Tetrazine-based targeting agents for in vivo delivery of payloads

Abstract

This disclosure generally relates to tetrazine-based targeting agents for bioorthogonal delivery of payloads to targeted sites in a subject, which have applications, for example, in the treatment of cancer, tumor growth, and immunotherapy.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 63 / 500,246, filed on 4 May 2024 under 35 U.S. Code § 119(e), and U.S. Provisional Application No. 63 / 536,292, filed on 1 September 2023, each of which is incorporated herein by reference in whole.

[0002] This disclosure generally relates to tetrazine-based targeting agents for bioorthogonal delivery of payloads to targeted sites in a subject, which have applications, for example, in the treatment of cancer, tumor growth, and immunotherapy. [Background technology]

[0003] Bioorthogonal conjugations, or click reactions, are selective and orthogonal (non-interacting) functions found in biological systems that have found use in a variety of applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine. They can be used to facilitate the selective manipulation of molecules, cells, particles, and surfaces, as well as the tagging and tracking of biomolecules in vitro and in vivo. These reactions include Staudinger ligation, azido-cyclooctin ring addition, and reverse electron-demanded Diels-Alder reactions. [Overview of the project]

[0004] Tetrazine-based targeting agents are provided herein, optionally comprising one or more targeting moieties covalently conjugated to one or more tetrazine moieties via a linker, provided that at least one of the targeting moieties is a non-antibody or non-antibody fragment targeting agent selected from nectin-4 peptide targeting agents, PSMA peptide targeting agents, CCK2R peptide targeting agents, PD-L1 peptide targeting agents, integrin alpha-V beta-3 targeting agents, integrin alpha-V beta-6 targeting agents, integrin alpha-V beta-8 targeting agents, FAP targeting agents, CEACAM5 (also known as CEA) targeting agents, and carbonic anhydrase IX (CAIX) targeting agents.

[0005] The tetrazine-based targeting agents described herein are designed to localize to a target site within a subject upon administration. The tetrazine-based targeting agents can be administered topically or systemically. Upon administration, a prodrug comprising a payload or therapeutic agent and one or more complementary bioorthogonal components (i.e., trans-cyclooctene moieties) can be administered, which, upon contact with the tetrazine-based targeting agent in vivo, enables targeted delivery of the payload or therapeutic agent. In some embodiments, the tetrazine-based targeting agent comprises a therapeutic targeting agent.

[0006] In some embodiments, the tetrazine-based targeting agents described herein include a diagnostic agent so that the tetrazine-based targeting agents described herein can be used for the diagnosis of a condition or disease, whether or not a payload or therapeutic agent is administered.

[0007] In some embodiments, a method for treating cancer is provided, comprising administering a tetrazine-based targeting agent described herein to a subject in need thereof, and administering a conjugate, or a pharmaceutically acceptable salt or composition thereof, to the subject thereof.

[0008] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal cancer / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

[0009] In some embodiments, cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, cancer is a solid tumor. In some embodiments, cancer is lymphoma or leukemia. In some embodiments, cancer is a hematological malignancy. [Brief explanation of the drawing]

[0010] [Figure 1A] Compound 111 binds to both the human (A) and mouse (B) isoforms of recombinant nectin-4 in ligand-binding assays, while compound 111-C does not bind in either case. [Figure 1B] Compound 111 binds to both the human (A) and mouse (B) isoforms of recombinant nectin-4 in ligand-binding assays, while compound 111-C does not bind in either case. [Figure 2] This shows that compounds B and F are converted to MMAE and exatecan, respectively, after contact with compound 111. [Figure 3] This shows the tumor response of compound 111 + compound B in NCI-H292 tumors in BALB / c nude mice. [Modes for carrying out the invention]

[0011] The following description illustrates exemplary embodiments of the Technology. However, such descriptions should be understood not as limiting the scope of the Disclosure, but rather as being provided solely for illustrative purposes.

[0012] 1.Definition For clarity, certain features of the Disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any preferred partial combination. All combinations of embodiments belonging to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if each and all combinations were individually and expressly disclosed to the extent that such combinations encompass subject matter that is, for example, a stable compound (i.e., a compound that can be prepared, isolated, characterized, and tested for biological activity). Furthermore, all partial combinations of various embodiments and their elements (e.g., elements of chemical groups enumerated in embodiments describing such variables) are also specifically encompassed by the Disclosure and are disclosed herein as if each and all such partial combinations were individually and expressly disclosed herein.

[0013] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In case of any conflict, this specification, including its definitions, shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0014] The terms “comprise(s),” “include(s),” “have,” “possess,” “can have,” and “contain,” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include multiple references unless the context otherwise explicitly indicates. This disclosure also contemplates other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0015] The modifier "approximately" used in relation to quantity includes the stated value and has a meaning determined by the context (for example, at least the degree of error associated with measuring a particular quantity). The modifier "approximately" should also be considered to disclose a range defined by the absolute values ​​of two endpoints. For example, the expression "approximately 2 to approximately 4" also discloses a range of "2 to 4". The term "approximately" can refer to plus or minus 10% of the stated number. For example, "approximately 10%" may indicate a range of 9% to 11%, and "approximately 1" may mean 0.9 to 1.1. Other meanings of "approximately" may become apparent from contexts such as rounding, so for example, "approximately 1" may also mean 0.5 to 1.4.

[0016] The conjunction “or” includes any and all combinations of the one or more enumerated elements associated by the conjunction. For example, the phrase “a device containing A or B” could mean a device containing A without B, a device containing B without A, or a device containing both A and B. The phrases “at least one of A, B, ... and N” or “at least one of A, B, ... N, or any combination thereof” are defined in the broadest sense to mean one or more elements selected from the group containing A, B, and N, i.e., any combination of one or more elements A, B, ... or N that includes any one element alone or in combination with one or more of the other elements, and may also include additional elements not enumerated.

[0017] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are defined as follows: Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Identified according to the ed. and inside cover, specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional parts and reactivity, are referred to in *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999, and *Smith and March March's Advanced Organic Chemistry*, 5 th Edition, John Wiley&Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modern Methods of Organic Synthesis, 3 rdThis information is contained in Edition, Cambridge University Press, Cambridge, 1987, and the entirety of each of its contents is incorporated herein by reference.

[0018] As used herein, the term "alkyl" means a straight or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. 1- The term "C6-alkyl" refers to a linear or branched hydrocarbon containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" refers to a linear or branched hydrocarbon containing 1 to 3 carbon atoms. Typical examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0019] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to the parent molecule via an oxygen atom. Typical examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0020] As used herein, the term "alkenyl" means a hydrocarbon chain containing 2 to 30 carbon atoms having at least one carbon-carbon double bond. The alkenyl group may be substituted or unsubstituted. For example, the alkenyl group may be substituted with an aryl group such as phenyl.

[0021] As used herein, the term "alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 30 carbon atoms, e.g., 2 to 20 or 2 to 10 carbon atoms, and having at least one triple-unsaturated site. The term "alkyne" also includes non-aromatic cycloalkyl groups having 5 to 20 carbon atoms, e.g., 5 to 10 carbon atoms, and having one or more rings and at least one triple bond. Examples of such alkynyl groups include, but are not limited to, acetylenyl (-C≡CH) and propargyl (-CH2C≡CH), and cycloalkynyl moieties such as substituted or unsubstituted cyclooctin moieties.

[0022] When used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein, attached to the parent molecule via an alkyl group as defined herein.

[0023] As used herein, the term "alkylene" refers to a divalent group derived from a linear or branched hydrocarbon having 1 to 30 carbon atoms, for example, 2 to 10 carbon atoms. Typical examples of alkylenes include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH(CH3)CH2-, -C(CH3)2CH2-, -CH2CH2CH2-, -CH(CH3)CH2CH2-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0024] The term "amino acid" refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, and amino acid analogs and mimics that function in a similar manner to naturally occurring amino acids. Naturally encoded amino acids include 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolidine and selenocysteine. Unnatural amino acids refer to amino acid analogs that have the same basic chemical structure as naturally occurring amino acids, i.e., α-carbons bonded to hydrogen, carboxyl groups, amino groups, and R groups, for example only. Such analogs may have modified R groups (e.g., norleucine, for example) or may retain a modified peptide skeleton while retaining the same basic chemical structure as natural amino acids. Non-exclusive examples of unnatural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formylglycine, phenylglycine, para-azidophenylglycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(1,2,4,5-tetrazine))-phenylglycine, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.

