Chemically selective mercapto coupling with olefin-or alkyne-thiophosphonates and-phosphonates

Abstract

The invention discloses a novel conjugate and a preparation method thereof. A process for the preparation of olefin-or alkyne-thiophosphonates and-phosphonates, comprising the step of reacting a compound of formula (I) with a mercapto group-containing molecule of formula (II), wherein # imgabs0 # represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C1-C8-alkyl group, an optionally substituted phenyl group or an optionally substituted aromatic 5-or 6-membered heterocyclic system; and obtaining the compound of the formula (III). # imgabs1 #

Description

Background Art

[0001] Chemoselective and bioorthogonal reactions have become powerful tools for site-specific modification of proteins (Hackenberger, CPR; Schwarzer, D. Angew. Chemie-Int. Ed. 2008, 47(52), 10030; Spicer, CD; Davis, BG Nat. Commun. 2014, 5, 4740). Through these reactions, various protein- and antibody-conjugates can be obtained, which carry functional modules such as fluorophores and other spectral markers, polymers, toxins, and small molecules and proteins similar to post-translational protein modifications. Therefore, from the study of protein biological functions and the development of new imaging technologies to promising new diagnostic medical methods, protein-based drug design and targeted drug delivery, chemically selective protein modification technology has greatly promoted these basic research.

[0002] In recent years, researchers have focused on two different aspects of bioorthogonal reaction engineering for modifying proteins (Sletten, EM; Bertozzi, CR Angew. Chemie-Int. Ed. 2009, 48 (38), 6974). On the one hand, much effort has been devoted to rapid reactions, which require highly reactive starting materials for the conversion of unique functionalities present in protein side chains (Patterson, DM; Nazarova, LA; Prescher, JAACS Chem. Biol. 2014, 9 (3), 592; Lang, K.; Chin, JWACS Chem. Biol. 2014, 9 (1), 16). Along with this approach came advanced amber suppression techniques that enabled site-specific labeling, resulting in a number of genetically encoded, highly reactive bioorthogonal reporters that undergo various types of cycloaddition reactions, including strain-promoted alkyne-azide cycloadditions or inverse-demand Diels-Alder reactions (Nikic, I.; Plass, T.; Schraidt, O.; Szymaski, J.; Briggs, JAG; Schultz, C.; Lemke, EA Angew. Chemie-Int. Ed. 2014, 53(8), 2245; Agard, NJ; Prescher, JA; Bertozzi, CRJA Am. Chem. Soc. 2004, 126(46), 15046). On the other hand, researchers have focused on developing and applying high-yield protein modification reactions, especially when large quantities of functional protein conjugates and ideally quantitative conversions are required to avoid tedious purification steps (1). To achieve this goal, high yields in protein expression are particularly important. Since amber repression can lead to low levels of expressed proteins, standard and auxotrophic expression systems are often preferred. A common approach to achieving site-specific labeling in combination with standard protein expression is to place unique cysteine ​​(Cys) residues in the selected protein by site-directed mutagenesis, followed by a cysteine ​​modification strategy (Chalker, JM; Bernardes, GJL; Lin, YA; Davis, BG Chem.-An Asian J. 2009, 4(5), 630).Alternatively, auxotrophic expression systems can be used to introduce azide- or alkyne-containing amino acids (Hoesl, MG; Budisa, N. Angew. Chemie-Int. Ed. 2011, 50(13), 2896), which can be modified using Staudinger ligation and copper-catalyzed azide-alkyne cycloaddition (CuAAC) (Artner, LM; Merkel, L.; Bohlke, N.; Beceren-Braun, F.; Weise, C.; Dernedde, J.; Budisa, N.; Hackenberger, CPR Chem. Commun. 2012, 48(4), 522; van Kasteren, S I; Kramer, HB; Jensen, HH; Campbell, SJ; Kirkpatrick, J.; Oldham, NJ; Anthony, DC; Davis, BGNature 2007, 446(7139), 1105).

[0003] Although both aspects have made significant progress in recent years, the universal and modular availability of highly reactive and complex functional modules for metal-free chemoselective modification reactions is still often challenging. This is due to the need for additional group protection operations when synthesizing reactive building blocks, which can be problematic due to the high reactivity and instability of the functional groups used. For example, the synthesis of a highly reactive cyclooctane-containing fluorescent peptide carrying Xe-cryptoxanthin for molecular imaging requires the use of orthogonal protecting groups that are cumbersome and have low yields (Witte, C.; Martos, V.; Rose, H.M.; Reinke, S.; Klippel, S.; L.; Hackenberger, CPR Angew. Chemie-Int. Ed. 2015, 54(9), 2806).

[0004] Previous Cys residue coupling techniques mainly rely on maleimide coupling. However, maleimide conjugates are often unstable, especially at high concentrations of thiol, which are often easily hydrolyzed and undergo sulfhydryl exchange. For the latest comprehensive review of Cys coupling technology, see Gunnoo, SB; Madder, A.; ChemBioChem. 2016, 17, 529-553. As an optional coupling method, WO 2015 / 169784 discloses a method for preparing C2-disulfide bridged peptides and proteins, wherein the bridging is achieved by a sulfhydryl-alkyne-reaction with an alkyne. US2535174 describes a base-catalyzed addition reaction of esters of saturated aliphatic thiols and vinyl phosphates. J. Bertran-Vicente et al., Nature Comm. 2016, 7, DOI: 10.1038 / ncomms12703 describe a sequence in which protected phosphorous acid is first reacted with an electrophilic disulfide to generate a phosphorothioate, which upon deprotection (e.g., by UV light or alkali) generates a phosphorylated cysteine. This approach has been applied to the synthesis of naturally occurring phosphorylated cysteine ​​peptides.

[0005] The object of the present invention is to provide other methods for preparing conjugates, and to provide other conjugates. DETAILED DESCRIPTION OF THE INVENTION

[0007] definition

[0008] Those skilled in the art know that the terms "a" or "an" used in the present application may refer to "one (1) one", "one (1) one or more", or "at least one (1) one", depending on the circumstances.

[0009] Halogen, unless otherwise defined: refers to an element of main group 7, preferably fluorine, chlorine, bromine and iodine, more preferably fluorine, chlorine and bromine, and even more preferably bromine when combined with magnesium (Mg).

[0010] Alkyl, unless otherwise defined elsewhere: refers to a group having preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 )-carbon atom saturated straight chain or branched hydrocarbon group. For example: methyl, ethyl, propyl, 1-methylethyl, butyl, etc.

[0011] Alkenyl, unless otherwise defined elsewhere, refers to an unsaturated straight or branched hydrocarbon group having a double bond. 2 -C 8 )-、(C 2 -C 6 )-or(C2 -C 4 )-alkenyl. For example: vinyl, 1-propenyl, 3-butenyl, etc.

[0012] Alkynyl, unless otherwise defined elsewhere, refers to an unsaturated straight or branched hydrocarbon group having a triple bond. Alkynyl is preferably (C 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkynyl. For example: ethynyl, 1-propynyl, etc.

[0013] Alkoxy (alkyl radical-O-), unless otherwise defined elsewhere: refers to an alkyl radical attached to the basic structure via an oxygen atom (-O-). Alkoxy is preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 )-alkoxy. For example: methoxy, ethoxy, propoxy, 1-methylethoxy, etc.

[0014] Similarly, unless otherwise defined elsewhere, alkenyloxy and alkynyloxy are alkenyl radicals and alkynyl radicals, respectively, attached to the base structure via -O-. 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkenyloxy. Alkynyloxy is preferably (C 3 -C 10 )-、(C 3 -C 6 )-or(C 3 -C 4 )-alkynyloxy.

[0015] Alkylcarbonyl (alkyl radical -C(=O)-), unless otherwise defined: Alkylcarbonyl is preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 Here, the number of carbon atoms refers to the alkyl radical in the alkylcarbonyl group.

[0016] Similarly, unless defined elsewhere, alkenylcarbonyl and alkynylcarbonyl are alkenyl radicals and alkynyl radicals, respectively, attached to the base structure via -C(=O)-.2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkenylcarbonyl. Alkynylcarbonyl is preferably (C 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkynylcarbonyl.

[0017] Alkoxycarbonyl (alkyl radical -OC(=O)-), unless otherwise defined elsewhere: Alkoxycarbonyl is preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 Here, the number of carbon atoms refers to the alkyl radical in the alkoxycarbonyl group.

[0018] Similarly, unless defined elsewhere, alkenyloxycarbonyl and alkynyloxycarbonyl are respectively alkenyl radicals and alkynyl radicals attached to the base structure via -OC(=O)-. 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkenyloxycarbonyl. Alkynyloxycarbonyl is preferably (C 3 -C 8 )-、(C 3 -C 6 )-or(C 3 -C 4 )-alkynyloxycarbonyl.

[0019] Alkylcarbonyloxy (alkyl radical -C(=O)-O-), unless otherwise defined elsewhere: refers to an alkyl radical attached to the basic structure via a carbonyloxy group (-C(=O)-O-) via an oxygen atom. Alkylcarbonyloxy is preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 )-alkylcarbonyloxy.

[0020] Similarly, unless defined elsewhere, alkenylcarbonyloxy and alkynylcarbonyloxy are alkenyl radicals and alkynyl radicals, respectively, attached to the base structure via (-C(=O)-O-). 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkenylcarbonyloxy. Alkynylcarbonyloxy is preferably (C 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkynylcarbonyloxy.

[0021] Alkylthio, unless otherwise defined elsewhere, refers to an alkyl radical attached to the base structure via -S-. Alkylthio is preferably (C 1 -C 8 )-、(C 1 -C 6 )-or(C 1 -C 4 )-alkylthio.

[0022] Similarly, unless otherwise specified elsewhere, alkenylthio and alkynylthio are respectively alkenyl radicals and alkynyl radicals attached to the base structure via -S-. 2 -C 8 )-、(C 2 -C 6 )-or(C 2 -C 4 )-alkenylthio. Alkynylthio is preferably (C 3 -C 8 )-、(C 3 -C 6 )-or(C 3 -C 4 )-alkynylthio.

[0023] Unless otherwise defined elsewhere, the term "substituted" or "optionally substituted" refers to a very broad range of substitution patterns. It can be seen from the disclosure of the present invention that, in particular, position R 1 , and ● allow substitution with a variety of organic (macro)molecules. The present invention considers that the structures of these molecules are irrelevant to the methods disclosed in the present invention and the conjugates produced therefrom. Therefore, it would be an undue limitation to limit the principle of this novel and inventive concept to only certain molecules. Nevertheless, the present invention considers that the terms refer to organic substituents or their salts, respectively, which can be substituted multiple times with other organic substituents or their salts, respectively. Examples of such complex substituents have been prepared and presented in this application (see, e.g., Schemes 7, 8, Figure 4 Preferably, the term "substituted" refers to a group substituted by one or more substituents selected from nitro, cyano, Cl, F, Cl, Br, -NH-R, NR 2 、COOH、-COOR、-OC(O)R-NH 2 、-OH、-CONH 2 CONHR, CON(R) 2 , -SR, -SH, -C(O)H, -C(O)R, (C 1 -C 20 )-alkyl, (C 1 -C 20 )-alkoxy, (C 2 -C 20 )-allyl, (hetero)cyclic rings of 3 to 8 ring members (wherein, if present, the heteroatom or atoms are independently selected from N, O and S), (hetero)aromatic systems having 5 to 12 ring atoms (e.g., phenyl, pyridyl, naphthyl, etc.), wherein R may again represent any of these substituents, and the substitution may be repeated multiple times, for example, the substitution may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times; see, the following ● substituents:

[0024] wherein # indicates the position of Y (sulfur or oxygen in the compounds used herein) if ● is already part of a compound such as formula (I) or (III). However, one skilled in the art will appreciate that a general substitution pattern cannot be used to simply describe an alkyl chain substituted with, for example, a polysaccharide having 40 units.

[0025] As used herein, the term "peptide" refers to an organic compound comprising two or more amino acids covalently linked by peptide bonds (amide bonds). Peptides can be named according to the number of constituent amino acids, i.e., a dipeptide contains two amino acid residues, a tripeptide contains three amino acid residues, etc. Peptides containing ten or fewer amino acids can be called oligopeptides, while peptides containing more than ten amino acid residues (e.g., having up to about 30 amino acid residues) are polypeptides. The amino acids can form at least one ring or a branched or unbranched chain or a mixture thereof. Proteins and antibodies are peptides and are therefore covered by this term, but may be named separately due to their importance.

[0026] As used herein, the term "amino acid" refers to a 3 )-COOH group. In one embodiment, the term "amino acid" refers to the naturally occurring amino acids: arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leucine (leicine), phenylalanine, valine, proline and glycine. However, the term also broadly encompasses non-naturally occurring amino acids.

[0027] The amino acids and peptides according to the invention may also be modified with functional groups. Non-limiting examples are sugars like N-acetylgalactosamine (GalNAc), or protecting groups like fluorenylmethoxycarbonyl (Fmoc)-modifications or esters.

[0028] The term "protein" refers to a peptide comprising one or more long chains of amino acid residues, such as peptides having greater than about 30 amino acid residues. Proteins perform a wide range of functions in vivo and in vitro, including catalysis of metabolic reactions, DNA replication, response to stimuli, and transport of molecules, catalysis of reactions. Proteins are folded into specific three-dimensional structures. The residues in proteins are often chemically modified, such as by post-translational modifications, thereby changing the physical and chemical properties, folding, stability, activity and ultimately the function of the protein. Sometimes proteins are attached with non-peptide groups, which may be referred to as prosthetic groups or cofactors. Various proteins, including enzymes and coenzymes, can also work together to perform a specific function, and they often combine to form stable protein complexes. All of these forms are encompassed by the term "protein".

[0029] The term "protein tag" as used herein refers to a peptide sequence that can be attached to a protein or other sulfhydryl-containing compound via a linker according to the present invention for various purposes. Non-limiting examples of protein tags are affinity tags, solubilization tags, chromatography tags, epitope tags, and reporter enzymes.

[0030] Affinity tags are attached to proteins and other sulfhydryl-containing compounds according to the invention via a linker so that they can be purified, for example, using affinity techniques. These tags include, for example, chitin binding protein (CBP), maltose binding protein (MBP) and glutathione-S-transferase (GST) or poly (histidine) tags.

[0031] Solubilizing tags can be used to help proteins fold correctly and prevent protein precipitation. These tags include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as solubilizing agents, such as MBP and GST.

[0032] Chromatographic tags are used to alter the chromatographic properties of proteins to provide different resolutions in specific separation techniques. Typically, these tags consist of polyanionic amino acids, such as the FLAG-tag.

[0033] Epitope tags are short peptide sequences that are chosen because they can reliably generate high-affinity antibodies in many different populations. These tags are often derived from viral genes. Epitope tags include V5-tags, Myc-tags, HA-tags, and NE-tags. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, and antibody purification.

[0034] As used herein, the term "reporter enzyme" refers to any known enzyme that enhances the signal in a biochemical assay. Non-limiting examples of such enzymes are colorant-forming enzymes such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or glucose oxidase (GOX); fluorescent proteins such as green fluorescent protein (GFP), redox-sensitive GFP (RoGFP), blue fluorescent protein (Azurite), or emerald fluorescent protein (Emerald); luciferase, a class of oxidases that produce bioluminescence (e.g., firefly luciferase (EC 1.13.12.7)); chloramphenicol acetyltransferase (CAT); β-galactosidase; or β-glucuronidase.

[0035] Non-limiting examples of protein tags are: AviTag, a peptide that allows a protein to be biotinylated by the BirA enzyme and thereby be isolated by streptavidin (GLNDIFEAQKIEWHE); calmodulin-tag, a peptide bound by calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL); polyglutamic acid tag, a peptide that binds efficiently to anion exchange resins such as Mono-Q (EEEEEE); E-tag, a peptide recognized by an antibody (GAPVPYPDPLEPR); FLAG-tag, a peptide recognized by an antibody (DYKDDDDK); HA-tag, a peptide derived from hemagglutinin recognized by an antibody (YPYDVPDYA); His- Tag, 5-10 histidines conjugated to nickel or cobalt chelates (HHHHHH); Myc-tag, a peptide derived from c-myc recognized by antibodies (EQKLISEEDL); NE-tag, a novel 18 amino acid synthetic peptide recognized by monoclonal IgG1 antibodies (TKENPRSNQEESYDDNES), which can be used in a wide range of applications including western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation and recombinant protein affinity purification; S-tag, a peptide derived from ribonuclease A (KETAAAKFERQHMDS); SBP-tag, a peptide conjugated to streptavidin (MDEKTTGWRGGHVVEGLAGELEQLRARLE HHPQGQREP); Softag 1, for mammalian expression (SLAELLNAGLGGS); Softag 3, for prokaryotic expression (TQDPSRVG); Strep-tag, a peptide that binds to streptavidin or a modified form of streptavidin called streptactin (Strep-tag II: WSHPQFEK); TC tag, a tetracysteine ​​tag recognized by FIAsH and ReAsH bisarsenic compounds (CCPGCC); V5 tag, a peptide recognized by an antibody (GKPIPNPLLGLDST); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK); Xpress tag (DLYDDDDK); Isopeptag, a peptide covalently bound to pilin-C protein (TDKDMTITFTNKKDAE); SpyTag, a peptide covalently bound to SpyCatcher protein (AHIVMVDAYKPTK); SnoopTag, a peptide covalently bound to SnoopCatcher protein (KLGDIEFIKVNK); BCCP (biotin carboxyl carrier protein), a protein domain biotinylated by BirA to enable recognition by streptavidin;Glutathione-S-transferase-tag, a protein that binds to immobilized glutathione; green fluorescent protein-tag, a protein that is autofluorescent and can be bound by nanobodies; Halo-tag, a protein that is covalently bound to HaloLink; TM Mutant hydrolases of resin (Promega); maltose binding protein-tag, a protein that binds to amylose agarose; Nus-tag; thioredoxin-tag; Fc-tag, derived from the immunoglobulin Fc domain, allows dimerization and solubilization. Can be used for purification on protein-A sepharose, designed intrinsic disorder tags contain disorder-promoting amino acids (P, E, S, T, A, Q, G, ...), alkaline phosphatase (AP), horseradish peroxidase (HRP), glucose oxidase (GOX), green fluorescent protein (GFP), redox-sensitive GFP (RoGFP), blue fluorescent protein (Azurite), emerald fluorescent protein (Emerald), firefly luciferase (EC 1.13.12.7)), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, tubulin-tyrosine ligase (TTL).

[0036] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule, preferably consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, which are generally interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. The heavy chain constant region may include, for example, three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (referred to herein as VL) and a light chain constant region. The light chain constant region consists of a domain (CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL is generally composed of three CDRs and up to four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0037] As used herein, the term "complementarity determining region" (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain whose presence is essential for antigen binding. Each variable domain typically has three CDR regions, designated CDR1, CDR2, and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain, about residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; and / or those residues from a "hypervariable loop" (e.g., about residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and about residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain). In some cases, the complementarity determining region may include amino acids from both the CDR regions and the hypervariable loops as defined by Kabat.

[0038] According to the amino acid sequence of the constant domain of its heavy chain, intact antibodies can be divided into different "classes". Intact antibodies are divided into five major classes: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The preferred class of immunoglobulins used in the present invention is IgG.

[0039] The heavy chain constant domains corresponding to different classes of antibodies are called [α], [δ], [ε], [γ] and [μ], respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known. The antibodies used herein are conventionally known antibodies and their functional fragments.

[0040] "Functional fragments" or "antigen-binding antibody fragments" of antibodies / immunoglobulins are defined herein as fragments of antibodies / immunoglobulins that retain the antigen binding region (e.g., the variable region of IgG). The "antigen binding region" of an antibody is generally located in one or more highly variable regions of the antibody, e.g., CDR1, -2 and / or -3 regions; however, variable "framework" regions may also play an important role in antigen binding, such as by providing a skeleton for the CDRs. Preferably, the "antigen binding region" comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and amino acid residues 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and amino acid residues 4 to 111 of VH, and complete VL and VH chains (amino acid positions 1 to 109 of VL and amino acid positions 1 to 113 of VH; amino acids are numbered according to WO 97 / 08320).

[0041] The "functional fragment", "antigen-binding antibody fragment" or "antibody fragment" of the present invention includes but is not limited to Fab, Fab', Fab'-SH, F(ab') 2 and Fv fragments; diabodies; single domain antibodies (DAb), linear antibodies; single chain antibody molecules (scFv); and multispecific antibodies composed of antibody fragments, such as bispecific and trispecific antibodies. Antibodies other than "multispecific" or "multifunctional" antibodies are understood to have the same binding site. F(ab') 2 Alternatively, the Fab can be engineered to reduce or completely remove the intermolecular disulfide interactions that exist between the CH1 and CL domains.

[0042] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the Fc region or constant region amino acid residues are numbered according to the EU numbering system (also referred to as the EU index).

[0043] The antibody variants or antigen-binding antibody fragment variants encompassed by the present invention are molecules that retain the binding activity of the antibody or antigen-binding antibody fragment.

[0044] "Binding proteins" encompassed in the present invention are, for example, antibody mimetics, such as Affibodies, Adnectins, Anticalins, DARPins, Avimers and Nanobodies.

[0045] "Human" antibodies or antigen-binding fragments thereof are defined herein as those that are non-chimeric (e.g., non-"humanized") and non-derived from non-human species (in whole or in part). Human antibodies or antigen-binding fragments thereof may be derived from humans or may be synthetic human antibodies. "Synthetic human antibodies" are defined herein as antibodies having sequences that are derived in whole or in part from computer-simulated synthetic sequences based on analysis of known human antibody sequences. For example, a database of human antibody or antibody fragment sequences may be analyzed and a polypeptide sequence may be designed using the data obtained therefrom, thereby achieving computer simulation design of human antibody sequences or fragments thereof. Another example of a human antibody or antigen-binding fragment thereof is those encoded by nucleic acids isolated from a human antibody sequence library (e.g., such libraries are based on antibodies obtained from natural sources of humans).

[0046] A "humanized antibody" or a humanized antigen-binding fragment thereof is defined herein as an antibody that: (i) is derived from a non-human source (e.g., a transgenic mouse with a heterologous immune system) and is based on a human germline sequence; (ii) wherein the amino acids in the framework region of a non-human antibody are partially replaced with human amino acid sequences by genetic engineering; or (iii) is CDR-grafted, wherein the CDRs of the variable domain are from a non-human source, while one or more frameworks of the variable domain are of human origin, and the constant domains (if any) are of human origin.

[0047] A "chimeric antibody" or antigen-binding fragment thereof is defined herein as an antibody in which the variable domains are derived from a non-human source and part or all of the constant domains are derived from a human source.

[0048] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody group, that is, except for possible mutations such as naturally occurring mutations that may be present in small amounts, the individual antibodies that make up the group are all identical. Therefore, the term "monoclonal" indicates that the characteristic of an antibody is not a mixture of discrete antibodies. Different from polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. In addition to its specificity, the advantage of a monoclonal antibody preparation is that they are generally not contaminated by other immunoglobulins. The term "monoclonal" should not be construed as requiring the production of antibodies by any ad hoc method. The term "monoclonal antibody" specifically includes chimeric antibodies, humanized antibodies, and human antibodies.

[0049] An "isolated" antibody is one that has been identified and separated from components of the cell in which it is expressed. Contaminants of the cell are substances that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous lysates.

[0050] As used herein, an antibody "specifically binds to" means "specifically binds to / targets" or "specifically recognizes" a target antigen, such as a tumor-associated polypeptide antigen target, and is an antibody that binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent targeting cells or tissues expressing the antigen, and does not significantly cross-react with other proteins or with proteins other than orthologs and variants (such as mutants, splice variants, or proteolytic truncations) of the above-mentioned antigen target. The term "specifically recognizes" or "specifically binds to" or "specifically binds to / targets" a specific polypeptide or an epitope on a specific polypeptide target, for example, can be represented by an antibody or an antigen-binding fragment thereof, wherein the monovalent K of the antibody or antigen-binding fragment to the antigen is 1. D Less than about 10 -4 M, or less than about 10 -5M, or less than about 10 -6 M, or less than about 10 -7 M, or less than about 10 -8 M, or less than about 10 -9 M, or less than about 10 -10 M, or less than about 10 -11 M, or less than about 10 -12 M, or less. If the antibody is able to distinguish the antigen from one or more reference antigens, the antibody "specifically binds to", "specifically binds to / targets" or "specifically recognizes" the antigen. In its most common form, "specific binding", "specifically binds to", "specifically binds to / targets" or "specifically recognizes" refers to the ability of an antibody to distinguish a target antigen from an unrelated antigen, for example, as determined by one of the following methods. These methods include, but are not limited to, surface plasmon resonance (SPR), protein blotting, ELISA-, RIA-, ECL-, RMA-experiments and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be performed by standard chromogenic methods (such as a second antibody with horseradish peroxidase and hydrogen peroxide with tetramethylbenzidine). The reaction in certain wells is scored by, for example, the optical density at 450nm. A typical background (= negative reaction) can be 0.1OD; a typical positive reaction can be 1OD. This means that the positive / negative difference is greater than 5 times, 10 times, 50 times, and preferably greater than 100 times. Typically, rather than using a single reference antigen, a panel of about three to five unrelated antigens, such as milk powder, BSA, transferrin, etc., is used for determination of binding specificity.

[0051] "Binding affinity" or "affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The dissociation constant "K D "Affinity" is often used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), that is, how tightly a ligand binds to a specific protein. Ligand-protein affinity is affected by non-covalent intermolecular interactions between the two molecules. Affinity can be determined by common methods known in the art, including those described herein. In one embodiment, the "K" according to the present invention is D ” or “K DThe "value" is determined by surface plasmon resonance analysis using a suitable instrument, including but not limited to a Biacore instrument such as Biacore T100, Biacore T200, Biacore 2000, Biacore 4000, Biacore 3000 (GE Healthcare Biacore, Inc.) or a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.).

[0052] As used herein, the terms "nucleoside" and "nucleoside moiety" refer to a nucleic acid subunit comprising a sugar group and a heterocyclic base, and analogs of the subunit, such as modified or naturally occurring deoxyribonucleosides or ribonucleosides or any chemical modifications thereof. Other groups (e.g., protecting groups) may be attached to any component of the nucleoside. Modifications of nucleosides include, but are not limited to, sugar modifications at the 2'-, 3'-, and 5'-positions, pyrimidine modifications at the 5- and 6-positions, purine modifications at the 2-, 6-, and 8-positions, modifications at the exocyclic amine, substitution of 5-bromouracil, and the like. The synthesis of oligonucleotides may be accomplished by appropriately protecting and derivatizing nucleosides by methods known in the art, such as solid phase automated synthesis using nucleoside phosphoramidite monomers, H-phosphonate coupling, or phosphotriester coupling.

