Drugs and conjugate products for directed conjugation techniques

Abstract

Among other things, the present disclosure provides techniques for site-specific conjugation of various moieties of interest to targeted agents. In some embodiments, the present disclosure utilizes target-binding moieties to provide high conjugation efficiency and selectivity. In some embodiments, the provided techniques are useful for preparing antibody conjugates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 189,522, filed May 17, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to conjugate therapeutic enhancers that are useful for preventing and / or treating various conditions, disorders, or diseases. In particular, the present invention relates to protein conjugates, such as antibody-drug conjugates, that can function as therapeutic enhancers. [Background technology]

[0003] Conjugate therapeutic enhancers are widely used to prevent and / or treat various conditions, disorders, and diseases. Such enhancers typically include a therapeutically active molecule, such as an antibody, linked to a moiety that has affinity for a specific target involved in the condition, disorder, or disease. However, most of the known conjugation techniques are not directed to a specific site of the therapeutically active molecule and usually result in a mixture of conjugates. There is still a need in the development of site-specific conjugation techniques that provide reaction products with a high degree of homogeneity. Summary of the Invention

[0004] The present disclosure relates to compositions comprising a therapy enhancer agent that contains a moiety of interest conjugated to a targeting agent moiety at a specific location.

[0005] In one embodiment, a compound having the structure of formula RI: LG-RG-L RM -MOI (RI) or a salt thereof, wherein LG is a group comprising a target binding moiety that binds to a targeting agent; RG is a compound of formula -L LG2 -L LG3 -LLG4 -L RG1 -L RG2 - reactive group, L LG2 is -NH-C(O)OC(R')-, where each R' is independently H or C1-C10 alkyl, and R' are optionally joined to form a ring; L LG3 is an optionally substituted aryl ring; L LG4- is -NH- or -O-, L RG1 is -C(O)-, -S(O)-, -OS(O)2-, or -OP(O)(OR)2-, L RG2 - is a covalent bond or [-C(R")2C(R")=C(R")]C(O)-, where each R" is independently H or C1-C10 alkyl, and any two R" are optionally joined to form a ring; L RM is the linker, The MOI is provided with a compound or a salt thereof that is the moiety of interest.

[0006] In another embodiment, a composition is provided that includes the above compound.

[0007] These and / or other aspects will become apparent and more readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0008] [Figure 1] Schematic diagram of the conjugation process of targeted binding agent and target (top) and the targeted binding agent interacting with Lys246 of the immunoglobulin target. [Diagram 2] Binding specificity data for linear peptide IgG binders. Data for the GSYWYDVWF peptide (SEQ ID NO: 1) is shown. [Diagram 3] Binding specificity data for cyclic peptide IgG binders. Data for the DCAWXLGELVWCT (SEQ ID NO: 2) peptide is shown. [Figure 4] 4A shows a targeted conjugation where the reactive compound has a reactive group that is an aza-Michael acceptor, and 4B shows a targeted conjugation where the reactive compound has a reactive group that releases CO2 upon conjugation. [Diagram 5] A targeted binding agent comprising the peptide DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER (SEQ ID NO:3). [Figure 6] Exemplary targeting binding groups according to some embodiments: Ac-DCAWNLGELVWCT (SEQ ID NO: 4), Ac-DCAWHLGELVWCT-R (SEQ ID NO: 5), R-DCAWHLGELVWCT (SEQ ID NO: 6), ASYHLGELVW-Tic-Aib-CE-R (SEQ ID NO: 7). [Figure 7] Synthesis of exemplified target binding groups. [Figure 8] Synthesis of exemplified target binding groups. [Figure 9] Exemplary LG-RG groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The following detailed description is provided to assist those skilled in the art in the present disclosure. Hereinafter, exemplified embodiments are described in detail. However, these embodiments are merely examples, and the present disclosure is not limited thereto, but rather defined by the scope of the appended claims. Those skilled in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure.

[0010] Accordingly, embodiments are described below by reference to structures and schemes merely to illustrate aspects of the specification. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. The term "or" means "and / or." A phrase such as "at least one of" preceding a list of elements modifies the entire list of elements and not the individual elements of the list.

[0011] When an element is referred to as being "on" another element, it will be understood that it may be in direct contact with the other element, or there may be intervening elements between them. In contrast, when an element is referred to as being "directly on" another element, there are no intervening elements present.

[0012] Terms such as first, second, third, etc. may be used herein to describe various elements, components, regions, layers, and / or sections, but it should be understood that these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section discussed below can be referred to as a second element, component, region, layer, or section without departing from the teachings of the present embodiment.

[0013] As used herein, the terms "comprises" and / or "comprising", or "includes" and / or "including", specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The technical terms used herein are intended only to describe specific embodiments and are not intended to be limiting. It is further understood that terms such as those defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the relevant technical field and the context of this disclosure, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0015] As used in this application, unless expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application. If a term is not specifically defined herein, the term will be given its art-recognized meaning by one of ordinary skill in the art applying the term in the context in which it is used in this disclosure.

[0016] The articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless the context clearly dictates otherwise. By way of example, "an element" means one element or more than one element.

[0017] As used herein, unless a specific definition is provided otherwise, the term “substituted” includes any of the following: deuterium, halogen (-F, -Cl, -Br, -I), hydroxyl group (-OH), amino group (-NH), carboxyl group (-COH), substituted or unsubstituted C1-C6 10 Amine group, nitro group (-NO2), C1~C 10 Alkyl groups, C3-C 10 Cycloalkyl groups, C6-C 12 Aryl groups, C1-C 10 C1-C such as alkoxy group, trifluoromethyl group (-CF3) 10It refers to a trifluoroalkyl group or a group in which at least one hydrogen of a substituent or compound is replaced with a cyano group (-CN).

[0018] Additional aspects are set forth in part in the description that follows, and in part will be apparent from the description.

[0019] The starting materials useful for making the pharmaceutical compositions of the present disclosure are readily commercially available or can be prepared by one of ordinary skill in the art.

[0020] The present disclosure relates to compositions comprising a therapy enhancer agent that contains a moiety of interest conjugated to a targeting agent moiety at a specific location.

[0021] In one embodiment, a compound having the structure of formula RI: LG-RG-L RM -MOI (RI) or a salt thereof, wherein LG is a group comprising a target binding moiety that binds to a targeting agent; RG is a compound of formula -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - reactive group, L LG2 is -NH-C(O)OC(R')-, where each R' is independently H or C1-C10 alkyl, and R' are optionally joined to form a ring; L LG3 is an optionally substituted aryl ring; L LG4- is -NH- or -O-, L RG1 is -C(O)-, -S(O)-, -OS(O)2-, or -OP(O)(OR)2-, L RG2- is a covalent bond or [-C(R")2C(R")=C(R")]C(O)-, where each R" is independently H or C1-C10 alkyl, and any two R" are optionally joined to form a ring; L RM is the linker, The MOI is provided with a compound or a salt thereof that is the moiety of interest.

[0022] target After reading this disclosure, those skilled in the art will understand that the techniques provided herein are useful for conjugating a variety of targeting agents to many types of moieties of interest. In some embodiments, the techniques provided are particularly useful for conjugating protein agents to a variety of moieties of interest. In some embodiments, the targeting agent is or includes a protein agent, a nucleic acid, or a combination thereof.

[0023] In some embodiments, the targeting agent is or comprises a protein drug. In some embodiments, the targeting agent is a protein drug. In some embodiments, the targeting agent is a native protein in a cell, tissue, organ, or organism. In some embodiments, the targeting agent is an endogenous protein. In some embodiments, the targeting agent is an exogenous protein. In some embodiments, the targeting agent is a manufactured protein, e.g., a protein produced using various biotechnologies. In some embodiments, the targeting agent is an antibody drug. In some embodiments, the targeting agent is an antibody useful as a therapeutic agent. A variety of such antibodies are known in the art and may be utilized as targeting agents. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is IVIG (in some embodiments, pooled from healthy donors). In some embodiments, the protein comprises an Fc region. In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region comprises a single heavy chain or fragment thereof. In some embodiments, the Fc region comprises two heavy chains or fragments thereof. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a murine antibody.

[0024] In some embodiments, when characterizing polyclonal antibody or IVIG agents, digestion, e.g., enzymatic digestion using IdeZ, IdeS, etc., is performed either before, during, or after conjugation, resulting in removal of certain regions of the antibody (e.g., the Fab) and providing a composition with improved homogeneity for characterization (e.g., by MS).