[0025] The term "aryl," as used herein, refers to an aromatic carbocyclic group having a single ring (e.g., monocyclic) or multiple rings (e.g., bicyclic or tricyclic) including a condensed system. Typical examples of aryls include, but are not limited to, phenyl, naphthyl, and anthracenyl. Monocyclic, bicyclic, and tricyclic aryls are connected to the parent molecule via any carbon atoms contained within the ring and can be unsubstituted or substituted. Aromatic bicyclic or aromatic tricyclic systems do not contain a non-aromatic ring. Therefore, if a bicyclic or tricyclic system contains a non-aromatic ring, the system is cycloalkyl or heterocyclyl, depending on whether the heteroatom is present on the non-aromatic ring, regardless of the bonding site to the rest of the molecule.

[0026] In some embodiments, the term “aryl” as used herein refers to a phenyl group, or a bicyclic aryl or tricyclic aryl fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to the parent molecule and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to the parent molecule and fused to two other phenyl groups. Typical examples of bicyclic aryls include, but are not limited to, naphthyl. Typical examples of tricyclic aryls include, but are not limited to, anthracenyl. Monocyclic, bicyclic, and tricyclic aryls can be attached to the parent molecule via any carbon atoms contained within the ring and can be unsubstituted or substituted.

[0027] As used herein, the term "azide" refers to the functional group -N3.

[0028] As used herein, the term "cycloalkyl" refers to a non-aromatic carbocyclic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10 carbon atoms and zero heteroatoms. A cycloalkyl ring system may contain one or more double bonds, provided the ring is not aromatic; therefore, the term cycloalkyl includes cycloalkenyl ring systems. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl. "Cycloalkyl" also includes carbocyclic ring systems in which the cycloalkyl group is condensed to an aryl or heteroaryl as defined herein, regardless of its bonding site to the rest of the molecule.

[0029] In some embodiments, the term “cycloalkyl” refers, as used herein, to a carbocyclic ring system containing 3 to 10 carbon atoms, zero heteroatoms, and zero double bonds. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. “Cycloalkyl” also includes carbocyclic ring systems in which a cycloalkyl group is added to a parent molecule and condensed into an aryl group, a heteroaryl group, or a heterocycle as defined herein.

[0030] As used herein, the term "cycloalkenyl" means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Examples of monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0031] As used herein, the term "cyclooctene" refers to a substituted or unsubstituted non-aromatic cyclic alkyl group comprising eight carbon atoms and having a single ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0032] As used herein, the term "fluoroalkyl" means an alkyl group as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by fluorine. Typical examples of fluoroalkyls include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, for example, 3,3,3-trifluoropropyl.

[0033] When used herein, the term "alkoxyfluoroalkyl" refers to an alkoxy group as defined herein that is attached to the parent molecule via a fluoroalkyl group, as defined herein.

[0034] As used herein, the term "fluoroalkoxy" means at least one fluoroalkyl group as defined herein, attached to the parent molecule via an oxygen atom. Typical examples of fluoroalkyloxys include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0035] The terms "halogen" or "halo" as used herein mean Cl, Br, I, or F.

[0036] As used herein, the term "haloalkyl" means an alkyl group as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogens.

[0037] When used herein, the term "haloalkoxy" means at least one haloalkyl group as defined herein, which is attached to the parent molecule via an oxygen atom.

[0038] As used herein, the term "heteroalkyl" means an alkyl group as defined herein, in which one or more carbon atoms are replaced by heteroatoms selected from S, Si, O, P, and N. Heteroatoms can be oxidized. Typical examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkylamines, and alkyl sulfides.

[0039] As used herein, the term "heteroaryl" refers to an aromatic group having a monocyclic, polycyclic, or multiple fused ring, where one or more ring heteroatoms are independently selected from nitrogen, oxygen, and sulfur. In some embodiments, as used herein, the term "heteroaryl" refers to an aromatic monocyclic ring, an aromatic bicyclic ring system, or an aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridine-2-yl, pyridine-3-yl, and pyridine-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryls include, but are not limited to, clomenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranil, indolyl, quinolinyl, imidazopyridine, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranil and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are linked to the parent molecule via any carbon or nitrogen atom contained within the ring and can be unsubstituted or substituted. In some embodiments, aromatic bicyclic or aromatic tricyclic ring systems do not contain a non-aromatic ring. Therefore, if a bicyclic or tricyclic ring system contains a non-aromatic ring, the ring system is cycloalkyl or heterocyclyl, depending on whether the heteroatom is present in the non-aromatic ring, regardless of the bonding site to the rest of the molecule.

[0040] In some embodiments, a five-membered aromatic monocyclic ring has two double bonds, and a six-membered aromatic monocyclic ring has three double bonds. In some embodiments, exemplary bicyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings fused to a parent molecule and a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein. In some embodiments, tricyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings fused to two of a parent molecule and a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle as defined herein.

[0041] As used herein, the terms “heterocyclyl,” “heterocycle,” or “heterocyclic” refer to a non-aromatic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10 carbon atoms, at least one heteroatom (e.g., 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1), and optionally one or more oxo and / or double bonds. The terms “heterocyclyl,” “heterocycle,” or “heterocyclic” include monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridging ring systems, provided that at least one non-aromatic ring system containing at least one heteroatom is present. In some embodiments, a monocyclic heterocycle is a 3, 4, 5, 6, 7, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. In some embodiments, the 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. In some embodiments, the 7- and 8-membered rings contain zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S.Typical examples of monocyclic heterocycles include azetidinyl, azepanyl, azilidinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, and oxetanyl. Examples include, but are not limited to, piperazinil, piperidinil, pyranil, pyrazolinil, pyrazolidinil, pyrrolinil, pyrrolidinil, tetrahydrofuranil, tetrahydropyranil, tetrahydropyridinil, tetrahydrothienyl, thiadiazolinil, thiadiazolidinil, 1,2-thiadinil, 1,3-thiadinil, thiazolinil, thiazolidinil, thiomorpholinil, 1,1-dioxidethiomorpholinil (thiomorpholine sulfone), thiopyranil, and trithianil. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl group, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiroheterocyclic group, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hepta-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl.Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl group, or bicyclic heterocycles fused to a monocyclic cycloalkenyl group, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of a bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, and aza-adamantane(1-azatricyclo[3.3.1.1). 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 Examples include, but are not limited to, decanes. Monocyclic, bicyclic, and tricyclic heterocyclic rings are connected to the parent molecule via any carbon or nitrogen atoms contained within the ring and can be unsubstituted or substituted.

[0042] As used herein, the term "hydroxyl" means the -OH group.

[0043] As used herein, the term "hydroxyalkyl" means an alkyl group as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by a hydroxyl group.

[0044] The term "substituted" refers to a group that can be further substituted with one or more nonhydrogen substituent groups. Examples of substituent groups include, but are not limited to, halogens, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketones, amides, carbamates, and acyls.

[0045] The term "tetrazine" refers to a substituted or unsubstituted aromatic cyclic group having two carbon atoms and four nitrogen atoms, with a single ring having three double bonds. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2,4,5-tetrazine is referred to as the "Tz" group. The term "tetrazine-based" generally refers to a moiety that can be converted to tetrazine as defined herein, or to tetrazine such as optionally substituted dihydrotetrazine.

[0046] The term “selective delivery” refers to the delivery of a drug (e.g., payload) to an organ or tissue (or part thereof) requiring treatment or diagnosis without significant binding to other non-target organs or tissues (or parts thereof). In some embodiments, the tetrazine-based targeting agents described herein do not have a therapeutic effect themselves, but rather are designed to enable the selective or targeted delivery of a therapeutic agent. However, tetrazine-based targeting agents may have a therapeutic effect, and such constructs are therefore not excluded by this disclosure.

[0047] The term "payload" refers to an agent for delivery to a target site in a subject. In some embodiments, the payload includes a therapeutic agent or a diagnostic agent.

[0048] The term "therapeutic agent" refers to an agent capable of treating and / or ameliorating a condition or disease of a subject, or one or more symptoms thereof. The therapeutic agents of the present disclosure also include prodrug forms of therapeutic agents.