[0053] "Nucleotide" or "nucleotide moiety" refers to a nucleic acid subunit comprising a phosphate group, a sugar group and a heterocyclic base, and analogs of the subunit. Other groups (e.g., protecting groups) may be attached to any component of the nucleotide. The term "nucleotide" may refer to a modified or naturally occurring deoxyribonucleotide or ribonucleotide. In some cases, nucleotides comprise purines and pyrimidines, including thymine, cytosine, guanine, adenine and uracil. The term "nucleotide" is intended to include those moieties that contain not only known purine and pyrimidine bases, such as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U), but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. Such modifications include, for example, diaminopurine and its derivatives, inosine and its derivatives, alkylated purine or pyrimidine, acylated purine or pyrimidine, thiolated purine or pyrimidine, etc., or adding a protecting group, such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamide, dibutylformamide, dimethylacetamide, N,N-diphenylcarbamate, etc. The purine or pyrimidine base may also be an analog of the aforementioned group; suitable analogs are known to those skilled in the art and are described in relevant textbooks and literature. Common analogs include, but are not limited to: 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-mercaptoguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil Uracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-mercaptouracil, 5-methyl-2-mercaptouracil, 5-(2-bromovinyl)uracil, uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-mercaptopurine and 2,6-diaminopurine.

[0054] As used herein, the term "oligonucleotide" refers to a polynucleotide formed by a plurality of nucleotide units connected as defined above. The nucleotide units each include a nucleoside unit linked together by a phosphate linking group or its analogs. The term "oligonucleotide" also refers to a plurality of nucleosides linked together by a linking group (e.g., a thiophosphate linking group or a square amide linking group) other than a phosphate linking group. The oligonucleotide may be naturally occurring or non-naturally occurring. In some cases, the oligonucleotide may include ribonucleotide monomers (i.e., may be oligoribonucleotides) and / or deoxyribonucleotide monomers. As an illustrative example, an oligonucleotide may include 2 to 50 nucleotide units, such as including 2 to 40 nucleotide units, such as including 5 to 35 nucleotide units, such as including 10 to 35 nucleotide units, such as including 15 to 30 nucleotide units.

[0055] The term "monosaccharide" as used herein refers to a sugar of the general formula C m (H 2 O) n open-chain or cyclic compounds wherein m is 3, 4, 5, 6, 7 or 8 and n is 2, 3, 4, 5, 6, 7 or 8. However, the term also covers derivatives of these basic compounds in which one hydroxyl group (OH) is replaced by an amino group (NH 2 ) replaced by a hydroxyl group (e.g., glucosamine), a deoxy sugar in which at least one hydroxyl group (OH) is replaced by a hydrogen atom (H) (e.g., deoxyribose). Preferred examples of monosaccharides are D-(+)-glyceraldehyde; D-(-)-erythrose; D-(-)-threose; D-(-)-ribose; D-(-)-arabinose; D-(+)-xylose; D-(-)-lyxose; D-(+)-allose; D-(+)-altrose; D-(+)-glucose; D-(+)-mannose; D-(-)-glucose; D-(-)-idose; D-(+)-galactose; D-(+)-talose; dihydroxyacetone; D-erythrolose; D-ribulose; D-xylulose; D-psicose; D-fructose; D-sorbose; and D-tagatose. The term "monosaccharide" also includes monosaccharides in which 1, 2, 3 or 4 hydroxyl groups are substituted.

[0056] The term "polysaccharide" refers to a molecule comprising at least 2, preferably at least 5, more preferably at least 10 monosaccharides, which are linked by glycosidic bonds.

[0057] Carbohydrates as used herein encompass monosaccharides and polysaccharides and their derivatives.

[0058] As used herein, a polymer refers to a macromolecule composed of many repeating organic subunits, but these subunits are not polysaccharides, oligonucleotides or peptides. Examples of polymers are polyethylene glycol (PEG), polyoxyethylene (PEO) or polyglycerol (e.g., polyglycerol-polyricinoleate (PGPR)).

[0059] The term "fluorophore" is well known to those skilled in the art and refers to a chemical compound that re-emits light when excited by light. Non-limiting examples are CY 5 , EDANS, xanthine derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red), cyanine derivatives (e.g., indocarbocyanine, oxacarbocyanine, merocyanine), squaramide derivatives (e.g., Seta dyes, Se Tau dyes, Square dyes), naphthalene derivatives (e.g., dansyl or prodan derivatives), coumarin derivatives, oxadiazole derivatives, anthracene derivatives (e.g., anthraquinones such as DRAQ5, DRAQ7, CyTRAK Orange), pyrene derivatives (e.g., Cascade Blue), oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet), acridine derivatives (e.g., diaminoacridine, acridine orange, acridine yellow), arylmethine derivatives (e.g., auramine, crystal violet, malachite green) or tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin).

[0060] As used herein, the term "aliphatic or aromatic residue" refers to an aliphatic substituent, such as an alkyl residue, which however may be optionally further substituted with an aliphatic and / or aromatic substituent, such as an aliphatic residue may be a nucleic acid, a peptide, a protein, an enzyme, an isozyme, an antibody, a nucleotide, an oligonucleotide, a monosaccharide, a polysaccharide, a polymer, a fluorophore, an optionally substituted benzene, etc., as long as the molecule is directly linked to the core structure (at R 1 In the case of a substituted phenyl radical, for example a direct bond to the corresponding oxygen of a compound of formula (I), (I*), (III) or (III*) is aliphatic. An aromatic residue is a substituent in which the direct bond to the core structure is part of an aromatic system, for example an optionally substituted phenyl or pyridyl radical or a peptide if the direct bond of the peptide to the core structure is, for example, via a phenyl residue.

[0061] The term "antibody drug conjugate" or the abbreviation ADC is well known to those skilled in the art and, as used herein, refers to the connection of an antibody or an antigen-binding fragment thereof to a drug (e.g., a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc.). As used herein, a "linker" is any chemical moiety that covalently connects an antibody or an antigen-binding fragment thereof to a drug. The linker may be any linker known to those skilled in the art. As used herein, the term "linker drug conjugate" refers to a molecule or chemical group comprising or consisting of a linker as defined above and a drug and a drug. In this regard, the term "linker drug conjugate" generally refers to that portion of an antibody-drug conjugate that is not an antibody or an antigen-binding fragment thereof. Typically, in a linker-drug conjugate, the linker is covalently linked to the drug. As an illustrative example, the linker used in the present invention may include a self-cleaving peptide that can be cleaved by an enzyme such as cathepsin B. In particular, the linker comprising a self-cleaving peptide used in the present invention may include valine-citrulline-p-aminobenzyloxycarbonyl (VC-PAB, ), valine-alanine-p-aminobenzyloxycarbonyl (VA-PAB, ), lysine-phenylalanine-p-aminobenzyloxycarbonyl (KF-PAB, ) or valine-lysine-p-aminobenzyloxycarbonyl (VK-PAB, ). For example, the linker comprising a self-cleaving peptide may be Best wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), * represents the position of the drug, m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. Linkers with self-cleaving peptides are disclosed in, for example, U.S. Patent Application Publication No. US 2006 / 0074008; G.M. Dubowchik et al., Bioconjuate Chem. 2002, 13, 855-869 or S.O. Doronina et al., Nature Biotechnology, vol. 21, 778-784 (2003), the entire contents of which are incorporated herein by reference. The linker-drug conjugate can be

[0062] Best Wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. As an illustrative example, the drug used in the present invention may be auristatin, preferably monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Preferably, auristatins, in particular MMAE or MMAF, may be used in combination with self-cleaving peptides such as VC-PAB, VA-PAB, KF-PAB or VK-PAB. Therefore, the linker drug conjugate used herein may comprise VC-PAB-MMAE, VC-PAB-MMAF, VA-PAB-MMAE, VA-PAB-MMAF, KF-PAB-MMAE, KF-PAB-MMAF, VK-PAB-MMAE or VK-PAB-MMAF. In particular, the linker drug conjugate may be

[0063] Preferably More preferably wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1.

[0064] Also described herein is an "antibody-fluorophore conjugate" or AFC for short, which refers to a linker of an antibody or antigen-binding fragment thereof and a fluorophore (such as, for example, Cy5). The fluorophore can be linked to the antibody or antigen-binding fragment thereof via a linker. The linker can be any linker known to those skilled in the art. The antibody-fluorophore conjugate may comprise a "linker-fluorophore conjugate". As used herein, the term "linker-fluorophore conjugate" refers to a molecule or chemical group comprising or consisting of a linker as defined above and a fluorophore. In this regard, the term "linker-fluorophore conjugate" generally refers to that portion of the antibody-fluorophore conjugate that is not an antibody or antigen-binding fragment thereof. Typically, in a linker-fluorophore conjugate, the linker is covalently linked to the fluorophore.

[0065] The term "small molecule" as used herein refers to an organic molecule comprising at least two carbon atoms, but preferably comprising no more than 7, 12, 15 or 20 rotatable carbon bonds, more preferably comprising no more than 7, 12 or 15 rotatable carbon bonds, even more preferably comprising no more than 7 or 12 rotatable carbon bonds, having a molecular weight of 100 to 2000 Daltons, preferably 100 to 1000 Daltons, and optionally including one or two metal atoms. As illustrative examples of small molecules only, biotin and the fluorophores EDANS and Cy5 can be mentioned.

[0066] method

[0067] The present invention provides a novel reaction of thiol-containing compounds with olefin or alkyne phosphonothiolates and phosphonates. Scheme 1 describes the general reaction according to the present invention and uses vinyl and ethynyl phosphonothioates and phosphonates by way of illustrative example.

[0068] Scenario 1:

[0069]

[0070] =aliphatic or aromatic residues: e.g., biotin, fluorophores, small molecules, amino acids, peptides, proteins, antibodies, nucleotides, oligonucleotides;

[0071] For example, amino acids, peptides, proteins, antibodies, nucleotides, oligonucleotides;

[0072] R 1 =aliphatic or aromatic residue;

[0073] Y=S (sulfur), O (oxygen).

[0074] The present invention proposes that the method described herein allows the 1 、●and In particular, when When it is an amino acid, peptide, protein, antibody, nucleotide or oligonucleotide, the method according to the present invention is suitable for generating a conjugate. Advantageously, the sulfhydryl group present in such amino acid, peptide, protein or antibody (such as the sulfhydryl group of a cysteine ​​residue) or the sulfhydryl group present in a nucleotide or oligonucleotide reacts chemoselectively with an alkene or alkyne phosphonothioate or phosphonate, thereby providing a chemoselective modification method. Due to this chemoselectivity, sulfhydryl-containing compounds, particularly amino acids, peptides, proteins, antibodies, nucleotides or oligonucleotides can be unprotected, which means that no protecting group is required. Alkenes or alkyne phosphonothioates or phosphonates can be electron-deficient alkene or alkyne phosphonothioates or phosphonates.

[0075] Furthermore, the method according to the invention allows the coupling of two complex molecules. For example, a protein can be coupled to an antibody or another protein.

[0076] This article has demonstrated that:

[0077] Synthesis of electrophilic olefin- and alkyne-phosphonothioates and phosphonates;

[0078] Coupling reactions of electron-deficient olefin- and alkyne-phosphonothioates and phosphonates with sulfhydryl-containing molecules including amino acids, peptides, proteins and antibodies;

[0079] The stability of these conjugates under physiologically relevant conditions;

[0080] - The coupling occurs under physiologically relevant conditions, such as physiological pH.

[0081] The present invention features several innovative aspects that further simplify the accessibility of conjugates such as antibody or protein conjugates through new conjugation chemistry:

[0082] A reaction used to modify sulfhydryl groups in a variety of compounds such as small molecules, proteins, and antibodies;

[0083] Direct coupling can be used to connect two complex molecules (such as fluorophores and proteins or antibodies). For peptides, proteins and antibodies, this connection is cysteine ​​selective.

[0084] Compared with conventional maleimide reagents, the conjugate has high stability and fast coupling reaction;

[0085] Unlike other methods for modifying or coupling peptides, proteins and antibodies, due to the selectivity for cysteine, no manipulation of protecting groups is required after the preparation of the olefin phosphonothioate or alkyne phosphonothioate, or after the preparation of the olefin or alkyne phosphonate and / or after the chemoselective coupling;

[0086] Unsaturated thiophosphonates show important advantages, since they react much faster than the corresponding phosphonates in the thiol addition. For bioconjugation reactions, fast reaction rates are highly desirable, thereby increasing conversion and yield. The resulting thiol-thiophosphonate-conjugates show good stability under physiologically relevant conditions.

[0087] • The high stability of thiophosphonates under acidic conditions is often used to cleave the peptide from the solid support after solid phase synthesis.

[0088] Some examples of phosphonothioates and the addition of thiols to phosphonoesters have been reported, see for example patent documents US3904710A, GB917085, GB863434, DE1064512, and public documents Gao et al., Chemistry Eur. J. 2009, 15 (9), 2064-2070; Khusinova et al., Russian Chemical Bulletin, International Edition, 2004, vol. 53, no. 10, pp. 2253-2256 and Acheson et al., Journal of Chemical Research, Synopses, 1986. However, these documents do not report the addition of thiols to phosphonothioates. In addition, these documents do not involve the modification of biomolecules such as peptides, proteins, antibodies or oligonucleotides, and these documents do not mention the problem of reaction rate and stability under physiologically relevant conditions.

[0089] Generally, the method according to the invention can be implemented to couple different compounds, such as small molecules (such as optionally substituted alkyl, phenyl or heterocyclic), peptides, proteins, antibodies, oligonucleotides or polysaccharides with tags, protein oligonucleotides, etc. Therefore, the present invention relates to a method for preparing a compound of formula (III), the method comprising the following steps:

[0090] The compound of formula (I)

[0091]

[0092] in,

[0093] represents a double or triple bond;

[0094] when When it is a triple bond, X represents R 3 -C;

[0095] when When it is a double bond, X represents (R 3 R 4 )C;

[0096] Y stands for S or O;

[0097] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0098] R 3 Represents H or C 1 -C 8 -alkyl;

[0099] R 4Represents H or C 1 -C 8 -alkyl; and

[0100] ● represents an aliphatic or aromatic residue;

[0101] Reaction with a thiol-containing molecule of formula (II)

[0102]

[0103] in represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 - alkyl, optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system;

[0104] Obtaining a compound of formula (III)

[0105]

[0106] Wherein, if the compound of formula (I) represents a double bond, then represents a single bond; or

[0107] If the compound of formula (I) represents a triple bond, then represents a double bond; and

[0108] ●、R 1 , X and Y are as defined above for the compounds of formula (I) and (II).

[0109] In some embodiments of any of the methods of the present invention, the method wherein the compound of formula (I) reacts with the compound of formula (II) to obtain a compound of formula (III), represents a double bond, X represents (R 3 R 4 )C,R 3 and R 4 Independently represents H or C 1 -C 8 -alkyl, and Preferably, R 3 and R 4 Independently represents H or C 1 -C 6 -alkyl, more preferably representing H or C 1 -C 4 -alkyl, more preferably H or C 1 -C 2 In a preferred embodiment, R3 and R 4 In a preferred embodiment, R 3 and R 4 Both are H.

[0110] Alternatively, in some embodiments of any of the methods of the present invention, the compound of formula (I) reacts with the compound of formula (II) to obtain a compound of formula (III), represents triple bond, X represents R 3 -C, R 3 Represents H or C 1 -C 8 -alkyl, and Preferably, R 3 Table H or C 1 -C 6 -alkyl, more preferably representing H or C 1 -C 4 -alkyl, more preferably H or C 1 -C 2 In a preferred embodiment, R 3 For H.

[0111] In any of the methods of the present invention, the compound of formula (I) in the method is reacted with the compound of formula (II) to obtain the compound of formula (III), and Y can be S (sulfur) or O (oxygen). When Y is S, compounds (I) and (III) represent thiophosphonates. When Y is O, compounds (I) and (III) represent phosphonates. Therefore, in some embodiments, Y is S. In some embodiments, Y is O. In a preferred embodiment of any of the methods, the compound of formula (I) in the method is reacted with the compound of formula (II) to obtain the compound of formula (III), and Y is S. The inventors have found that the addition of a thiol of formula (II) to a triple bond or double bond of a thiophosphonate (i.e., when Y is S) is much faster than the addition to the corresponding phosphonate (i.e., when Y is O). This faster reaction rate is very desirable because it increases conversion and yield.

[0112] The preparation of the compound of formula (I) may comprise:

[0113] The compound of formula (IV)

[0114]

[0115] Where R 1 , X, Y, and ● as defined above and below;

[0116] The oxidant may be selected from tert-butyl hydroperoxide (tBu-OOH), meta-chloroperbenzoic acid (mCPBA), hydrogen peroxide (H 2 O 2 ), iodine (I 2 ), potassium persulfate or oxygen (O 2 ), such as oxygen from air. Preferably, the oxidant is tert-butyl hydroperoxide (tBu-OOH). The preparation of the compound of formula (IV) may include:

[0117] Make phosphorus trihalide (X), preferably PCl 3 Sequentially with (i)R 1 -OH(XI);(ii) (XII), where R 2 Independently represents C 1 -C 8 -alkyl; (iii) (XIII), wherein Hal represents a halogen selected from Cl, Br and I, preferably Br; and (iv) ●-YH (XIV) to produce a compound of formula (IV); wherein R 1 , X, Y, and ● as defined above and below. The phosphorus trihalide may be PCl 3 , PBr 3 or PI 3 , preferably PCl 3 . R in (XII) 2 Independently represents C 1 -C 8 -alkyl, preferably C 1 -C 6 -alkyl, more preferably C 1 -C 4 -alkyl, even more preferably C 1 -C 3 -alkyl. Preferably, the two R 2 Even more preferably, the two R 2 is isopropyl. Preferably, step (iv) (wherein ●-YH(XIV) reacts) is carried out in the presence of tetrazole. The tetrazole may be unsubstituted tetrazole or substituted tetrazole.

[0118] Alternatively, when Y is S, the preparation of the compound of formula (I) may include:

[0119] The compound of formula (V)

[0120]

[0121] reacting with a compound of formula (VIa) or (VIb) to produce a compound of formula (I);

[0122]

[0123] Wherein EWG represents an electron withdrawing group, preferably, the electron withdrawing group is selected from wherein # indicates the position of S; Hal represents a halogen selected from Cl, Br and I, preferably Cl; and

[0124] Where R 1 ,X, and ● are as defined above and below. Preferably, in the compound of formula (V) both R 1 More preferably, when the compound of formula (VIa) is used, EWG is The compound of formula (V) can be prepared, for example, by a process comprising:

[0125] make (XX) and (XXI) to obtain a compound of formula (V), wherein R 1 , X and As defined above and below; Hal 1 is a halogen selected from Cl, Br and I, preferably Cl; Hal 2 is a halogen selected from Cl, Br and I, preferably Br. The compound of formula (VIa) can be prepared, for example, by reacting the compound (XXX) is reacted with ●-SH (XXXI) to obtain a compound of formula (VIa), wherein EWG is an electron withdrawing group, preferably selected from An electron withdrawing group, wherein # represents the position of S; more preferably Preferably, in the compounds of formula (XXX), both EWG are identical; and wherein ● is as defined above and below. Compounds of formula VIb can be prepared according to methods known from the literature, for example by reacting thiol ●-SH(XXXI) with sulfuryl chloride (see, for example, Allared, F. et al., Synthetic Metals, 120(1-3), 1061-1062; 2001) or thionyl chloride (see, for example, Masaki, Yukio et al., Chemical & Pharmaceutical Bulletin, 33(5), 1930-40; 1985), or by reacting thiol ●-SH(XXXI) with N-chlorosuccinimide (NCS) (see, for example, Kawamura, Takamasa et al., European Journal of Organic Chemistry, 2015(4), 719-722; 2015) or chlorine (see, for example, E. Schneider, Chemische Berichte 84, 911-916 (1951)), wherein ● is as defined above and below.

[0126] Preferably, when the compound of formula (I) is prepared by reacting a compound of formula (V) with a compound of formula (VIa) or (VIb), is a double bond and X represents (R 3 R 4 )C, where R 3 and R 4 As defined above and below.

[0127] The present invention also relates to a method for preparing a compound of formula (III*), the method comprising the steps of:

[0128] The compound of formula (I*)

[0129]

[0130] in,

[0131] V is for C 1 -C 8 - alkyl, preferably methyl, ethyl or propyl, more preferably methyl;

[0132] X represents (R 3 R 4 )C;

[0133] Y stands for S or O;

[0134] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0135] R 3 Represents H or C 1 -C 8 -alkyl;

[0136] R 4 Represents H or C 1 -C 8 -alkyl; and

[0137] ● represents an aliphatic or aromatic residue;

[0138] Reaction with a thiol-containing molecule of formula (II)

[0139]

[0140] in represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 - alkyl, optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system;

[0141] Obtaining a compound of formula (III*)

[0142]

[0143] in ●、V、R 1 , X and Y are as defined for the compounds of formula (I*) and (II).

[0144] In any of the methods of the present invention, in the method, the compound of formula (I*) is reacted with the compound of formula (II) to obtain the compound of formula (III*), Y can be S (sulfur) or O (oxygen), that is, when Y is S, the compounds (I*) and (III*) represent thiophosphonates, and when Y is O, the compounds (I*) and (III*) represent phosphonates. Therefore, in some embodiments, Y is S. In some embodiments, Y is O. In a preferred embodiment of any of the methods, in the method, the compound of formula (I*) is reacted with the compound of formula (II) to obtain the compound of formula (III*), Y is S, because the inventors have found that the addition of thiols to the triple or double bonds of thiophosphonates (i.e., when Y is S) is much faster than the addition to the corresponding phosphonates (i.e., when Y is O).

[0145] The preparation of the compound of formula (I*) may comprise:

[0146] The compound of formula (IV*)

[0147]

[0148] Where X represents (R 3 R 4 )C, and Y, R 1 , R 3 , R 4 , V and ● are as defined above and below;

[0149] The oxidant may be selected from tert-butyl hydroperoxide (tBu-OOH), meta-chloroperbenzoic acid (mCPBA), hydrogen peroxide (H 2 O 2 ), iodine (I 2 ), potassium persulfate or oxygen (O 2 ), such as oxygen from air. Preferably, the oxidant is tert-butyl hydroperoxide (tBu-OOH). The preparation of the compound of formula (IV*) may include:

[0150] Make phosphorus trihalide (X), preferably PCl 3 Sequentially with (i)R 1 -OH(XI);(ii) (XII), where R 2 Independently represents C 1 -C 8 -alkyl; (iii) (XIII*), wherein Hal represents a halogen selected from Cl, Br and I, preferably Br, and X represents (R 3 R 4 )C; and (iv) ●-YH (XIV) to produce a compound of formula (IV *);

[0151] Where R 1 , R 3 , R 4 , V, Y and ● are as defined above and below. The phosphorus trihalide may be PCl 3 , PBr 3 or PI 3 , preferably PCl 3 . R in (XII) 2 Independently represents C 1 -C 8 -alkyl, preferably C 1 -C 6 -alkyl, more preferably C 1 -C 4 -alkyl, even more preferably C 1 -C 3 -alkyl. Preferably, the two R 2 Even more preferably, the two R 2is isopropyl. Preferably, step (iv) (wherein ●-YH(XIV) reacts) is carried out in the presence of tetrazole. The tetrazole may be unsubstituted tetrazole or substituted tetrazole.

[0152] Alternatively, when Y is S, the preparation of the compound of formula (I*) may comprise:

[0153] The compound of formula (V*)

[0154]

[0155] reacting with a compound of formula (VIa) or (VIb) to produce a compound of formula (I*);

[0156]

[0157] Wherein EWG represents an electron withdrawing group, preferably, the electron withdrawing group is selected from wherein # indicates the position of S; Hal represents a halogen selected from Cl, Br and I, preferably Cl; and

[0158] Where X represents (R 3 R 4 )C, and R 1 , R 3 , R 4 , V and ● are as defined above and below. Preferably, in the compound of formula (V*) two R 1 More preferably, when the compound of formula (VIa) is used, EWG is The compound of formula (V*) can be prepared, for example, by a process comprising:

[0159] make (XX) and (XXI*) to obtain a compound of formula (V*), wherein X represents (R 3 R 4 )C, and R 1 , R 3 , R 4 and V are as defined above and below; and Hal 1 is a halogen selected from Cl, Br and I, preferably Cl; Hal 2 is a halogen selected from Cl, Br and I, preferably Br. The compounds of formula (VIa) and (VIb) can be prepared, for example, as described above and below.

[0160] In some embodiments of any of the methods of the invention, R 1 represents C optionally substituted by at least one of the following 1 -C8 -alkyl: wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n 、F、Cl、Br、I、-NO 2 、-N(C 1 -C 8 -alkyl)H, -NH 2 、-N 3 、-N(C 1 -C 8 -alkyl) 2 , =O, C 3 -C 8 -cycloalkyl, -SS-(C 1 -C 8 -alkyl), wherein n is 1, 2, 3, 4, 5 or 6 hydroxy-(C 1 -C 8 -alkoxy) n , C 2 -C 8 -alkenyl or C 2 -C 8 -alkynyl.

[0161] In some embodiments of any of the methods of the invention, R 1 represents an optionally substituted phenyl group, such as Where # represents the position of O.

[0162] In some embodiments of any of the methods of the invention, R 1 represents a phenyl group which is optionally independently substituted by at least one of the following: 1 -C 8 -alkyl, wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n 、F、Cl、I、Br、-NO 2 、-N(C 1 -C 8 -alkyl)H, -NH 2 or -N(C 1 -C 8 -alkyl) 2 .

[0163] In some embodiments of any of the methods of the invention, R 1 represents a 5- or 6-membered heteroaromatic system such as pyridyl.

[0164] In some embodiments of any of the methods of the invention, R 1 Represents C 1-C 8 -alkyl; 1 -C 8 -alkyl) substituted C 1 -C 8 -alkyl; wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n Substituted C 1 -C 8 -alkyl; C substituted by optionally substituted phenyl 1 -C 8 -alkyl; or phenyl; or -NO 2 Substituted phenyl.

[0165] In some embodiments of any of the methods of the invention, R 1 represents methyl, ethyl, propyl or butyl, preferably methyl or ethyl.

[0166] In some embodiments of any of the methods of the invention, R 1 represents optionally -SS-(C 1 -C 8 -alkyl)-substituted aliphatic or aromatic residue. In a preferred embodiment, R 1 represent Where R 10 , R 11 , R 12 and R 13 Each independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 10 , R 11 , R 12 and R 13 Each independently represents hydrogen, methyl or ethyl. In a preferred embodiment, R 1 represent Where R 10 and R 11 independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 10 and R 11 In a further preferred embodiment, R 1 represent Where R 10 and R 11 independently represents hydrogen, methyl or ethyl; and # represents the position of O. In some of these embodiments, R 10 and R11 In some of these embodiments, R 10 is hydrogen, R 11 C 1 -C 6 -alkyl. In some of these embodiments, R 10 is hydrogen, R 11 is methyl or ethyl. In some of these embodiments, R 10 and R 11 In a preferred embodiment, R 1 represent More preferably represents Where R 10 and R 11 As defined above. In another preferred embodiment, R 1 represent Where R 12 and R 13 independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 12 and R 13 R independently represents hydrogen, methyl or ethyl. In an even more preferred embodiment, R 1 represent Where R 12 and R 13 independently represents hydrogen, methyl or ethyl; and # represents the position of O. In some of these embodiments, R 12 and R 13 In some of these embodiments, R 12 is hydrogen, R 13 C 1 -C 6 -alkyl. In some of these embodiments, R 12 is hydrogen, R 13 is methyl or ethyl. In some of these embodiments, R 12 and R 13 same.