[0025] In some embodiments, the antibody is a therapeutic antibody, e.g., an FDA approved antibody for therapeutic use. In some embodiments, the therapeutic antibody is useful for the treatment of cancer. In some embodiments, the antibody is selected from the group consisting of adalimumab, alemtuzumab, atezolizumab, avelumab, ipilimumab, cetuximab, daratumumab, dinutuximab, elotuzumab, ibritumomab tiuxetan, imgatuzumab, infliximab, ipilimumab, necitumumab, obinutuzumab, ofatumumab, pertuzumab, reslizumab, rituximab, trastuzumab, mogamulizumab, AMP-224, FS-102, GSK-2857916, ARGX-111, ARGX-110, AFM-13, APN-301, BI-83 6826, BI-836858, enoblitzumab, otlertuzumab, veltuzumab, KHK-4083, BIW-8962, ALT-803, carotuximab, epratuzumab, inebilizumab, isatuximab, margetuximab, MOR-208, ocaratuzumab, talatuzumab, tremelimumab, benralizumab, lumiliximab, MOR-208, ifibatuzumab, GSK2831781, SEA-CD40, KHK-2823, or BI836858. In some embodiments, the antibody is selected from the group consisting of rituximab, basiliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoxaximab, daclizumab, palivizumab, trastuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, eculizumab, mogamulizumab, pertuzumab, obinutuzumab, bedrigravir, cefotaxime ... In some embodiments, the antibody is daratumumab. In some embodiments, the antibody is cetuximab.In some embodiments, provided compounds or drugs that comprise an antibody drug moiety are useful for treating a condition, disorder or disease that can be treated by an antibody drug.

[0026] According to the present disclosure, antibodies may be prepared by several techniques according to the present disclosure. In some embodiments, the antibodies may have an engineered structure compared to a natural immunoglobulin. In some embodiments, the antibodies may include specific tags for purification, identification, evaluation, etc. In some embodiments, the antibodies may contain fragments (e.g., CDRs and / or Fc, etc.) and may not contain the entire immunoglobulin. Those skilled in the art will understand that when antibody sites are cited in the present disclosure (e.g., K246, K248, K288, K290, K317, etc., human antibodies according to EU numbering unless otherwise indicated), the amino acid residue may not be at the exact numbered site, but may be present at the site corresponding to that numbered site and / or sequence homology (e.g., homologs of the same or different species), for example, according to EU numbering.

[0027] As will be appreciated by those of skill in the art, among other things, the provided techniques can provide for directed conjugation with natural targets (e.g., natural antibodies). In some embodiments, the targeting agent is or comprises a natural antibody drug. In some embodiments, the targeting agent is or comprises an engineered antibody drug. In some embodiments, the targeting agent (e.g., an antibody) does not comprise an engineered non-natural amino acid residue.

[0028] target binding moiety In some embodiments of formulas (LG-I) and (RI), LG is R LG -L LG and R LG teeth, [ka] , R c -(Xaa)z-, a nucleic acid moiety, or a small molecule moiety; each Xaa is independently a residue of an amino acid or amino acid analog; t is 0 to 50; z is 1 to 50; Each R c is independent of -L a -R', Each L a are independently a covalent bond or C 20 Aliphatic or C1-C with 1-5 heteroatoms 20 heteroaliphatic, one or more methylene units of the group are optionally and independently replaced by C(R')-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted divalent monocyclic, bicyclic, or polycyclic group, and each monocyclic ring is independently selected from the group consisting of C 3~20 Alicyclic ring, C 6~20 an aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; L LG -L LG1 -, -L LG1 -L LG2 -, -L LG1 -L LG2 -L LG3 - or -L LG1 -L LG2 -L LG3 -L LG4 - and L LG1 , L LG2 , L LG3 , and L LG4each independently represents a covalent bond, or one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1 to 20 heteroatoms, heteroaromatic moieties each independently having 1 to 20 heteroatoms, or any combination of any one or more of such moieties; 1~100 is a group, one or more methylene units of the group are optionally and independently selected from C 1~6 Alkylene, C 1~6 Alkenylene, divalent C with 1-5 heteroatoms 1~6 Heteroaliphatic group, -C≡C-, -Cy-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')- , -C(O)C(R')2N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S- , -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S)(SR')-, -P( S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R')2-C(R')2-) n ]- (wherein n is 1 to 20), each R' is independently -R, -C(O)R, -CO2R, or -SO2R; Each R is independently -H or C 1~30 Aliphatic, C with 1-10 heteroatoms 1~30 Heteroaliphatic, C 6~30 Aryl, C 6~30 Arylaliphatic, C with 1-10 heteroatoms 6~30 an optionally substituted group selected from arylheteroaliphatic, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms; or two R groups, optionally and independently, taken together form a covalent bond, or two or more R groups on the same atom optionally and independently, taken together with the atom, form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having, in addition to the atom, 0-10 heteroatoms; or Two or more R groups on two or more atoms optionally and independently, taken together with their intervening atoms, form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

[0029] In some embodiments, the LG is or comprises a target binding moiety that binds to a targeting agent, and the targeting agent is an antibody agent.

[0030] In some embodiments, LG is or comprises a target binding moiety that binds to the Fc region, and / or R LG is or comprises DCAWXLGELVWCT (SEQ ID NO: 2), wherein the two cysteine ​​residues optionally form a disulfide bond, and X is an amino acid residue.

[0031] In some embodiments, LG is or includes a target binding moiety having a structure of Formulas A-1 through A-50. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0032] Purpose Those of skill in the art reading this disclosure will appreciate that various types of object moieties can be utilized for various purposes in accordance with the disclosure.

[0033] In some embodiments, the moiety of interest is or comprises a detectable moiety. Such moieties may be useful for, among other things, detection, quantification, diagnosis, treatment, and the like. In some embodiments, the moiety of interest is or comprises a radioactive label. In some embodiments, the moiety of interest is or comprises a label that can be detected by spectroscopy. In some embodiments, the moiety of interest is or comprises a fluorophore, such as a FITC moiety.

[0034] The moiety of interest may be a moiety that has affinity for a particular target involved in a medical condition, disorder, or disease. In some embodiments, the moiety of interest is or comprises a therapeutic drug moiety. In some embodiments, the moiety of interest is or comprises a drug moiety, e.g., a drug moiety in an antibody-drug conjugate. In some embodiments, the moiety of interest is or comprises a toxic drug. In some embodiments, the moiety of interest is or comprises a cytotoxic drug. In some embodiments, the moiety of interest is or comprises an anti-cancer drug. In some embodiments, the anti-cancer drug is a chemotherapeutic drug.

[0035] In some embodiments, the moiety of interest is or includes a moiety that can interact with and / or recruit other agents, such as proteins, nucleic acids, cells, etc. In some embodiments, the moiety of interest interacts with proteins expressed by a particular cell type, e.g., immune cells, diseased cells, etc. In some embodiments, the moiety of interest is an immune cell binding agent. In some embodiments, the moiety of interest recruits immune cells. In some embodiments, the moiety of interest induces, promotes, and / or enhances one or more immune activities, e.g., to eliminate, kill, and / or inhibit a desired target (e.g., cancer cells, antigens, etc.). In some embodiments, the moiety of interest interacts, recruits, and / or binds to diseased cells and induces, promotes, and / or enhances the elimination, killing, and / or inhibition of diseased cells.

[0036] In some embodiments, the moiety of interest is or comprises a small molecule drug (e.g., capable of specifically binding to its protein target, cellular target, etc.). In some embodiments, the moiety of interest is or comprises a peptide or protein drug (e.g., scFv, peptide binders to specific targets, etc.). In some embodiments, the moiety of interest is or comprises a nucleic acid drug (e.g., oligonucleotides, mRNA, etc.). In some embodiments, the moiety of interest is or comprises a carbohydrate drug. In some embodiments, the moiety of interest is or comprises a lipid drug.

[0037] In some embodiments, the moiety of interest is or comprises a protein complex (e.g., a Fab). In some embodiments, the moiety of interest is or comprises a fluorophore. In some embodiments, the moiety of interest is or comprises a cytotoxic small molecule drug. In some embodiments, the moiety of interest is or comprises a cytotoxic peptide drug.

[0038] In some embodiments, the moiety of interest is an adjuvant. Those skilled in the art will understand that various adjuvants can be used as the moiety of interest according to the present disclosure. In some embodiments, the adjuvant is one described in US2019 / 0015516, the entirety of which is incorporated herein by reference. In some embodiments, the moiety of interest stimulates the immune system.

[0039] In some embodiments, the moiety of interest is or comprises a particle, hi some embodiments, the particle is or comprises a nanoparticle.

[0040] In some embodiments, the moiety of interest is or comprises a nucleic acid moiety. In some embodiments, the moiety of interest is or comprises an oligonucleotide. In some embodiments, the moiety of interest is or comprises an aptamer.

[0041] In some embodiments, the moiety of interest is an antibody drug. In some embodiments, the moiety of interest is or comprises an antibody fragment. In some embodiments, the moiety of interest is an antibody drug moiety that does not contain the region to which the target binding moiety binds. In some embodiments, the moiety of interest is an antibody drug that does not contain an Fc region. In some embodiments, the moiety of interest is or comprises an scFv. In some embodiments, the scFv is for a different antigen than the antibody targeting drug.