[0049] The term "diagnostic agent" refers to an agent that aids in the diagnosis of a condition or disease. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, and radionuclides. A paramagnetic agent is an imaging agent that is magnetic under an externally applied magnetic field. Examples of paramagnetic agents include, but are not limited to, iron particles including iron nanoparticles and iron microparticles. An optical probe is a fluorescent compound that can be detected by excitation with radiation of one wavelength and detection with radiation of a different second wavelength. Examples of optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. A radionuclide is an element that undergoes detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include 3 H, 11 C, 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 89 Zr, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu, and 241 Examples include Am, but are not limited to these.

[0050] The term “targeting agent” refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue) and thereby forms a stable association between the targeting agent and the specific target. “Stable association” means that a compound covalently, noncovalently, or otherwise associates with another part or structure, for example, under standard physiological conditions. Stable associations, or bindings, may include covalent and noncovalent interactions, such as, but are not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), and dipole-dipole interactions. Examples of targeting agents include ligands that specifically (or substantially specifically) bind to a particular clinically relevant target receptor or cell surface target. Specific targeting agents are described herein.

[0051] The term "targeted organ or tissue" refers to an organ or tissue that is targeted for payload delivery. Typical organs and tissues for targeting include those that can be targeted by chemical or biological targeting agents, as well as those that cannot be targeted by chemical or biological targeting agents.

[0052] The term "transplantation" refers to the surgical insertion of a target into the body.

[0053] The term "contact" or "to come into contact" refers to the process of bringing at least two different species into contact so that they can interact with each other, such as in non-covalent or covalent interactions or bonding reactions. However, it should be understood that the resulting complex or reaction product can be formed directly from interactions or reactions between added reagents, or from intermediates from one or more of the added reagents or parts that can be formed in the contact mixture.

[0054] The term "binder" refers to a drug having a functional group capable of forming a covalent bond with a complementary functional group of another binder in a biological environment. The bonding between binders in a biological environment may also be referred to as bioconjugation. Examples of binders include bioorthogonal binders, which are binders having bioorthogonal functional groups. The bioorthogonal functional groups of a bioorthogonal binder selectively react with complementary bioorthogonal functional groups of another bioorthogonal bonding partner. Selective reactions between bioorthogonal bonding partners minimize side reactions with other binders, biological compounds, or other non-complementary bioorthogonal binders or non-complementary bioorthogonal functional groups. Examples of bioorthogonal moieties or functional groups of bioorthogonal binders include, but are not limited to, azides and alkynes, trans-cyclooctene (TCO) and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine) for forming triazoles via click reactions, and others. Binders useful in this disclosure may have high reactivity with the corresponding binder so that the reaction is rapid.

[0055] The term "functionalized" refers to a part that has a functional group attached to it, for example, a part that has a binder functional group attached to it (e.g., a bioorthoth functional group).

[0056] The term “administering” refers to any preferred route of administration to the subject, including, but not limited to, oral administration, suppository administration, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal administration, or subcutaneous administration, subarachnoid administration, or implantation of a sustained-release device to the subject, such as a mini osmotic pump.

[0057] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0058] The term "leaving group" refers to an electron-withdrawing atom (or group of atoms) that can replace a bonded electron with a stable species. Suitable examples of leaving groups include halides (e.g., Br, Cl, I), sulfonic acid esters (e.g., triflates, mesylates, tosylates, and brosylates), and nitrophenols.

[0059] The terms “pharmaceutical effective dose” and “therapeutic effective dose” refer to the amount of a compound sufficient to treat one or more of a particular disorder or disease or its symptoms, and / or to prevent or reduce the risk of the onset or recurrence of the disease or disorder or its symptoms. With respect to oncogenic proliferative disorders, the pharmaceutical or therapeutic effective dose includes, among other things, the amount sufficient to shrink the tumor or reduce the rate of tumor growth.

[0060] As used herein, the terms “subject,” “patient,” or “organism” include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which the drugs of this disclosure may be administered may include mammals, in particular primates, and especially humans. For veterinary use, suitable subjects may include, for example, livestock such as cattle, sheep, goats, and pigs; poultry such as chickens, ducks, geese, and turkeys; and domesticated animals, in particular pets such as dogs and cats. For diagnostic or research use, suitable subjects may include mammals such as rodents (e.g., mice, rats, and hamsters), rabbits, primates, and pigs such as inbred pigs.

[0061] When used herein, “to treat” or “treatment” means treating or treating a disease or medical condition or symptom(s) in a patient, such as a mammal (in particular, a human), which includes (a) improving the disease or medical condition or symptom(s) in a patient, for example, eliminating or causing regression of the disease or medical condition or symptom(s) in a patient; (b) suppressing the disease or medical condition or symptom(s) in a patient, for example, by delaying or stopping the onset of the disease or medical condition or symptom(s) in a patient; or (c) reducing the symptoms of the disease or medical condition or symptom(s) in a patient.

[0062] The term "physiological conditions" means that these conditions are suitable for living cells, including, for example, aqueous conditions such as temperature, pH, and salinity, which are primarily suitable for living cells.

[0063] For the compounds described herein, the groups and substituents may be selected according to the allowable valencies of the atoms and substituents, and the selection and substitution result in stable compounds that do not undergo spontaneous transformations such as rearrangement, cyclization, or elimination.

[0064] Where a numerical range is indicated, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of that range (up to one-tenth of the lower limit), as well as any other values ​​or intermediate values ​​indicated within that described range, should be understood to be included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also included in this disclosure, subject to any specifically excluded limits within the described range. If the described range includes one or both limit values, the range excluding one or both of those limit values ​​is also included in this disclosure.

[0065] For the purposes of enumerating numerical ranges as defined herein, each number intervening between them is explicitly intended with the same degree of precision. For example, for the range 6–9, the digits 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the digits 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0066] Compounds may exist as stereoisomers containing an asymmetric or chiral center. Stereoiomers are designated "R" or "S" depending on the arrangement of substituents around the chiral carbon atom. As used herein, the terms "R" and "S" refer to the arrangements defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45:13-30. This disclosure envisions various stereoisomers and mixtures thereof, which are specifically included within the scope of this disclosure. Examples of stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds may be prepared synthetically from commercially available starting materials containing an asymmetric or chiral center, or by the preparation of racemic mixtures followed by separation methods well known to those skilled in the art. These separation methods include (1) attachment of the enantiomer mixture to a chiral auxiliary, separation of the diastereomer mixture obtained by recrystallization or chromatography, and optional release of optically pure products from the auxiliary (Furniss, Hannaford, Smith, and Tatchell, “Vogel’s Textbook of Practical Organic Chemistry,” 5 th As exemplified by (1989 edition, Longman Scientific & Technical, Essex CM20 2JE, England), or (2) direct separation of a mixture of optical enantiomers on a chiral chromatography column, or (3) fractional recrystallization methods.

[0067] It should be understood that the compound may have tautomers and geometric isomers, and these also constitute aspects of this disclosure.

[0068] This disclosure also includes isotope-labeled compounds that are identical to those enumerated herein, but are not identical in that one or more atoms are replaced by atoms having atomic masses or mass numbers different from those normally found in nature. Examples of isotopes suitable for inclusion in the compounds of this disclosure include, but are not limited to, those listed above. 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 These include hydrogen (such as Cl), carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine. Deuterium, that is, 2 Substitution with heavier isotopes such as 1H can offer certain therapeutic benefits resulting from higher metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and may therefore be preferable in some situations. The compound may also incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated are: 11 C, 13 N, 15 O, and 18 F is the isotope-labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the appended examples, using appropriate isotope-labeled reagents instead of non-isotope-labeled reagents.

[0069] B. Tetrazine-based targeting agents The tetrazine-based targeting agents described herein are designed to localize to a target site within a subject upon administration. The tetrazine-based targeting agents can be administered topically or systemically. Upon administration, a prodrug containing a complementary bioorthogonal component (e.g., a payload or therapeutic agent having a trans-cyclooctene moiety) can be administered, which, upon contact with the tetrazine-based targeting agent in vivo, enables targeted drug delivery of the payload or therapeutic agent. In some embodiments, the tetrazine-based targeting agents described herein include a diagnostic agent, so that the tetrazine-based targeting agents described herein can be used for the diagnosis of a condition or disease, regardless of whether a payload or therapeutic agent is administered.