[0167] In some embodiments of any of the methods of the invention, R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further substituted, wherein Z is O or NH, and wherein # represents the position of the phenyl group. In some embodiments Z is O. In some embodiments Z is NH. C 1 -C 8-alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl. In a preferred embodiment, R 1 represent Wherein C 1 -C 8 -alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl; wherein Z is O or NH, and wherein # represents the position of O. In another preferred embodiment, R 1 represent Wherein C 1 -C 8 -Alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl; wherein Z is O or NH, and wherein # represents the position of O.

[0168] In some embodiments of any of the methods of the invention, R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further substituted, and wherein # represents the position of the phenyl group. In some embodiments, R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further substituted, and wherein # represents the position of the phenyl group. In a preferred embodiment, R 1 represent wherein # represents the position of O. In another preferred embodiment, R 1 represent Where # represents the position of O.

[0169] In some embodiments of any of the methods of the invention, R 1 represent wherein # represents the position of O and n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably n=0, 1, 2, 3, 4, 5, 6; more preferably n=0, 1, 2, 3, 4; even more preferably n=0, 1, 2, 3, even further preferably n=0, 1, 2, even further preferably n=0, 1; and most preferably n=1.

[0170] In some embodiments of any of the methods of the invention, R 1 Represents optionally C 2 -C 8-alkynyl-substituted aliphatic or aromatic residue. In a preferred embodiment, R 1 It is a high propargyl group.

[0171] In some embodiments of any of the methods of the invention, R 1 represent wherein # represents the position of O, n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably n=0, 1, 2, 3, 4, 5, 6; more preferably n=0, 1, 2, 3, 4; even more preferably n=0, 1, 2, 3, even more preferably n=0, 1, 2, even more preferably n=0, 1; and most preferably n=1, i.e. when n is 1, R 1 represent

[0172] In some embodiments of any of the methods of the invention, ● represents a small molecule; wherein optionally said ● further comprises a linker bound to Y.

[0173] In some embodiments of any of the methods of the invention, ● represents a small molecule such as, for example, an optionally substituted C 1 -C 8 -alkyl, -CH 2 -phenyl,

[0174] wherein # indicates the position of Y. In a preferred embodiment, ● represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C 4 -alkyl, even more preferably C 1 -C 2 -alkyl. In some embodiments, ● represents -CH 2 -phenyl, i.e. benzyl. In a preferred embodiment, ● represents Wherein # indicates the position of Y. In a preferred embodiment, → represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In some embodiments, ● represents wherein # indicates the position of Y. In some embodiments, ● represents wherein # indicates the position of Y. In some embodiments, ● represents Where # indicates the position of Y.

[0175] In some embodiments of any of the methods of the present invention, → represents an optionally substituted phenyl group, preferably Where # indicates the position of Y.

[0176] In some embodiments of any of the methods of the invention, ● represents a radioactive or non-radioactive nuclide, biotin, a reporter enzyme, a nucleotide, an oligonucleotide, such as CY 5 or EDANS fluorophore, amino acid, peptide, optionally substituted 5- or 6-membered heteroaromatic system; wherein optionally, said ● further comprises a linker bound to Y. Thus, in some embodiments, ● represents a radioactive or non-radioactive nuclide. In preferred embodiments, ● represents biotin. In some embodiments, ● represents a reporter enzyme. In preferred embodiments, ● represents a nucleotide. In preferred embodiments, ● represents an oligonucleotide. In preferred embodiments, ● represents a nucleotide such as CY 5 In a preferred embodiment, ● represents an amino acid. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents an optionally substituted 5- or 6-membered heteroaromatic system. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0177] Throughout the specification, regardless of any method or compound regarding "optionally said ● further comprises a linker bound to Y", etc., ● may actually further comprise any linker, and the linker is bound to Y, as described herein for compounds of formula (I), (I*), (III) or (III*), wherein Y is S (sulfur) or O (oxygen), preferably S. The linker may be any linker known to those skilled in the art, such as a peptide linker or a linear or branched hydrocarbon-based moiety. The linker may also comprise a cyclic moiety. The peptide linker may comprise, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 2 or 1 amino acids. If the linker is a hydrocarbon-based moiety, the backbone of the linker may comprise only carbon atoms, but may also comprise heteroatoms such as oxygen (O), nitrogen (N) or sulfur (S) atoms, and / or may comprise a carbonyl group (C=O). The linker may be, for example, C 1 -C 20 A chain of carbon atoms or a 2 -CH 2)-repeat units of a polyethylene glycol-based chain. In typical embodiments of hydrocarbon-based linkers, the linking moiety comprises 1 to about 150, 1 to about 100, 1 to about 75, 1 to about 50, or 1 to about 40, or 1 to about 30, or 1 to about 20, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 backbone atoms. As illustrative examples, the linker can be Wherein # represents the position of Y, and * represents the position of the other part of ●, wherein as a non-limiting example, the other part can be an amino acid, a peptide, an antibody, a protein, a nucleotide, an oligonucleotide or a small molecule; and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. The aforementioned exemplary linkers can also be used, for example, when the specification itself refers to a "linker", or when, for example, in the context of an antibody drug conjugate, it refers to a "linker-drug conjugate", or when, for example, in the context of an antibody fluorophore conjugate, it refers to a "linker-fluorophore conjugate". Those skilled in the art know how to select a suitable linker.

[0178] In some embodiments of any of the methods of the invention, ● a cyclic RGD peptide (c(RDGfK)) representing structure (VII)

[0179]

[0180] Where * represents the position of Y.

[0181] In some embodiments of any of the methods of the present invention, ● represents a phenyl group, wherein the phenyl group is optionally independently selected from C 1 -C 8 -alkyl, C 1 -C 8 -alkoxy, halogen, -CN, -NO 2 、-NH 2 、-N(C 1 -C 8 -alkyl), -N(C 1 -C 8 -alkyl) 2 、-COOH、-COO(C 1 -C 8 -alkyl), -OC(O)-(C 1 -C 8 -alkyl), -C(O)N-(C 1 -C 8 -alkyl), -N(H)-C(O)-(C 1 -C 8-alkyl) is substituted with 1, 2, 3, 4 or 5 substituents, preferably, the phenyl group is optionally selected from C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 substituted with 1 substituent.

[0182] In some embodiments of any of the methods of the invention, ● represents C 1 -C 8 -alkyl, the C 1 -C 8 -alkyl is optionally substituted by at least one substituent selected from the group consisting of: 3 -C 8 -cycloalkyl; heterocyclyl having 3 to 8 ring members, wherein the heteroatoms are selected from N, O, S; C 1 -C 8 -alkoxy; halogen; -CN; -NO 2 ;-NH 2 ;-N(C 1 -C 8 -alkyl); -N(C 1 -C 8 -alkyl) 2 ;-COOH;-COO(C 1 -C 8 -alkyl); -OC(O)-(C 1 -C 8 -alkyl); -CONH 2 ; -C(O)N(C 1 -C 8 -alkyl) 2 ; -C(O)NH-(C 1 -C 8 -alkyl); -N(H)-C(O)-(C 1 -C 8 -alkyl), preferably C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 , phenyl or heteroaromatic system, monosaccharide, polysaccharide, peptide, protein, antibody, nucleotide, oligonucleotide, polymer, amino acid, fluorophore, protein tag (first generation substituent), wherein the first generation substituent can be optionally substituted again by the following groups: C 3 -C 8 -cycloalkyl; heterocyclyl having 3 to 8 ring members, wherein the heteroatoms are selected from N, O, S; C 1 -C8 -alkoxy; halogen; -CN; -NO 2 ;-NH 2 ;-N(C 1 -C 8 -alkyl); -N(C 1 -C 8 -alkyl) 2 ;-COOH;-COO(C 1 -C 8 -alkyl); -OC(O)-(C 1 -C 8 -alkyl); -CONH 2 ; -C(O)N(C 1 -C 8 -alkyl) 2 ; -C(O)NH-(C 1 -C 8 -alkyl); -N(H)-C(O)-(C 1 -C 8 -alkyl), preferably C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 , phenyl or a heteroaromatic system (2nd generation substituent), and wherein the 2nd generation substituent may be substituted again by at least one substituent selected from the same group, and wherein such substitution may be carried out to 3, 4, 5, 6, 7, 8, 9 or 10 generations.

[0183] In some embodiments of any of the methods of the invention, ● represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 -alkyl, an optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system; wherein optionally said ● further comprises a linker bound to Y. Thus, in a preferred embodiment, ● represents an amino acid. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents a nucleotide. In a preferred embodiment, ● represents an oligonucleotide. In some embodiments, ● represents a sugar. In some embodiments, ● represents a polysaccharide. In some embodiments, ● represents a polymer. In some embodiments, ● represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C4 -alkyl, even more preferably optionally substituted C 1 -C 2 -alkyl. In some embodiments, ● represents an optionally substituted phenyl. In some embodiments, ● represents an optionally substituted aromatic 5- or 6-membered heterocyclic ring system. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0184] In a preferred embodiment of any of the methods of the present invention, ● represents an amino acid, a peptide, a protein, an antibody, a nucleotide or an oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. In a more preferred embodiment, ● represents a peptide, a protein, an antibody or an oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. In a preferred embodiment, ● represents an amino acid. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents a nucleotide. In a preferred embodiment, ● represents an oligonucleotide. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0185] In a preferred embodiment of any of the methods of the present invention, ● represents a drug, a protein tag, such as CY 5 or EDANS fluorophore, biotin, protein, peptide, antibody or oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, ● represents a drug. In a preferred embodiment, ● represents a protein tag. In a preferred embodiment, ● represents a linker-drug conjugate. In a preferred embodiment, ● represents a CY 5 In a preferred embodiment, ● represents biotin. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents an oligonucleotide. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0186] In a preferred embodiment of any of the methods of the present invention, ● represents a linker or a linker-drug conjugate. In a preferred embodiment, ● represents a linker, such as a linker comprising VC-PAB, VA-PAB, KF-PAB or VK-PAB, preferably a linker comprising VC-PAB. In a preferred embodiment, ● represents a linker-drug conjugate, such as

[0187] Best wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. More preferably, the linker drug conjugate is Even more preferably Most preferably wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1.

[0188] According to any one of the methods of the present invention, may represent amino acids, peptides, proteins, antibodies, nucleotides, oligonucleotides, sugars, polysaccharides, polymers, optionally substituted C 1 -C 8 -alkyl, optionally substituted phenyl or optionally substituted aromatic 5- or 6-membered heterocyclic ring system. In a preferred embodiment, Represents an amino acid, a peptide, a protein, an antibody, a nucleotide or an oligonucleotide. In a more preferred embodiment, Represents a peptide, protein, antibody or oligonucleotide. In a preferred embodiment, In a preferred embodiment, In a preferred embodiment, Represents a protein. In a preferred embodiment, In a preferred embodiment, In a preferred embodiment, In some embodiments, represents a sugar. In some embodiments, represents a polysaccharide. In some embodiments, Represents a polymer. In some embodiments, represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C 4 -alkyl, even more preferably optionally substituted C 1 -C 2 -alkyl. In some embodiments, represents an optionally substituted C 3 -C 8-alkyl, preferably optionally substituted C 3 -C 6 -alkyl, more preferably optionally substituted C 3 -C 4 -alkyl. In some embodiments, represents an optionally substituted C 5 -C 8 -alkyl, preferably optionally substituted C 6 -C 7 -alkyl. In some embodiments, represents an optionally substituted phenyl group. In some embodiments, represents an optionally substituted aromatic 5- or 6-membered heterocyclic ring system.

[0189] In a preferred embodiment of any of the methods of the present invention, represents an antibody, preferably an IgG antibody, more preferably cetuximab or trastuzumab or brentuximab; a protein, preferably a GFP protein or an eGFP protein, an albumin, a tripeptide, preferably of formula (VIII) (VIII), or formula (IX) (IX) wherein # represents the position of S. Therefore, in a preferred embodiment, In a preferred embodiment, the antibody is an IgG antibody, and even more preferably, cetuximab or trastuzumab or brentuximab. In a preferred embodiment, Represents a protein, more preferably a GFP protein or an eGFP protein. In a preferred embodiment, In a preferred embodiment, Represents a tripeptide. In a preferred embodiment, Representative formula (VIII) (VIII) In a preferred embodiment, Representative formula (IX) (IX) In a preferred embodiment of any of the methods of the present invention, represents an antibody (such as cetuximab, trastuzumab, or brentuximab) and ● represents a protein tag or a 5 or a fluorophore, biotin, peptide, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents an antibody and ● represents a protein tag. In a preferred embodiment, represents antibodies and ● represents CY 5Or EDANS fluorophore. In a preferred embodiment, represents an antibody and ● represents biotin. In a preferred embodiment, represents an antibody and ● represents a peptide. In a preferred embodiment, represents an antibody and ● represents a protein. In a preferred embodiment, represents an antibody and ● represents an oligonucleotide. In a preferred embodiment, represents an antibody and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0190] In a preferred embodiment of any of the methods of the present invention, represents a protein (such as GFP protein or eGFP protein) and ● represents a protein tag or a protein tag such as CY 5 or a fluorophore, biotin, peptide, antibody, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents a protein and ● represents a protein tag. In a preferred embodiment, represents proteins and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents protein and ● represents biotin. In a preferred embodiment, represents a protein and ● represents a peptide. In a preferred embodiment, represents a protein and ● represents an antibody. In a preferred embodiment, represents a protein and ● represents a protein. In a preferred embodiment, represents a protein and ● represents an oligonucleotide. In a preferred embodiment, represents a protein and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0191] In a preferred embodiment of any of the methods of the present invention, represents peptides and ● represents protein tags or such as CY 5 or a fluorophore, biotin, peptide, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents a peptide and ● represents a protein tag. In a preferred embodiment, represents peptides and ● represents such as CY 5 Or EDANS fluorophore. In a preferred embodiment, represents a peptide and ● represents biotin. In a preferred embodiment, represents a peptide and ● represents a peptide. In a preferred embodiment, represents a peptide and ● represents a protein. In a preferred embodiment, represents a peptide and ● represents an oligonucleotide. In a preferred embodiment, represents a peptide and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0192] In a preferred embodiment of any of the methods of the present invention, represents amino acids and ● represents protein tags or such as CY 5 or a fluorophore, biotin, peptide, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents an amino acid and ● represents a protein tag. In a preferred embodiment, represents amino acids and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents an amino acid and ● represents biotin. In a preferred embodiment, represents an amino acid and ● represents a peptide. In a preferred embodiment, represents amino acids and ● represents proteins. In a preferred embodiment, represents an amino acid and ● represents an oligonucleotide. In a preferred embodiment, represents an amino acid and ● represents a small molecule. Optionally, in any one of these embodiments, said ● further comprises a linker bound to Y.

[0193] In a preferred embodiment of any of the methods of the present invention, represents an antibody (such as cetuximab, trastuzumab or brentuximab) and ● represents a linker, a drug or a linker-drug conjugate. Therefore, in a preferred embodiment, represents an antibody and ● represents a linker, such as a linker comprising VC-PAB, VA-PAB, KF-PAB or VK-PAB, preferably a linker comprising VC-PAB. In a preferred embodiment, represents an antibody and ● represents a drug. In a preferred embodiment, represents an antibody and ● represents a linker-drug conjugate, such as

[0194] Best wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. In a more preferred embodiment, represents an antibody and ● represents a linker-drug conjugate, such as

[0195] Even more preferably Most preferably wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. In any of these embodiments, the antibody may be cetuximab, trastuzumab or brentuximab, preferably brentuximab.

[0196] In a preferred embodiment of any of the methods of the present invention, represents nucleotides and represents peptides, proteins, protein tags, antibodies, oligonucleotides, such as CY 5 or a fluorophore, biotin or a small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. In a preferred embodiment, represents a nucleotide and ● represents a peptide. In a preferred embodiment, represents nucleotides and ● represents proteins. In a preferred embodiment, represents a nucleotide and ● represents a protein tag. In a preferred embodiment, represents a nucleotide and ● represents an antibody. In a preferred embodiment, represents a nucleotide and ● represents an oligonucleotide. In a preferred embodiment, represents nucleotides and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents a nucleotide and ● represents biotin. In a preferred embodiment, represents a nucleotide and ● represents a small molecule. Optionally, in any one of these embodiments, said ● further comprises a linker bound to Y.

[0197] In a preferred embodiment of any of the methods of the present invention, represents nucleotides and ● represents linkers.

[0198] In a preferred embodiment of any of the methods of the present invention, represents oligonucleotides and represents peptides, proteins, protein tags, antibodies, oligonucleotides, such as CY 5 or a fluorophore, biotin or a small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. In a preferred embodiment, represents an oligonucleotide and ● represents a peptide. In a preferred embodiment, represents an oligonucleotide and ● represents a protein. In a preferred embodiment, represents an oligonucleotide and ● represents a protein tag. In a preferred embodiment, represents an oligonucleotide and ● represents an antibody. In a preferred embodiment, represents an oligonucleotide and ● represents an oligonucleotide. In a preferred embodiment, represents oligonucleotides and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents an oligonucleotide and ● represents biotin. In a preferred embodiment, represents an oligonucleotide and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0199] In a preferred embodiment of any of the methods of the present invention, represents oligonucleotide and ● represents linker.

[0200] In a preferred embodiment of any of the methods of the present invention, ● represents an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, a peptide, a nucleotide or an oligonucleotide is bound to a solid support. In some embodiments, ● represents an amino acid or a peptide bound to a solid support. In some embodiments, ● represents a nucleotide or an oligonucleotide bound to a solid support. In a preferred embodiment, ● represents a peptide bound to a solid support. As an advantage, the inventors have found that the thiophosphonate of the present invention is highly stable under acidic conditions (such as 90% trifluoroacetic acid (TFA)), which are commonly used to cleave peptides from a solid support. The solid support may be any solid support suitable for solid phase peptide synthesis known to those skilled in the art, or any solid support suitable for solid phase oligonucleotide synthesis. Such solid supports are also referred to as resins. Illustrative examples of solid supports suitable for solid phase peptide synthesis include organic and inorganic supports, such as Merrifield polystyrene resin (copolymer of styrene and 1-2% divinylbenzene), polyacrylamide resin, TentaGel (grafted polymer with polyethylene glycol grafted onto polystyrene), Wang resin (generally based on cross-linked polystyrene, such as in Merrifield resin) or porous glass with defined pore size as examples of inorganic solid supports. Illustrative examples of commercially available solid supports for solid phase peptide synthesis are Rink amide resins or TGR resin. Illustrative examples of solid supports suitable for solid phase oligonucleotide synthesis include glass (controlled pore glass, CPG) and polystyrene with a defined pore size, such as macroporous polystyrene (MPPS). Optionally, in the above-mentioned embodiments in which amino acids, peptides, nucleotides or oligonucleotides are bound to a solid support, the ● further comprises a linker that is bound to Y in the structural formula described herein, particularly compounds (I), (III), (I*) and (III*). In addition, the linker is bound to an amino acid, peptide, nucleotide or oligonucleotide. Therefore, the ● can have a structure of a linker-amino acid-solid support, a linker-peptide-solid support, a linker-nucleotide-solid support or a linker-oligonucleotide-solid support. The "linker" can actually be any linker, and the linker is bound to Y, for example, as described herein for compounds of formula (I), (I*), (III) or (III*), wherein Y is S (sulfur) or O (oxygen), preferably S. The linker can be any linker known to those skilled in the art, such as a peptide linker or a linear or branched hydrocarbon-based moiety. The linker can also contain a cyclic moiety. The peptide linker can contain, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 2 or 1 amino acids. If the linker is a hydrocarbon-based moiety, the backbone of the linker can contain only carbon atoms, but can also contain heteroatoms such as oxygen (O), nitrogen (N) or sulfur (S) atoms, and / or can contain a carbonyl group (C=O). The linker can be, for example, C 1 -C 20 A chain of carbon atoms or a 2 -CH 2 )-repeat units. In typical embodiments of hydrocarbon-based linkers, the linking moiety comprises 1 to about 150, 1 to about 100, 1 to about 75, 1 to about 50, or 1 to about 40, or 1 to about 30, or 1 to about 20, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 backbone atoms. Those skilled in the art will appreciate the selection of appropriate linkers. For example, in some embodiments, the linker may be wherein # represents the position of Y, and * represents the position of an amino acid, peptide, nucleotide or oligonucleotide; and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. The amino acid, peptide, nucleotide or oligonucleotide may be bound to the linker via an N (nitrogen) atom.

[0201] In a preferred embodiment of any of the methods of the present invention, represents an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, a peptide, a nucleotide or an oligonucleotide is bound to a solid support. In some embodiments, Represents an amino acid or peptide bound to a solid support. In some embodiments, Represents a nucleotide or oligonucleotide bound to a solid support. In a preferred embodiment, represents the peptide bound to a solid support.

[0202] The present invention also relates to a method for preparing a compound of formula (I) or formula (I*), the method comprising:

[0203] (I) making the compound of formula (Ia)

[0204] or

[0205] Compounds of formula (I*a)

[0206]

[0207] in

[0208] L represents a linker suitable for binding to an amino acid, a peptide, a nucleotide or an oligonucleotide; and

[0209] X, Y, V, and R 1 As defined above and below,

[0210] reacting with an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, a peptide, a nucleotide or an oligonucleotide is bound to a solid support,

[0211] Obtaining a compound of formula (Ib) or (I*b)

[0212]

[0213] wherein Z represents an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, peptide, nucleotide or oligonucleotide is bound to a solid support; and

[0214] (II) cleaving the compound of formula (Ib) or (I*b) from the solid support to obtain a compound of formula (I) or (I*):

[0215]

[0216] wherein ● represents an amino acid, peptide, nucleotide or oligonucleotide bound to Y via a linker L, and

[0217] X, Y, V, and R 1As defined in step (I) above. In some embodiments, the linker L is Wherein # represents the position of Y, and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. The amino acid, peptide, nucleotide or oligonucleotide is bound to such a linker via a carboxyl group. In some embodiments, the compound of formula (Ia) or (I*a) reacts with a peptide bound to a solid support or with an oligonucleotide bound to a solid support, preferably the compound of formula (Ia) or (I*a) reacts with a peptide bound to a solid support. In some embodiments, the cleavage of step (II) is carried out under acidic conditions, such as using an aqueous solution of trifluoroacetic acid (such as 90% TFA). In this regard, the inventors have found that the thiophosphonate (Y=S) of the present invention is highly stable under acidic conditions, which are particularly useful for cleaving peptides from a solid support. Optionally, in any of these embodiments, the process may further comprise reacting a compound of formula (I) or (I*) with a compound of formula (II) as defined above and below.

[0218] In one embodiment of the method according to the present invention, and stated In the same molecule. Therefore, the present invention also relates to a method, wherein the compound of formula (L)

[0219]

[0220] where as shown by connecting ● and The arc and stated In the same molecule,

[0221] The compound of formula (IIIa) is obtained by reaction:

[0222]

[0223] Among them, if the compound of formula (XX) represents a double bond, then represents a single bond, and X represents (R 3 R 4 )C; or

[0224] If the compound of formula (XX) represents a triple bond, then represents a double bond, and X represents R 3 -C; and

[0225] ●、R 1 , R 3 , R4 and Y are as defined above and below.

[0226] The present invention also relates to a method, wherein the compound of formula (L*)

[0227]

[0228] where as shown by connecting ● and The arc and stated In the same molecule,

[0229] The compound of formula (III*a) is obtained by reaction:

[0230]

[0231] Where X is (R 3 R 4 )C, and ●、V、R 1 , R 3 , R 4 and Y are as defined above and below.

[0232] In some embodiments, in the same molecule having The compound (L) is a peptide, such as, for example, a BCL9 peptide. Thus, the compound of formula (IIIa) obtained by the method may be a cyclic peptide, such as, for example, a cyclic peptide derived from a BCL9 peptide. In some embodiments, in the same molecule, The compound (L*) is a peptide, such as, for example, a BCL9 peptide. Thus, the compound of formula (III*a) obtained by the method may be a cyclic peptide, such as, for example, a cyclic peptide derived from a BCL9 peptide.

[0233] All methods described herein for compounds of Formula (I), (I*), (II), (III) and (III*) can be similarly performed for compounds of Formula (L), (L*), (IIIa) and (III*a).

[0234] Compound

[0235] The present invention also relates to compounds obtainable or obtained by any of the methods described herein. In addition, the present invention relates to compounds useful in any of the methods described herein, such as as starting materials or intermediates. In particular, the present invention also relates to compounds of formula (I), (I*), (III), (III*), (L), (L*), (IIIa) and (III*a).

[0236] Therefore, the present invention also relates to a compound of formula (I)

[0237]

[0238] in

[0239] represents a double or triple bond;

[0240] when When it is a triple bond, X represents R 3 -C;

[0241] when When it is a double bond, X represents (R 3 R 4 )C;

[0242] Y stands for S or O;

[0243] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0244] R 3 Represents H or C 1 -C 8 -alkyl;

[0245] R 4 Represents H or C 1 -C 8 -alkyl; and

[0246] ●Represents an aliphatic or aromatic residue.

[0247] In some embodiments of the compound of Formula (I), represents a double bond, X represents (R 3 R 4 )C, and R 3 and R 4 Independently represents H or C 1 -C 8 -alkyl. Preferably, R 3 and R 4 Independently represents H or C 1 -C 8 -alkyl, more preferably representing H or C 1 -C 6 -alkyl, even more preferably representing H or C 1 -C 4 -alkyl, more preferably H or C 1 -C 2 In a preferred embodiment, R 3 and R 4 In a preferred embodiment, R 3 and R 4 Both are H.

[0248] Alternatively, in some embodiments of the compound of formula (I), represents triple bond, X represents R 3 -C, R 3 Represents H or C 1 -C 8 -alkyl. Preferably, R 3 Table H or C 1 -C 8 -alkyl, more preferably representing H or C 1 -C 6 -alkyl, even more preferably representing H or C 1 -C 4 -alkyl, more preferably H or C 1 -C 2 In a preferred embodiment, R 3 For H.

[0249] The present invention also relates to a compound of formula (I*)

[0250]

[0251] in,

[0252] V is for C 1 -C 8 - alkyl, preferably methyl, ethyl or propyl, more preferably methyl;

[0253] X represents (R 3 R 4 )C;

[0254] Y stands for S or O;

[0255] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0256] R 3 Represents H or C 1 -C 8 -alkyl;

[0257] R 4 Represents H or C 1 -C 8 -alkyl; and

[0258] ●Represents an aliphatic or aromatic residue.