[0042] In some embodiments, the moiety of interest is or comprises a reactive moiety, in particular a reaction partner for bioorthogonal reactions. Suitable reactive moieties, including those for bioorthogonal reactions, are widely known in the art and can be utilized herein. In some embodiments, the bioorthogonal reaction is a cycloaddition reaction, e.g., click chemistry. In some embodiments, the moiety of interest is or comprises -N3. In some embodiments, the moiety of interest is or comprises an alkyne.

[0043] In some embodiments, the moiety of interest may be a moiety that binds to the SARS-CoV-2 virus, which is involved in COVID-19 disease. For example, the moiety that binds to the SARS-CoV-2 virus may be a polypeptide disclosed in L. Cao et al., “De novo design of picomolar SARS-CoV-2 mini-protein inhibitors” Science 370, 426-431 (2020), which is incorporated by reference in its entirety. Such a polypeptide moiety may cause binding to the SARS-CoV-2 spike protein, inhibition, reduction and prevention of binding and / or infection of cells, inhibition, killing and elimination of the SARS-CoV-2 virus and / or cells infected thereby, etc. Various moieties of interest that interact with the SARS-CoV-2 virus are described in International Patent Application No. PCT / US21 / 24186, filed March 25, 2021, U.S. Provisional Patent Application No. 63 / 146584, filed February 6, 2021, and U.S. Provisional Patent Application No. 63 / 182098, filed April 30, 2021, each of which is incorporated by reference in its entirety herein.

[0044] In some embodiments, the moiety of interest improves one or more properties and / or activities of the targeted agent. In some embodiments, the moiety of interest is or includes a stability enhancer. In some embodiments, the moiety of interest improves one or more pharmacodynamic and / or pharmacokinetic properties of the targeted agent.

[0045] In some embodiments, the following conditions (a) the moiety of interest is or contains a therapeutic agent; (b) the moiety of interest is or comprises a moiety capable of binding to a protein, a nucleic acid, or a cell; and / or (c) the moiety of interest is or contains a reactive moiety suitable for a bioorthogonal reaction.

[0046] In some embodiments, the MOI is or comprises a therapeutic drug moiety and / or the MOI is or comprises an antibody drug.

[0047] linking group In some embodiments, the moieties are optionally connected to one another via a linker moiety. For example, in some embodiments, the reactive group (e.g., RG) is linked to a linker (e.g., L RM In some embodiments, the moiety (e.g., LG) is also connected to the moiety of interest (e.g., MOI) via one or more linkers, e.g., L, for linking to various moieties. LG1 , L LG2 , L LG3 , L LG4 In some embodiments, L LG is a linker moiety as described herein. In some embodiments, L LG1 is a linker moiety as described herein. In some embodiments, L LG2 is a linker moiety as described herein. In some embodiments, L LG3 is a linker moiety as described herein. In some embodiments, L LG4 is a linker moiety as described herein. In some embodiments, L RM is a linker moiety as described herein. In some embodiments, L PM is L, as described herein. In some embodiments, L PM is a linker moiety as described herein. In some embodiments, L PM is L as described herein.

[0048] Linker moieties of various types and / or for various purposes (such as those utilized in antibody-drug conjugates) may be utilized in accordance with the present disclosure.

[0049] Linker moieties can be either bivalent or multivalent depending on how they are used. In some embodiments, the linker moiety is bivalent. In some embodiments, the linker is multivalent and connects more than two moieties.

[0050] In some embodiments, L LM is one or more -[(CH2) n -O] m -, wherein each n is independently 1 to 20 and m is 1 to 100.

[0051] In some embodiments, L RM The linker consists of one or more -[(CH2) n -O] m -, wherein each n is independently 1 to 20 and m is 1 to 100.

[0052] Reactive Groups In some embodiments, provided compounds (e.g., those useful as reaction partners) include a reactive group (e.g., RG). As exemplified herein, in many embodiments, in provided compounds, the reactive group (e.g., RG) is located between a first group (e.g., LG) and a moiety of interest (e.g., MOI), and is optionally and independently linked to the first group and the moiety of interest via a linker. In some embodiments, RG is a reactive group as described herein.

[0053] In some embodiments, as shown herein, reactive groups, when utilized in compounds that do not include a target binding moiety, react slowly, resulting in low levels of conjugation of the moiety of interest with the targeting agent, and in some embodiments, substantially no conjugation. As shown herein, the combination of a reactive group and a target binding moiety in the same compound, such as in a compound of formula RI or a salt thereof, can, among other things, facilitate the reaction between the reactive group and the targeting agent, enhance the reaction efficiency, reduce side reactions, and / or improve the reaction selectivity (e.g., with respect to the target site at which conjugation of the moiety of interest with the targeting agent occurs).

[0054] The reactive groups in the provided compounds can react with various types of groups in the targeting agent. In some embodiments, the reactive groups in the provided compounds selectively react with amino groups of the targeting agent, for example, the -NH2 group on the side chain of a lysine residue of a protein. In some embodiments, the reactive groups, when utilized in the provided compounds (e.g., those of formula RI or salts thereof), selectively react with specific sites of the targeting agent, for example, one or more of K246, K248, K288, K290, K317, etc. of IgG1, K251, K253, etc. of IgG2, and K239, K241, etc. of IgG4, as shown in the examples herein. In some embodiments, the site is K246 or K248 of the antibody heavy chain. In some embodiments, the site is K246 and / or K248 of the antibody heavy chain. In some embodiments, the site is K246 of the antibody heavy chain. In some embodiments, the site is K248 of the antibody heavy chain. In some embodiments, the site is K288 or K290 of the antibody heavy chain. In some embodiments, the site is K288 of the antibody heavy chain. In some embodiments, the site is K290 of the antibody heavy chain. In some embodiments, the site is K317. In some embodiments, the site is K414 of the antibody heavy chain. In some embodiments, the site is K185 of the antibody light chain. In some embodiments, the site is K187 of the antibody light chain. In some embodiments, the site is K251 and / or K253 of an IgG2 heavy chain. In some embodiments, the site is K251 of an IgG2 heavy chain. In some embodiments, the site is K253 of an IgG2 heavy chain. In some embodiments, the site is K239 and / or K241 of an IgG4 heavy chain. In some embodiments, the site is K239 of an IgG4 heavy chain. In some embodiments, the site is K241 of an IgG4 heavy chain. In some embodiments, conjugation occurs preferentially at one or more heavy chain sites over light chain sites. In some embodiments, for technologies that do not have target binding moieties, conjugation occurs at light chain sites rather than heavy chain sites.

[0055] In some embodiments, the reactive group (e.g., RG) is or includes an ester group. In some embodiments, the reactive group (e.g., RG) is or includes an electrophilic group (e.g., a Michael acceptor).

[0056] In some embodiments, RG is of the formula -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - reactive group, In the formula, L LG2 is -NH-C(O)OC(R')-, where each R' is independently H or C1-C10 alkyl, and R' are optionally joined to form a ring; L LG3 is an optionally substituted aryl ring; L LG4- is -NH- or -O-, L RG1 is -C(O)-, -S(O)-, -OS(O)2-, or -OP(O)(OR)2-, L RG2 - is a covalent bond, or [-C(R")C(R")=C(R")]C(O)-, where each R" is independently H or C1-C10 alkyl, and any two R" are optionally joined to form a ring.

[0057] In some embodiments, RG is or includes a reactive group having the formula: [ka] wherein ARYL is a substituted or unsubstituted para-phenylene ring. In some embodiments, ARYL is [ka] wherein R sis independently selected at each occurrence from halogen, -NO2, -F, -L-R', -C(O)-L-R', -S(O)-L-R', -S(O)2-L-R', and -P(O)(-L-R')2, where R' is H or C1-C6 alkyl.

[0058] In some embodiments, the reactive group has the formula: [ka] It is or contains one of the following:

[0059] In some embodiments, RG is or includes a reactive group having the formula: [ka] wherein ARYL is a substituted or unsubstituted para-phenylene ring. In some embodiments, ARYL is [ka] wherein R s is independently selected at each occurrence from halogen, -NO2, -F, -L-R', -C(O)-L-R', -S(O)-L-R', -S(O)2-L-R', and -P(O)(-L-R')2, where R' is H or C1-C6 alkyl.

[0060] In some embodiments, the reactive group has the formula: [ka] It is or contains one of the following:

[0061] composition In some embodiments, compositions are provided that include one or more of the above compounds.