[0070] Tetrazine-based targeting agents are provided herein, optionally comprising one or more targeting moieties covalently conjugated to one or more tetrazine moieties via a linker, provided that at least one targeting moiety is a non-antibody or non-antibody fragment targeting agent selected from nectin-4 peptide targeting agents, PSMA peptide targeting agents, CCK2R peptide targeting agents, PD-L1 peptide targeting agents, integrin alpha-V beta-3 targeting agents, integrin alpha-V beta-6 targeting agents, integrin alpha-V beta-8 targeting agents, FAP targeting agents, CEACAM5 (also known as CEA) targeting agents, and carbonic anhydrase IX (CAIX) targeting agents. In some embodiments, at least one targeting moiety is selected from Table 1.

[0071] In some embodiments, each tetrazine portion is independently of formula I, [ka] During the ceremony, R in each appearance 1These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR Selected from the group consisting of 'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected from 1 to 3 Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4 Each of these is a group that can be removed independently after hydrogen or a trigger event. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 This represents an optional double bond that forms dihydrotetrazine when both are present. Y in each appearance 1 These are independently, directly bonded, O, S, NR a , or CR 31a R 31b NR a Selected from the group consisting of, Y in each appearance 2Each R is independently a directly bonded alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently and optionally has 1 to 4 R 21 It has been replaced with, R in each appearance a , R 31a , and R 31b These are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl, R in each appearance 21 These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, each Z 1These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. In each occurrence, R''' is independently selected from alkyl and aryl groups.

[0072] In some embodiments, R 3 and R 4 Both are absent, and the dotted line represents an optional double bond that forms tetrazine.

[0073] In some embodiments, R 3 and R 4 Each of these groups can be removed independently after a hydrogen or trigger event, and the dotted line represents an optional double bond that forms dihydrotetrazine.

[0074] Several embodiments, R in each occurrence 2 Each is independently a cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently, optionally, 1 to 3 Z 1 It has been replaced with.

[0075] In some embodiments, each tetrazine portion is independently of the formula II, [ka] During the ceremony, R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR Selected from the group consisting of 'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected from 1 to 3 Z 1 It has been replaced with, R in each appearance 21These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, In each occurrence, ring A is independently a cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each cycloalkylene, heterocyclylene, arylene, or heteroarylene independently has 1 to 3 Z of any choice. 1 It has been replaced with, In each occurrence, t is independently 0, 1, 2, 3, or 4. each Z 1These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. In each occurrence, R''' is independently selected from alkyl and aryl groups.

[0076] In some embodiments, the linker is a branch.

[0077] In some embodiments, the tetrazine-based targeting agent comprises two or more targeting moieties.

[0078] In some embodiments, the tetrazine-based targeting agent comprises two or more tetrazine moieties.

[0079] In some embodiments, the linker is a straight chain.

[0080] It should be understood that linkers can be branched or linear, and independently, they can be one or more (e.g., 1 to 16) linkers covalently bonded to the targeting moiety. Furthermore, two or more targeting moieties can be covalently bonded via branched linkers.

[0081] In some embodiments, a tetrazine-based targeting agent of Formula IX is provided,

Chemical formula

[0082] In some embodiments, a tetrazine-based targeting agent of formula IX is provided, where X is a targeting agent selected from Table 1.

[0083] In some embodiments, a tetrazine-based targeting agent of formula III is provided. [ka] During the ceremony, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. L is a linker, p is between 1 and 16. R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR Selected from the group consisting of 'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected from 1 to 3 Z1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4 Each of these is a group that can be removed independently after hydrogen or a trigger event. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 This represents an optional double bond that forms dihydrotetrazine when both are present. Y in each appearance 1 These are independently, directly bonded, O, S, NR a , or CR 31a R 31b NR a Selected from the group consisting of, Y in each appearance 2 Each R is independently a directly bonded alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently and optionally has 1 to 4 R 21 It has been replaced with, R in each appearance a , R 31a , and R 31b These are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl, R in each appearance 21These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. In each occurrence, R''' is independently selected from alkyl and aryl groups.

[0084] In some embodiments, a tetrazine-based targeting agent of formula III is provided. [ka] During the ceremony, X is a targeting agent selected from Table 1, L is a linker, p is between 1 and 16. R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR Selected from the group consisting of 'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected from 1 to 3 Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4 Each of these is a group that can be removed independently after hydrogen or a trigger event. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4This represents an optional double bond that forms dihydrotetrazine when both are present. Y in each appearance 1 These are independently, directly bonded, O, S, NR a , or CR 31a R 31b NR a Selected from the group consisting of, Y in each appearance 2 Each R is independently a directly bonded alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently and optionally has 1 to 4 R 21 It has been replaced with, R in each appearance a , R 31a , and R 31b These are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl, R in each appearance 21 These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, each Z 1These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. In each occurrence, R''' is independently selected from alkyl and aryl groups.

[0085] In some embodiments, tetrazine-based targeting agents of formula IA, formula IIA, or formula VA are provided. [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 This represents an additional bond that forms dihydrotetrazine when both are present. X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl may optionally have 1 to 3 Z 1 It has been replaced with, R in each appearance 2These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4 Each of these is a group that can be removed independently after hydrogen or a trigger event. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 22It is independently and optionally comprises a linker of 1 to 100 linked atoms containing one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups. R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0086] In some embodiments, part: [ka] teeth, [ka] [ka] It is represented by [this].

[0087] In some embodiments, the tetrazine portion [ka] It is represented by [this].

[0088] Several embodiments, R in each occurrence 1 Each is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, and each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with.

[0089] Several embodiments, R in each occurrence 1 Independently, hydrogen or of any choice, 1 to 3 Z 1 It is an alkyl group substituted with [a specific compound].

[0090] In some embodiments, Z in each occurrence 1 These are independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0091] Several embodiments, R in each occurrence 21 These are independently halo, alkyl, or haloalkyl.

[0092] In some embodiments, p is 1-5, or 1-4, or 1-3, or 1-2, or 1.

[0093] In some embodiments, t is 0 in each occurrence.

[0094] In some embodiments, R 2 Alternatively, ring A is pyridyl or phenyl.

[0095] In some embodiments, R 2 Alternatively, ring A is something other than pyridyl or phenyl.

[0096] In some embodiments of formula II, part: [ka] That is the case.

[0097] In some embodiments of formula II, part: [ka] [ka] [ka] That is the case.

[0098] In some embodiments, tetrazine-based targeting agents of formula IID are provided. [ka] In the formula, X is a targeting agent disclosed herein. In some embodiments, X is a targeting agent selected from Table 1.

[0099] In some embodiments, a tetrazine-based targeting agent of formula IIE is provided. [ka] In the formula, X is a targeting agent disclosed herein. In some embodiments, X is a targeting agent selected from Table 1.

[0100] In some embodiments, tetrazine-based targeting agents of formula IA, formula II-A, or formula VA are provided. [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 This represents an additional bond that forms dihydrotetrazine when both are present. X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 1These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl may optionally have 1 to 3 Z 1 It has been replaced with, R in each appearance 2 These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4Each of these is a group that can be removed independently after hydrogen or a trigger event. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 22 It is independently and optionally comprises a linker of 1 to 100 linked atoms containing one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups. R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. each Z 1These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0101] In a particular embodiment, if ring A is aryl, then R 3 and R 4 Both exist.

[0102] In one embodiment, a tetrazine-based targeting agent of formula IA is provided. [ka] During the ceremony, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 22 It is independently and optionally comprises a linker of 1 to 100 linked atoms containing one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups.

[0103] In one embodiment, a tetrazine-based targeting agent of formula IIA-a is provided, [ka] During the ceremony, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. t is independently 0, 1, 2, 3, or 4.

[0104] One embodiment: R in each occurrence 22 These are independently linkers of 1 to 100 linked atoms and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.

[0105] In one embodiment, a tetrazine-based targeting agent of formula IIA-b is provided, [ka] In the formula, L, p, X, and R 20 Each of these is independent and as defined herein.

[0106] In one embodiment, a tetrazine-based targeting agent of formula IIB-a is provided, [ka] In the formula, L, p, and X are each independently as defined herein.

[0107] In one embodiment, a tetrazine-based targeting agent of formula IIC-a is provided. [ka] In the formula, L, p, and X are each independently as defined herein.

[0108] In one embodiment, a tetrazine-based targeting agent of formula III-a is provided, [ka] During the ceremony, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 30 These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. t is independently 0, 1, 2, 3, or 4.