[0259] The present invention also relates to a compound of formula (III)

[0260]

[0261] in

[0262] represents a single bond, and X represents (R 3 R 4 )C; or

[0263] represents a double bond, and X represents R 3 -C;

[0264] Y stands for S or O;

[0265] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0266] R 3 Represents H or C 1 -C 8 -alkyl;

[0267] R 4 Represents H or C 1 -C 8 -alkyl; and

[0268] ● represents an aliphatic or aromatic residue;

[0269] represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 -alkyl, optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system.

[0270] In some embodiments of the compound of formula (III), represents a single bond, X represents (R 3 R 4 )C, and R 3 and R 4 Independently represents H or C 1 -C 8 -alkyl. Preferably, R 3 and R 4 Independently represents H or C 1 -C 6 -alkyl, more preferably representing H or C 1 -C 4 -alkyl, even more preferably representing H or C 1 -C 2 In a preferred embodiment, R 3 and R 4 In a preferred embodiment, R 3 and R 4 Both are H.

[0271] Alternatively, in some embodiments of the compound of formula (III), represents a double bond, and X represents R 3 -C, and R 3 Represents H or C 1 -C 8 -alkyl. Preferably, R 3 Table H or C 1 -C 6 -alkyl, more preferably representing H or C 1 -C 4 -alkyl, even more preferably representing H or C 1 -C 2 In a preferred embodiment, R 3 For H.

[0272] The present invention also relates to compounds of formula (III*)

[0273]

[0274] in

[0275] X represents (R 3 R 4 )C

[0276] Y stands for S or O;

[0277] R 1 represents an optionally substituted aliphatic or aromatic residue;

[0278] R 3 Represents H or C 1 -C 8 -alkyl;

[0279] R 4 Represents H or C 1 -C 8 -alkyl;

[0280] V is for C 1 -C 8 - alkyl, preferably methyl, ethyl or propyl, more preferably methyl;

[0281] ● represents an aliphatic or aromatic residue; and

[0282] represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 -alkyl, optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system.

[0283] In some embodiments of the compound of Formula (III*), R 3 and R 4Independently represents H or C 1 -C 8 -alkyl. Preferably, R 3 and R 4 Independently represents H or C 1 -C 6 -alkyl, more preferably representing H or C 1 -C 4 -alkyl, even more preferably representing H or C 1 -C 2 In a preferred embodiment, R 3 and R 4 In a preferred embodiment, R 3 and R 4 Both are H.

[0284] In any of the compounds of formula (I), (I*), (III) or (III*), Y may be S (sulfur) or O (oxygen), i.e. when Y is S, the compound (I), (I*), (III) or (III*) represents a phosphonothioate, and when Y is O, the compound (I), (I*), (III) or (III*) represents a phosphonothioate. Thus, in some embodiments, Y is S. In some embodiments, Y is O. As an advantage, the inventors have demonstrated that phosphonothioates and phosphonates are both stable under physiologically relevant conditions. In a preferred embodiment of any of the compounds of formula (I), (I*), (III) or (III*), Y is S. It has been shown that phosphonothioates of formula (III) or (III*) in which Y is S can be obtained by a faster thiol addition than the corresponding phosphonate. Such a faster reaction rate is highly desirable because it increases the conversion and yield of the phosphonothioate.

[0285] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents C optionally substituted by at least one of the following 1 -C 8 -alkyl: wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n 、F、Cl、Br、I、-NO 2 、-N(C 1 -C 8 -alkyl)H, -NH 2 、-N 3 、-N(C 1 -C 8 -alkyl) 2 , =O, C 3 -C 8-cycloalkyl, -SS-(C 1 -C 8 -alkyl), wherein n is 1, 2, 3, 4, 5 or 6 hydroxy-(C 1 -C 8 -alkoxy) n , C 2 -C 8 -alkenyl or C 2 -C 8 -alkynyl.

[0286] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents an optionally substituted phenyl group, such as Where # represents the position of O.

[0287] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents a phenyl group which is optionally independently substituted by at least one of the following: 1 -C 8 -alkyl, wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n 、F、Cl、I、Br、-NO 2 、-N(C 1 -C 8 -alkyl)H, -NH 2 or -N(C 1 -C 8 -alkyl) 2 .

[0288] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents a 5- or 6-membered heteroaromatic system such as pyridyl.

[0289] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 Represents C 1 -C 8 -alkyl; 1 -C 8 -alkyl) substituted C 1 -C 8 -alkyl; wherein n is 1, 2, 3, 4, 5 or 6 (C 1 -C 8 -alkoxy) n Substituted C 1 -C8 -alkyl; C substituted by optionally substituted phenyl 1 -C 8 -alkyl; or phenyl; or -NO 2 Substituted phenyl.

[0290] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents methyl, ethyl, propyl or butyl, preferably methyl or ethyl.

[0291] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents optionally -SS-(C 1 -C 8 -alkyl)-substituted aliphatic or aromatic residue. In a preferred embodiment, R 1 represent Where R 10 , R 11 , R 12 and R 13 Each independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 10 , R 11 , R 12 and R 13 Each independently represents hydrogen, methyl or ethyl. In a preferred embodiment, R 1 represent Where R 10 and R 11 independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 10 and R 11 In a further preferred embodiment, R 1 represent Where R 10 and R 11 independently represents hydrogen, methyl or ethyl; and # represents the position of O. In some of these embodiments, R 10 and R 11 In some of these embodiments, R 10 is hydrogen, R 11 C 1 -C 6 -alkyl. In some of these embodiments, R 10 is hydrogen, R 11is methyl or ethyl. In some of these embodiments, R 10 and R 11 In a preferred embodiment, R 1 represent More preferably represents Where R 10 and R 11 As defined above. In a preferred embodiment, R 1 represent Where R 12 and R 13 independently represents hydrogen or C 1 -C 8 -alkyl; and # represents the position of O. In a more preferred embodiment, R 12 and R 13 R independently represents hydrogen, methyl or ethyl. In an even more preferred embodiment, R 1 represent Where R 12 and R 13 independently represents hydrogen, methyl or ethyl; and # represents the position of O. In some of these embodiments, R 12 and R 13 In some of these embodiments, R 12 is hydrogen, R 13 C 1 -C 6 -alkyl. In some of these embodiments, R 12 is hydrogen, R 13 is methyl or ethyl. In some of these embodiments, R 12 and R 13 same.

[0292] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further substituted, wherein Z is O or NH, and wherein # represents the position of the phenyl group. In some embodiments Z is O. In some embodiments Z is NH. C 1 -C 8 -alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl. In a preferred embodiment, R 1 represent Wherein C 1 -C 8-alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl; wherein Z is O or NH, and wherein # represents the position of O. In another preferred embodiment, R 1 represent Wherein C 1 -C 8 -Alkyl can be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl; wherein Z is O or NH, and wherein # represents the position of O.

[0293] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further substituted, and wherein # represents the position of the phenyl group. In some embodiments, R 1 represents C substituted by phenyl 1 -C 8 -alkyl, the phenyl group is further In a preferred embodiment, R 1 represent wherein # represents the position of O. In another preferred embodiment, R 1 represent Where # represents the position of O.

[0294] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represent wherein # represents the position of O, n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably n=0, 1, 2, 3, 4, 5, 6; more preferably n=0, 1, 2, 3, 4; even more preferably n=0, 1, 2, 3, even further preferably n=0, 1, 2, even further preferably n=0, 1; and most preferably n=1.

[0295] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 Represents optionally C 2 -C 8 -alkynyl-substituted aliphatic or aromatic residue. In a preferred embodiment, R 1 It is a high propargyl group.

[0296] In some embodiments of compounds of any of Formula (I), (I*), (III) or (III*), R 1 represent wherein # represents the position of O, n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably n=0, 1, 2, 3, 4, 5, 6; more preferably n=0, 1, 2, 3, 4; even more preferably n=0, 1, 2, 3, even more preferably n=0, 1, 2, even more preferably n=0, 1; and most preferably n=1, i.e. when n is 1, R 1 represent

[0297] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents a small molecule; wherein optionally said ● further comprises a linker bound to Y.

[0298] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents a small molecule such as, for example, an optionally substituted C 1 -C 8 -alkyl, -CH 2 -phenyl,

[0299]

[0300] wherein # indicates the position of Y. In a preferred embodiment, ● represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C 4 -alkyl, even more preferably C 1 -C 2 -alkyl. In some embodiments, ● represents -CH 2 -phenyl, i.e. benzyl. In a preferred embodiment, ● represents Wherein # indicates the position of Y. In a preferred embodiment, → represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In a preferred embodiment, ● represents wherein # indicates the position of Y. In some embodiments, ● represents wherein # indicates the position of Y. In some embodiments, ● represents wherein # indicates the position of Y. In some embodiments, ● represents Where # indicates the position of Y.

[0301] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents an optionally substituted phenyl group, preferably Where # indicates the position of Y.

[0302] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents a radioactive or non-radioactive nuclide, biotin, a reporter enzyme, a nucleotide, an oligonucleotide, such as CY 5 or EDANS fluorophore, amino acid, peptide or optionally substituted 5- or 6-membered heteroaromatic system; wherein optionally, said ● further comprises a linker bound to Y. Thus, in some embodiments, ● represents a radioactive or non-radioactive nuclide. In some preferred embodiments, ● represents biotin. In some embodiments, ● represents a reporter enzyme. In some preferred embodiments, ● represents a nucleotide. In some preferred embodiments, ● represents an oligonucleotide. In some preferred embodiments, ● represents a nucleotide such as CY 5 In some preferred embodiments, ● represents an amino acid. In some preferred embodiments, ● represents a peptide. In some preferred embodiments, ● represents an optionally substituted 5- or 6-membered heteroaromatic system. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0303] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● a cyclic RGD peptide (c(RDGfK)) representing structure (VII)

[0304]

[0305] Where * represents the position of Y.

[0306] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents a phenyl group, which is optionally independently selected from C 1 -C 8 -alkyl, C 1 -C 8 -alkoxy, halogen, -CN, -NO 2 、-NH 2 、-N(C1 -C 8 -alkyl), -N(C 1 -C 8 -alkyl) 2 、-COOH、-COO(C 1 -C 8 -alkyl), -OC(O)-(C 1 -C 8 -alkyl), -C(O)N-(C 1 -C 8 -alkyl), -N(H)-C(O)-(C 1 -C 8 -alkyl) is substituted with 1, 2, 3, 4 or 5 substituents, preferably, the phenyl group is optionally selected from C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 substituted with 1 substituent.

[0307] In some embodiments of compounds of any one of Formulas (I), (I*), (III) or (III*), ● represents C 1 -C 8 -alkyl, the C 1 -C 8 -alkyl is optionally substituted by at least one substituent selected from the group consisting of: 3 -C 8 -cycloalkyl; heterocyclyl having 3 to 8 ring members, wherein the heteroatoms are selected from N, O, S; C 1 -C 8 -alkoxy; halogen; -CN; -NO 2 ;-NH 2 ;-N(C 1 -C 8 -alkyl); -N(C 1 -C 8 -alkyl) 2 ;-COOH;-COO(C 1 -C 8 -alkyl); -OC(O)-(C 1 -C 8 -alkyl); -CONH 2 ; -C(O)N(C 1 -C 8 -alkyl) 2 ; -C(O)NH-(C 1 -C 8 -alkyl); -N(H)-C(O)-(C 1 -C8 -alkyl), preferably C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 , phenyl or heteroaromatic system, monosaccharide, polysaccharide, peptide, protein, antibody, nucleotide, oligonucleotide, polymer, amino acid, fluorophore, protein tag (first generation substituent), wherein the first generation substituent can be optionally substituted again by the following groups: C 3 -C 8 -cycloalkyl; heterocyclyl having 3 to 8 ring members, wherein the heteroatoms are selected from N, O, S; C 1 -C 8 -alkoxy; halogen; -CN; -NO 2 ;-NH 2 ;-N(C 1 -C 8 -alkyl); -N(C 1 -C 8 -alkyl) 2 ;-COOH;-COO(C 1 -C 8 -alkyl); -OC(O)-(C 1 -C 8 -alkyl); -CONH 2 ; -C(O)N(C 1 -C 8 -alkyl) 2 ; -C(O)NH-(C 1 -C 8 -alkyl); -N(H)-C(O)-(C 1 -C 8 -alkyl), preferably C 1 -C 8 -alkoxy, -COOH, -COO(C 1 -C 8 -alkyl) and NO 2 , phenyl or a heteroaromatic system (2nd generation substituent), and wherein the 2nd generation substituent may be substituted again by at least one substituent selected from the same group, and wherein such substitution may be carried out to 3, 4, 5, 6, 7, 8, 9 or 10 generations.

[0308] In some embodiments of any one of the compounds of Formula (I), (I*), (III) or (III*), ● represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8-alkyl, an optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system; wherein optionally said ● further comprises a linker bound to Y. Thus, in a preferred embodiment, ● represents an amino acid. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents a nucleotide. In a preferred embodiment, ● represents an oligonucleotide. In some embodiments, ● represents a sugar. In some embodiments, ● represents a polysaccharide. In some embodiments, ● represents a polymer. In some embodiments, ● represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C 4 -alkyl, even more preferably optionally substituted C 1 -C 2 -alkyl. In some embodiments, ● represents an optionally substituted phenyl. In some embodiments, ● represents an optionally substituted aromatic 5- or 6-membered heterocyclic ring system. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0309] In a preferred embodiment of any one of the compounds of formula (I), (I*), (III) or (III*), ● represents an amino acid, a peptide, a protein, an antibody, a nucleotide or an oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. In a more preferred embodiment, ● represents a peptide, a protein, an antibody or an oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. In a preferred embodiment, ● represents an amino acid. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents a nucleotide. In a preferred embodiment, ● represents an oligonucleotide. Optionally, in any one of these embodiments, said ● further comprises a linker bound to Y.

[0310] In preferred embodiments of any of the compounds of formula (I), (I*), (III) or (III*), ● represents a drug, a protein tag, such as CY 5 or EDANS fluorophore, biotin, protein, peptide, antibody or oligonucleotide; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, ● represents a drug. In a preferred embodiment, ● represents a protein tag. In a preferred embodiment, ● represents a linker-drug conjugate. In a preferred embodiment, ● represents a CY 5In a preferred embodiment, ● represents biotin. In a preferred embodiment, ● represents a protein. In a preferred embodiment, ● represents a peptide. In a preferred embodiment, ● represents an antibody. In a preferred embodiment, ● represents an oligonucleotide. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0311] In preferred embodiments of any of the compounds of formula (I), (I*), (III) or (III*), ● represents a linker or a linker-drug conjugate. In preferred embodiments, ● represents a linker, such as a linker comprising VC-PAB, VA-PAB, KF-PAB or VK-PAB, preferably a linker comprising VC-PAB. In preferred embodiments, ● represents a linker-drug conjugate, such as

[0312] Best wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. More preferably, the linker drug conjugate is

[0313] Even more preferably Most preferably wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1.

[0314] Any one of the compounds according to formula (III) or (III*), may represent amino acids, peptides, proteins, antibodies, nucleotides, oligonucleotides, sugars, polysaccharides, polymers, optionally substituted C 1 -C 8 -alkyl, optionally substituted phenyl or optionally substituted aromatic 5- or 6-membered heterocyclic ring system. In a preferred embodiment, Represents an amino acid, a peptide, a protein, an antibody, a nucleotide or an oligonucleotide. In a more preferred embodiment, Represents a peptide, protein, antibody or oligonucleotide. In a preferred embodiment, In a preferred embodiment, In a preferred embodiment, Represents a protein. In a preferred embodiment, In a preferred embodiment, In a preferred embodiment, In some embodiments, represents a sugar. In some embodiments, represents a polysaccharide. In some embodiments, Represents a polymer. In some embodiments, represents an optionally substituted C 1 -C 8 -alkyl, preferably optionally substituted C 1 -C 6 -alkyl, more preferably optionally substituted C 1 -C 4 -alkyl, even more preferably optionally substituted C 1 -C 2 -alkyl. In some embodiments, represents an optionally substituted C 3 -C 8 -alkyl, preferably optionally substituted C 3 -C 6 -alkyl, more preferably optionally substituted C 3 -C 4 -alkyl. In some embodiments, represents an optionally substituted C 5 -C 8 -alkyl, preferably optionally substituted C 6 -C 7 -alkyl. In some embodiments, represents an optionally substituted phenyl group. In some embodiments, represents an optionally substituted aromatic 5- or 6-membered heterocyclic ring system.

[0315] In any preferred embodiment of the compound of formula (III) or (III*), represents an antibody, preferably an IgG antibody, more preferably cetuximab or trastuzumab or brentuximab; a protein, preferably a GFP protein or an eGFP protein, an albumin, a tripeptide, preferably of formula (VIII) Or formula (IX) A peptide wherein # represents the position of S. Therefore, in a preferred embodiment, In a preferred embodiment, the antibody is an IgG antibody, and even more preferably, cetuximab or trastuzumab or brentuximab. In a preferred embodiment, Represents a protein, more preferably a GFP protein or an eGFP protein. In a preferred embodiment, In a preferred embodiment, Represents a tripeptide. In a preferred embodiment, Representative formula (VIII) In a preferred embodiment, Representative formula (IX) In a preferred embodiment of any one of the compounds of formula (III) or (III*), represents an antibody (such as cetuximab, trastuzumab, or brentuximab) and ● represents a protein tag or a 5 or a fluorophore, biotin, peptide, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents an antibody and ● represents a protein tag. In a preferred embodiment, represents antibodies and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents an antibody and ● represents biotin. In a preferred embodiment, represents an antibody and ● represents a peptide. In a preferred embodiment, represents an antibody and ● represents a protein. In a preferred embodiment, represents an antibody and ● represents an oligonucleotide. In a preferred embodiment, represents an antibody and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0316] In any preferred embodiment of the compound of formula (III) or (III*), represents a protein (such as GFP protein or eGFP protein) and ● represents a protein tag or a protein tag such as CY 5 or a fluorophore, biotin, peptide, antibody, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents a protein and ● represents a protein tag. In a preferred embodiment, represents proteins and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents protein and ● represents biotin. In a preferred embodiment, represents a protein and ● represents a peptide. In a preferred embodiment, represents a protein and ● represents an antibody. In a preferred embodiment, represents a protein and ● represents a protein. In a preferred embodiment, represents a protein and ● represents an oligonucleotide. In a preferred embodiment, represents a protein and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0317] In any preferred embodiment of the compound of formula (III) or (III*), represents peptides and ● represents protein tags or such as CY 5 or a fluorophore, biotin, peptide, antibody, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents a peptide and ● represents a protein tag. In a preferred embodiment, represents peptides and ● represents such as CY 5 Or EDANS fluorophore. In a preferred embodiment, represents a peptide and ● represents biotin. In a preferred embodiment, represents a peptide and ● represents a peptide. In a preferred embodiment, represents a peptide and ● represents an antibody. In a preferred embodiment, represents a peptide and ● represents a protein. In a preferred embodiment, represents a peptide and ● represents an oligonucleotide. In a preferred embodiment, represents a peptide and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0318] In any preferred embodiment of the compound of formula (III) or (III*), represents amino acids and ● represents protein tags or such as CY 5 or a fluorophore, biotin, peptide, protein, oligonucleotide or small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents an amino acid and ● represents a protein tag. In a preferred embodiment, represents amino acids and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents an amino acid and ● represents biotin. In a preferred embodiment, represents an amino acid and ● represents a peptide. In a preferred embodiment, represents amino acids and ● represents proteins. In a preferred embodiment, represents an amino acid and ● represents an oligonucleotide. In a preferred embodiment, represents an amino acid and ● represents a small molecule. Optionally, in any one of these embodiments, said ● further comprises a linker bound to Y.

[0319] In any preferred embodiment of the compound of formula (III) or (III*), represents an antibody (such as cetuximab, trastuzumab or brentuximab) and ● represents a linker, a drug or a linker-drug conjugate. Therefore, in a preferred embodiment, represents an antibody and ● represents a linker, such as a linker comprising VC-PAB, VA-PAB, KF-PAB or VK-PAB, preferably a linker comprising VC-PAB. In a preferred embodiment, represents an antibody and ● represents a drug. In a preferred embodiment, represents an antibody and ● represents a linker-drug conjugate, such as

[0320] Best wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. In a more preferred embodiment, represents an antibody and ● represents a linker-drug conjugate, such as

[0321] Even more preferably Most preferably wherein # represents the position of Y (O (oxygen) or S (sulfur), preferably S), m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. In any of these embodiments, the antibody may be cetuximab, trastuzumab or brentuximab, preferably brentuximab.

[0322] In any preferred embodiment of the compound of formula (III) or (III*), represents nucleotides and represents peptides, proteins, protein tags, antibodies, oligonucleotides, such as CY 5 or a fluorophore, biotin or a small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. In a preferred embodiment, represents a nucleotide and ● represents a peptide. In a preferred embodiment, represents nucleotides and ● represents proteins. In a preferred embodiment, represents a nucleotide and ● represents a protein tag. In a preferred embodiment, represents a nucleotide and ● represents an antibody. In a preferred embodiment, represents a nucleotide and ● represents an oligonucleotide. In a preferred embodiment, represents nucleotides and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents a nucleotide and ● represents biotin. In a preferred embodiment, represents a nucleotide and ● represents a small molecule. Optionally, in any one of these embodiments, said ● further comprises a linker bound to Y.

[0323] In any preferred embodiment of the compound of formula (III) or (III*), represents nucleotides and ● represents linkers.

[0324] In any preferred embodiment of the compound of formula (III) or (III*), represents oligonucleotides and represents peptides, proteins, protein tags, antibodies, oligonucleotides, such as CY 5 or a fluorophore, biotin or a small molecule of EDANS; wherein optionally said ● further comprises a linker bound to Y. Therefore, in a preferred embodiment, represents an oligonucleotide and ● represents a peptide. In a preferred embodiment, represents an oligonucleotide and ● represents a protein. In a preferred embodiment, represents an oligonucleotide and ● represents a protein tag. In a preferred embodiment, represents an oligonucleotide and ● represents an antibody. In a preferred embodiment, represents an oligonucleotide and ● represents an oligonucleotide. In a preferred embodiment, represents oligonucleotides and ● represents CY 5 Or EDANS fluorophore. In a preferred embodiment, represents an oligonucleotide and ● represents biotin. In a preferred embodiment, represents an oligonucleotide and ● represents a small molecule. Optionally, in any of these embodiments, said ● further comprises a linker bound to Y.

[0325] In any preferred embodiment of the compound of formula (III) or (III*), represents oligonucleotide and ● represents linker.

[0326] In preferred embodiments of any one of the compounds of formula (I), (I*), (III) or (III*), ● represents an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, peptide, nucleotide or oligonucleotide is bound to a solid support. In some embodiments, the compound is a compound of formula (I) or a compound of formula (I*). In some embodiments, ● represents an amino acid or a peptide bound to a solid support. In some embodiments, ● represents a nucleotide or an oligonucleotide bound to a solid support. In a preferred embodiment, ● represents a peptide bound to a solid support. As an advantage, the inventors have found that the thiophosphonates (Y=S) of the present invention are highly stable under acidic conditions (such as 90% trifluoroacetic acid (TFA)), which are typically used to cleave peptides from a solid support. As described above in the context of the method, the solid support can be any solid support known to those skilled in the art to be suitable for solid phase peptide synthesis, or any solid support suitable for solid phase oligonucleotide synthesis. Optionally, in the above embodiments wherein the amino acid, peptide, nucleotide or oligonucleotide is bound to a solid support, the ● further comprises a linker bound to Y in the formulae described herein, in particular compounds (I), (III), (I*) and (III*). In addition, the linker is bound to the amino acid, peptide, nucleotide or oligonucleotide. Thus, the ● may have a structure of linker-amino acid-solid support, linker-peptide-solid support, linker-nucleotide-solid support or linker-oligonucleotide-solid support. The "linker" may be virtually any linker, and the linker is bound to Y, for example, as described herein for compounds of formula (I), (I*), (III) or (III*), wherein Y is S (sulfur) or O (oxygen), preferably S. The linker may be any linker known to those skilled in the art, such as a peptide linker or a linear or branched hydrocarbon-based moiety. The linker may also comprise a cyclic moiety. The peptide linker may comprise, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3 or 2 or 1 amino acid. If the linker is a hydrocarbon-based moiety, the backbone of the linker may comprise only carbon atoms, but may also comprise heteroatoms such as oxygen (O), nitrogen (N) or sulfur (S) atoms, and / or may comprise a carbonyl group (C=O). The linker may be, for example, C 1 -C 20 A chain of carbon atoms or a 2 -CH 2)-repeat units. In typical embodiments of hydrocarbon-based linkers, the linking moiety comprises 1 to about 150, 1 to about 100, 1 to about 75, 1 to about 50, or 1 to about 40, or 1 to about 30, or 1 to about 20, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 backbone atoms. Those skilled in the art will appreciate the selection of appropriate linkers. For example, in some embodiments, the linker may be wherein # represents the position of Y, and * represents the position of an amino acid, peptide, nucleotide or oligonucleotide; and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. The amino acid, peptide, nucleotide or oligonucleotide may be bound to the linker via an N (nitrogen) atom.

[0327] In any preferred embodiment of the compound of formula (I), (I*), (III) or (III*), represents an amino acid, a peptide, a nucleotide or an oligonucleotide, wherein the amino acid, a peptide, a nucleotide or an oligonucleotide is bound to a solid support. In some embodiments, Represents an amino acid or peptide bound to a solid support. In some embodiments, Represents a nucleotide or oligonucleotide bound to a solid support. In a preferred embodiment, represents the peptide bound to a solid support.

[0328] The present invention also relates to a kit comprising a solid support, and

[0329] Compounds of formula (I)

[0330]

[0331] and / or a compound of formula (I*)

[0332]

[0333] wherein ● is a linker suitable for binding to an amino acid, a peptide, a nucleotide or an oligonucleotide; and

[0334] in R 1, X, Y and V are as defined above and below. Such a kit is suitable for solid phase peptide synthesis and / or solid phase oligonucleotide synthesis. Preferably, since the inventors have found that the thiophosphonate (Y=S) of the present invention is highly stable under acidic conditions (such as 90% trifluoroacetic acid (TFA)), which are typically used to cleave peptides from a solid support, the kit can be used for solid phase peptide synthesis. In solid phase synthesis, an amino acid, a peptide, a nucleotide or an oligonucleotide is bound to a solid support, and a linker of a compound of formula (I) or formula (I*) is bound to an amino acid, a peptide, a nucleotide or an oligonucleotide. In a preferred embodiment of the kit, the linker is wherein # represents the position of Y, and m and n are each independently an integer such as 0 to 20, 0 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 1, preferably m is 1 and n is 1. The amino acid, peptide, nucleotide or oligonucleotide is bound to this linker via a carboxyl group. In some embodiments, the compound of formula (I) has structure.