[0062] In some embodiments, the composition comprises: Structure of formula (P-II) PNL PM-MOI(P-II) A first compound having the formula: PN is a protein drug moiety that contains a lysine residue; L PM is the linker, The MOI is the moiety of interest, the first compound, The following structure: LG-OH(LG-I) and a second compound having the formula:

[0063] In some embodiments, the composition comprises: Formula (RI) LG-RG-L RM -MOI(RI) wherein LG is a group comprising a target binding moiety that binds to a targeting agent and is identical to LG in formula (LG-I); RG is a compound of formula -L LG2 -L LG3 -L LG4 -L RG1 -L RG2 - reactive group, L LG2 is -NH-C(O)OC(R')-, where each R' is independently H or C1-C10 alkyl, and R' are optionally joined to form a ring; L LG3 is an optionally substituted aryl ring; L LG4- is -NH- or -O-, L RG1 is -C(O)-, -S(O)-, -OS(O)2-, or -OP(O)(OR)2-, L RG2 - is a covalent bond or [-C(R")2C(R")=C(R")]C(O)-, where each R" is independently H or C1-C10 alkyl, and any two R" are optionally joined to form a ring; L RM is a linker, and is the same as in formula (P-II), The MOI is the moiety of interest, the third compound, Formula (R-III) HO-RG-L RM -MOI(R-III) or a fourth compound having the formula:

[0064] In some embodiments, the composition may include an equimolar amount of the first compound and the second compound. In some embodiments, the amount of the second compound may be 50 mole percent (mole%) or less, based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 50 mole% or less, 45 mole% or less, 40 mole% or less, 35 mole% or less, 30 mole% or less, 25 mole% or less, 20 mole% or less, 15 mole% or less, 10 mole% or less, or 5 mole% or less, based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 0.10% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, or 0.01% or less based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 0.010% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, 0.001% or less based on the total number of moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 0.0010% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, 0.0001% or less based on the total number of moles of the first compound and the second compound in the composition.In some embodiments, the amount of the second compound may be 0.00010% or less, 0.00009% or less, 0.00008% or less, 0.00007% or less, 0.00006% or less, 0.00005% or less, 0.00004% or less, 0.00003% or less, 0.00002% or less, or 0.00001% or less based on the total moles of the first compound and the second compound in the composition. In some embodiments, the amount of the second compound may be 0.000010% or less, 0.000009% or less, 0.000008% or less, 0.000007% or less, 0.000006% or less, 0.000005% or less, 0.000004% or less, 0.000003% or less, 0.000002% or less, or 0.000001% or less based on the total moles of the first compound and the second compound in the composition.

[0065] In some embodiments, the composition may further include a third compound, a fourth compound, or a combination thereof. In some embodiments, the amount of the third compound, the fourth compound, or a combination thereof may be 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less based on the moles of the first compound in the composition. In some embodiments, the amount of the third compound, the fourth compound, or a combination thereof may be 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less based on the moles of the first compound in the composition. In some embodiments, the amount of the third compound, the fourth compound, or a combination thereof, based on the number of moles of the first compound in the composition, may be 0.10% or less, 0.09% or less, 0.08% or less, 0.07% or less, 0.06% or less, 0.05% or less, 0.04% or less, 0.03% or less, 0.02% or less, 0.01% or less. In some embodiments, the amount of the third compound, the fourth compound, or a combination thereof, based on the number of moles of the first compound in the composition, may be 0.010% or less, 0.009% or less, 0.008% or less, 0.007% or less, 0.006% or less, 0.005% or less, 0.004% or less, 0.003% or less, 0.002% or less, 0.001% or less. In some embodiments, the amount of the third compound, the fourth compound, or a combination thereof may be 0.0010% or less, 0.0009% or less, 0.0008% or less, 0.0007% or less, 0.0006% or less, 0.0005% or less, 0.0004% or less, 0.0003% or less, 0.0002% or less, 0.0001% or less based on the number of moles of the first compound in the composition. In some embodiments, the amount of the third compound, the fourth compound, or combinations thereof may be 0.00010% or less, 0.00009% or less, 0.00008% or less, 0.00007% or less, 0.00006% or less, 0.00005% or less, 0.00004% or less, 0.00003% or less, 0.00002% or less, 0.00001% or less based on the number of moles of the first compound in the composition.In some embodiments, the amount of the third compound, the fourth compound, or combinations thereof may be 0.000010% or less, 0.000009% or less, 0.000008% or less, 0.000007% or less, 0.000006% or less, 0.000005% or less, 0.000004% or less, 0.000003% or less, 0.000002% or less, 0.000001% or less based on the number of moles of the first compound in the composition.

[0066] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0067] In some embodiments, the target binding moiety may be an immunoglobulin binding moiety that comprises K246 and / or K248, and the process of directional conjugation may be represented by the following diagram shown in FIG. 1.

[0068] In some embodiments, the targeting binding group has a K d The peptide may be a linear IgG binder comprising a directing group having the formula: [ka]

[0069] The binding specificity data for the linear peptide IgG binders is shown in FIG.

[0070] In some embodiments, the targeting binding group has a K d The cyclic peptide IgG binder may also comprise a directing group having the formula: [ka]

[0071] The binding specificity data for the cyclic peptide binders is shown in FIG.

[0072] In some embodiments, the compound may have a reactive group that is an aza-Michael acceptor, as shown in Figure 4A. In some embodiments, the compound may have a reactive group that releases CO2 upon conjugation, as shown in Figure 4B.

[0073] In some embodiments, the compound may have the structure shown in FIG.

[0074] In some embodiments, the targeting binding group may comprise one of the following sequences shown in FIG. [ka]

[0075] The syntheses of some of these groups are shown in Figures 7-8.

[0076] FIG. 9 illustrates exemplary LG-RG groups according to some embodiments.

[0077] Throughout this application, various publications are referenced by author name and date, or patent or patent publication number. The disclosures of these publications are incorporated by reference in their entireties into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described herein and claimed herein. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention.

[0078] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the following claims. For example, pharma- ceutical acceptable salts other than those specifically disclosed in the description and examples herein may be used. Furthermore, it is contemplated that any particular item in a list of items, or any subset of items within a larger group of items, may be combined with any other particular item, subset of items, or larger group of items, regardless of whether there is a specific disclosure herein specifying such combination. [Table 1]

[0079] Example 1. Procedure for the preparation of compound 1290. [ka] Preparation of compound 1290: Peptides were synthesized using standard Fmoc chemistry (CTC resin).

[0080] Resin preparation: DIEA (4.00 equiv.) was added dropwise to a vessel containing CTC resin (1.00 g, 1.00 mmol, 1.00 mmol / g) and Fmoc-Thr(tBu)-OH (397.0 mg, 1.00 mmol, 1.00 equiv.) in DCM (10 mL) and mixed at 25° C. for 2 h with N2 bubbling. MeOH (1.0 mL) was then added and N2 was bubbled for another 30 min. The resin was washed with DMF (20 mL), followed by 20% piperidine in DMF (10 mL) and N2 was bubbled for 30 min at 25° C. for Fmoc deprotection. The mixture was filtered and the resin was washed with DMF (10 mL) before proceeding to the next step.

[0081] Coupling: A solution of Fmoc-Cys(Trt)-OH (1.76 g, 3.0 mmol, 3.00 equiv.), HBTU (0.82 g, 2.86 mmol, 2.85 equiv.) in DMF (10 mL) was added to the resin while bubbling with N2. Then, DIEA (6.00 equiv.) was added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (20 mL).

[0082] Deprotection: 20% piperidine in DMF (20 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (20 mL).

[0083] Steps 2 and 3 were repeated for the following amino acid extensions: numbers 3-13, Table 1.

[0084] After all steps were completed, the resin was washed with DMF (50 mL), MeOH (50 mL), and then dried under reduced pressure to give the resin-bound peptide intermediate 1 (CTC resin, 2.40 g, 1.00 mmol). [Table 2]

[0085] Peptide cleavage and cyclization: Cleavage: A solution of TFA / TIS / H2O / 3-mercaptopropanoic acid (92.5 / 2.5 / 2.5 / 2.5, v / v / v, 40 mL) was added to the above resin (Intermediate 1, 0.50 mmol, another 0.5 mmol was used for compound 1291) at room temperature and stirred for 2 h. After filtration, the filtrate was collected and precipitated with cold isopropyl ether (200 mL), then filtered off, the solid was washed twice with isopropyl ether (100 mL), and the crude peptide was dried under reduced pressure for 2 h to give Intermediate 2 (0.50 mmol, crude) as a white solid.

[0086] Cyclization: To the crude peptide (intermediate 2) in MeCN / H2O (1 / 1, v / v, 500 mL) was added 0.1 M I2 / AcOH dropwise until the yellow color persisted, and then the mixture was stirred at 25 °C for 5 min. The mixture was quenched by adding 0.1 M aqueous Na2S2O3 dropwise until the yellow color disappeared. After filtration, the filtrate was purified by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) and then lyophilized to give compound 1290 (93.0 mg, 94.6% purity, 15.5% yield) as a white solid. LCMS: RT = 0.81 min, MS calculated: M 平均 = 1521.76, observed mass: [M+H] + =1522.70, [M+2H] 2+ =761.50. [ka]

[0087] Example 2. Procedure for the preparation of compound 1291. [ka] Preparation of compound 1291: Intermediate 3 (peptide-resin) was synthesized by performing acetylation on peptide-resin (intermediate 1, 0.50 mmol).