[0109] In some embodiments, a tetrazine-based targeting agent represented by formula IID-a is provided, [ka] In the formula, X and R 20 Each of these is independently defined as herein. In some embodiments, R 20 It is methyl.

[0110] Furthermore, a tetrazine-based targeting agent of formula IIE-a is provided. [ka] In the formula, p and X are each independently as defined herein.

[0111] Furthermore, a tetrazine-based targeting agent of formula IIF-a is provided. [ka] In the formula, p and X are each independently as defined herein.

[0112] Furthermore, a tetrazine-based targeting agent of formula IIG-a is provided. [ka] In the formula, p and X are each independently as defined herein.

[0113] Furthermore, a tetrazine-based targeting agent of formula IIH is provided. [ka] In the formula, X is as defined herein.

[0114] In some embodiments, a tetrazine-based targeting agent of formula IIH is provided. [ka] In the formula, X is a targeting agent selected from Table 1.

[0115] In some embodiments, a tetrazine-based targeting agent of formula IIH-a is provided. [ka] In the formula, X is a targeting agent selected from Table 1.

[0116] In some embodiments, X is a nectin-4 targeting agent.

[0117] In some embodiments of formula IIA-a, [ka] [ka] That is the case.

[0118] In some embodiments of formula IIA-a, [ka] And in the formula, R 20 This is defined herein.

[0119] In some embodiments of formula IIA-a, [ka] That is the case.

[0120] In some embodiments of formula IIA-a, [ka] And in the formula, R 20 This is defined herein.

[0121] In some embodiments of formula IIA-a, [ka] That is the case.

[0122] In one embodiment, a tetrazine-based targeting agent of formula VA is provided herein, [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 1These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', C(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl may optionally have 1 to 3 Z 1 It has been replaced with, R in each appearance 2 These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4Each of these is a group that can be removed independently after hydrogen or a trigger event. each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0123] In one embodiment, a tetrazine-based targeting agent of formula VA is provided. [ka] During the ceremony, Ring A is a cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 This represents an additional bond that forms dihydrotetrazine when both are present. X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', C(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl may optionally have 1 to 3 Z 1 It has been replaced with, R in each appearance 2These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, R 3 and R 4 Both are either absent or R 3 It is a base that can be removed after the trigger event, R 4 is hydrogen or R 3 And, each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4.

[0124] In one embodiment, a tetrazine-based targeting agent of formula VI-A is provided. [ka] In the formula, R 1 , R 2 , R 3 , R 4 Each of the rings A, L, p, t, and X is independently defined as herein.

[0125] In some embodiments, R 4 It is hydrogen.

[0126] In some embodiments of the tetrazine-based targeting agents described herein, R 3 This is a base that can be removed after the trigger event. In some embodiments, the trigger event occurs in vivo. When the trigger event occurs, R 3 When removed, the dihydrotetrazine portion is oxidized to obtain tetrazine as shown in formula VII-A. [ka] In the formula, R 1 , R 2 Each of the rings A, L, p, t, and X is independently defined as herein.

[0127] The trigger event is initiated after administration of a tetrazine-based targeting agent to the target and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of functional groups, optionally followed by degradation) or external means (e.g., a photocleavable linker).

[0128] In some embodiments, R 3 It contains an amino acid sequence specific to cleavage by proteases or esterases.

[0129] In some embodiments, R 3 As shown in Table 1A, it contains an amino acid sequence specific to cleavage by proteases. TIFF2026518126000042.tif226170TIFF2026518126000043.tif158170TIFF2026518126000044.tif220170

[0130] In some embodiments, R 3 This includes an amino acid sequence specific to cleavage by cathepsin, matrix metalloproteinase (MMP), or PSMA. For example, in some embodiments, R 3 This includes Val-Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cathepsin-mediated cleavage. In some embodiments, R 3 This comprises Ac-γE-PLG-S(OBn)YL or Ac-PLG-HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMP. In some embodiments, R 3 It contains the amino acid sequence shown in Table 1B. TIFF2026518126000045.tif232170TIFF2026518126000046.tif228170TIFF2026518126000047.tif235170TIFF2026518126000048.tif227170TIFF2026518126000049.tif62170↓ indicates the cutting site Abbreviations for special amino acids: Cit: Citrulline, Cha: β-Cyclohexylalanine, Hof: Homophenylalanine, Nva: Aminosuberic acid, Dpa: D-Phenylalanine, Nle: Norleucine, Smc: S-Methylcysteine *The list of multiple amino acids before, between, or after the slash indicates alternative amino acids that can be substituted at that position, and "-" indicates that any amino acid can be substituted with the corresponding amino acid shown in the middle column. **x is any L-amino acid other than proline. Hy is any hydrophobic L-amino acid. γ indicates that the bond is a gamma-carboxyl bond.

[0131] Additional cleavable groups are described in Choi, et al., Theranostics. 2012;2(2):156-178, and Table 2 therein is incorporated herein by reference.

[0132] In some embodiments, R 3 It is photodegradable. In some embodiments, the photodegradable group is unstable or decomposes upon exposure to light of a wavelength that matches the absorbance profile of the photodegradable group.

[0133] In some embodiments, R 3 teeth, [ka] And, L 5 This is a direct link or linker, X 1 This is an optionally substituted peptide unit containing -NO2, an optionally substituted sugar moiety, or one or more natural or unnatural amino acids.

[0134] In some embodiments, at least one of the following parts: [ka] teeth, [ka] [ka] It is expressed by an expression selected from, where R 1 , R 2 , R 3 , and R 4 Each of them is independent as defined herein, and optionally, ring A portion may be one or more R 2 It can be replaced in parts.

[0135] In some embodiments, at least one of the following parts: [ka] teeth, [ka] It is expressed by an expression selected from, where X 2 is optionally an alkyl (e.g., methyl) substituted with PEG, amino acids, esters, amides, amines, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility-enhancing substituents, and L, ring A, R 1 , R 2 Each of t, p, and X is independently defined as herein.

[0136] In some embodiments, ring A is a cycloalkyl group. In some embodiments, ring A is a heterocyclyl group. In some embodiments, ring A is a heteroaryl group. In some embodiments, ring A is an aryl group.

[0137] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl.

[0138] In some embodiments, ring A is phenyl.

[0139] In some embodiments, at least one of the following parts: [ka] teeth, [ka] It is expressed by an expression selected from, where R 1 and R 2 Each of these is independent and as defined herein.

[0140] Several embodiments, R in each occurrence 1 Each is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, and each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with.

[0141] Several embodiments, R in each occurrence 1 Independently, hydrogen or of any choice, 1 to 3 Z 1 It is an alkyl group substituted with [a specific compound].

[0142] In some embodiments, Z in each occurrence 1 These are independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0143] Several embodiments, R in each occurrence 2 R is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments, R in each appearance 2R is independently a halo, alkyl, or haloalkyl. In some embodiments, R in each occurrence 2 These are independently halo or alkyl.

[0144] In some embodiments, t is 0 in each occurrence.

[0145] Furthermore, a tetrazine-based targeting agent of formula VA-a is provided. [ka] In the formula, p and X are each independently as defined herein. Furthermore, a tetrazine-based targeting agent of formula VB-a is provided. [ka] In the formula, p and X are each independently as defined herein.

[0146] In some embodiments, ring A is something other than pyridyl. In some embodiments, ring A is something other than aryl. In some embodiments, ring A is something other than phenyl.

[0147] B. Targeting moiety Provided herein are tetrazine-based targeting agents comprising one or more targeting moieties covalently bonded to one or more tetrazine moieties. The targeting moieties described herein are designed to localize to a target site within a subject upon administration. Upon administration of a tetrazine-based targeting agent, a prodrug comprising a complementary bioorthogonal component (i.e., a trans-cyclooctene moiety) can be administered, which, upon contact with the tetrazine-based targeting agent in vivo, enables targeted drug delivery of a payload or therapeutic agent.

[0148] In some embodiments, the targeting moiety or X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, and carbonic anhydrase IX (CAIX) targeters.

[0149] The targeting moiety, when used herein, is derived from a known compound (i.e., a targeting agent) that targets (or binds to) a desired target. The parent or known targeting agent (e.g., a compound or peptide) is optionally modified to be conjugated to a tetrazine moiety via a linking moiety as defined herein. Even after being modified to reach the compounds described herein, the targeting moiety maintains biological activity equivalent to that observed with the original unmodified targeting agent. In certain embodiments, the targeting moiety exhibits binding activity that is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of that observed with the original unmodified targeting agent.