[0335] The kit may further comprise one or more amino acids, one or more peptides, one or more nucleotides and / or one or more oligonucleotides that can be used in solid phase synthesis. In particular, since solid phase peptide synthesis is preferred, the kit may comprise one or more amino acids.

[0336] The present invention also relates to a compound of formula (IIIa)

[0337]

[0338] where as shown by connecting ● and The arc of ● and In the same molecule, X, Y, and R 1 As defined above and below, in particular as defined for compound (III). Preferably, compound (IIIa) is a cyclic peptide, such as for example a cyclic peptide derived from the BCL9 peptide.

[0339] The present invention also relates to a compound of formula (III*a):

[0340]

[0341] where as shown by connecting ● and The arc of ● and In the same molecule, V, X, Y, and R 1 As defined above and below, in particular as defined for compound (III*). Preferably, compound (III*a) is a cyclic peptide, such as for example a cyclic peptide derived from the BCL9 peptide.

[0342] Furthermore, the compounds provided herein as examples in the Examples section for compounds of formula (I), (I*), (III), (III*), (IIIa) or (III*a) are preferred.

[0343] It should be understood by those skilled in the art that the embodiments according to the present invention can be combined with each other as long as combinations that violate any laws of nature are excluded.

[0344] Any embodiments, features, definitions, etc. described herein for any method may also be applied mutatis mutandis to any compound described herein. Similarly, any embodiments, features, definitions, etc. described herein for any compound may also be applied mutatis mutandis to any method described herein.

[0345] Synthesis of compounds of formula (IV), (IV*), (I) and (I*) starting from phosphorus trihalides

[0346] The following sections provide some general characteristics of the synthesis of compounds of formula (IV), (IV*), (I) and (I*) starting from phosphorus trihalides. Generally, those skilled in the art will know to select appropriate reaction conditions to implement the synthesis. Further details are provided in the Examples section hereinafter.

[0347] As shown above and below, the compound of formula (IV) or (IV*) can be prepared by a sequence comprising steps (i) to (iv). Thus, the compound of formula (IV) or (IV*) can be synthesized by: (i) reacting a phosphorus trihalide (X), preferably PCl 3 With R 1 The invention relates to a process for preparing a compound of formula (IV) or (IV*) by reacting the product obtained in step (i) with an alcohol (XI) containing a residue, (ii) reacting the product obtained in step (i) with an amine (XII), (iii) reacting the product obtained in step (ii) with an alkenyl magnesium halide or an alkynyl magnesium halide (XIII) or with an alkenyl magnesium halide (XIII*), and (iv) reacting the product obtained in step (iii) with an alcohol or thiol (XIV) containing a residue to provide a compound of formula (IV) or (IV*). The compound of formula (I) or (I*) can be obtained by oxidation from the compound of formula (IV) or (IV*).

[0348] Step (i)

[0349] Step (i) can be carried out, for example, by reacting a phosphorus trihalide (X), preferably PCl 3With alcohol (XI) in a suitable solvent such as diethyl ether or tetrahydrofuran, at a low temperature below -10 ° C, and then the reaction mixture is warmed; for example, the temperature range can be -50 ° C to +50 ° C; more specifically, the reaction can be carried out at about -40 ° C or -30 ° C, and then warmed to room temperature. Preferably, the reaction of phosphorus trihalide with alcohol is carried out in the presence of a weak base (such as an amine base such as triethylamine). The molar ratio of alcohol to phosphorus trihalide should be 5: 1 to 1: 5, preferably 2: 1 to 1: 2, more preferably, the molar ratio is 1: 1. When a weak base (such as an amine base such as triethylamine) is used, the molar ratio of the base to phosphorus trihalide can be 5: 1 to 1: 5, for example 2: 1 to 1: 2, preferably about 1: 1. Of course, the reaction time depends on the reaction volume and the amount of substance. As a guide, the reaction time at low temperature before heating can be 2 minutes to 2 hours, such as about 10 minutes, and the reaction time after heating can be 15 minutes to 6 hours, such as about 1 hour. Preferably, the reaction is carried out under an inert gas such as argon. In this context, "inert" refers to a gas that does not react with any starting material or reaction product under given reaction conditions. Preferably, the mixture obtained after the reaction of step (i) is directly used in step (ii) without separating the product. Optionally, after step (i) and before step (ii), the mixture can be purified such as by filtration through diatomaceous earth.

[0350] Step (ii)

[0351] Step (ii) can be carried out, for example, by reacting the product obtained in step (i) with an amine (XII) (preferably diisopropylamine) in a suitable solvent such as diethyl ether or tetrahydrofuran. In this regard, the solvent can be the same as the solvent in step (i). The reaction of step (ii) can be carried out at a low temperature below -10°C, and then the reaction is heated; for example, the temperature range can be -50°C to +50°C; more specifically, the reaction can be carried out at about -40°C or -30°C, and then warmed to room temperature. The molar ratio of amine (XII) can be based on the molar amount of phosphorus trihalide (X) used in step (i). Therefore, the molar ratio of amine (XII) to phosphorus trihalide (X) should be 5:1 to 1:5, preferably the molar ratio of amine (XII) to phosphorus trihalide (X) should be about 2:1. As a guide, the reaction time at low temperature before heating can be 2 minutes to 2 hours, such as about 10 minutes, and the reaction time after heating can be 15 minutes to 6 hours, such as about 1 hour. Preferably, the reaction is carried out under an inert gas such as argon. The mixture obtained after the reaction of step (ii) is directly used in step (iii) without isolating the product. Optionally, after step (ii) and before step (iii), the mixture can be purified, such as by filtration through diatomaceous earth. In step (ii), a product having the general structure wherein Hal is a halogen such as Cl, Br or I, depending on the phosphorus trihalide used in step 1, such as when PCl is used 3 When Hal is Cl; and R 1 and R 2 As defined above and below.

[0352] Step (iii)

[0353] Step (iii) can be carried out, for example, as follows: react the product obtained in step (ii) with alkenyl magnesium halide or alkynyl magnesium halide (XIII) or with alkenyl magnesium halide (XIII *) in a suitable solvent such as ether or tetrahydrofuran. In this regard, the solvent can be the same as the solvent in step (ii). The reaction of step (ii) can be carried out at a low temperature below -50 ° C, and then the reaction is heated; for example, the temperature range can be -100 ° C to +50 ° C; more specifically, the reaction can be carried out at about -78 ° C and then warmed to room temperature. The molar ratio of alkenyl magnesium halide or alkynyl magnesium halide (XIII), or alkenyl magnesium halide (XIII *) can be based on the molar amount of phosphorus trihalide (X) used in step (i). Therefore, the ratio of alkenyl magnesium halide or alkynyl magnesium halide (XIII) or alkenyl magnesium halide (XIII*) to phosphorus trihalide (X) should be 5:1 to 1:5, preferably 2:1 to 1:2, and more preferably, the molar ratio of alkenyl magnesium halide or alkynyl magnesium halide (XIII) to phosphorus trihalide (X) should be about 1:1. As a guide, the reaction time at low temperature before heating can be 2 minutes to 1 hour, such as about 10 minutes, and the reaction time after heating can be 15 minutes to 6 hours, such as about 1 hour. Preferably, the reaction is carried out under an inert gas such as argon. The mixture obtained by the reaction of step (iii) can be post-treated, and the product can then be separated by methods generally known to those skilled in the art, such as silica gel chromatography or vacuum distillation. In step (iii), when alkenyl magnesium halide or alkynyl magnesium halide (XIII) is used, a compound having the general structure can be obtained. or, when an alkenyl magnesium halide (XIII*) is used, a product having the general structure The product of , where in any of these structures, R 1 and R 2 , X and V are as defined above and below.

[0354] Step (iv)

[0355] Step (iv) can be carried out, for example, by reacting the product obtained in step (iii) with an alcohol or thiol (XIV) containing a ● residue in a suitable solvent such as acetonitrile. The reaction of step (ii) can be carried out at a low temperature below -20°C, and then the reaction is heated; for example, the temperature range can be -50°C to +50°C; more specifically, the reaction can be carried out at about -40°C and then warmed to room temperature. Preferably, the reaction of the product obtained in step (iii) with the alcohol or thiol (XIV) is carried out in the presence of tetrazole. The tetrazole can be an unsubstituted tetrazole or a substituted tetrazole. The molar ratio of the alcohol or thiol (XIV) to the product obtained in step (iii) should be 5:1 to 1:5, preferably 2:1 to 1:2, and more preferably the molar ratio is about 1:1. When using tetrazole, the molar ratio of the product obtained by the tetrazole and step (iii) should be 5:1 to 1:5, preferably, the molar ratio of the product obtained by the tetrazole and step (iii) should be about 2:1 to 1:2. As a guide, the reaction time at low temperature before heating can be 2 minutes to 2 hours, such as about 10 minutes, and the reaction time after heating can be 15 minutes to 6 hours, such as about 30 minutes or about 1 hour. Reaction can be carried out under an inert gas such as argon.

[0356] Oxidation to synthesize compounds of formula (I) or (I*)

[0357] Step (iv) obtains a compound of formula (IV) or (IV*). The compound of formula (I) or (I*) is then obtained by oxidizing the compound of formula (IV) or (IV*) on phosphorus. The mixture obtained after the reaction of step (iv) can be directly used in the oxidation without further post-treatment or purification, that is, the oxidant can be directly added to the mixture obtained after the reaction of step (iv). Various suitable oxidants can be used, such as tert-butyl hydroperoxide (tBu-OOH), meta-chloroperbenzoic acid (mCPBA), hydrogen peroxide (H 2 O 2 ), iodine (I 2 ), potassium persulfate or oxygen (O 2), such as oxygen from air. The skilled person will readily determine a suitable oxidant. Preferably, tert-butyl hydroperoxide (tBu-OOH) can be used as the oxidant. The reaction can be carried out at a temperature of 0 to 60° C., such as at room temperature. The molar ratio of the oxidant can be based on the molar amount of the product obtained in step (iii). Therefore, the molar ratio of the oxidant to the product obtained in step (iii) should be 2:1 to 1:2, preferably the molar ratio of the oxidant to the product obtained in step (iii) should be 1:1. As a guide, the reaction time of the oxidation can be 2 minutes to 2 hours, such as about 10 minutes. The reaction can be carried out under an inert gas such as argon. The mixture obtained by oxidation can be post-treated, and the obtained compound of formula (V) or (V*) can be separated by methods generally known to those skilled in the art, such as silica gel chromatography.

[0358] Synthesis of compounds of formula (I) or (I*) starting from electrophilic disulfides

[0359] The following section provides some general features of the synthesis of compounds of formula (I) and (I*) starting from electrophilic disulfides, wherein Y is S (sulfur). Generally, one skilled in the art will know to select appropriate reaction conditions to carry out the synthesis. Further details are given in the Examples section below.

[0360] As shown above and below, compounds of formula (I) or (I*) (wherein Y is S) can be obtained by reacting an electrophilic disulfide (XXX) with a thiol (XXXI) containing a ● residue to give a compound of formula (VIa). Compounds of formula (VIb) can be prepared according to methods known in the literature. Compounds of formula (VIa) or (VIb) are then reacted with a thiol containing R 1 The compound of formula (V) can be prepared by reacting a phosphorus (III) compound of formula (V) or (V*) with a residue to obtain a compound of formula (I) or (I*). 1 The residue can be prepared by reacting a halogenated phosphite of formula (XX) with a Grignard compound of formula (XXI), and the compound of formula (V*) can be prepared by reacting a halogenated phosphite of formula (XX) with a Grignard compound of formula (XXI*).

[0361] Synthesis of compounds of formula (V) or (V*)

[0362] The compounds of formula (V) or (V*) can be prepared, for example, by reacting an alkenyl magnesium halide or alkynyl magnesium halide of formula (XXI) - preferably an alkenyl magnesium halide of formula (XXI) - or an alkenyl magnesium halide of formula (XXI*) with a 1The halogenated phosphite of formula (XX) of the residue is reacted. Preferably, the alkenyl magnesium halide of formula (XXI) or the alkenyl magnesium halide or the alkenyl magnesium halide of formula (XXI*) is alkenyl magnesium bromide or alkynyl magnesium bromide. Preferably, the halogenated phosphite of formula (XX) is a chlorophosphite. It can be used, for example, ether or tetrahydrofuran as a suitable solvent. Preferably, the reaction is carried out at a temperature below -20°C, such as -100°C to -40°C, preferably -90°C to -50°C (such as about -78°C). Preferably, the reaction is carried out under an inert gas such as argon. As a guide, the reaction time should be 2 minutes to 4 hours, such as 2 hours. The molar ratio of the alkenyl magnesium halide or alkynyl magnesium halide or alkenyl magnesium halide of formula (XXI*) to the halophosphite of formula (XXI) should be 5:1 to 1:5, such as 2:1 to 1:2, such as about 1:1, for example, the ratio of the alkenyl magnesium halide or alkynyl magnesium halide or alkenyl magnesium halide of formula (XXI*) to the halophosphite of formula (XXI) is about 1:1.2. For details, please refer to, for example, MRJ Vallée, Angewandte Chemie Int. Ed., 2013, 52(36), 9504.

[0363] Synthesis of compounds of formula (VIa) or (VIb)

[0364] Compounds of formula (VIa) or (VIb) can be prepared, for example, by reacting an electrophilic disulfide of formula (XXX) with a thiol (XXXI) comprising a ● residue. The reaction can be carried out in a suitable solvent, such as, for example, tetrahydrofuran or N,N-dimethylformamide (DMF). Preferably, the reaction of the electrophilic disulfide of formula (XXX) with the thiol (XXXI) is carried out in the presence of a weak base, such as an amine base such as triethylamine. The reaction can be carried out at a temperature of 0° C. to 60° C., such as, for example, at room temperature. The molar ratio of the electrophilic disulfide of formula (XXX) to the thiol (XXXI) should be 5:1 to 1:5, preferably 2:1 to 1:2, more preferably about 1:1, such as a ratio of the electrophilic disulfide of formula (XXX) to the thiol (XXXI) of 1:1.2. When a weak base (such as an amine base such as triethylamine) is used, the molar ratio of the base to the thiol of formula (XXXI) can be 5:1 to 1:5, preferably, the molar ratio of the base to the thiol of formula (XXXI) is about 3:1. The reaction time can be, for example, 2 minutes to 6 hours, such as about 1 hour, or about 20 minutes or about 10 minutes. Suitable methods can be used, such as thin layer chromatography to monitor the reaction. The reaction is carried out under an inert gas such as argon. The compound of formula (VIa) containing the residue can be separated by methods generally known to those skilled in the art, such as silica gel chromatography. Compounds of formula VIb can be prepared according to methods known from the literature, for example, by reacting thiol ●-SH(XXXI) with sulfuryl chloride (see, for example, Allared, F. et al., Synthetic Metals, 120(1-3), 1061-1062; 2001) or thionyl chloride (see, e.g., Masaki, Yukio et al., Chemical & Pharmaceutical Bulletin, 33(5), 1930-40; 1985), or by reacting thiol ●-SH(XXXI) with N-chlorosuccinimide (NCS) (see, e.g., Kawamura, Takamasa et al., European Journal of Organic Chemistry, 2015(4), 719-722; 2015) or chlorine (see, e.g., E. Schneider, Chemische Berichte 84, 911-916 (1951)), wherein ● is as defined above and below.

[0365] Synthesis of compounds of formula (I) or (I*)

[0366] The compound of formula (I) or (I*) (wherein Y is S) can be prepared by reacting a compound of formula (V) or (V*) with a compound of formula (VIa) or (VIb). Preferably, the compound of formula (V) or (V*) is an olefin phosphonite, i.e., is a double bond. The reaction is carried out in a suitable organic solvent, such as tetrahydrofuran or in N,N-dimethylformamide (DMF) or in a solvent mixture, such as in a mixture of tetrahydrofuran and toluene. The reaction can be carried out at a temperature of 0°C to 60°C, such as at room temperature. Preferably, the reaction can be carried out under an inert gas such as argon. The molar ratio of the compound of formula (V) or (V*) to the compound of formula (VIa) or (VIb) should be 5:1 to 1:5, preferably 2:1 to 1:1, more preferably about 1:1, such as the molar ratio of the compound of formula (V) or (V*) to the compound of formula (VIa) or (VIb) is about 1.2:1. As a guide, the reaction time can be, for example, 2 minutes to 6 hours, such as about 2 hours, about 1 hour, about 30 minutes or about 10 minutes. The compound of formula (I) or (I*) can be separated by methods generally known to those skilled in the art, such as silica gel chromatography.

[0367] Hydrothiolation of compound (I) or (I*) with a compound of formula (II)

[0368] The thiophosphonate or phosphonate of formula (I) or (I*) can be subjected to a hydrothiolation reaction with a thiol of formula (II) in a suitable solvent. The solvent system can be selected from a variety of solvents. The solvent can be a polar aprotic solvent system, such as tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile (MeCN), acetone, dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), N-ethylpyrrolidone or a mixture thereof, preferably THF, DMF, DMSO; a non-polar solvent, such as hexane, toluene, benzene, 1,4-dioxane, chloroform, ether or dichloromethane (DCM), preferably DCM; a polar protic solvent, such as water, ethanol, isopropanol, methanol, n-butanol, preferably ethanol; or a mixture thereof. For example, the hydrothiolation can be carried out in DMF or a DMF / water mixture. In particular, when a biomolecule such as a protein, antibody, peptide, nucleotide or oligonucleotide is reacted, the hydrothiolation can be carried out in DMF or a DMF / water mixture. The solvent may also be an aqueous medium, such as water or an aqueous buffer, such as phosphate buffered saline (PBS), tris(hydroxymethyl)-aminomethane (TRIS), bicarbonate, EDTA / NH 4 HCO 3Buffer, EDTA / NH in phosphate buffered saline (PBS) 4 HCO 3 or borate-containing phosphate buffered saline. If a biomolecule such as a protein, antibody, peptide, nucleotide or oligonucleotide is used in the hydrothiolation reaction, it is preferred to carry out the reaction in a buffer. The hydrothiolation can also be carried out in a mixture of any of the aforementioned aqueous buffers and DMF. Those skilled in the art can easily select suitable solvents and buffers.

[0369] Preferably, the hydrothiolation reaction of the thiophosphonate or phosphate is carried out under alkaline conditions, in particular under slightly alkaline conditions, such as at a pH of 7.2 to 9, such as at a pH of 8 or 8.5. Such alkaline conditions can be established by using a suitable buffer system, such as, for example, by using any of the above-mentioned buffers. Additionally or alternatively, alkaline conditions for the hydrothiolation reaction can also be established by using a weak base. Suitable bases are, for example, (NH 4 ) 2 CO 3 、Na 2 CO 3 , Rb 2 CO 3 , K 2 CO 3 or Cs 2 CO 3 carbonates or their related bicarbonates (such as NaHCO 3 etc.); and weak nitrogenous bases, such as trimethylamine Et 3 N (pKa 10.76 at 25°C). Preferably, a base with a pKa value of 7.5 to 11.5 is used. A person skilled in the art will easily select a suitable base.

[0370] The reaction temperature of the hydrothiolation is not particularly limited. For example, the hydrothiolation can be carried out at a temperature of 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 0°C to 30°C, such as at room temperature (i.e., about 25°C), such as at about 5°C, or under physiologically relevant conditions such as about 37°C. The reaction time depends on the temperature, the reaction volume and the amount of the substance. As a guide, the reaction can be carried out in a time frame such as 1 minute to 24 hours, such as in a time frame of 1 minute to 20 hours, in a time frame of 1 minute to 10 hours, in a time frame of 1 minute to 3 hours, or even in a time frame of 1 minute to 1 hour. Suitable reaction temperatures and reaction times can be easily determined by those skilled in the art.

[0371] Further details regarding the hydrothiolation reaction are given in the Examples section below. Example

[0372] Example 1: Synthesis of olefin-thiophosphonate

[0373] The inventors have developed two different synthetic routes to obtain olefin-thiophosphonates: a one-step reaction from electrophilic disulfides or alternatively from phosphine trichloride (PCl) or thiocyanate (PcCl). 3 ) of a phosphorus trihalide. Both pathways and isolated compounds are described herein (see Example 10 below for details of the synthesis and characterization) and depicted in Scheme 2. As an illustrative example only, it is demonstrated that R 1 Derivatives of methyl or ethyl (O-ethyl and O-methyl derivatives (R 1 = methyl, ethyl); see Scheme 2) for the synthesis. However, neither synthetic route is limited to this scope. As used in the Examples section, “R 2 ” or in some cases just “R”, i.e. a residue bound to S, corresponding to ● used in this specification.

[0374] Disulfide pathway: EWG: electron-withdrawing group PCl 3 way:

[0375] Scheme 2: Synthesis of olefin-thiophosphonates

[0376] Electrophilic disulfide pathway

[0377] Olefin-phosphonothioates can be obtained in a one-step reaction from an electrophilic disulfide and an alkenyl phosphite. Without wishing to be bound by any theory, it is hypothesized that the free lone pair of the phosphorus atom of the phosphite attacks the electrophilic sulfur of the disulfide, thereby generating a highly reactive intermediate that is rapidly oxidized to the phosphonothioate.

[0378] Diethyl olefin phosphinates were synthesized according to a published protocol (MRJ Vallée, Angewandte Chemie Int. Ed., 2013, 52 (36), 9504) and reacted with different aliphatic electrophilic disulfides, see Scheme 3. The electrophilic disulfides were obtained by reaction of the corresponding thiols with 2,2'-dithiobis(5-nitropyridine) (PNP). O-ethyl olefin-thiophosphonates were separated by silica gel column chromatography. The yields are given in Table 1.

[0379]

[0380] Scheme 3: Synthesis of O-ethyl olefin-phosphonothioates via reaction of diethyl olefin phosphinates with electrophilic disulfides.

[0381]

[0382] Table 1: O-Ethyl olefin-thiophosphonates synthesized via the disulfide route and isolated yields.

[0383] PCl 3 way

[0384] Alternatively, in some substitution reactions, PCl can then be oxidized by oxidation with an oxidizing agent such as tBuOOH. 3 Conversion to olefin-phosphonothioate (Scheme 4).

[0385]

[0386] Solution 4: From PCl 3 Synthesis of olefin-thiophosphonates.

[0387] Following this approach, benzyl and Boc protected amine derivatives were obtained and separated by silica gel chromatography (see Table 2 for structures and yields).

[0388]

[0389] Table 2: PCl 3 O-Ethyl olefin-phosphonothioate synthesized via the described route and its isolated yield.

[0390] Example 2: Synthesis of Alkyne-Phosphonothioate

[0391] Similar to olefin-thiophosphonates (see Example 1 above and Scheme 5 below), PCl 3 Initial access to alkyne-phosphonothioates.

[0392] PCl 3 way:

[0393]

[0394] Option 5: Through PCl 3 Route to synthesize alkyne-phosphonothioates.

[0395] Using PCl 3 The inventors were able to isolate the desired product by silica gel chromatography (see Scheme 6 and Table 3 for structure and yield).

[0396]

[0397] Scheme 6: Synthesis of alkyne-phosphonothioates.

[0398]

[0399]

[0400] Table 3: Synthesis of O-methylalkynes-PT and isolated yields. *(starting from N,N-diisopropylmethylphosphonamidic chloride)

[0401] Example 3: Further functionalization of the general thiophosphonate building block

[0402] The generic carboxylic acid or amine phosphonothioate derivatives can be further modified by means such as amine coupling with functionalized building blocks such as fluorophores EDANS or biotin. Two illustrative examples are given in Scheme 7 (see Example 14 below for more compounds and alkyne derivatives).

[0403]

[0404] Scheme 7: Examples of functionalization of the general thiophosphonate building block. A) Carboxylic acid derivatives can be activated to NHS esters and further reacted with amines such as the fluorophore EDANS. B) Alternatively, the reactivating agent can be exchanged by coupling the amine PT to a carboxylic acid such as biotin.

[0405] Example 4A: Thiol Addition of Glutathione to Thiophosphonate

[0406] As a proof of principle for the reaction of unsaturated phosphonothioates with thiols, the present inventors performed coupling with the model thiol glutathione in alkaline aqueous buffer to obtain water-soluble phosphonothioate-conjugates. 31 The reaction was monitored by P-NMR (see Figure 1 of olefin-phosphonothioate).

[0407] Figure 1 Shown are: A) Synthesis of glutathione-olefin-phosphonothioate conjugate 14. Analysis by B) UV LC-MS and C) 31 The reaction was monitored by P-NMR, indicating the formation of a single product. P: product 14, SM: starting material = olefin-phosphonothioate 2, PMe 4 Br = Tetramethylphosphonium bromide, internal standard 31 P-NMR.

[0408] pass 31 The reaction was monitored by P-NMR and UPLC-MS, indicating a clean reaction to a single product (P).

[0409] Similar to the above, the present inventors carried out the coupling of glutathione with an alkyne derivative, namely with S-benzyl O-methylalkyne-PT 7 from Example 11 below (Table 3, entry 1) (see Figure 2A). The reaction is much faster than that with olefin-phosphonothioates (kinetic studies are also compared, see Example 5 below). Under the specified conditions, the inventors observed that the alkyne-phosphonothioate was completely converted after about 1 minute. The two double bond isomers are formed in a ratio of about 3:1.

[0410] Figure 2 Shown are: A) the synthesis of glutathione-alkyne-PT-conjugate 15. The reaction was monitored by B) UV LC-MS, indicating the formation of two double bond isomers in a ratio of approximately 3: 1. Inosine was used as an internal standard.

[0411] Example 4B: Thiol Addition of Glutathione to Olefin Phosphonates

[0412] The present inventors carried out the coupling of diethyl olefin phosphonate and glutathione by the thiol addition method to obtain a water-soluble conjugate (see Scheme 1).

[0413]

[0414] Scheme 1: Synthesis of glutathione-phosphonate conjugates.

[0415] pass 31 The reaction was monitored by P-NMR and UPLC-MS, indicating a clean reaction to a single product. The conjugate could be isolated by semi-preparative HPLC (acidic conditions) in 60% yield.

[0416] Example 5: Kinetic Study of Thiol Addition to Thiophosphonates and Phosphonates

[0417] In the next step, the inventors set out to study the kinetics of the addition of thiols to unsaturated thiophosphonates in order to obtain kinetic data. To this end, the inventors carried out addition reactions of olefin- and alkyne-thiophosphonates with fluorescent EDANS at room temperature (about 25° C.) in the presence of 1 equivalent of reduced glutathione (Scheme 8). The inventors also carried out the same studies using the corresponding phosphonate derivatives. For the preparation of these compounds, see Example 14 below.

[0418]

[0419] Scheme 8: Coupling of glutathione with fluorescent alkene- and alkyne-phosphonothioates and phosphonates.

[0420] The decay of the starting material was monitored by HPLC using a fluorescence detector over the course of 8 hours. The results of this study are shown below. Figure 3The following two trends are evident: First, in the thiol addition, alkyne derivatives react much faster than olefin derivatives. Second, phosphonothioates react faster than phosphonates. This is an important advantage of phosphonothioates because it will allow the user to perform the reaction at lower concentrations and obtain higher conversions in a shorter time, ultimately increasing the yield of the reaction. This is critical in the production of, for example, antibody-drug conjugates, which typically require a high drug to antibody ratio.