[0088] Acetylation: A solution of Ac2O / NMM / DMF (10 / 5 / 85, v / v / v, 40 mL) was added to the resin and the mixture was bubbled with N2 for 20 min. The acetylation reaction was monitored by ninhydrin test. The resin was then washed with DMF (20 mL), MeOH (20 mL), and then dried under reduced pressure to give the resin-bound peptide intermediate 4 (CTC resin, 1.23 g, 0.50 mmol). [Table 3]

[0089] Peptide cleavage and cyclization were carried out according to the procedure described in Peptide Cleavage and Cyclization Reaction in Example 1. From 0.50 mmol of resin, compound 1291 (148.0 mg, 95.5% purity, 18.1% yield) was obtained as a white solid. LCMS: RT=1.53 min, MS calculated: M 平均 = 1563.79, observed mass: [M+H] + =1564.40, [M+2H] 2+ =782.80. [ka]

[0090] Example 3. Procedure for the preparation of compound 1292. [ka] [ka] Preparation of intermediate 6: [ka] A mixture of intermediate 5 (1.40 g, 3.09 mmol, 1.00 equiv.) and intermediate 5A (1.30 g, 9.26 mmol, 3.00 equiv.), HOBt (1.25 g, 9.26 mmol, 3.00 equiv.), DMAP (188.56 mg, 1.54 mmol, 0.50 equiv.) and EDCI (1.78 g, 9.26 mmol, 3.00 equiv.) in DMF (0.2 mL) was stirred for 16 h at 25° C. The mixture was directly purified by Flash (C18, A: 0.075% TFA / HO, B: MeCN) and then lyophilized to give intermediate 6 (1.10 g, 1.91 mmol, 61.9% yield) as a yellow oil.

[0091] Preparation of intermediate 7: [ka] A mixture of intermediate 6 (1.85 g, 3.21 mmol, 1.00 equiv) was dissolved in MeOH (5 mL) and to the reaction mixture was added a mixture of NaBH4 (145.91 mg, 3.86 mmol, 1.20 equiv) in MeOH (1 mL) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. After completion, the reaction was monitored by LC-MS and the mixture was acidified to pH = 5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) followed by lyophilization to give intermediate 7 (1.35 g, 2.34 mmol, 72.7% yield) as a colorless oil. LCMS: RT = 9.3 min, MS calculated: M 平均 = 577.64, observed mass: [M+H] + =578.30, [M+H2O+H] + =595.4, [M-Boc+H] + =478.37.

[0092] Preparation of intermediate 8: [ka] To a solution of intermediate 7 (1.20 g, 2.08 mmol, 1.00 equiv), TEA (420.43 mg, 4.15 mmol, 578.30 μL, 2.00 equiv) in DCM (10 mL) was added intermediate 7A (460.61 mg, 2.29 mmol, 1.10 equiv). The reaction was stirred at 25° C. for 4 h. After completion as monitored by LC-MS, the mixture was directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) and then lyophilized to give intermediate 8 (900.0 mg, 1.21 mmol, 58.3% yield) as a yellow oil. LCMS: RT=9.3 min, MS calculated: M 平均 = 742.74, observed mass: [M+H] + =743.2, [M+H2O+H] + =761.3, [M-Boc+H] + =643.3.

[0093] Preparation of intermediate 9: [ka] A mixture of intermediate 8 (500.00 mg, 667.50 μmol, 2.00 equiv.) and compound 1291 (521.5 mg, 333.70 μmol, 1.00 equiv.), DIEA (129.0 mg, 174.3 μL, 1.00 mmol, 3.00 equiv.) in DMF (5 mL) was stirred at 25° C. for 2 h. After completion as monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give intermediate 9 (517.3 mg, 238.69 μmol, 81.2% purity, 71.5% yield) as a white solid. LCMS: RT=1.05 min, MS calculated: M 平均 = 2167.43, observed mass: [M-Boc+2H] 2+ =1034.58.

[0094] Preparation of intermediate 10: [ka] A mixture of intermediate 9 (517.3 mg, 238.69 μmol) in TFA / DCM (3 / 7, v / v, 3 mL) was stirred at 0° C. for 2 h. After completion monitored by LC-MS, the solvent was removed under reduced pressure. The residue was purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) and then lyophilized to give intermediate 10 (429.27 mg, 207.65 μmol, 87.0% yield, TFA salt) as a white solid. UPLC: RT=0.92 min, MS calculated: M 平均 = 2067.43, observed mass: [M+2H] 2+ =1034.12.

[0095] Preparation of compound 1292: [ka] To a mixture of intermediate 10 (429.27 mg, 207.65 μmol, 1.00 equiv) and FITC (121.28 mg, 311.47 μmol, 1.50 equiv) in DMF (0.2 mL) was added DIEA (20.76 mg, 934.42 μmol, 162.75 μL, 4.50 equiv) at 25° C. The mixture was stirred at 25° C. for 2 h. After completion monitored by LC-MS, the mixture was acidified to pH=5 with 1M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give compound 1292 (115.0 mg, 92.0% purity, 22.5% yield) as a yellow solid. LCMS: RT=2.08 min, MS calculated: M 平均 = 2456.69, observed mass: [M+2H] 2+ =1229.1, [M+3H] 3+ =819.7, [2M+3H] 3+ =1637.9.

[0096] Example 4. Procedure for the preparation of compound 1294. [ka] [ka] [ka] [ka] Preparation of intermediates 6, 7 and 8: [ka] Intermediates 6, 7, and 8 were synthesized according to the procedures described in the preparation of intermediates 6, 7, and 8 in Example 3.

[0097] Preparation of intermediate 13: [ka] Peptides were synthesized using standard Fmoc chemistry (CTC resin).

[0098] Resin preparation: DIEA (4.00 equiv.) was added dropwise to a vessel containing CTC resin (1.00 g, 1.00 mmol, 1.00 mmol / g) and Fmoc-Thr(tBu)-OH (397.0 mg, 1.00 mmol, 1.00 equiv.) in DCM (10 mL) and mixed at 25° C. for 2 h with N2 bubbling. MeOH (1.0 mL) was then added and N2 was bubbled for another 30 min. The resin was washed with DMF (20 mL), followed by 20% piperidine in DMF (10 mL) and N2 was bubbled for 30 min at 25° C. for Fmoc deprotection. The mixture was filtered and the resin was washed with DMF (10 mL) before proceeding to the next step.

[0099] Coupling: A solution of Fmoc-Cys(Trt)-OH (1.76 g, 3.0 mmol, 3.00 equiv.), HBTU (0.82 g, 2.86 mmol, 2.85 equiv.) in DMF (10 mL) was added to the resin while bubbling with N2. Then, DIEA (6.00 equiv.) was added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (20 mL).

[0100] Deprotection: 20% piperidine in DMF (20 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (20 mL).

[0101] Steps 2 and 3 were repeated for the following amino acid extensions: numbers 3-13, Table 3.

[0102] Alloc-Cl coupling on the N-terminus: The resin was washed with DCM (20 mL). A solution of Allo-Cl (0.72 g, 6.0 mmol, 6.00 equiv.) in DCM (10 mL) was added to the resin while bubbling with N2. DIEA (12.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 30 min at 25°C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DCM (50 mL) x 3, DMF (50 mL) x 3, MeOH (50 mL) x 3, and then dried under reduced pressure to give resin-bound peptide intermediate 11 (CTC resin, 2.35 g, 1.00 mmol). [Table 4]

[0103] Peptide cleavage and cyclization: Cleavage: A solution of TFA / TIS / H2O / 3-mercaptopropanoic acid (92.5 / 2.5 / 2.5 / 2.5, v / v / v, 40 mL) was added to the above resin (Intermediate 11, 1.00 mmol) at room temperature and stirred for 2 h. After filtration, the filtrate was collected and precipitated with cold isopropyl ether (400 mL), then filtered off, the solid was washed twice with isopropyl ether (200 mL), and the crude peptide was dried under reduced pressure for 2 h to give Intermediate 12 (1.00 mmol, crude) as a white solid.

[0104] Cyclization: To the crude peptide (Intermediate 12) in MeCN / H2O (1 / 1, v / v, 1000 mL) was added 0.1 M I2 / AcOH dropwise until the yellow color persisted, and then the mixture was stirred at 25 °C for 5 min. The mixture was quenched by adding 0.1 M aqueous Na2S2O3 dropwise until the yellow color disappeared. After filtration, the filtrate was purified by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) and then lyophilized to give Intermediate 13 (280.5 mg, 17.4% yield) as a white solid. LCMS: RT = 7.9 min, MS calculated: M 平均 = 1605.83, observed mass: [M+H] +=1606.80, [M+2H] 2+ =803.52. [ka]

[0105] Preparation of intermediate 14: [ka] A mixture of intermediate 8 (475.28 mg, 0.63 mmol, 2.00 equiv) and intermediate 13 (500.0 mg, 0.31 mmol, 1.00 equiv), DIEA (120.5 mg, 162.9 μL, 0.93 mmol, 3.00 equiv) in DMF (5 mL) was stirred at 25° C. for 2 h. After completion as monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give intermediate 14 (517.3 mg, 74.6% yield) as a white solid. LCMS: RT=0.960 min, MS calculated: M 平均 = 2209.46, observed mass: [M-Boc+2H] 2+ =1055.28.