[0150] In certain embodiments, the terms “modified” and “derived” as used with respect to the targeting agent mean that one or more atoms of the original unmodified targeting agent (i.e., a known targeting agent) are optionally replaced by covalent bonds to the tetrazine moiety via a linking moiety. In certain embodiments, a heteroatom (e.g., N, O, or S) or a hydrogen atom bonded to a reactive functional group (e.g., -OH) of the original unmodified targeting agent (i.e., a known targeting agent) is optionally replaced by covalent bonds to the tetrazine moiety via a linking moiety. In certain embodiments, in particular, a synthetic handle is provided when the targeting agent contains an additional amino acid or an amino acid with a conservative substitution (e.g., introducing lysine or cysteine).

[0151] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment nectin-4 peptide targeting agent. See, for example, Table 1, Entry 1.

[0152] The tetrazine-based targeting agents disclosed herein can be tested for nectin-4 binding using assays known in the art (e.g., Babeker, et al., Journal of Nuclear Medicine Jun 2022, 63(supplement 2)2865).

[0153] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment PSMA peptide targeting agent.

[0154] The tetrazine-based targeting agents disclosed herein can be tested for PSMA binding using assays known in the art (e.g., Rogers, et al. Targeted delivery of cytotoxic proteins to prostate cancer via conjugation to small molecule urea-based PSMA inhibitors. Sci Rep. 2021 Jul 21;11(1):14925. doi:10.1038 / s41598-021-94534-5).

[0155] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment cholecystokinin-2 receptor (CCK2R) peptide targeter.

[0156] The tetrazine-based targeting agents disclosed herein can be tested for CCK2R binding using assays known in the art (e.g., Hormann et al., Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177. Molecules. 2020 Oct 8;25(19):4585. doi:10.3390 / molecules25194585). In certain embodiments, the tetrazine-based targeting agents disclosed herein can be used in methods for treating cancer or gastrointestinal disorders.

[0157] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment PD-L1 peptide targeting agent.

[0158] The tetrazine-based targeting agents disclosed herein can be tested for PD-L1 binding using assays known in the art (e.g., Liu, et al. Clinical applications of PD-L1 bioassays for cancer immunotherapy. J Hematol Oncol 10,110(2017). https: / / doi.org / 10.1186 / s13045-017-0479-y).

[0159] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 3 targeting agent.

[0160] The tetrazine-based targeting agents disclosed herein can be tested for integrin alpha(v)beta(3) binding using assays known in the art (e.g., Danhier et al., RGD-Based Strategies To Target Alpha(v)Beta(3)Integrin in Cancer Therapy and Diagnosis, Mol.Pharmaceutics 2012, 9, 11, 2961-2973).

[0161] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 6 targeting agent.

[0162] The tetrazine-based targeting agents disclosed herein can be tested for integrin alpha-V-beta-6 binding using assays known in the art (e.g., Monieri et al., A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins, Int. J. Biol. Sci., 2023; 19(1): 156-166. doi: 10.7150 / ijbs.76148, and Nardelli, M., et al. (2019). Switching on phosphorescence in [Ir(C^N)2(N^N)]+ complexes by trifluoromethylation of the N^N ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039 / C9CC07666E).

[0163] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 8 targeting agent.

[0164] The tetrazine-based targeting agents disclosed herein can be tested for integrin alpha-beta-8 binding using assays known in the art (e.g., Monieri et al., A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins, Int. J. Biol. Sci., 2023; 19(1): 156-166. doi: 10.7150 / ijbs.76148, and Nardelli, M., et al. (2019). Switching on phosphorescence in [Ir(C^N)2(N^N)]+ complexes by trifluoromethylation of the N^N ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039 / C9CC07666E).

[0165] In some embodiments, the targeting portion, or X, is a non-antibody or non-antibody fragment FAP targeting agent.

[0166] The tetrazine-based targeting agents disclosed herein can be tested for FAP binding using assays known in the art (e.g., Zhang, et al. (2020). A cell-based fluorescent assay for FAP inhibitor discovery. Bioorganic & Medicinal Chemistry Letters. 30.127253.10.1016 / j.bmcl. 2020.127253).

[0167] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment carbonic anhydrase IX (CAIX) targeting agent.

[0168] The tetrazine-based targeting agents disclosed herein can be tested for carbonic anhydrase IX (CAIX) binding using assays known in the art (e.g., US10016511B2).

[0169] As used herein, the term “non-antibody or non-antibody fragment” is intended to refer to targeting agents that are considered antibodies or antibody fragments, such as peptide-containing compounds, generally having a size of less than 15 kDa, or less than 10 kDa, or less than 9 kDa, or less than 8 kDa, or less than 7 kDa, or less than 6 kDa, or less than 5 kDa.

[0170] In some embodiments, the targeting portion, or X, is selected from Table 1. For representative procedures for preparing various targeting segments, see, for example, Cazzamalli, et al., Clin Cancer Res; 24(15) August 1, 2018; Wayua, et al., Mol. Pharmaceutics 2015, 12, 2477-2483; Pastorino, et al., Current Radiopharmaceuticals, 2020, Vol. 13, No. 1, 63-79; Lerchen, et al., Cancers 2022, 14, 391; Millul, et al., PNAS 2021, Vol. 118, No. 16, e2101852118; Patel, et al., New J. Chem., 2021, 45, 5291; WO2019 / 243832; and Zana, et al., Bioconjugate See Chem.2023,34,7,1205-1211. TIFF2026518126000060.tif225170TIFF2026518126000061.tif224170TIFF2026518126000062.tif210170TIFF20265181260 00063.tif167170TIFF2026518126000064.tif202170TIFF2026518126000065.tif221170TIFF2026518126000066.tif115170

[0171] Linker In the tetrazine-based targeting agents disclosed herein, the linker may be linear or branched (including, but not limited to, divalent, trivalent, and tetravalent branching). Thus, in a single tetrazine-based targeting agent, there may be one or more targeting moieties covalently bonded to one or more tetrazine moieties. In the formulas disclosed herein, "L" may be inside or outside the notation of "p", however, the linker may be linear having one bond to a tetrazine moiety and one bond to a targeting moiety, and it is intended that there may be two or more [tetrazine-L]- moieties bonded to a single targeting moiety, or the linker may be branched to have two bonded tetrazine moieties and one bond to a targeting moiety.

[0172] In some embodiments, L, or the linker, comprises one or more of the following: hydrazone, hydrazide, disulfide, N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acids, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

[0173] In some embodiments, L, or the linker, comprises 1 to 100 linked atoms, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0174] In some embodiments, L, or linker, comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety independently and optionally oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0175] In some embodiments, L, or the linker, is bound to X via a cysteine ​​or lysine residue on X.

[0176] In some embodiments, L, or the linker, is an uncuttable linker.

[0177] In some embodiments, L, or the linker, is a severable linker.

[0178] In some embodiments, L, or the linker, comprises one or more amino acids.

[0179] In some embodiments, L, or the linker, comprises a polypeptide.

[0180] In some embodiments, L, or the linker, is an alkylene linker comprising, optionally, one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

[0181] In some embodiments, L, or linker, is of the following formula: -Y 10 -(CHR 130 ) n’ -Y 20 -(CHR 140 ) n’’ -Y30 -(CHR 150 ) m’’ -Y 40 - During the ceremony, Y 10 , Y 20 , Y 30 , and Y 40 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O) 1-5 -, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 130 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. Each R 140 Hydrogen and C are independent of each other.1-4 Alkyl, C 1-4 The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. Each R 150 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0182] In some embodiments, L, or linker, is of the following formula: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - During the ceremony, Y 10 , Y 20 , and Y 30 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R 110Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. n' and m'' are independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0183] In a particular embodiment, each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 It is a haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. In certain embodiments, the linker is not a bond.

[0184] L, or linker, may contain one or more of the following: polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diyrbis(methyl carbamate), arylene (ee, phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0185] In some embodiments, the linker is [ka] Includes one or more of the following.

[0186] In some embodiments, the linker is [ka] Includes one or more of the following.

[0187] In some embodiments, the linker is [ka] Includes one or more of the following.

[0188] In some embodiments, the linker is one or more [ka] This includes. In some embodiments, the linker is one or more [ka] Includes.

[0189] In some embodiments, the linker is one or more [ka] It is either or includes it.