[0421] Figure 3 Shown is a kinetic study of the addition of glutathione to olefins and alkynes phosphonothioates and phosphonates at pH 8.5. Shown is the decay of the starting material over time. The fluorescence intensity of the remaining starting material relative to the internal standard was measured using HPLC with a fluorescence detector. Each reaction was performed three times. Shown is the average with standard deviation. An Area starting material of 1.0 means 100% starting material.

[0422] Example 6: Stability study of thiophosphonate conjugates

[0423] When using unsaturated phosphonothioates as bioconjugate handles, an important consideration is whether the resulting thiol addition product is stable under physiologically relevant conditions. To address this issue, the inventors utilized the Dabcyl-EDANS quencher pair and synthesized the corresponding olefin- and alkyne-phosphonothioate conjugates, as well as the corresponding olefin- and alkyne-phosphonothioate conjugates ( Figure 4 ). These compounds allow us to monitor the stability of the conjugates in complex matrices such as cell lysates or serum.

[0424] When the conjugate is intact, Dabcyl and EDANS are in close proximity and the fluorescence of EDANS is quenched. Once the conjugate is cleaved, EDANS is released, and its fluorescence can be detected and quantified.

[0425] These quencher pairs allow monitoring not only the stability of the PS bond, but also the stability of the thiol conjugates. This is very important, since potential retro-thiol additions or exchanges with other thiols can also be detected.

[0426] Figure 4 Shown: Design of an EDANS-Dabcyl quencher pair linked via thiophosphonate or phosphonate coupling chemistry. If the enclosed moiety is cleaved, then EDANS loses its accessibility to Dabcyl and thus the fluorescence of EDANS will no longer be quenched.

[0427] The inventors monitored the stability of olefin- and alkyne-phosphonothioates and olefin- and alkyne-phosphonates under the following conditions: PBS (phosphate buffered saline) pH=7.4, Hela cell lysate, human serum. The temperature was room temperature (about 25°C). The fluorescence was monitored over the course of about 3 days. As positive controls, uncoupled EDANS and free Dabcyl-peptide (1:1) were measured under the same conditions. The results are shown in Figure 5 The right column shows a zoom of the same data set as the left column. Again, as a control, phosphonothioates and phosphonates were treated with 1 M NaOH, which resulted in rapid cleavage of phosphonothioates and phosphonates. This confirms that cleavage, if any, occurred only to a very low extent in the assays using PBS, HeLa cell lysate, or human serum at pH = 7.4.

[0428] All derivatives showed good stability in phosphate buffered saline (PBS, pH 7.4). They also showed good stability in Hela cell lysates and serum, indicating that thiophosphonate-based bioconjugates and phosphonate-based bioconjugates are stable under physiologically relevant conditions.

[0429] Figure 5 Shown are the stability of phosphonothioate conjugates and phosphonate conjugates under the indicated conditions. Increased fluorescence indicates conjugate cleavage.

[0430] Example 7: Protein conjugation with thiophosphonates

[0431] Next, the present inventors coupled alkene-alkyne-phosphonothioate to the model protein bovine serum albumin (BSA) and the monoclonal antibody cetuximab.

[0432] Example 7A1: Coupling of bovine serum albumin with thiophosphonates

[0433] As a model protein, the inventors selected bovine serum albumin (BSA) to couple to olefins- and alkyne-phosphonothioates. This experiment demonstrated that olefins- and alkyne-phosphonothioates are suitable bioconjugation treatment agents for selective modification of cysteine ​​at the protein level. BSA has a reduced cysteine ​​residue (Cys58) that can be alkylated by PT. In theory, since other cysteines are present in disulfide bridges, they do not react with phosphonothioates.

[0434] For modification, BSA solution was reacted with excess PT at pH = 7.4-8.5 at 4°C for 18 h (see Scheme 9).

[0435]

[0436] Scheme 9: BSA modification using PT

[0437] After the reaction, the modified proteins were subjected to SDS-PAGE, followed by in-gel trypsin digestion, and the obtained peptides were then subjected to MS / MS analysis.

[0438] The results of MS / MS analysis are summarized in Table 4. Both derivatives obtained good BSA sequence coverage. Moreover, the degree of modification was high (>50% for olefin-phosphonothioate; and>90% for alkyne-phosphonothioate). For olefin-phosphonate derivatives, other amino acids (His, Ser, Thr, Arg) were also modified to some extent. However, using less equivalents and / or reducing the pH to 7.4 reduced the reaction with these amino acids. It was observed that the reaction of alkyne-phosphonothioate with cysteine ​​was more selective than that of olefin-phosphonothioate.

[0439]

[0440]

[0441] Table 4: MS / MS analysis results of thiophosphonate-modified BSA protein.

[0442] In summary, the results demonstrate the selectivity of cysteine, i.e., the cysteine ​​of BSA is selectively modified with alkene or alkyne phosphonothioates as desired.

[0443] Example 7A2: Coupling of bovine serum albumin with phosphonates

[0444] The inventors could further demonstrate that diethyl olefin phosphonates can be coupled to the model protein bovine serum albumin (BSA) (Scheme 10) in a cysteine-selective manner (see Table 5), as shown by MS / MS analysis of the conjugates. BSA has one reduced cysteine ​​residue (Cys58) which is available for alkylation.

[0445]

[0446] Scheme 10: Modification of BSA using diethyl olefin phosphonate.

[0447]

[0448] Table 5: Results of MS / MS analysis of phosphonate-modified BSA protein.

[0449] Together, these results demonstrate cysteine ​​selectivity, i.e., the cysteine ​​of BSA is selectively modified with either alkene or alkyne phosphonates, as desired.

[0450] Example 7B: Conjugation of Antibodies to Phosphonothioates

[0451] Finally, the inventors used this new cysteine ​​selective reaction sequence event for the coupling of IgG antibodies (Scheme 11). The modification strategy relies on the two-step reduction-alkylation method previously applied to maleimide coupling (SO Doronina, BE Toki, MY Torgov, BA Mendelsohn, CG Serveny, DF Chace, RL De Blanc, RP Gearing, TD Bovee, CB Siegall, JA Francisco, AF Wahl, DL Meyer, PD Senter, Nat Biotech 2003, 21, 778-784.). In the first step, the interchain disulfide bridges that hold the antibody together are reduced by treating with dithiothreitol (DTT). The free sulfhydryl groups can then react with thiophosphonates in a thiol addition reaction. The first experiments were carried out with cetuximab, which is a monoclonal IgG1 antibody against human epidermal growth factor.

[0452]

[0453] Scheme 11: Two-step reduction and alkylation method for cysteine-selective antibody modification using biotin-modified olefin-phosphonothioate 4.

[0454] As proof of principle, antibodies were modified with biotin olefin-derivative 4 and analyzed by SDS-PAGE followed by anti-biotin Western blotting.

[0455] The results of anti-biotin Western blot analysis are shown in Figure 6 . It can be confirmed that the antibody fragments (heavy chain and light chain) are modified by the biotin phosphonothioate derivative 4 at pH = 7.4 (lane 3) and pH = 8.5 (lane 5). The yield is higher at a higher pH = 8.5 (compare lanes 3 and 5). It should be noted that if the disulfide bonds are not reduced with DTT in advance, the modification cannot be detected (lanes 4 and 6). For comparison, the antibody was also labeled with biotin-maleimide at pH = 7.4 using the same protocol. Although the degree of modification in the case of maleimide is higher than PT (compare lanes 1 and 3), since modification is also detected in this case for the non-reduced antibody (lane 2), the reaction is obviously not chemically selective for cysteine.

[0456] Figure 6Shown: Western blot analysis of cetuximab conjugate after reduction of SDS-gel. Upper panel: Ponceau S stain of membrane after blotting shows equal amounts of blotted antibody. Lower panel: Chemiluminescent detection of biotin-modified antibody fragments (Strep-HRP). Reaction of reduced antibody with biotin compound was performed at pH = 7.4 (lanes 1-4) or at pH = 8.5 (lanes 5-6). M = marker (protein ladder).

[0457] Optionally, the inventors also performed the coupling of cetuximab with biotin-phosphonothioate derivatives 28 (olefin) and 13 (alkyne) at pH 8.5 ( Figure 7 A), and conjugation of cetuximab to 28 was performed at pH 7.4, 8.0, and 8.5 as a control ( Figure 7 B) Alkyne-phosphonothioates are more efficient than alkene-phosphonothioates in labeling antibodies ( Figure 7 A). With respect to olefin-phosphonothioate 4 ( Figure 6 ) similarly, for alkyne-phosphonothioate 28, more labeled antibody was obtained with alkyne-phosphonothioate as the pH increased.

[0458] Figure 7 Shown are: Western blot analysis of cetuximab conjugation after reducing SDS-gel. A) Modification with compounds 13 and 28 at pH 8.5. B) Modification with compound 28 at pH 7.4-8.5.

[0459] In summary, the results demonstrate cysteine ​​selectivity, i.e., antibody cysteines are selectively modified with olefin or alkyne phosphonothioates as desired.

[0460] Example 8: Synthesis Procedure of Electrophilic Disulfides

[0461] General Procedure A: Synthesis of mixed disulfides from 2,2'-dithiobis(5-nitropyridine)

[0462]

[0463] In a flame-dried round-bottom flask, 1.0 mmol (1.0 eq.) of thiol in 10 ml (c = 0.1 M) of THF was added. Subsequently, 3.0 mmol (3.0 eq.) of triethylamine and 1.2 mmol (1.2 eq.) of the disulfide 2,2'-dithiobis(5-nitropyridine) were added, and the reaction mixture was stirred at room temperature for 10 minutes. The reaction was monitored by TLC. When complete conversion was reached (about 10 minutes), the volatiles were evaporated under reduced pressure and the residue was purified by flash column chromatography on silica gel.

[0464] 2-(Ethyldisulfanyl)-5-nitropyridine

[0465]

[0466] 2-(Ethyldisulfanyl)-5-nitropyridine (103 μl, 1.34 mmol) was prepared from ethanethiol according to the general procedure A. The crude product was purified by flash column chromatography (hexanes / EtOAc=10:1) to give the title compound (228 mg, 1.05 mmol, 78%) as a yellow oil.

[0467] 1 H NMR (300 MHz, CHLOROFORM-d) δ = 9.24 (d, J = 2.6, 1H), 8.39 (dd, J = 8.9, 2.6, 1H), 7.92 (d, J = 8.9, 1H), 2.85 (q, J = 7.3, 2H), 1.34 (t, J = 7.3, 3H) ppm.

[0468] 13 C NMR (75M, chloroform-d) δ = 169.42, 145.15, 141.99, 131.67, 119.18, 33.01, 14.36 ppm.

[0469] HRMS(ESI):C 7 H 9 N 2 O 2 S 2 [M+H + ] calculated value: 217.0100; measured value: 217.0106.

[0470] 2-(Benzyldisulfanyl)-5-nitropyridine

[0471] 2-(Benzyldisulfanyl)-5-nitropyridine (48 μl, 0.41 mmol) was prepared from benzyl mercaptan according to the general procedure A. The crude product was purified by flash column chromatography (hexanes / EtOAc=10:1) to give the title compound (91 mg, 0.33 mmol, 80%) as a colorless solid.

[0472] 1 H NMR (300 MHz, CHLOROFORM-d) δ = 9.18 (d, J = 2.6, 1H), 8.15 (dd, J = 8.9, 2.6, 1H), 7.49 (d, J = 8.9, 1H), 7.30-7.24 (m, 2H), 7.24-7.17 (m, 3H), 4.04 (s, 2H) ppm.

[0473] 13C NMR (75 MHz, chloroform-d) δ = 168.90, 144.86, 141.81, 136.10, 131.17, 129.50 (2C), 128.83 (2C), 128.04, 119.03, 77.36, 43.86 ppm.

[0474] HRMS(ESI):C 12 H 11 N 2 O 2 S 2 [M+H + ] calculated value: 279.0256; measured value: 279.0271.

[0475] 3-((5-Nitropyridin-2-yl)disulfanyl)propanoic acid

[0476]

[0477] 3-((5-Nitropyridin-2-yl)disulfanyl)propanoic acid (250 μl, 2.85 mmol) was prepared from mercaptopropionic acid according to the general procedure A. The crude product was purified by flash column chromatography (hexane / EtOAc=1:1+0.1% formic acid) to give the title compound (80 mg, 1.54 mmol, 54%) as a yellow oil.

[0478] 1 H NMR (300MHz, chloroform-d) δ = 9.26 (d, J = 2.6 Hz, 1H), 8.40 ( dd, J = 8.8, 2.7 Hz, 1H), 7.87 ( d, J = 8.8 Hz, 1H), 3.09 ( t, J = 6.8 Hz, 2H), 2.82 ( t, J = 6.8 Hz, 2H) ppm.

[0479] 13 C NMR (75 MHz, chloroform-d) δ = 177.25, 168.05, 145.34, 142.35, 131.91, 119.68, 33.69, 33.33.

[0480] HRMS: n.d.

[0481] 2-(Biotinyldisulfanyl)-5-nitropyridine

[0482]

[0483] 2-(Biotinyldisulfanyl)-5-nitropyridine (44 mg, 0.179 mmol) was prepared from biotinthiol according to general procedure A. The crude product was purified by flash column chromatography (DCM / MeOH=20:1 to 10:1) to give the title compound (53 mg, 0.134 mmol, 75%) as a yellow solid.

[0484] 1 H NMR (300MHz, chloroform-d) δ = 9.27 (d, J = 2.6Hz, 1H), 8.42 (dd, J = 8.9, 2.6Hz, 1H), 7.91 (d, J = 8.9Hz, 1H), 4.51 (s, 1H) ,4.37-4.24(m,1H),3.14(d,J=6.4Hz,1H),2.97-2.68(m,4H),1.76-1.61(m,4H),1.44(d,J=3.5Hz,4H)ppm.

[0485] HRMS(ESI):C 15 H 21 N 4 O 3 S 3 [M+H + ] calculated value: 401.0770; measured value: 401.0791.

[0486] Example 9: Synthesis procedure of phosphorus (III) precursor

[0487] 1-Ethoxy-1-ethynyl-N,N-diisopropylphosphine

[0488]

[0489] Phosphorus trichloride (12.5 mmol, 1090 μl) and anhydrous ether (50 ml) were added to a flame-dried round-bottom Schlenk flask under an argon atmosphere and cooled to -30°C in a dry ice bath. Ethanol (12.5 mmol, 728 μl) and triethylamine (12.5 mmol, 1.733 ml) were added, and the solution was stirred at -30°C for 10 minutes, then warmed to room temperature and stirred for another hour. The resulting white suspension was filtered through celite. The filtrate was collected in a flame-dried round-bottom Schlenk flask under an argon atmosphere and cooled to -30°C again. Diisopropylamine (25 mmol, 3.528 ml) was added, and the reaction mixture was stirred at -30°C for 10 minutes, then warmed to room temperature and stirred for another hour. The resulting suspension was filtered through celite again. Under an argon atmosphere, the clear filtrate was cooled to -78°C, and ethynylmagnesium bromide solution (0.5M in THF, 13.75 mmol, 27.5 ml) was added, and stirred at -78°C for 10 minutes, and then stirred at room temperature for 1 hour. The reaction mixture was then concentrated under reduced pressure to about 20 ml and washed with saturated NaHCO 3 The solution was diluted with aqueous solution (60 ml) and extracted with EtOAc (3 x 120 ml). The combined organic layers were washed with NaSO 4 Dry, filter, and remove the solvent under reduced pressure. The resulting oily residue was purified by silica gel chromatography to give the title compound (1.236 g, 6.14 mmol, 49%) as a yellow oil. 1 H and 31 The purity was judged to be about 80% by P NMR. The compound was used in the next step without further purification.

[0490] 1 H-NMR (300 MHz, acetonitrile-d 3 )δH=3.84-3.57(m,4H),3.35(d,J=1.8 Hz,1H),1.20(m,15H)ppm.

[0491] 13 C-NMR (75 MHz, acetonitrile-d 3 )δC=91.78(d,J=7.4 Hz), 85.72(d,J=17.8Hz), 61.9(d,J=16.2 Hz), 47.4, 23.5, 16.5(d,J=16.5)ppm.

[0492] 31 P-NMR (122 MHz, acetonitrile-d 3 )δP=92.25 ppm.

[0493] HRMS(ESI):NaC 10 H 20 NOP[M+Na + ] calculated value: 224.1180; measured value: 224.1270.

[0494] 1-Ethoxy-N,N-diisopropyl-1-vinylphosphine

[0495]

[0496] Phosphorus trichloride (12.5 mmol, 1090 μl) and anhydrous ether (50 ml) were added to a flame-dried round-bottom Schlenk flask under an argon atmosphere and cooled to -30°C in a dry ice bath. Ethanol (12.5 mmol, 728 μl) and triethylamine (12.5 mmol, 1.733 ml) were added, and the solution was stirred at -30°C for 10 minutes, then warmed to room temperature and stirred for another hour. The resulting white suspension was filtered through celite. The filtrate was collected in a flame-dried round-bottom Schlenk flask under an argon atmosphere and cooled to -30°C again. Diisopropylamine (25 mmol, 3.528 ml) was added, and the reaction mixture was stirred at -30°C for 10 minutes, then warmed to room temperature and stirred for another hour. The resulting suspension was filtered through celite again. Under argon atmosphere, the clear filtrate was cooled to -78°C, and vinylmagnesium bromide solution (1.0 M in THF, 13.75 mmol, 13.75 ml) was added, stirred at -78°C for 10 minutes, and then stirred at room temperature for 1 hour. The reaction mixture was then concentrated under reduced pressure to about 20 ml and washed with saturated NaHCO 3 The mixture was diluted with aqueous solution (60 ml) and extracted with EtOAc (3 x 120 ml). The combined organic layers were washed with NaSO 4 Dry, filter, and remove the solvent under reduced pressure. The resulting oily residue was purified by silica gel chromatography to give the title compound (852 mg, 4.19 mmol, 34%) as a colorless oil. 1 H and 31 Purity was >95% as judged by P NMR.

[0497] 1 H-NMR (300 MHz, acetonitrile-d 3 )δH=6.36-6.14(m,1H),5.77-5.53(m,2H),3.81-3.61(m,2H),3.47(dt,J=9.8,6.7Hz,2H),1.23-1.04(m,15H)ppm.

[0498] 13 C-NMR (75 MHz, acetonitrile-d3 )δC=142.30(d,J=6.9Hz), 124.41(d,J=19.6Hz), 62.1(d,J=20.7Hz), 45.5(d,J=9.50Hz), 23.90(m), 16.66(d,J=7.7Hz)ppm.

[0499] 31 P-NMR (122 MHz, acetonitrile-d 3 )δP=113.79ppm.

[0500] HRMS(ESI):C 10 H 23 NOP[M+H + ] calculated value: 204.1517; measured value: 204.1596.

[0501] Example 10: Synthesis Procedure of Olefin Thiophosphonates

[0502] General Procedure B: Synthesis of Olefin-PT via the Disulfide Route

[0503]

[0504] Under argon atmosphere, 0.5 mmol (1.0 eq.) of mixed disulfide was placed in a flame-dried Schlenk tube and dissolved in a mixture of anhydrous THF / toluene (2:1, 5 ml). Diethyl olefin phosphite solution (about 0.6 M in THF, 0.6 mmol, 0.6 ml, 1.2 eq.) was added dropwise to the stirred mixture at room temperature and stirred for 10 minutes. The reaction mixture was then dry-loaded on silica gel and purified by flash column chromatography.

[0505] O,S-Diethylolefin-PT (Compound 1)

[0506]

[0507] O,S-Diethylolefin-PT was prepared from mixed disulfide 2-(ethyldisulfanyl)-5-nitropyridine (100 mg, 0.462 mmol) according to General Procedure B. The crude product was purified by flash column chromatography (hexanes / EtOAc=4:1 to 1:1 gradient) to give the title compound (44 mg, 0.244 mmol, 53%) as a colorless oil.

[0508] 1H NMR (300 MHz, chloroform-d) δ = 6.40-5.98 (m, 3H), 4.30-4.06 (m, 2H), 2.89-2.72 (m, 2H), 1.41-1.30 (m, 6H) ppm.

[0509] 13 C NMR (75 MHz, chloroform-d) δ = 133.92, 131.82, 129.90, 61.83, 24.98, 16.51 ppm.

[0510] 31 P NMR (122 MHz, chloroform-d) δ = 42.12 ppm.

[0511] HRMS(ESI):C 6 H 14 O 2 PS[M+H + ] calculated value: 181.0447; measured value: 181.0460.

[0512] S-Benzyl O-ethylolefin-PT (Compound 2)

[0513]

[0514] S-Benzyl O-ethylolefin-PT was prepared from mixed disulfide 2-(benzyldisulfanyl)-5-nitropyridine (100 mg, 0.359 mmol) according to General Procedure B. The crude product was purified by flash column chromatography (hexane / EtOAc=4:1 to 1:1 gradient) to give the compound 2 (55 mg, 0.227 mmol, 63%) as a yellow oil.

[0515] 1 H NMR (300 MHz, chloroform-d) δ = 7.42-7.19 (m, 5H), 6.40-5.91 (m, 3H), 4.28-3.39 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H) ppm.

[0516] 13 C (75 MHz, chloroform-d) δ = 162.92, 137.01, 134.54, 130.53, 128.87, 128.59, 128.57, 127.56, 62.23, 34.37, 16.08 ppm.

[0517] 31 P NMR (122 MHz, chloroform-d) δ = 42.69 ppm.

[0518] HRMS(ESI):C 11 H15 O 2 PS[M+H + ] calculated value: 242.0525; measured value: 242.0551.

[0519] O-Ethyl S-biotin olefin-PT (Compound 4)

[0520]

[0521] O-Ethyl S-biotinene-PT was prepared from mixed disulfide 2-(biotinyldisulfanyl)-5-nitropyridine (40 mg, 0.100 mmol) in solvent mixture THF / DMF=5:1 according to General Procedure B. The crude product was purified by flash column chromatography (100% DCM to DCM / MeOH=9:1 gradient) to give the compound 4 (21 mg, 0.0576 mmol, 58%) as a yellow solid.

[0522] 1 H NMR (600MHz, chloroform-d) δ = 6.35-6.05 (m, 3H), 5.83 (d, J = 24.7Hz, 1H), 5.24 (s, 1H) ,4.52(dd,J=7.8,4.9Hz,1H),4.32(dd,J=7.8,4.6Hz,1H),4.26-4.11(m,2H) ,3.17-3.12(m,1H),2.92(dd,J=12.8,5.0Hz,1H),2.87-2.72(m,3H),2.15(s ,2H),1.75-1.60(m,4H),1.49-1.39(m,4H),1.36(td,J=7.1,1.5Hz,3H)ppm.

[0523] 13 C NMR (151 MHz, chloroform-d) δ = 163.43, 133.85, 130.75 (dd, J = 145.2, 9.9 Hz), 62.10-61.69 (m, 2C), 60.17, 55.52, 40.53, 30.59 (t, J = 5.2 Hz), 30.12, 28.51 (d, J = 8.7 Hz), 28.30 (d, J = 25.5 Hz, 2C), 16.32 (d, J = 6.8 Hz) ppm.

[0524] 31 P NMR (122 MHz, chloroform-d) δ = 42.57 ppm.

[0525] HRMS(ESI):C 14 H 26 N 2 O3 PS 2 [M+H + ] calculated value: 365.1117; measured value: 365.1141.

[0526] 3-((Ethoxy(olefin)phosphoryl)thio)propionic acid (Compound 3)

[0527]

[0528] 3-((ethoxy(olefin)phosphoryl)thio)propanoic acid was prepared from mixed disulfide 3-((5-nitropyridin-2-yl)disulfanyl)propanoic acid (146 mg, 0.561 mmol) according to General Procedure B. The crude product was purified by flash column chromatography (hexane / EtOAc without acid addition = 4:1 to EtOAc + 0.1% formic acid) to give compound 3 (33.7 mg, 0.150 mmol, 27%) as a colorless oil. An analytically pure sample was obtained by subsequent HPLC purification (5-40% MeCN in 30 min) *.

[0529] 1 H NMR (600 MHz, chloroform-d) δ = 9.25 (s, 1H), 6.38-6.08 (m, 3H), 4.26-4.13 (m, 2H), 3.09-2.96 (m, 2H), 2.74 (t, J = 7.2, 2H), 1.35 (t, J = 7.1, 3H) ppm.

[0530] 13 C NMR (151MHz, chloroform-d) δ = 174.98, 134.92, 130.18 (d, J = 145.9Hz), 62.52 (d, J = 6.9Hz), 35.73 (d, J = 3.5Hz), 25.02 (d, J = 2.8Hz), 16.38 (d, J = 6.9Hz) ppm.

[0531] 31 P NMR (122 MHz, chloroform-d) δ = 43.17 ppm.

[0532] HRMS(ESI):C 7 H 14 O 4 PS 2 [M+H + ] calculated value: 225.0345; measured value: 225.0358.

[0533] 2,5-Dioxopyrrolidin-1-yl 3-((ethoxy(olefin)phosphoryl)thio)propionate (Compound 10)

[0534]

[0535] 10 mg of carboxylic acid 3-((ethoxy(olefin)phosphoryl)thio)propionic acid 3 (0.0446 mmol, 1.0 eq) and N-hydroxysuccinimide (5.1 mg, 0.0446 mg, 1.0 eq.) were dissolved in 400 μl of a mixture of dioxane / EtOAc=1:1 and cooled to 0°C. 9.2 mg of dicyclohexylcarbodiimide (0.0446 mmol, 1.0 eq.) was added to the stirred solution and the mixture was allowed to warm to room temperature. After 1 hour, the suspension was filtered and the clear residue was dried under reduced pressure to obtain 12 mg of the product 10 (0.0374 mmol, 84%).

[0536] 1 H NMR (600 MHz, chloroform-d) δ = 6.39-6.09 (m, 3H), 4.29-4.13 (m, 2H), 3.19-2.99 (m, 4H), 1.37 (t, J = 7.1 Hz, 3H) ppm.

[0537] 13 C NMR (151MHz, chloroform-d) δ = 168.92, 166.85, 134.76, 130.50 (d, J = 146.2Hz), 62.41 (d, J = 6.8Hz), 33.03 (d, J = 3.0Hz), 25.71, 24.57 (d, J = 2.9Hz), 16.40 (d, J = 6.9Hz) ppm.

[0538] 31 P NMR (122 MHz, chloroform-d) δ = 41.44 ppm.

[0539] HRMS(ESI):C 11 H 17 NO 6 PS[M+H + ] calculated value: 322.0509; measured value: 322.0540.