[0106] Preparation of intermediate 15: A mixture of intermediate 14 (517.3 mg) in TFA / DCM (3 / 7, 5 mL) was stirred for 2 h at 0° C. After completion monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give compound 1579 (437.0 mg, 97.9% purity, 88.4% yield, TFA salt) as a white solid. [ka]

[0107] Preparation of intermediate 16: [ka] To a mixture of intermediate 15 (437.0 mg, 207.2 μmol, 1.00 equiv) and FITC (120.8 mg, 310.80 μmol, 1.50 equiv) in DMF (0.2 mL) was added DIEA (120.7 mg, 162.5 μL, 932.4 μmol, 4.50 equiv) at 25° C. The mixture was stirred at 25° C. for 2 h. After completion monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give intermediate 16 (300.0 mg, 92.5% purity, 57.9% yield) as a yellow solid. LCMS: RT=9.3 min, MS calculated: M 平均 = 2498.73, observed mass: [M+2H] 2+ =1249.87.

[0108] Preparation of compound 1294: [ka] To a mixture of intermediate 16 (300.0 mg, 120.06 μmol, 1.00 equiv.) dissolved in DMF (3 mL), Pd(PPh3)4 (20.81 mg, 18.01 μmol, 0.15 equiv.) and phenylsilane (129.92 mg, 1.20 mmol, 148.14 μL, 10.00 equiv.) were added. The mixture was stirred at 20° C. for 1 h, and the resulting reaction was stirred at 0° C. for 5 min. After completion monitored by LC-MS. The mixture was acidified to pH=5 by 1 M HCl, then purified directly by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) followed by lyophilization to give compound 1294 (98.3 mg, 87.0% purity, 29.5% yield) as a yellow solid. LCMS: RT=2.02 min, MS calculated: M 平均 = 2414.65, observed mass: [2M+3H] 3+ =1610.6, [M+2Na] 2+ =1230.5, [M+H+Na] 2+ =1219.3, [M+2H] 2+ =1208.1, [M+3H] 3+ =805.8.

[0109] Example 5. Procedure for the preparation of compound 1295. [ka] [ka]

[0110] Preparation of intermediate 19: Peptides were synthesized using standard Fmoc chemistry (CTC resin).

[0111] Resin preparation: DIEA (4.00 equiv.) was added dropwise to a vessel containing CTC resin (0.50 g, 0.50 mmol, 1.00 mmol / g) and Fmoc-Thr(tBu)-OH (198.5 mg, 0.50 mmol, 1.00 equiv.) in DCM (5 mL) and mixed at 25° C. for 2 h with bubbling with N2. MeOH (0.5 mL) was then added and bubbled with N2 for another 30 min. The resin was washed with DMF (10 mL), followed by addition of 20% piperidine in DMF (10 mL) and bubbled with N2 for 30 min at 25° C. for Fmoc deprotection. The mixture was filtered and the resin was washed with DMF (10 mL) before proceeding to the next step.

[0112] Coupling: A solution of Fmoc-Cys(Trt)-OH (0.88 g, 1.5 mmol, 3.00 equiv.), HBTU (0.41 g, 1.43 mmol, 2.85 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. Then, DIEA (6.00 equiv.) was added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (20 mL).

[0113] Deprotection: 20% piperidine in DMF (10 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (10 mL).

[0114] Steps 2 and 3 were repeated for the following amino acid extensions: Nos. 3-9, Table 4.

[0115] Coupling: A solution of 2-(3-fluoro-4-hydroxyphenyl)acetic acid (253.5 mg, 1.50 mmol, 3.00 equiv.), HOBt (189.0 mg, 202.5 mg, 1.50 mmol, 3.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (10 mL).

[0116] Dde deprotection: 3% hydrazine hydrate in DMF (10 mL) was added to the resin while bubbling with N2 for 30 min. The deprotection reaction was then monitored by ninhydrin test, and the reaction was complete when it showed blue or brownish red color. The resin was then washed with DMF (10 mL).

[0117] Coupling: A solution of Fmoc-Trp-OH (639.0 mg, 1.50 mmol, 3.00 equiv.), HOBt (202.5 mg, 1.50 mmol, 3.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 30 min at 25°C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (10 mL).

[0118] Deprotection: 20% piperidine in DMF (10 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (10 mL).

[0119] Steps 7 and 8 were repeated for the following amino acid extensions: Nos. 11-14, Table 5.

[0120] Alloc-Cl coupling on the N-terminus: The resin was washed with DCM (20 mL). A solution of Allo-Cl (0.36 g, 3.0 mmol, 6.00 equiv.) in DCM (10 mL) was added to the resin while bubbling with N2. Then, DIEA (6.00 equiv.) was added dropwise to the mixture and bubbled with N2 for 30 min at 25°C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. Then, the resin was washed with DCM (50 mL), DMF (50 mL).

[0121] Coupling: A solution of BocHN-PEG6-CH2CH2COOH (700.0 mg, 1.50 mmol, 3.00 equiv.), HOBt (202.5 mg, 1.50 mmol, 3.00 equiv.), DMAP (61.0 mg, 0.50 mmol, 1.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 16 h at 25 °C. The coupling reaction was monitored by ninhydrin test and was complete when it showed no color. The resin was then washed with DMF (10 mL), MeOH (50 mL), and then dried under reduced pressure to give resin-bound peptide intermediate 17 (CTC resin, 1.30 g, 0.50 mmol). [Table 5]

[0122] Peptide cleavage and cyclization: Cleavage: A solution of TFA / TIS / H2O / 3-mercaptopropanoic acid (92.5 / 2.5 / 2.5 / 2.5, v / v / v, 30 mL) was added to the resin (Intermediate 17, 0.50 mmol) at room temperature and stirred for 2 h. After filtration, the filtrate was collected and precipitated with cold isopropyl ether (150 mL), then filtered off, the solid was washed twice with isopropyl ether (100 mL) and the crude peptide was dried under reduced pressure for 2 h to give Intermediate 18 (0.50 mmol, crude) as a white solid.

[0123] Cyclization: To the crude peptide (intermediate 18) in MeCN / H2O (1 / 1, v / v, 500 mL) was added 0.1 M I2 / AcOH dropwise until the yellow color persisted, and then the mixture was stirred at 25 °C for 5 min. The mixture was quenched by adding 0.1 M aqueous Na2S2O3 dropwise until the yellow color disappeared. After filtration, the filtrate was purified by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) and then lyophilized to give intermediate 19 (150.1 mg, 94.6% purity, 14.3% yield) as a white solid. LCMS: RT = 1.00 min, MS calculated: M 平均 = 2093.35, observed mass: [M+2H] 2+ =1048.2, [M+3H] 3+ =880.56. [ka]

[0124] Preparation of intermediate 20: [ka] To a mixture of intermediate 19 (150.1 mg, 71.7 μmol, 1.00 equiv) and FITC (41.9 mg, 107.55 μmol, 1.50 equiv) in DMF (2 mL) was added DIEA (27.7 mg, 37.5 μL, 515.1 μmol, 3.00 equiv) at 25° C. The mixture was stirred at 25° C. for 2 h. After completion monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give intermediate 20 (80.3 mg, 77.4% purity, 34.9% yield) as a yellow solid. LCMS: RT=1.06 min, MS calculated: M 平均 = 2482.73, observed mass: [2M+3H] 3+ =1655.79, [M+2H] 2+ =1242.10, [M+3H] 3+ =828.31, [M+4H] 4+ =621.72.

[0125] Preparation of compound 1295: [ka] To a mixture of intermediate 20 (80.30 mg, 32.30 μmol, 1.00 equiv.) dissolved in DMF (1 mL) was added Pd(PPh3)4 (5.60 mg, 4.84 μmol, 0.15 equiv.) and phenylsilane (35.0 mg, 323.0 μmol, 10.00 equiv.). The mixture was stirred at 20° C. for 1 h and the resulting reaction was stirred at 0° C. for 5 min. After completion monitored by LC-MS. The mixture was acidified to pH=5 by 1M HCl and then purified directly by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) followed by lyophilization to give compound 1295 (17.0 mg, 94.9% purity, 15.9% yield) as a yellow solid. LCMS: RT=2.02 min, MS calculated: M 平均 = 2398.65, observed mass: [2M+3H] 3+ =1599.8, [M+H+Na] 2+ =1211.6, [M+2H] 2+ =1200.1, [M+3H]3+ =800.4.

[0126] Example 6. Procedure for the preparation of compound 1293. [ka] [ka] Preparation of intermediate 23: [ka] Peptides were synthesized using standard Fmoc chemistry (CTC resin).

