conjugated heterodiene, 1,2-quinone and triazine, wherein when F 1 When Q is an azido group, an oxynitrile group, a nitrone group, an nitrile imine group, a diazo group, a conjugated diene group, a conjugated heterodiene group, a 1,2-quinone group, or a triazine group, 1 is an alkenyl, alkynyl, cycloalkynyl, heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9-yl] group or cycloalkenyl, and vice versa.

38. The method of claim 37, wherein Q 1 is alkenyl, alkynyl, and F 1 It is an azido group.

39. The method of claim 38, wherein Q 1 It is a cycloalkenyl group, a cycloalkynyl group or a heterocycloalkynyl group.

40. The method of claim 39, wherein Q 1 is a bicyclo[6.1.0]non-4-yn-9-yl] group.

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