[0190] In some embodiments, the linker is one or more [ka] It is either or includes it.

[0191] In some embodiments, the linker may comprise one or more natural or non-natural amino acids, which may be referred to as peptide linkers. The linker may also be a peptide linker composed of a carboxylacyl unit and one or more amino acids that constitute a protein or peptide sequence. The linker may also contain self-immolating spacers that space the drug and protein peptide sequences.

[0192] In some embodiments, the linker is "AYZX 2The peptide may contain a linker represented as "-W", where "A" is a carboxylate acyl unit, and "Y" and "Z" are each one or more natural or non-natural amino acids, together forming a peptide sequence, "X 2 " and "W" are optional additional linkers having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms, that space the peptide and payload, D, or bioorthogonal portion. In certain embodiments, one or more amino acids in the peptide linker are N-methylated.

[0193] In some embodiments, Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In some embodiments, Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0194] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, acetyl or formyl-protected lysine, arginine, tosyl or nitro-protected arginine, histidine, ornithine, acetyl or formyl-protected ornithine, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0195] Examples of YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0196] In some embodiments, A is -OC(O)-.

[0197] In some embodiments, X 2 It is -OC(O)-.

[0198] In some embodiments, W is -OC(O)-. In some embodiments, X 2 It is absent, and W is -OC(O)-.

[0199] In some embodiments, part-X 2 -W is, [ka] Includes. In some embodiments, part-X 2 teeth, [ka] That is the case.

[0200] In some embodiments, -XW is [ka] That is the case.

[0201] In some embodiments, -XW is [ka] That is the case.

[0202] In some embodiments, the peptide linker is specifically modified to be selectively cleaved (e.g., enzymatically cleaved) to release a drug, such as by one or more tumor-associated proteases.

[0203] In some embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0204] In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -Tosyl-Arg, or Phe-N 9 It is -nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0205] In some embodiments, L, or linker, [ka] [ka] [ka] [ka] It is one or more of the following, or includes one of them.

[0206] In some embodiments, the linker L is [ka] Includes one or more of the following.

[0207] In some embodiments, the linker L is [ka] Includes one or more of the following.

[0208] The aforementioned linker is an amino acid side chain present on X, such as lysine or cysteine ​​(for example, [ka] It can be combined with other things.

[0209] In some embodiments, L, or the linker, is -C(O)L 4 -or -C(O)C 1-6 Alkilen C(O)L 4 -and, L 4 This is a bond, -N(R 12 )-C 2-3 Alkylene-N(R) 13 )C(O)-,-CH(NHC(O)R 14 )C 1-4 Alkylene-SSC 1-4 Alkylene-OC(O)-,-NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1-6 Alkylene-CH(G x )OC(O)-, [ka] And, R 12 , R 13 , R 14 , R 15 , and R 19 Each of them independently consists of hydrogen or C 1-4 It is alkyl, R 16 is hydrogen, C 1-4 Alkyl, -C 1-4 Alkylene-OH,-C 1-4 Alkylene-OC 1-4 Alkyl, -C 1-4 Alkylene-CO2H, or -C 1-4 It is alkylene-CONH2, G x The following are optional choices: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C1-4 This is a phenyl compound substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0210] In some embodiments, L, or the linker, includes a carbonyl moiety for conjugating the tetrazine moiety to the linker or X. For example, the linker may include a polypeptide moiety (PPM) having a lysine residue and a lysine side chain, and the PPM may also include additional lysine or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, the linker L is [ka] It may include.

[0211] In some embodiments, L, or linker, [ka] [ka] It is one or more of the following, or includes one of them.

[0212] In some embodiments, L, or linker, [ka] It is one or more of the following, or includes one of them.

[0213] In some embodiments, L, or linker, [ka] It is one or more of the following, or includes one of them.

[0214] In some embodiments, L, or linker, [ka] It is one or more of the following, or includes one of them.

[0215] In some embodiments, L, or linker, [ka] That is the case.

[0216] In some embodiments, L, or linker, [ka] That is the case.

[0217] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0218] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0219] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0220] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0221] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0222] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0223] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0224] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0225] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0226] In some embodiments, L, or linker, is one or more [ka] It is either or includes it.

[0227] In some embodiments, the linker L is one or more [ka] It is either or includes it.

[0228] In one embodiment, a tetrazine-based targeting agent of formula I is provided. [ka] During the ceremony, p is between 1 and 16. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 22 It is independently and optionally comprises a linker of 1 to 100 linked atoms containing one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. In each occurrence, L is independent. [ka] This linker is selected from the group consisting of the following:

[0229] In one embodiment, a tetrazine-based targeting agent of formula II is provided. [ka] During the ceremony, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. R in each appearance 20 These are independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R”', SC(=O)R'”, OC(=S)R”', SC(=S)R”', S(=O)R', S(=O)2R”', S(=O)2NR'R”, C(=O)O-R', C(=O)S-R', Selected from the group consisting of C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', R in each appearance 30These are independently halogens, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R a , R 31a , and R 31b Each of these is independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. t is independently 0, 1, 2, 3, or 4. In each occurrence, L is independent. [ka] This linker is selected from the group consisting of the following:

[0230] In one embodiment, a tetrazine-based targeting agent of formula V is provided, [ka] During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R 3 and R 4 If both are absent, then tetrazine, or R 3 and R 4 R represents an additional bond that forms dihydrotetrazine when both are present, however, when ring A is aryl. 3 and R 4 Assuming that both exist, X is a non-antibody or non-antibody fragment targeter selected from nectin-4 peptide targeters, PSMA peptide targeters, CCK2R peptide targeters, PD-L1 peptide targeters, integrin alpha-V beta-3 targeters, integrin alpha-V beta-6 targeters, integrin alpha-V beta-8 targeters, FAP targeters, CEACAM5 (also known as CEA) targeters, and carbonic anhydrase IX (CAIX) targeters. p is between 1 and 16. R in each appearance 1 These are independently hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R'', C(=S)NR'R'', NR'R'', Selected from the group consisting of NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', C(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl may optionally have 1 to 3 Z 1 It has been replaced with, R in each appearance 2These are independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally one to three Z 1 It has been replaced with, R 3 and R 4 Both are absent, or R 3 and R 4 Each of these is a group that can be removed independently after hydrogen or a trigger event. each Z 1 These are independently halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S -R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R'', and NR'C(=S)NR"R'' are selected. In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. In each occurrence, t is independently 0, 1, 2, 3, or 4. In each occurrence, L is independent. [ka] This linker is selected from the group consisting of the following:

[0231] In some embodiments, ring A is pyrimidinyl, triazinyl, oxazolyl, isoxazole, imidazolyl, oxadiazolyl, 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidinyl, or 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidinyl, each of which may be optionally substituted.

[0232] In some embodiments, p is 1-5, or 1-4, or 1-3, or 1-2.

[0233] In a particular embodiment, p depends on the size and / or number of available bonding sites on X for forming a covalent bond to L.

[0234] In one embodiment, the tetrazine-based targeting agent has the following formula: [ka] In the formula, n is between 1 and 10.

[0235] In one embodiment, the tetrazine-based targeting agent is as shown in Table 2. TIFF2026518126000112.tif238170TIFF2026518126000113.tif203170TIFF2026518126000114.tif125170TIFF2026518126000115.tif161170TIFF2026518126000116.tif165170TIFF2026518126000117.tif167170TIFF2026518126000118.tif151170TIFF2026518126000119.tif106170TIFF2026518126000120.tif200170TIFF2026518126000121.tif194170TIFF2026518126000122.tif220170TIFF2026518126000123.tif227170TIFF2026518126000124.tif242170TIFF2026518126000125.tif228170TIFF2026518126000126.tif243170TIFF2026518126000127.tif241170TIFF2026518126000128.tif203170TIFF2026518126000129.tif194170TIFF2026518126000130.tif161170TIFF2026518126000131.tif159170TIFF2026518126000132.tif156170TIFF2026518126000133.tif159170

[0236] C. Support Composition A support composition comprises a support. In certain embodiments, the support composition is a therapeutic support composition. The support may be a biocompatible support composition, i.e., compatible with the body of the subject. In some examples, the support is non-toxic to the subject and substantially does not react with the subject's tissue or biological compounds. For example, the support may be a hydrogel, among other things. The support can be implanted in the body of the subject and support a binder (e.g., a TCO-containing group), as well as a payload after the binder has been conjugated. Typical supports include, but are not limited to, polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, crosslinked polymer matrices, metals, ceramics, plastics, bone graft materials, alginates, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, and heparin. Supports may also include particles such as nanoparticles and microparticles.