[0127] Resin preparation: DIEA (4.00 equiv.) was added dropwise to a vessel containing CTC resin (0.50 g, 0.50 mmol, 1.00 mmol / g) and Fmoc-Thr(tBu)-OH (198.5 mg, 0.50 mmol, 1.00 equiv.) in DCM (5 mL) and mixed at 25° C. for 2 h with bubbling with N2. MeOH (0.5 mL) was then added and bubbled with N2 for another 30 min. The resin was washed with DMF (10 mL), followed by addition of 20% piperidine in DMF (10 mL) and bubbled with N2 for 30 min at 25° C. for Fmoc deprotection. The mixture was filtered and the resin was washed with DMF (10 mL) before proceeding to the next step.

[0128] Coupling: A solution of Fmoc-Cys(Trt)-OH (0.88 g, 1.5 mmol, 3.00 equiv.), HBTU (0.41 g, 1.43 mmol, 2.85 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. Then, DIEA (6.00 equiv.) was added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (20 mL).

[0129] Deprotection: 20% piperidine in DMF (10 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (10 mL).

[0130] Steps 2 and 3 were repeated for the following amino acid extensions: Nos. 3-9, Table 6.

[0131] Coupling: A solution of 2-(3-fluoro-4-hydroxyphenyl)acetic acid (253.5 mg, 1.50 mmol, 3.00 equiv.), HOBt (189.0 mg, 202.5 mg, 1.50 mmol, 3.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 30 min at 25° C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (10 mL).

[0132] Dde deprotection: 3% hydrazine hydrate in DMF (10 mL) was added to the resin while bubbling with N2 for 30 min. The deprotection reaction was then monitored by ninhydrin test, and the reaction was complete when it showed blue or brownish red color. The resin was then washed with DMF (10 mL).

[0133] Coupling: A solution of Fmoc-Trp-OH (639.0 mg, 1.50 mmol, 3.00 equiv.), HOBt (202.5 mg, 1.50 mmol, 3.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 30 min at 25°C. The coupling reaction was monitored by ninhydrin test, and the coupling was complete when it showed no color. The resin was then washed with DMF (10 mL).

[0134] Deprotection: 20% piperidine in DMF (10 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 25° C. The deprotection reaction was monitored by ninhydrin test, and the reaction was complete when it showed a blue or brownish red color. The resin was then washed with DMF (10 mL).

[0135] Steps 7 and 8 were repeated for the following amino acid extensions: Nos. 11-14, Table 6.

[0136] Acetylation: A solution of Ac2O / NMM / DMF (2 / 1 / 17, v / v / v, 40 mL) was added to the resin and the mixture was bubbled with N2 for 20 min. The acetylation reaction was monitored by the ninhydrin test. The resin was then washed with DMF (20 mL).

[0137] Coupling: A solution of BocHN-PEG6-CH2CH2COOH (700.0 mg, 1.50 mmol, 3.00 equiv.), HOBt (202.5 mg, 1.50 mmol, 3.00 equiv.), DMAP (61.0 mg, 0.50 mmol, 1.00 equiv.) in DMF (5 mL) was added to the resin while bubbling with N2. DIC (1.50 mmol, 3.00 equiv.) was then added dropwise to the mixture and bubbled with N2 for 16 h at 25 °C. The coupling reaction was monitored by ninhydrin test and was complete when it showed no color. The resin was then washed with DMF (10 mL), MeOH (50 mL), and then dried under reduced pressure to give resin-bound peptide intermediate 21 (CTC resin, 1.35 g, 0.50 mmol). [Table 6]

[0138] Peptide cleavage and cyclization: Cleavage: A solution of TFA / TIS / H2O / 3-mercaptopropanoic acid (92.5 / 2.5 / 2.5 / 2.5, v / v / v, 30 mL) was added to the resin (Intermediate 21, 0.50 mmol) at room temperature and stirred for 2 h. After filtration, the filtrate was collected and precipitated with cold isopropyl ether (150 mL), then filtered off, the solid was washed twice with isopropyl ether (100 mL) and the crude peptide was dried under reduced pressure for 2 h to give Intermediate 22 (0.50 mmol, crude) as a white solid.

[0139] Cyclization: To the crude peptide (Intermediate 22) in MeCN / H2O (1 / 1, v / v, 500 mL) was added 0.1 M I2 / AcOH dropwise until the yellow color persisted, and then the mixture was stirred at 25 °C for 5 min. The mixture was quenched by adding 0.1 M aqueous Na2S2O3 dropwise until the yellow color disappeared. After filtration, the filtrate was purified by preparative HPLC (A: 0.075% TFA / H2O, B: MeCN) and then lyophilized to give Intermediate 23 (160.1 mg, 90.0% purity, 15.6% yield) as a white solid. LCMS: RT = 7.9 min, MS calculated: M 平均 = 2051.31, observed mass: [M+2H] 2+ =1026.60, [M+3H] 3+ =684.59. [ka]

[0140] Preparation of compound 1293: [ka] To a mixture of intermediate 23 (120.0 mg, 58.50 μmol, 1.00 equiv) and FITC (34.08 mg, 87.51 μmol, 1.50 equiv) in DMF (2 mL) was added DIEA (27.7 mg, 30.59 μL, 175.5 μmol, 3.00 equiv) at 25° C. The mixture was stirred at 25° C. for 2 h. After completion monitored by LC-MS, the mixture was acidified to pH=5 with 1 M HCl and then directly purified by preparative HPLC (A: 0.075% TFA / HO, B: MeCN) followed by lyophilization to give compound 1293 (33.8 mg, 91.4% purity, 23.7% yield) as a yellow solid. LCMS: RT=2.02 min, MS calculated: M 平均 = 2440.69, observed mass: [M+Na+H] 2+ =1232.00, [M+2H] 2+ =1221.60, [M+3H] 3+ =814.30.

Claims

1. Formula RI: LG-RG-L ＲＭ -MOI (R-I) [During the ceremony, LG is a group that comprises a target binding moiety that binds to a targeting agent; RG is a group represented by the formula -L ＬＧ２ -L ＬＧ３ -L ＬＧ４ -L ＲＧ１ -L ＲＧ２ -reactive group, L ＬＧ２ is -NH-C(O)OC(R') ２ -, each R' is independently H or C1-C10 alkyl, and R' may be connected to form a ring; L ＬＧ３ is an optionally substituted aryl ring, L ＬＧ４－ is —NH— or —O—, L ＲＧ１ -C(O)-, -S(O)-, -OS(O) ２ -, or -OP(O)(OR) ２ - and L ＲＧ２ - is a covalent bond or [-C(R") ２ C(R″)═C(R″)]C(O)—, each R″ is independently H or C1-C10 alkyl, and any two R″ may be joined to form a ring; L ＲＭ is the linker, MOI is the moiety of interest. A compound having the structure: or a salt thereof.

2. RG has the following formula: 【Chemistry 1】 is or comprises a reactive group having the formula wherein ARYL is a substituted or unsubstituted para-phenylene ring; The compound according to claim 1 or a salt thereof.

3. ARYL is, 【Chemistry 2】 wherein R ｓ represents independently at each occurrence a halogen, -NO ２ , -F, -LR', -C(O)-LR', -S(O)-LR', -S(O) ２ -L-R', and -P(O)(-L-R') ２ and R' is selected from H or C １ ~C ６ The compound or salt thereof according to claim 2, wherein the aryl group is alkyl.

4. The reactive group has the following formula: 【Chemistry 3】 3. The compound according to claim 2, which is or comprises one of:

5. RG has the following formula: 【Chemistry 4】 is or comprises a reactive group having the formula wherein ARYL is a substituted or unsubstituted para-phenylene ring; The compound according to claim 1 or a salt thereof.

6. ARYL is, 【Chemistry 5】 wherein R ｓ represents independently at each occurrence a halogen, -NO ２ , -F, -LR', -C(O)-LR', -S(O)-LR', -S(O) ２ -L-R', and -P(O)(-L-R') ２ and R' is selected from H or C １ ~C ６ is alkyl, The compound or salt thereof according to claim 5.