[0540] 5-((2-(3-((ethoxy(olefin)phosphoryl)thio)propionamido)ethyl)amino)naphthalene-1-sulfonic acid (compound Object 11)

[0541]

[0542] 30 mg of NHS ester 2,5-dioxopyrrolidin-1-yl 3-((ethoxy(olefin)phosphoryl)thio)propanoate 10 (0.0934 mmol, 1.0 eq.) and 30 mg of EDANS (0.103 mmol, 1.1 eq.) were dissolved in 470 μl DMF placed in an Eppendorf tube. 33 μl (0.189 mmol, 2.0 eq.) of DIPEA were added and the suspension was stirred at room temperature for 15 minutes. The volatiles were evaporated under reduced pressure and the crude product was purified by preparative HPLC (5-60% MeCN in 40 min, flow rate 16 ml / min) to give 24 mg of the compound 11 (0.0508 mmol, 54%) as a white powder after lyophilization.

[0543] 1 H NMR (600 MHz, DMSO-d 6 )δ=8.32(d,J=8.6Hz,1H),8.22(t,J=5.8Hz,1H),8.07(d,J=8.5Hz,1H),7.96(d,J=7.0Hz ,1H),7.40(dd,J=8.5,7.1Hz,1H),7.34(t,J=8.1Hz,1H),6.81(d,J=7.3Hz,1H),6.38(dd d,J=28.0,18.4,12.4Hz,1H),6.25-6.11(m,2H),4.14-3.98(m,2H),3.41(t,J=6.2Hz,2H ), 3.32 (t, J = 6.5 Hz, 2H), 2.93 (ddt, J = 18.0, 12.9, 6.6 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H) ppm.

[0544] 13 C NMR (151 MHz, DMSO-d 6 )δ=170.42,144.21,134.28,130.60(d,J=142Hz),130.09,125.92,124.59,124.07,123.23,122.62 , 61.37 (d, J = 6.7Hz), 44.79, 37.23, 36.23 (d, J = 4.0Hz), 25.42 (d, J = 2.8Hz), 16.10 (d, J = 6.5Hz) ppm.

[0545] 31 P NMR (243 MHz, DMSO-d 6 )δ=41.29ppm.

[0546] HRMS(ESI):C 19H 26 N 2 O 6 PS 2 [M+H + ] calculated value: 473.0964; measured value: 473.0984.

[0547] General Procedure C: via PCl 3 Pathway to synthesize olefins-PT

[0548] 1-Ethoxy-N,N-diisopropyl-1-vinylphosphinamine (as 0.3 mmol) was added to a round bottom flask, dissolved in anhydrous acetonitrile (3 ml) and cooled to -40°C. A solution of thiol (0.3 mmol) in tetrazole (0.45 M in MeCN, 0.6 mmol, 1.33 ml) was prepared separately and added to the stirred mixture at -40°C. The reaction mixture was stirred at -40°C for 10 minutes, then warmed to room temperature and stirred for another 30 minutes. To this mixture was added tert-butyl hydroperoxide solution (H 2 O (70 wt.%, 0.3 mmol, 86 μl) and stirred for 10 minutes. The reaction mixture was then washed with H 2 O (10 ml) and extracted with DCM (3 x 30 ml). The combined organic layers were washed with Na 2 SO 4 Dry, filter, and remove the solvent under reduced pressure.Purify the crude product by silica gel chromatography.

[0549] Tert-butyl (2-((ethoxy(vinyl)phosphoryl)thio)ethyl)carbamate (Compound 6)

[0550]

[0551] Compound 6 was prepared starting from 1-ethoxy-N,N-diisopropyl-1-vinylphosphinamine (93 mg, 0.465 mmol) and tert-butyl (2-mercaptoethyl)carbamate (82 mg, 0.465 mmol) according to general procedure C. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (30 mg, 0.10 mmol, 22%) as a colorless oil.

[0552] Example 11: Procedure for the Synthesis of Alkyne Phosphonothioates

[0553] S-Benzyl O-methylacetylene-PT (Compound 7)

[0554]

[0555] Under argon atmosphere, anhydrous THF (17 mL) was added to a flame-dried Schlenk tube equipped with a stirring bar, followed by a THF solution of ethynylmagnesium bromide (0.5 M, 4 mL, 2 mmol). The solution was then cooled to -78 °C, 1-chloro-N,N-diisopropyl-1-methoxyphosphinamine (0.39 mL, 2 mmol) was added, stirred for 10 min, then warmed to 0 °C for 35 min, and then to room temperature (0.5 mL of the reaction mixture was then taken for 31 P-NMR). The reaction mixture was then transferred to a flame-dried RBF under argon, the solvent was removed in vacuo, leaving the solid, and the flask was filled with argon. The solid was cooled to -40 °C, anhydrous tetrazole in MeCN (11 mL, 5 mmol) was added, anhydrous benzyl mercaptan (0.6 mL, 1.7 mmol) was added, and stirred overnight to obtain a solid suspension. 70% tert-butyl hydroperoxide in H 2 O solution (1.6 mL, 12 mmol) and then stirred for 30 min. The solvent was then removed in vacuo, diluted with water, extracted with DCM, washed with brine, and washed with MgSO 4 The solvent was removed in vacuo to afford a brown oil which was loaded onto a silica gel column using hexane / ethyl acetate (1:0 to 1:1) as eluent to afford the pure product (82 mg, 0.362 mmol, 18%) as a yellow oil.

[0556] 1 H-NMR (300MHz, CDCl 3 )δH=7.40-7.28(5H,m,5xaromatic C(sp 2 )-H),4.17(1H,d, 3 JHP=3.2Hz,S-CHa),4.11(1H,d, 3 JHP=1.8Hz,S-CHb),3.71(3H,d, 3 JHP=14.0Hz,O-CH3),3.20(1H,d, 3 JHP=12.6Hz,CCH)ppm.

[0557] 13 C-NMR (76 MHz, CDCl 3 )δC=135.39(d, 3 JCP = 6.1 Hz, IPSO aromatic C (sp 2 )), 129.07(s, meta-aromatic C(sp 2 )), 128.76(s, ortho aromatic C(sp 2 )), 127.88(s, para-aromatic C(sp 2),89.76(d, 2 JCP=43.2Hz,C(sp)-H),76.66(d, 1 JCP=239.2Hz,C(sp)),52.93(d, 2 JCP=6.2Hz,O-CH3),34.96(d, 2 JCP=3.5Hz).

[0558] 31 P-NMR (122MHz, CDCl 3 )δP=17.59(m)ppm.

[0559] HRMS(ESI):C 10 H 11 O 2 PS[M+H + ] calculated value: 227.0290; measured value: 227.0293.

[0560] General Procedure D: Via PCl 3 Alkyne-phosphonothioate synthesis

[0561] 1-Ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (as 0.3 mmol) was added to a round bottom flask, dissolved in anhydrous acetonitrile (3 ml) and cooled to -40°C. A solution of thiol (0.3 mmol) in tetrazole (0.45 M MeCN, 0.6 mmol, 1.33 ml) was prepared separately and added to the stirred mixture at -40°C. The reaction mixture was stirred at -40°C for 10 minutes, then warmed to room temperature and stirred for another 30 minutes. To the mixture was added tert-butyl hydroperoxide solution (H 2 O (70 wt.%, 0.3 mmol, 86 μl) and stirred for 10 minutes. The reaction mixture was then washed with H 2 O (10 ml) and extracted with DCM (3 x 30 ml). The combined organic layers were washed with Na 2 SO 4 Dry, filter, and remove the solvent under reduced pressure.Purify the crude product by silica gel chromatography.

[0562] S-Benzyl O-ethylethynylphosphonothioate (Compound 8)

[0563]

[0564] Compound 8 was prepared starting from 1-ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (25 mg, 0.123 mmol) and mercaptobenzyl (14.4 μl, 0.123 mmol) according to general procedure C. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (10 mg, 0.042 mmol, 34%) as a colorless oil.

[0565] 1 H-NMR (300MHz, CDCl3) δH = 7.45-7.25 (m, 5H), 4.28-4.02 (m, 4H), 3.12 (d, J = 12.5Hz, 1H), 1.35 (t, J = 7.1Hz, 3H) ppm.

[0566] 13 C-NMR (76MHz, CDCl3) δC=136.47(d,J=6.4Hz),129.09,128.91,127.84,88.90(d,J =43.0Hz), 63.50 (d, J = 3.3Hz), 35.04 (d, J = 3.3Hz), 28.27, 16.05 (d, J = 7.7Hz) ppm.

[0567] 31 P-NMR (122MHz, CDCl3) δP=15.36ppm.

[0568] HRMS: n.d.

[0569] Tert-butyl (2-((ethoxy(ethynyl)phosphoryl)thio)ethyl)carbamate (Compound 9)

[0570]

[0571] Compound 9 was prepared starting from 1-ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (93 mg, 0.465 mmol) and tert-butyl (2-mercaptoethyl)carbamate (82 mg, 0.465 mmol) according to general procedure D. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (50 mg, 0.17 mmol, 37%) as a colorless oil.

[0572] Example 12: Procedure for the Synthesis of Olefin-Phosphonates

[0573] General Procedure E: Via PCl 3 Olefin-phosphonate synthesis

[0574] 1-Ethoxy-N,N-diisopropyl-1-vinylphosphinamine (as 0.3 mmol) was added to a round bottom flask, dissolved in anhydrous acetonitrile (3 ml) and cooled to -40°C. A tetrazole solution (0.45 M in MeCN, 0.6 mmol, 1.33 ml) of the alcohol (0.3 mmol) was prepared separately and added to the stirred mixture at -40°C. The reaction mixture was stirred at -40°C for 10 minutes, then warmed to room temperature and stirred for another 30 minutes. To this mixture was added tert-butyl hydroperoxide solution (H 2 O (70 wt.%, 0.3 mmol, 86 μl) and stirred for 10 minutes. The reaction mixture was then washed with H 2 O (10 ml) and extracted with DCM (3 x 30 ml). The combined organic layers were washed with Na 2 SO 4 Dry, filter, and remove the solvent under reduced pressure.Purify the crude product by silica gel chromatography.

[0575] Benzyl ethyl vinyl phosphonate

[0576]

[0577] Benzyl ethyl vinyl phosphonate was prepared starting from 1-ethoxy-N,N-diisopropyl-1-vinylphosphinamine (25 mg, 0.123 mmol) and benzyl alcohol (12.3 μl, 0.123 mmol) according to General Procedure E. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (2 mg, 0.0088 mmol, 7%) as a colorless oil. (The reason for the low yield may be the loss of product in the workup step during purification).

[0578] tert-Butyl (2-((ethoxy(vinyl)phosphoryl)oxy)ethyl)carbamate

[0579]

[0580] The title compound was prepared starting from 1-ethoxy-N,N-diisopropyl-1-vinylphosphinamine (201 mg, 1.00 mmol) and tert-butyl (2-hydroxyethyl)carbamate (161 mg, 1.00 mmol) according to general procedure E. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (100 mg, 0.358 mmol, 36%) as a colorless oil.

[0581] HRMS(ESI):NaC11H22NO5P[M+Na + ] calculated value: 302.1133; measured value: 302.1120.

[0582] Example 13: Procedure for the synthesis of alkyne-phosphonates

[0583] General Procedure F: Via PCl 3 Route to synthesize alkyne-phosphonates

[0584] 1-Ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (as 0.3 mmol) was added to a round bottom flask, dissolved in anhydrous acetonitrile (3 ml) and cooled to -40°C. A solution of the alcohol (0.3 mmol) in tetrazole (0.45 M in MeCN, 0.6 mmol, 1.33 ml) was prepared separately and added to the stirred mixture at -40°C. The reaction mixture was stirred at -40°C for 10 minutes, then warmed to room temperature and stirred for another 30 minutes. To the mixture was added tert-butyl hydroperoxide solution (H 2 O (70 wt.%, 0.3 mmol, 86 μl) and stirred for 10 minutes. The reaction mixture was then washed with H 2 O (10 ml) and extracted with DCM (3 x 30 ml). The combined organic layers were washed with Na 2 SO 4 Dry, filter, and remove the solvent under reduced pressure.Purify the crude product by silica gel chromatography.

[0585] Benzyl ethyl ethynyl phosphonate

[0586]

[0587] Benzylethyl ethynylphosphonate was prepared starting from 1-ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (25 mg, 0.123 mmol) and benzyl alcohol (12.3 μl, 0.123 mmol) according to general procedure F. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (27 mg, 0.120 mmol, 49%) as a colorless oil.

[0588] tert-Butyl (2-((ethoxy(ethynyl)phosphoryl)oxy)ethyl)carbamate

[0589]

[0590] The title compound was prepared starting from 1-ethoxy-N,N-diisopropyl-1-ethynylphosphinamine (201 mg, 1.00 mmol) and tert-butyl (2-hydroxyethyl)carbamate (161 mg, 1.00 mmol) according to General Procedure F. Purification by silica gel chromatography (100% ethyl acetate) gave the desired compound (168 mg, 0.601 mmol, 60%) as a colorless oil.

[0591] HRMS(ESI): C11H21NO5P[M+H + ] calculated value: 278.1157; measured value: 278.1149.

[0592] Example 14: Coupling Procedure of Carboxylic Acids with Olefin- and Alkyne-Phosphonothioates and Phosphonates

[0593] General Procedure G: Preparation of functionalized phosphonothioates and phosphonates via amide bond formation

[0594]

[0595] Boc-protected amine derivatives of olefin- and alkyne-phosphonothioates (X=S) or phosphonates (X=O) (see above scheme) (e.g. 0.2 mmol) were dissolved in TFA / H 2 O (95:5, xx ml) and the resulting clear solution was stirred at room temperature for 15 minutes. The solvent was then removed by bubbling nitrogen through the solution. The residue was redissolved in H 2 O and lyophilized to give a colorless oil. The product can be used in the next step without further purification. To the deprotected amine (e.g. 0.05 mmol) was added a solution of HATU (0.055 mmol), carboxylic acid (0.055 mmol) and DIPEA (0.15 mmol) in DMF (250 μl). The resulting mixture was stirred at room temperature for 30 minutes in an Eppendorf tube and then concentrated under reduced pressure. The residue was redissolved in H 2 O / MeCN (9:1, 5 ml) and purified by preparative HPLC.

[0596] 5-((2-(4-((2-((ethoxy(ethynyl)phosphoryl)oxy)ethyl)amino)-4-oxobutyrylamide (4-(2-(4-(2-amino)ethyl)naphthalene-1-sulfonic acid) (Compound 20)

[0597]

[0598] Compound 20 was prepared from the deprotected amine (14 mg, 0.079 mmol) according to the general procedure G. The pure product was obtained as a colorless powder after purification by HPLC () and lyophilization (18 mg, 0.0342 mmol, 43%).

[0599] HRMS(ESI):C22H29N3O8PS[M+H + ] calculated value: 526.1413; measured value: 526.1387.

[0600] Figure 8 The UV-LC trace of purified compound 20 is shown.

[0601] 5-((2-(4-((2-((ethoxy(vinyl)phosphoryl)oxy)ethyl)amino)-4-oxobutyrylamino)ethyl 1-(4-(2-amino)naphthalenesulfonic acid (Compound 21)

[0602]

[0603] Compound 21 was prepared from the deprotected amine (12.9 mg, 0.072 mmol) according to the general procedure G. The pure product was obtained as a colorless powder after purification by HPLC () and lyophilization (10 mg, 0.0190 mmol, 26%).

[0604] HRMS(ESI):C22H31N3O8PS[M+H + ] calculated value: 528.1570; measured value: 528.1543.

[0605] Fig. 9 The UV-LC trace of purified compound 21 is shown.

[0606] 5-((2-(4-((2-((ethoxy(ethynyl)phosphoryl)thio)ethyl)amino)-4-oxobutyrylamino)ethyl 1-(4-(2-amino)naphthalenesulfonic acid (Compound 22)

[0607]

[0608] Compound 22 was prepared from the deprotected amine (11.2 mg, 0.058 mmol) according to the general procedure G. The pure product was obtained as a colorless powder after purification by HPLC () and lyophilization (10 mg, 0.0185 mmol, 32%).

[0609] HRMS(ESI):C22H29N3O7PS2[M+H + ] calculated value: 542.1184; measured value: 542.1156.

[0610] Fig.10 The UV-LC trace of purified compound 22 is shown.

[0611] 5-((2-(4-((2-((ethoxy(vinyl)phosphoryl)thio)ethyl)amino)-4-oxobutyrylamino)ethyl 1-(4-(2-amino)naphthalenesulfonic acid (Compound 23)

[0612]

[0613] Compound 23 was prepared from the deprotected amine (9.6 mg, 0.049 mmol) according to the general procedure G. The pure product (11 mg, 0.0202 mmol, 41%) was obtained as a colorless powder after purification by HPLC () and lyophilization.

[0614] HRMS(ESI):C22H31N3O7PS2[M+H + ] calculated value: 544.1341; measured value: 544.1316.

[0615] Fig.11 The UV-LC trace of purified compound 23 is shown.

[0616] O-ethyl S-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl (amino)ethyl)vinyl phosphonothioate (Compound 13)

[0617]

[0618] Compound 13 was prepared from the deprotected amine (14.8 mg, 0.076 mmol) according to the general procedure G. The pure product was obtained as a colorless powder after purification by HPLC () and lyophilization (14 mg, 0.033 mmol, 44%).

[0619] O-ethyl S-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl (amino)ethyl)ethynylphosphonothioate (Compound 28)

[0620]

[0621] The title compound was prepared from the deprotected amine (20 mg, 0.104 mmol) according to the general procedure G. The pure product was obtained as a colorless powder after purification by HPLC () and lyophilization (7 mg, 0.0167 mmol, 16%).

[0622] Example 15A: Hydrothiolation Procedure of Olefin-Phosphonothioates Using Glutathione

[0623] (Ethyl-S-benzyl-P-ethyl-phosphonothioate)-S-glutathione conjugate (Compound 14)

[0624]

[0625] Reduced glutathione (20.3 mg, 0.066 mmol, 2.0 eq.) was dissolved in 1.32 ml of aqueous buffer (1 mM EDTA, 50 mM NH 4 HCO 3 , pH 8.0) and then added to a solution of S-benzyl O-ethyl olefin-PT 2 (8 mg, 0.033 mmol, 1.0 eq.) in 0.33 ml DMF, and the mixture was then stirred at room temperature for 90 minutes. The reaction was monitored by UPLC-MS (gradient: 3-60% MeCN in 5 minutes), indicating complete conversion after 90 minutes. The solvent was then removed under reduced pressure and the residue was redissolved in 5 ml H 2 O and purified by semi-preparative HPLC (acidic conditions, gradient: 20-60% MeCN in 40 minutes, product eluted with 35% MeCN, flow rate: 16 ml / min). After freeze-drying, the compound 14 (14 mg, 0.026 mmol, 77%) was obtained as a white powder.

[0626] 1H NMR (300 MHz, deuterium oxide) δ = 7.46-7.28 (m, 5H), 4.50 (dd, J = 8.4, 5.3 Hz, 1H), 4.19-3.93 (m, 7H), 2.90 (dd, J = 14.1, 5.3 Hz, 1H), 2.76 (ddd, J = 14.1, 8.4, 1.9 Hz, 1H), 2.69-2.47 (m, 4H), 2.25-2.01 (m, 4H), 1.26 (t, J = 7.1 Hz, 3H) ppm.

[0627] 13 C NMR (75 MHz, deuterium oxide) δ = 174.29, 172.82, 172.51, 172.08, 137.34, 128.95, 128.84, 127.95, 63.30, 63.20, 52.79, 52.60, 41.06, 33.90, 32.66, 31.37, 30.93, 25.58, 23.95, 15.38 (d, J = 6.5 Hz) ppm.

[0628] 31 P NMR (122 MHz, deuterium oxide) δ 60.51 (d, J = 1.9 Hz) ppm.

[0629] HRMS(ESI):C 21 H 33 N 3 O 8 PS[M+H + ] calculated value: 550.1441; measured value: 550.1451.

[0630] Example 15B: Hydrothiolation Procedure of Olefin-Phosphonates Using Glutathione

[0631] (Diethyl-phosphonate)-S-glutathione conjugate

[0632]

[0633] Diethyl olefin phosphonate (20 mg, 0.147 mmol, 1.0 eq.) was dissolved in 1 ml of buffer (50 mM NH 4 HCO 3 , 1 mM EDTA, pH 8.0) and 1 ml DMF. To the resulting solution was added a solution of glutathione (90.3 mg, 0.294 mmol, 2.0 eq.) in 9 ml of the same buffer (the pH was adjusted to 8.0 after dissolving GSH), and the mixture was stirred at room temperature. When complete conversion was achieved (by 31P-NMR judgment), the reaction mixture was frozen in liquid nitrogen and then lyophilized. The residue was purified by semi-preparative HPLC to give the title compound (41.6 mg, 0.0882 mmol, 60%) as a colorless powder.

[0634] 1 H NMR (300 MHz, DMSO-d 6 )δ=8.48(t,J=5.9Hz,1H),8.38-8.25(m,4H),4.49(td,J=8.9,4.8Hz,1H),4.07-3.90(m,5H),3.76(d,J=5.8Hz,2H) ,2.91(dd,J=13.8,4.8Hz,1H),2.71-2.55(m,3H),2.45-2.23(m,2H),2.12-1.89(m,4H),1.23(t,J=7.0Hz,6H)ppm.

[0635] 13 C NMR (75 MHz, DMSO-d 6 )δ=171.45,171.39,171.27,171.01,61.70,61.62,52.27,52.07,34.13,31.09,26.95,26.50,25.17,24.82,16.76,16.69ppm.

[0636] 31 P NMR (122 MHz, DMSO-d 6 )δ=28.58ppm.

[0637] HRMS: ESI-MS (positive mode) m / z 472.1496 [(M+H) + ; C 16 H 31 N 3 O 9 PS + Calculated value: 472.1513].

[0638] Example 16: Hydrothiolation Procedure of Alkyne-Phosphonothioates Using Glutathione

[0639] (Methyl-S-benzyl-P-ethyl-phosphonothioate)-S-glutathione conjugate (Compound 15)

[0640]

[0641] In a buffered aqueous solution (1 mM EDTA, 50 mM NH 4 HCO 3A DMF solution of compound 7 (0.75 mL, 100 mM) was added to a mixture of glutathione (0.15 mmol, 23 mg) in 4% paraformaldehyde (pH = 8.5), and the mixture was vortexed for 4 minutes. The mixture was then lyophilized and the remaining residue was dissolved in MeCN / H 2 O and purified by HPLC (5-45% MeCN in 50 min). Product-containing fractions were freeze-dried to give the title compound 15 (16.4 mg, ca. 33%) as a colorless powder.

[0642] 1H-NMR (600MHz, DMSO-d 6 )δ=8.47-8.45(1H,m),8.39-8.37(1H,m),7.62-7.51(1H,ddd,J=49.9Hz,J=12.3 Hz,J=4.8Hz),7.37-7.25(5H,m),5.83-5.78(1H,ddd,J=21.3Hz,J=12.2Hz,J=6.4 Hz),4.56-4.54(1H,m),4.01-3.91(3H,m),3.78-3.77(2H,m),3.77-3.53(3H,m) ,3.22-3.19(1H,m),2.96-2.92(1H,m),2.38-2.31(2H,m),2.07-1.98(2H,m)ppm.

[0643] 13 C-NMR (151 MHz, DMSO-d 6 )δ=171.07,170.87,170.75,170.02,137.80,137.76,128.87,128.50,127.32,118.07,116.09,1 13.62,112.63,52.89,51.63,51.20,51.17,51.13,40.77,36.81,33.37,30.66,30.62,25.88ppm.

[0644] 31 P-NMR (122 MHz, DMSO-d 6 )δ=41.6ppm.

[0645] Example 17: Procedure for the synthesis of Dabcyl-EDANS quencher pairs for stability studies

[0646] General procedure for the synthesis of Dabcyl-EDANS quencher pairs

[0647]

[0648] The DABCYL-containing peptides were synthesized on Rink-amide resin under standard solid phase peptide synthesis conditions (Fmoc strategy). Dabcyl (as carboxylic acid) was coupled to the free N-terminus on the resin using HATU as coupling agent. TFA / TIS / H 2 The peptide was cleaved from the resin with a mixture of O / DTT = 95 / 2 / 2 / 1. The peptide was purified by semi-preparative HPLC.

[0649] The purified Dabcyl peptide (e.g., 2.35 μmol) was dissolved in aqueous buffer (50 mM NH 4 HCO 3 ,1mM EDTA, pH8.5) and mixed with EDANS compounds 20-23 (2.82μmol) dissolved in DMF (280μl) in an Eppendorf tube. The reaction mixture was stirred at room temperature and the reaction was monitored by UV-LC-MS. The reaction mixture was then diluted with water (4.7ml) and purified by semi-preparative HPLC (10-50% MeCN, 0.1% TFA in 45 minutes, flow rate: 16ml / min), and the fractions containing the pure product were combined and lyophilized.

[0650] EDANS-Dabcyl FRET pair (alkyne phosphonate derivative) (Compound 24)

[0651]

[0652] Compound 24 was obtained from Edans compound 20 (1.48 mg, 2.82 μmol) and Dabcyl peptide (2.5 mg, 2.35 μmol) according to general procedure H. The product was obtained as a red powder (3 mg, 2.01 μmol, 86%) after purification by semi-preparative HPLC (10-50% MeCN, 0.1% TFA, 16 ml / min) and lyophilization.

[0653] Fig.12 The UV LC trace of purified compound 24 is shown.

[0654] EDANS-Dabcyl FRET pair (olefin phosphonate derivative) (Compound 25)

[0655]

[0656] Compound 25 was obtained from Edans compound 21 (1.49 mg, 2.82 μmol) and Dabcyl peptide (2.5 mg, 2.35 μmol) according to general procedure H. The product was obtained as a red powder (3 mg, 2.01 μmol, 86%) after purification by semi-preparative HPLC (10-50% MeCN, 0.1% TFA, 16 ml / min) and lyophilization.

[0657] Fig.13 The UV LC trace of purified compound 25 is shown.

[0658] EDANS-Dabcyl FRET pair (alkyne phosphonothioate derivative) (Compound 26)

[0659]

[0660] Compound 26 was obtained from Edans compound 22 (1.53 mg, 2.82 μmol) and Dabcyl peptide (2.5 mg, 2.35 μmol) according to general procedure H. The product was obtained as a red powder (3 mg, 1.87 μmol, 80%) after purification by semi-preparative HPLC (10-50% MeCN, 0.1% TFA, 16 ml / min) and lyophilization.

[0661] Fig.14 The UV LC trace of purified compound 26 is shown.

[0662] EDANS-Dabcyl FRET pair (olefin thiophosphonate derivative) (Compound 27)

[0663]

[0664] Compound 27 was obtained from Edans compound 23 (1.53 mg, 2.82 μmol) and Dabcyl peptide (2.5 mg, 2.35 μmol) according to general procedure H. The product was obtained as a red powder (3 mg, 1.87 μmol, 80%) after purification by semi-preparative HPLC (10-50% MeCN, 0.1% TFA, 16 ml / min) and lyophilization.

[0665] Fig.15 The UV LC trace of purified compound 27 is shown.