7. The reactive group has the following formula: 【Chemistry 6】 6. The compound according to claim 5, which is or comprises one of:

8. LG is R ＬＧ -L ＬＧ and R ＬＧ teeth, 【Chemistry 7】 R ｃ -(Xaa)z-, a nucleic acid moiety, or a small molecule moiety; each Xaa is independently a residue of an amino acid or amino acid analog; t is 0 to 50; z is 1 to 50; Each R ｃ are independently -L ａ -R', Each L ａ are independently a covalent bond or C １ ~C ２０ Aliphatic or C having 1 to 5 heteroatoms １ ~C ２０ an optionally substituted divalent group selected from heteroaliphatic, one or more methylene units of said group are independently selected from C(R') ２ -, -Cy-, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O) ２ -, -S(O) ２ may be replaced by -N(R')-, -C(O)S-, or -C(O)O-; Each -Cy- is independently an optionally substituted divalent monocyclic, bicyclic, or polycyclic group, and each monocyclic ring is independently ３～２０ alicyclic ring, C ６～２０ an aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; L ＬＧ Is, -L ＬＧ１ -, -L ＬＧ１ -L ＬＧ２ -, -L ＬＧ１ -L ＬＧ２ -L ＬＧ３ - or -L ＬＧ１ -L ＬＧ２ -L ＬＧ３ -L ＬＧ４ - and L ＬＧ１ , L ＬＧ２ , L ＬＧ３ , and L ＬＧ４ each independently represents a covalent bond or one or more aliphatic moieties, aryl moieties, heteroaliphatic moieties each independently having 1 to 20 heteroatoms, heteroaromatic moieties each independently having 1 to 20 heteroatoms, or any combination of any one or more of such moieties; １～１００ one or more methylene units of said group are independently １～６ Alkylene, C １～６ Alkenylene, divalent C having 1 to 5 heteroatoms １～６ Heteroaliphatic groups, -C≡C-, -Cy-, -C(R') ２ -, -O-, -S-, -S-S-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -C(O)C(R') ２ N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S(O)-, -S(O) ２ -, -S(O) ２ N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S )(SR')-, -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, amino acid residue, or -[(-OC(R') ２ -C(R') ２ -) ｎ ]- (wherein n is 1 to 20), Each R' is independently -R, -C(O)R, -CO ２ R or -SO ２ R, Each R is independently -H or C １～３０ Aliphatic, C with 1-10 heteroatoms １～３０ Heteroaliphatic, C ６～３０ Aryl, C ６～３０ Arylaliphatic, C with 1-10 heteroatoms ６～３０ an optionally substituted group selected from arylheteroaliphatic, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms; or The two R groups may independently be taken together to form a covalent bond, or two or more R groups on the same atom may independently, together with said atom, form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having, in addition to said atom, 0-10 heteroatoms; or two or more R groups on two or more atoms may independently be taken together with their intervening atoms to form an optionally substituted 3-30 membered monocyclic, bicyclic, or polycyclic ring having, in addition to said intervening atoms, 0-10 heteroatoms; The compound according to claim 1 or a salt thereof.

9. 2. The compound or salt thereof of claim 1, wherein LG is or comprises a target binding moiety that binds to a targeting agent, and the targeting agent is an antibody agent.

10. LG is or comprises a target binding moiety that binds to the Fc region, and / or ＬＧ The compound or salt thereof according to claim 1, wherein the amino acid sequence is or comprises DCAWXLGELVWCT (SEQ ID NO: 2), the two cysteine ​​residues may form a disulfide bond, and X is an amino acid residue.

11. The following conditions: (a) the moiety of interest is or comprises a therapeutic agent; (b) the moiety of interest is or comprises a moiety capable of binding to a protein, a nucleic acid, or a cell; and / or (c) the moiety of interest is or comprises a reactive moiety suitable for a bioorthogonal reaction; The compound or salt thereof according to claim 1, wherein at least one of the following is satisfied:

12. LG is a compound represented by the formulas A-1 to A-50: 【Chemistry 8】 【Chemical Formula 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemical Formula 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 2. The compound of claim 1, or a salt thereof, which is or comprises a target binding moiety having a structure selected from:

13. 2. The compound or salt thereof of claim 1, wherein the MOI is or comprises a therapeutic drug moiety and / or the MOI is or comprises an antibody drug.

14. L ＲＭ is one or more -[(CH ２ ) ｎ -O] ｍ -, each n is independently 1 to 20, and m is 1 to 100, or a salt thereof, of the compound according to claim 1 .

15. In formula (R-I), the targeting agent is an antibody comprising an IgG heavy chain comprising K246 or K248; The target binding moiety brings the reactive group into close proximity with K246 or K248 of the IgG heavy chain to allow a reaction between K246 or K248 and the reactive group, and ＲＭ 2. The compound or salt thereof of claim 1, configured to bind to the antibody in a manner that results in attachment of a moiety that comprises an -MOI and removal of a group that contains a target binding moiety from the compound or salt thereof.

16. P-I: P-L ＰＭ -MOI (P-I) [During the ceremony, P is a targeting drug moiety; L ＰＭ is the linker, MOI is the moiety of interest. or a salt thereof, comprising the steps of: 1) A compound of formula R-I: LG-RG-L ＲＭ -MOI (R-I) [During the ceremony, LG is a group that comprises a target binding moiety that binds to a targeting agent; RG is a group represented by the formula -L ＬＧ２ -L ＬＧ３ -L ＬＧ４ -L ＲＧ１ -L ＲＧ２ -reactive group, L ＬＧ２ is -NH-C(O)OC(R') ２ -, each R' is independently H or C1-C10 alkyl, and R' may be connected to form a ring; L ＬＧ３ is an optionally substituted aryl ring, L ＬＧ４－ is —NH— or —O—, L ＲＧ１ -C(O)-, -S(O)-, -OS(O) ２ -, or -OP(O)(OR) ２ - and L ＲＧ２ - is a covalent bond or [-C(R") ２ C(R″)═C(R″)]C(O)—, each R″ is independently H or C1-C10 alkyl, and any two R″ may be joined to form a ring; L ＲＭ is the linker, MOI is the moiety of interest. or a salt thereof; 2) forming a compound having the structure P-I or a salt thereof; A method comprising: or P-II: P-N-L ＰＭ -MOI (P-II) [During the ceremony, P-N is a protein drug moiety containing a lysine residue; L ＰＭ is the linker, MOI is the moiety of interest. or a salt thereof, comprising the steps of: P-N and a compound of formula R-I: LG-RG-L ＲＭ -MOI (R-I) [During the ceremony, LG is a group that contains a protein binding moiety that is attached to P-N; RG is a group represented by the formula -L ＬＧ２ -L ＬＧ３ -L ＬＧ４ -L ＲＧ１ -L ＲＧ２ -reactive group, L ＬＧ２ is -NH-C(O)OC(R') ２ -, each R' is independently H or C1-C10 alkyl, and R' may be connected to form a ring; L ＬＧ３ is an optionally substituted aryl ring, L ＬＧ４－ is —NH— or —O—, L ＲＧ１ -C(O)-, -S(O)-, -OS(O) ２ -, or -OP(O)(OR) ２ - and L ＲＧ２ - is a covalent bond or [-C(R") ２ C(R″)═C(R″)]C(O)—, each R″ is independently H or C1-C10 alkyl, and any two R″ may be joined to form a ring; L ＲＭ is the linker, MOI is the moiety of interest. or a salt thereof with a reaction partner having the structure:

17. 17. The method of claim 16, wherein the targeting agent is or comprises an antibody agent.

18. 18. The method of claim 17, wherein the moiety of interest selectively binds to the antibody drug at K246 or K248 of the IgG1 heavy chain, or a corresponding position.

19. 18. The method of claim 17, wherein the moiety of interest selectively binds to the antibody drug at K251 or K253 of the IgG2 heavy chain, or a corresponding position.

20. 18. The method of claim 17, wherein the moiety of interest selectively binds to the antibody drug at K239 or K241 of the IgG4 heavy chain, or a corresponding position.

21. 20. The method of claim 16, wherein the contacting and forming steps occur in one chemical reaction.

22. A composition comprising one or more compounds or salts thereof according to any one of claims 1 to 15.

23. 1. A composition comprising: Formula (P-II): P-N-L ＰＭ (a) [During the ceremony, P-N is a protein drug moiety containing a lysine residue; L ＰＭ is the linker, MOI is the part of interest a first compound having the structure of 〕 Structure: LG-OH (LG-I) 〔wherein LG is a group containing a target binding moiety that binds to the target agent〕 a second compound having 〕 A composition comprising 〕

24. Formula (R-I): LG-RG-L ＲＭ (a) 〔wherein LG is a group containing a target binding moiety that binds to the target agent and is the same as LG in formula (LG-I), RG is a group represented by the formula -L ＬＧ２ -L ＬＧ３ -L ＬＧ４ -L ＲＧ１ -L ＲＧ２ -reactive group, L ＬＧ２ is -NH-C(O)OC(R') ２ -, each R' is independently H or C1-C10 alkyl, and R' may be connected to form a ring; L ＬＧ３ is an optionally substituted aryl ring, L ＬＧ４－ is —NH— or —O—, L ＲＧ１ -C(O)-, -S(O)-, -OS(O) ２ -, or -OP(O)(OR) ２ - and L ＲＧ２ - is a covalent bond or [-C(R") ２ C(R″)═C(R″)]C(O)—, each R″ is independently H or C1-C10 alkyl, and any two R″ may be joined to form a ring; L ＲＭ is a linker and is the same as L RM in formula (P-II), MOI is the part of interest〕 a third compound having 〕 Formula (R-III): HO-RG-L ＲＭ -MOI(R---I) a fourth compound having 〕 or a combination thereof The composition according to claim 23, further comprising 〕

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