[0666] Example 18: Procedure for stability study of Example 6

[0667] Prepare 0.20mM stock solutions of EDANS-DABCYL conjugates 24-27 (PBS pH 7.4) respectively. Take 5μl from the stock solution and mix with 95μl of the corresponding matrix (PBS pH 7.4, freshly prepared HeLa cell lysate (1mg / ml), human serum) to test in 96-well plates (Corning, N°3615) at room temperature (about 25°C). For experiments in NaOH, these compounds (150μL of 200μM PBS stock solution) were mixed with 150μL of 1M NaOH solution in Eppendorf tubes at room temperature (about 25°C). At a given time point, 3μl of this solution was mixed with 5.6μl of 1M HCl solution and 91μl PBS for neutralization in 96-well plates. Fluorescence was monitored over the course of 3 days (for all conditions except NaOH) using a Safire / Tecan instrument (EDANS: λex=360 nm, λem=508 nm). Fluorescence intensity was corrected by background measurement (matrix only) and the mean and standard deviation of three independent experiments were calculated (n=3).

[0668] Example 19: Procedure for the kinetic studies of Example 5

[0669]

[0670] 2.5 μl of a 20 mM DMF solution of the corresponding phosphorus compounds 20-23 and 5 μl of a 1 mM DMF / buffer (1:1) solution of uncoupled EDANS (as an internal standard) were added to 488 μl of coupling buffer (50 mM NH 4 HCO 3 and 1mM EDTA in ultrapure water, adjusted to pH 8.5 with aqueous ammonia solution). 5μl of 10mM glutathione buffer solution was added to start the reaction. The reaction was carried out at room temperature (about 25°C). The first sample was taken before adding glutathione (t=0). Subsequent samples were collected after 15, 30, 60, 120, 240 and 480 minutes. The sample was drawn in a volume of 20μl and then immediately diluted into 80μl of 50mM NaOAc buffer with a pH of 3.5 to terminate the reaction. These samples were subjected to HPLC analysis using a fluorescence detector, with 20μl injected each time. (The results of this study are shown in Figure 3 ).

[0671] Example 20A: Procedure for coupling BSA with thiophosphonates

[0672] The cells were cultured in a buffer solution (PBS, 1 mM EDTA, pH 7.4 or 50 mM NH 4 HCO 3, 100mM NaCl, 1mM EDTA, pH8.5) was mixed with 1μl of 100mM (corresponding to 100eq.) or 1μl of a solution of compound 2 or 7 (50mM in DMF) at 4°C. The reaction mixture was stirred at 4°C for 18h. Excess compound was then removed by spin filtration (7kDa MWCO), whereby the buffer was changed to PBS at pH 7.4. 3μl of a PBS solution of the protein (10μM) was mixed with 27μl of Laemmli buffer containing mercaptoethanol, heated at 95°C for 10 minutes, and run on SDS-Gel (12% acrylamide, 250V, 40 minutes). The protein was then digested in-gel with chymotrypsin and analyzed by MS / MS (The results of the MS / MS analysis are summarized in Table 6.)

[0673] Example 20B: Procedure for coupling BSA with phosphonates

[0674] The 50 mM NH 4 HCO 3 , 100 mM NaCl, 1 mM EDTA, pH 8.5) was mixed with 1 μl of a 100 mM solution of diethyl olefin phosphonate in DMF at 4°C. The reaction mixture was stirred at 4°C for 18 h. The excess phosphonate compound was then removed by spin filtration (7 kDa MWCO), whereby the buffer was changed to PBS at pH 7.4. 3 μl of the protein in PBS (10 μM) was then mixed with 27 μl of Laemmli buffer containing mercaptoethanol, heated at 95°C for 10 min, and run on SDS-Gel (12% acrylamide, 250 V, 40 min). The protein was then digested in-gel with chymotrypsin and analyzed by MS / MS (The results of the MS / MS analysis are summarized in Table 1.)

[0675] Example 20: Procedure for coupling antibodies to thiophosphonates

[0676] 1.25 μl of 100 μM brentuximab antibody solution (in PBS) was mixed with 10 μl of borate-containing PBS (50 mM borate, pH 8.0) and 1.25 μl of DTT solution (100 mM DTT, pH 8.0 in the same borate buffer). Control samples were similarly prepared without the addition of DTT. These mixtures were incubated at 37 ° C for 30 minutes in a thermostatic oscillator. Excess DTT was then removed using a size exclusion chromatography column (Thermo Scientific, ZebaTM Micro Spin desalting column, 7K MWCO, product No. 89877). First, alkylation buffer (50 mM NH 4 HCO 3 , 1mM EDTA, pH=8.5 / 8.0 or PBS, 1mM EDTA, pH 7.4) to balance the size exclusion chromatography column (3x 50μl at 1000g for 1min). Then the antibody-DTT mixture (12.5μl) was loaded onto the balanced chromatography column and the antibody was collected into a new Eppendorf tube by centrifugation (2min, 1000g). 0.25μl of biotin-PT (alkene or alkynyl thiophosphonate) solution (25mM in DMSO) was immediately added to the solution, and the mixture was incubated overnight in a thermostatic oscillator at 4°C.

[0677] 3 μl of this mixture was mixed with 27 μl of Laemmli buffer containing mercaptoethanol and heated at 95°C for 10 minutes. Then 10 μl was loaded onto a 12% acrylamide SDS-Gel and run at 250 V for 35 minutes. The gel was then Western blotted using a commercially available streptavidin-HRP conjugate to hybridize with a biotin-modified antibody and indirectly detect biotin by chemiluminescence.

[0678] Example 21: Modification of eGFP using alkyne phosphonothioates

[0679] In a proof of principle experiment, the inventors employed an eGFP variant carrying a solvent accessible cysteine ​​and reacted it with a small molecule fluorescent alkyne phosphonothioate NA1 (corresponding to compound 22 of Example 14 above) at physiological pH ( Fig.16 A). The reaction was monitored by ESI-MS and when checked after 14 hours, complete conversion was observed without any detectable by-product formation ( Fig.16 B) This result demonstrates that alkyne phosphonothioates are suitable for modifying proteins under mild conditions at physiological pH.

[0680] Fig.16 Shown: Modification of eGFP with the alkyne phosphonothioate-Edans derivative NA1. A) Synthesis scheme, B) Complete conversion of GFP to the desired product observed by ESI-MS. The spectra shown here are from the reaction mixture without any purification. PT = phosphonothioate NA1.

[0681] Example 22: Synthesis of Antibody Drug Conjugates (ADCs) with Alkyne Phosphonothioates

[0682] The inventors have also developed methods for synthesizing phosphonothioate-linked antibody drug conjugates (ADCs) from the highly potent anti-mitotic, tubulin-binding cytotoxin monomethyl auristatin E (MMAE) and the CD30-adressing antibody brentuximab. To facilitate release of the toxic payload, an ADC with a cathepsin B cleavage side (valine-citrulline linker VC) between the antibody and the toxin was prepared, resulting in a commercially available ADC. (Brentuximab vedotin) (MA Stephen, Blood 2014, 124, 3197-3200) A direct analogue. As depicted in Scheme 12, the phosphonothioate-VC-PAB-MMAE construct NA3 was synthesized from the alkyne phosphonothioate carboxylic acid NA2 and VC-PAB-MMAE via amine coupling.

[0683]

[0684] Scheme 12: Synthetic route to construct the phosphonothioate-linked, cathepsin B-cleavable monomethyl auristatin E conjugate NA3. VC: valine-citrulline dipeptide, PAB: p-aminobenzyl.

[0685] Then, an antibody drug conjugate was synthesized by coupling NA3 to brentuximab. In order to make the activity of ADC containing phosphonothioate-linked ADC comparable to FDA-approved ADC In contrast, the inventors first screened to find ADCs with drug-to-antibody ratios (DARs) of 3-4. In the case of Adcetris, VC-PAB-MMAE was coupled to the cysteine ​​of brentuximab via maleimide, with a DAR of 4 (MA Stephen, Blood 2014, 124, 3197-3200.). The inventors first reduced the interchain-disulfide bonds of brentuximab with DTT to generate free sulfhydryl groups, which could then react with alkyne phosphonothioate NA3 (see Fig.17 A). by Zeba TM The excess DTT was removed by a spin desalting column. For the screening, the inventors varied the concentration of the brentuximab antibody (1 vs. 5 mg / ml), the pH in the coupling (pH 8.5 vs. 7.4), and the equivalents of the thiophosphonate-drug compound NA3 (8.8-100 eq.), and analyzed the resulting ADC by ESI-MS after deglycosylation and reduction with the enzyme PNGase-F. The DAR was estimated using the mass signal intensity of the heavy and light chain species with different degrees of modification. The results of this screening are shown in Figure 1. Fig.17As shown in B. Higher antibody concentrations and more equivalents of phosphonothioate generate higher DARs. Coupling with alkyne phosphonothioate is also efficient at physiological pH 7.4. This is an advantage of alkyne phosphonothioate for coupling proteins or antibodies, because for reasons such as stability, the proteins or antibodies need to be handled at physiological pH.

[0686] Fig.17 Shown: Modification of brentuximab with NA3 (phosphonothioate-VC-PAB-MMAE). A) Reaction scheme, reduction and alkylation of interchain disulfides. B) Screening of reaction conditions. C) Exemplary MS spectra of antibody fragments after deglycosylation with PNGase-F and reduction with DTT. Shown are deconvoluted spectra, and the inset shows the original data. LC: light chain; HC: heavy chain; mod: phosphonothioate-VC-PAB-MMAE NA3.

[0687] The inventors further characterized the ADC by size exclusion chromatography (SEC) and found DAR = 3.15 (see Fig.21 The ADC was then evaluated in a standard resazurin assay using the CD30-overexpressing cell line Karpas 299 (see Fig.18 A). Brentuximab-NA3 ADC showed strong growth inhibition (blue curve) and it showed a slightly lower DAR (3.15 vs. 4.0) has ADCs used in clinical practice (red curve). Brentuximab alone (green curve) showed no growth inhibitory effect. As a control to demonstrate CD30 selectivity, the cell line HL 60 ( Fig.18 B). In this case, no inhibition of cell proliferation was observed for all constructs. Taken together, these results demonstrate that alkyne phosphonothioates are suitable linkers for generating active ADCs that selectively kill antigen-positive cell lines.

[0688] Fig.18 Shown: A) MMAE-linked brentuximab ADC selectively enhances growth inhibition of the CD30-overexpressing cell line Karpas 299. The graph depicts cell viability based on antibody concentration after 96 hours of treatment. Brentuximab alone (green), brentuximab-NA3 (blue) and (red). Karpas 299: a cell line overexpressing CD30. B) Control. Brentuximab alone (green), Brentuximab-NA3 (blue) and (red). HL 60: a cell line with low CD30 expression levels.

[0689] Compared to maleimide-thiol conjugates, phosphonothioate-thiol conjugates are more stable, especially in the presence of excess free thiols. This is an important advantage particularly for ADCs where premature toxin release due to thiol exchange can lead to increased off-target toxicity.

[0690] As a further advantage, the inventors have observed that in the modification of antibodies, phosphonothioates have better cysteine ​​selectivity than maleimides when the same number of equivalents is used at physiological pH, see also Example 7B above.

[0691] Example 23: Introduction of phosphonothioates onto peptides on resin

[0692] The inventors have observed that the phosphonothioates described herein are highly stable under acidic conditions. Therefore, the phosphonothioates are introduced into the peptides via solid phase peptide synthesis (SPPS) on a resin. This allows the electrophile to be introduced into the peptide in a direct manner during SPPS and then cleaved from the resin under acidic conditions.

[0693] The model peptide is coupled to the carboxylic acid NA2 through the free N-terminus. After 2 hours of cleavage with 95% TFA, the product can be separated by semi-preparative HPLC. Therefore, this method allows the introduction of electrophiles on the resin during SPPS. As an advantage, under the strongly acidic conditions during the cleavage from the resin, the thiophosphonate is not hydrolyzed. Therefore, the thiophosphonate is highly stable under acidic conditions (e.g., under >90% trifluoroacetic acid (TFA) during the cleavage from the resin).

[0694] By UV-LC-MS and 31 P-NMR analysis of the alkyne phosphonothioate-peptide. It is noteworthy that the peptide exists in a linear form as shown in Scheme 13 and is not cyclized by intramolecular thiol addition via a cysteine ​​residue.

[0695]

[0696] Scheme 13: Solid phase peptide synthesis using alkyne phosphonothioates.

[0697] Example 24: Procedure for modification of eGFP using alkyne phosphonothioate NA1

[0698] This experiment used an eGFP mutant (eGFPC70MS174C). The protein has been expressed as a His-tagged variant with a protease cleavage site. After cleavage, the complete sequence is:

[0699] RGSHMGSIQMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQMFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKF EGDTLVNRIELKGIDFKEDGNILGHKLEYNYNCHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0700] Fig.19 Shown is the ESI-MS spectrum of eGFPC70MS174C used as a starting material for coupling with NA1.

[0701] Procedure for eGFP modification using NA1

[0702] To a solution of eGFP in PBS (pH 7.4) (50 μl, 1 mg / ml) in a low binding Eppendorf tube was added alkyne phosphonothioate NA1 (0.72 μl of a 25 mM DMSO stock solution, 10 eq.) and the mixture was kept on a thermostatic shaker at 14° C. and 800 rpm for 14 hours. Full conversion was achieved as judged by ESI-MS.

[0703] Example 25: Procedure for producing antibody drug conjugates (ADCs) using alkyne-phosphonothioates

[0704] 4-((2-((ethoxy(ethynyl)phosphoryl)thio)ethyl)amino)-4-oxobutanoic acid (Compound NA2) synthesis

[0705]

[0706] *Compound number in original application

[0707] Compound NA2 was generated in two steps from the Boc-protected precursor (compound No. 9 described in Example 11 above). 9 (94 mg, 0.321 mmol) was dissolved in TFA / H 2 O 95:5 (3 ml), and stirred at room temperature for 5 minutes. The reaction mixture was then washed with H 2The dried crude product was dissolved in DMF (1 ml) and added to a mixture of succinic acid (38 mg, 0.321 mmol), HATU (122 mg, 0.321) and DIPEA (167 ml, 0.962 mmol) in DMF (1 ml). The resulting mixture was stirred at room temperature for 30 minutes. The solvent was then removed under reduced pressure and the residue was dissolved in MeCN / H2O containing 0.1% TFA. 2 O (1:4) and purified by semi-preparative HPLC (20-60% MeCN in 60 min, flow rate = 10 ml / min). The product-containing fractions were freeze-dried to give 1 The purity of NA2 (34 mg, 0.116 mmol, 36%) in the form of lyophilized powder was about 90% by H-NMR. The compound was used in the subsequent reaction without further purification.

[0708] C 10 H 17 NO 5 PS 1+ [M+1H] 1+ HR-MS calculated value: 294.0560, found 294.0614.

[0709] 1 H-NMR (300MHz, CDCl 3 )δ H =7.22-7.13(m,1H),4.25(dq,J=9.8,7.1Hz,2H),3.76-3.74(m,2H),3.29(d, J=12.6Hz,1H),3.25-2.98(m,2H),2.72-2.51(m,4H),1.50-1.36(m,3H)ppm.

[0710] 31 P-NMR (122MHz, CDCl 3 )δ P =17.1ppm.

[0711] Synthesis of Alkyne-Phosphonothioate-Val-Cit-Pab-MMAE (Compound NA3)

[0712]

[0713] Compound NA2 (1.88 mg, 6.41 mmol), HATU (2.44 mg, 6.41 mmol) and DIPEA (4.7 ml, 26.7 mmol) were dissolved in DMF (100 ml) and added to a 1% HCl solution in an Eppendorf tube. 2N-Val-Cit-Pab-MMAE (6 mg, 5.34 mmol) was added to a DMF (150 ml) solution. After 30 minutes, the reaction mixture was treated with H 2 O (4.5 ml), filtered and purified by semi-preparative HPLC (30-99% MeCN in 50 min, flow rate = 5 ml / min). After lyophilization, the product NA3 (3 mg, 2.14 mmol, 40%) was obtained as a white powder.

[0714] C 68 H 109 N 11 O 16 PS 1+ [M+1H] 1+ HR-MS calculated value: 1398.7507, found value: 1398.7201.

[0715] Fig. 20 The UPLC-UV purity of phosphonothioate-Val-Cit-Pab-MMAE NA3 is shown.

[0716] Brentuximab production

[0717] According to previously published (A. Stengl, D. H. Leonhardt, J. Helma, SLAS Discov 2017, 22, 309-315.), and an additional final purification was performed by gel filtration on a Superdex 200 Increase 10 / 300 from GE (GE life sciences, USA) using PBS and a flow rate of 0.75 ml / min.

[0718] Procedure for modification of brentuximab by reduction / alkylation protocol

[0719]

[0720] Brentuximab modification was performed in a low binding Eppendorf by incubating Brentuximab (c = 5 mg / ml, 33.4 mM) in a total volume of 300 μl of a buffer containing 50 mM sodium borate and 34 mM DTT in PBS (pH 8.0) at 37°C for 30 min. Subsequently, 2 mL of Zeba TMExcess DTT was removed by using a Spin desalting column (Thermo Fisher Scientific, Waltham, United States) and the buffer was exchanged to PBS (pH 7.4) containing 1 mM EDTA. Then, 2.45 μl of 36 mM thiophosphonate NA3 in DMSO was quickly added and the mixture was shaken at 800 rpm and 14°C for 16 h. The precipitate was purified by size exclusion chromatography ( FPLC, Superose 6 Increase 10 / 300GL column, PBS, 0.8 ml / min) was used to remove excess thiophosphonate. The fractions containing the product were combined and sterile filtered. For analysis of ADC, 12 μl of 1 mg / ml solution was incubated with 1 μl of RapiGest at 60°C on a thermostatic oscillator for 30 minutes. Then 1 μl of PNGaseF was added and incubated for another 2 hours at 37°C. Finally, 1 μl of a 10 mM solution of DTT in PBS (pH 7.4) was added and the mixture was shaken at 37°C for 30 minutes. 10 μl of the mixture was washed with 30 μl of H 2 O dilution and then subjected to ESI-MS (results see Figure 2 B).

[0721] SEC of ADC

[0722] MAbPac SEC-1 was used on a Vanquish Flex UHPLC system with DAD detector, Split Sampler FT (4°C), Column Compartment H (25°C) and binary pump F (Thermo Fisher Scientific, USA). Analytical size exclusion chromatography (A-SEC) was performed using 4 x 300 mm columns (Thermo Fisher Scientific, USA) at a flow rate of 0.15 mL / min. Phosphate buffer (pH 7) (20 mM Na 2 HPO 4 / NaH 2 PO 4 ,300mM NaCl), 5% v / v isopropanol as the mobile phase was used to separate the different ADC / mAb populations during a 30-min isocratic gradient. 8μg of ADC / mAb was loaded onto the column for A-SEC analysis. UV chromatograms were recorded at 220 and 280nm. Quantification of monomers and HMWS was achieved after integration of the peak area at 220nm.

[0723] Fig.21Size exclusion chromatogram of brentuximab-NA3 ADC is shown.

[0724] Cell-based antiproliferation assay

[0725] HL60 and Karpas cell lines were cultured in RPMI-1640 supplemented with 10% FCS and 0.5% penicillin-streptomycin. SKBR3 and MDAMB468 cell lines were cultured in DMEM / F12 supplemented with 10% FCS and 0.5% penicillin-streptomycin. 3 cells / well (SKBR3, HL60 and Karpas) or 1*10 3 Cells were seeded at a density of 10 cells / well (MDAMB468) in 96-well cell culture microplates. 1:4 serial dilutions of ADC or antibody were made starting at a final concentration of 3 μg / mL in cell culture medium and then transferred in duplicate to individual wells of the microplate. Incubate at 37°C, 5% CO 2 The plates were incubated for 96 h at 37 °C and 5% CO. 2 The fluorescence signal (λ) of resorufin was measured on a Tecan Infinite M1000 microplate reader. EX =560nm and λ EM =590 nm) to quantify the metabolic conversion of resazurin to resorufin. Means and standard deviations were calculated from duplicate results, normalized to untreated controls, and plotted against antibody concentrations. Data analysis was performed using MatLab R2016 software.

[0726] Example 26: Procedure for the introduction of phosphonothioates onto peptides on resin

[0727]

[0728] The peptide with the sequence AYRCAK was synthesized on TentaGel S Rink amide resin with 0.22 g / mol loading. 5 eq. amino acids, 5 eq. HCTU, 5 eq. Oxyma and 10 eq. DIPEA were coupled in DMF for 1 h (concentration: 0.1 M based on 5 eq. amino acids). After each coupling, the remaining free amine groups were capped with Ac2O. Fmoc-deprotection was performed with 20% piperidine in DMF.

[0729] The amount of resin corresponding to 10 μM of the AYRCAK peptide with a free N-terminus was used for further modification with thiophosphonate building blocks. Therefore, in a peptide reactor, the peptide was swollen in DMF (1 ml) for 2 hours, and then a mixture of compound NA2 (120 μl of a 250 mM stock solution in DMF, 30 μmol, 3 eq.), HATU (11.4 mg, 30 μmol, 3 eq.) and DIPEA (10.4 μl, 60 μmol, 6 eq.) was added. The mixture was shaken at room temperature for 1 hour. TFA / H 2 Cleavage from the resin was performed with O / TIS (95:2.5:2.5; v:v:v, 2 ml) for 2 hours. Precipitation was performed in cold anhydrous ether. The product was purified by preparative reverse phase C18 HPLC (10-60% MeCN H 2 O solution + 0.1% TFA, flow rate: 10 ml / min) to purify the crude product. After freeze-drying, the product NA4 (2.44 mg, 2.48 μmol, 25%) was obtained as a white powder and was purified by 31 P-NMR, analytical UPLC (on a RP-C18 column, in H 2 The product NA4 was analyzed by ESI-MS.

[0730] C 40 H 66 N 12 O 11 PS 2 1+ [M+1H] 1+ HR-MS calculated value: 985.4148, found value: 985.4152.

[0731] 31 P-NMR (243 MHz, DMSO-d 6 )δ P =14.6(d,J=6.5Hz)ppm.

[0732] Fig. 22 The UPLC-UV purity of the phosphonothioate peptide NA4 is shown.

[0733] in conclusion

[0734] The inventors have demonstrated herein that unsaturated phosphonothioates and phosphonates (olefin- and alkyne-phosphonothioates and olefin- and alkyne-phosphonates) are suitable treatment agents for cysteine ​​selective bioconjugation reactions. Thiol addition is rapid under aqueous conditions and the conjugates formed are stable under physiologically relevant conditions. The inventors have shown that the method allows, for example, cysteine ​​selective modification of proteins (e.g., BSA) and antibodies (e.g., cetuximab, brentuximab). The results provided herein indicate that the method according to the present invention using phosphonothioates or phosphonates is superior to current cysteine ​​bioconjugation strategies (e.g., maleimide chemistry) because the reaction of the present invention is more selective for thiols and the conjugates formed exhibit better stability.

[0735] ***

[0736] It must be noted that, as used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may be modified or substituted for the methods described herein.

[0737] All publications and patents cited herein are incorporated herein in their entirety. To the extent material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will take precedence over any such material.

[0738] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments of the invention described herein. The present invention is intended to encompass such equivalents.

[0739] Throughout this specification and in the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprises" may be replaced by the terms "containing" or "including", or, at times as used herein, by the term "having".

[0740] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claimed element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In various instances herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms.

[0741] Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether before or after, is incorporated herein in its entirety. Nothing herein will be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

Claims

1. A method for preparing a thiophosphonate or a phosphonate, comprising the steps of: The compound of formula (I) in, represents a double or triple bond; when When it is a triple bond, X represents R 3 -C; when When it is a double bond, X represents (R 3 R 4 )C; Y stands for S or O; R 1 represents an optionally substituted aliphatic or aromatic residue; R 3 Represents H or C 1 -C 8 -alkyl; R 4 Represents H or C 1 -C 8 -alkyl; and ● represents an aliphatic or aromatic residue; Reaction with a thiol-containing molecule of formula (II) in represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a sugar, a polysaccharide, a polymer, an optionally substituted C 1 -C 8 - alkyl, optionally substituted phenyl or an optionally substituted aromatic 5- or 6-membered heterocyclic ring system; Obtaining a compound of formula (III) Wherein, if the compound of formula (I) represents a double bond, then represents a single bond; or If the compound of formula (I) represents a triple bond, then represents a double bond; and · R 1 , X and Y are as defined above.

2. The method according to claim 1, wherein represents a double bond, X represents (R 3 R 4 )C,R 3 and R 4 Independently represents H or C 1 -C 8 -alkyl and Represents a single bond.

3. The method according to claim 1, wherein represents triple bond, X represents R 3 -C, R 3 Represents H or C 1 -C 8 -alkyl and Represents a double bond.

4. A method according to any one of the preceding claims, wherein Y is S.

5. A method according to any one of the preceding claims, further comprising the preparation of a compound of formula (I) include: The compound of formula (IV) Where R 1 , X, Y, and as defined in any one of the preceding claims; With tert-butyl hydroperoxide (tBu-OOH), m-chloroperbenzoic acid (mCPBA), hydrogen peroxide (H 2 O 2 ), iodine (I 2 ), potassium persulfate or oxygen (O 2 ) to produce a compound of formula (I).

6. The method according to claim 5, further comprising the preparation of a compound of formula (IV): include: Make phosphorus trihalide (X), preferably PCl 3 Sequentially with (i)R 1 –OH(XI);(ii) Where R 2 Independently represents C 1 -C 8 -alkyl; (iii) wherein Hal represents a halogen selected from Cl, Br and I, preferably Br; and (iv) reacting to produce a compound of formula (IV); Where R 1 , X, Y, and ● as defined in any one of the preceding claims.

7. The method according to claim 4, further comprising the preparation of a compound of formula (I), wherein include: The compound of formula (V) reacting with a compound of formula (VIa) or (VIb) to produce a compound of formula (I); Wherein EWG represents an electron withdrawing group, preferably, the electron withdrawing group is selected from wherein # indicates the position of S; Hal represents a halogen selected from Cl, Br and I, preferably Cl; and Where R 1 ,X, and ● as defined in any one of the preceding claims.

8. The method according to claim 7, wherein is a double bond, and X represents (R 3 R 4 )C, where R 3 and R 4 As defined in any one of the preceding claims.

9. A method for preparing a thiophosphonate or a phosphonate, comprising the steps of: The compound of formula (I*) in, V is for C 1 -C 8 - alkyl, preferably methyl, ethyl or propyl, more preferably methyl; X represents (R 3 R 4 )C; Among them, ●, Y, R 1 , R 3 and R 4 As defined in any of the preceding claims; Reaction with a thiol-containing molecule of formula (II) in As defined in any of the preceding claims; Obtaining a compound of formula (III*) in ●、V、R 1 , X and Y are as defined in any one of the preceding claims.

10. The method according to claim 9, wherein Y is S.

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