[0237] The hydrogel may be a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosine, chitin, chondroitin sulfate, heparin, etc. Other suitable sugar-based biomaterials are described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that can be used as supports include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Typical polyethers include poly(ethylene glycol) (PEG), polypropylene glycol) (PPG), and triblock Pluronic ([PEG] n -[PPG] mSupports may also include, but are not limited to, PEG-n, PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). The support may also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(gamma-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), etc.

[0238] In some embodiments, the support is a hydrogel. In some embodiments, the support is an alginate. In some embodiments, the support is chitin. In some embodiments, the support is hyaluronic acid (e.g., substantially uncrosslinked non-hydrogel hyaluronic acid). In some embodiments, the support is chitosine.

[0239] In certain embodiments, the support is a particle. The particles of this disclosure may have a diameter of 2 cm or less, for example, 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles may be nanoparticles or microparticles. Nanoparticles include particles having an average size on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having an average size on the micrometer scale (e.g., 1000 μm or less). "Average" means the arithmetic mean. In some embodiments, the nanoparticles have a diameter in the range of 1 nm to 1 μm, for example, 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have diameters ranging from 1 μm to 1 mm, for example, 10 μm to 1 mm, or 25 μm to 1 mm, or 50 μm to 1 mm, or 75 μm to 1 mm, or 100 μm to 1 mm, or 150 μm to 1 mm, or 200 μm to 1 mm, or 250 μm to 1 mm, or 300 μm to 1 mm, or 350 μm to 1 mm, or 400 μm to 1 mm, or 450 μm to 1 mm, or 500 μm to 1 mm. In further embodiments, small particles with diameters of about 10 to 100 nm may be assembled to form larger complexes such as clusters or assemblies of about 1 to 10 μm. The particles of this disclosure may be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes may also be used, but are not limited to these, such as elliptical, cubic, cylindrical, conical, needle-shaped, or other irregular shapes.

[0240] "Particles" can take the form of any manufactured material, molecule, cryptophan, virus, phage, etc. Particles may be composed of materials such as metals, ceramics, plastics, glass, composites, polymers, and hydrogels, but are not limited to these. For example, particles may be made of inert materials such as alginates or iron oxides. In some embodiments, particles may be magnetic and can be formed from paramagnetic, superparamagnetic, or ferromagnetic materials, or other materials that respond to magnetic fields. Furthermore, particles may have any shape, such as spheres, rods, or asymmetric shapes. Particles, or groups of particles in a complex, may be functionalized with receptors that have a specific affinity for or interact with clinically relevant substrates. The receptors may be inherent to the particles themselves. For example, the particles themselves may be viruses or phages that have a specific affinity for a particular substrate. Additionally or alternatively, particles may be functionalized by covalently or otherwise binding or associating with receptors that specifically bind to or otherwise recognize a particular clinically relevant substrate. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule having a defined affinity for the target substrate. Examples of materials that may be used for “particles” and / or “carriers” include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphatidines, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, polyoxomers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. These examples are not limited to their concentrations, their crosslinking with different agents, their administration methods, their tuned degradation profiles, and other characteristics known to those skilled in the art.

[0241] Particles, or groups of particles in a complex, may be functionalized with a tetrazine-based targeting agent (e.g., ligand or antibody) that specifically (or substantially specifically) binds to a target (e.g., a target receptor or cell surface target, e.g., a clinically relevant receptor or cell surface target (e.g., an antigen)). The tetrazine-based targeting agent may be directly bound to the particle itself. The tetrazine-based targeting agent may be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule having specific affinity for the target receptor or cell surface target. In some examples, the receptor or cell surface target may be PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, or other cell receptors or cell surface targets. Other compounds or molecules, such as fluorophores or autofluorescent or luminescent markers, which may aid in the detection of particles (e.g., in vivo detection), may also be attached to the particles. Ligands and / or detectable labels may be directly bound to the particles or bound to the particles via bioorthogonal functional groups, as described herein.

[0242] In certain embodiments, the support is a bone graft material, such as a bone graft substitute. The bone graft substitute is a material that is structurally similar to bone. In some examples, the bone graft substitute is bioabsorbable so that it can dissolve or be absorbed into the body over time. The bone graft substitute can be osteoconductive so as to promote new bone formation into blood vessels and the bone graft substitute. In some examples, the bone graft substitute is osteoinducible so as to promote new bone formation by the active recruitment of mesenchymal stem cells from surrounding tissues. Growth factors, such as osteomorphic proteins, may be included in the bone graft substitute. Examples of bone graft substitutes include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and combinations thereof.

[0243] In a particular embodiment, the support composition is given by the following formula: [ka] It includes a substituted alginate having units of the salt thereof, where the dashed line represents a bond to L.

[0244] In some embodiments, the support composition is given by the following formula: [ka] or comprising a substituted hyaluronic acid having units of its salt, In the formula, the dashed line indicates a connection to L.

[0245] Hyaluronic acid derivatives include hyaluronic acid having multiple glucuronic acid units and tetrazine-containing groups linked or directly conjugated to the glucuronic acid units of hyaluronic acid. Hyaluronic acid may also have multiple N-acetylglucosamine units. In certain embodiments, the N-acetylglucosamine units of hyaluronic acid are not linked or conjugated to the tetrazine-containing groups.

[0246] The tetrazine-containing group can be linked or directly linked to a carboxylic acid of the glucuronic acid unit. The tetrazine-containing group can be incorporated into hyaluronic acid in amounts ranging from approximately 0.1% to approximately 80%, measured by the percentage of carboxylic acid linked or conjugated to the L of the tetrazine-containing group, such as approximately 1% to approximately 75%, approximately 5% to approximately 75%, approximately 10% to approximately 50%, or approximately 40% to approximately 75%.

[0247] Additional support compositions are exemplified in WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, the entire contents of each thereof being incorporated herein by reference.

[0248] D. Trans-cyclooctene functionalized prodrug Trans-cyclooctene-functionalized prodrugs, including prodrugs for anticancer agents, are known to those skilled in the art as described in WO2018 / 187740, WO2014 / 205126, WO2015 / 139025, and WO2017 / 044983, which are incorporated herein by reference. Further embodiments using trans-cyclooctene-functionalized prodrugs are described below.

[0249] In some embodiments, the trans-cyclooctene functionalized prodrug is a conjugate consisting of a payload linked to one or more trans-cyclooctene moieties.

[0250] In some embodiments, the conjugate (or trans-cyclooctene functionalized prodrug) includes an immunomodulatory payload, such as an immunomodulatory payload selected from the group consisting of cytokines, chemokines, chemokine antagonists, therapeutic monoclonal antibodies, and immune checkpoint inhibitor payloads, or a pharmaceutically acceptable salt thereof.

[0251] In some embodiments, the immunomodulatory payload is an inhibitor of the cytokine payload or a pharmaceutically acceptable salt thereof.

[0252] In some embodiments, the cytokine payload inhibitor is an inhibitor of TNF-α, infliximab, certolizumab, TGF-β, garnicertib, fresolimmab, M7824, CSF-1, pexidartinib, or kabilizumab.

[0253] In some embodiments, the conjugate comprises a monoclonal antibody or a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, the conjugate comprises a therapeutic protein payload or a pharmaceutically acceptable salt thereof.

[0255] In some embodiments, the therapeutic protein payload is an antibody-based drug, an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an engineered protein scaffold, an enzyme, a growth factor, a hormone, an interferon, an interleukin, or a thrombolytic agent.

[0256] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0257] In some embodiments, the therapeutic protein payload may be, but is not limited to, erythropoietin (EPO), such as PROCRIT®, EPREX®, or EPOGEN® (epoetin-α), ARANESP® (darbepoetin-α), NEORECORMON®, EPOGIN® (epoetin-β), etc., natural EPO or synthetic EPO® (see, for example, US2003 / 0191291). (e.g., growth hormone (e.g., somatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); therapeutic monoclonal antibodies (e.g., atezolizumab, avelumab, bevacizumab, semiprimab, cetuximab, daratumumab, dinutuximab, durvalumab, elotuzumab, ipilimumab, isatuximab, moga) Murizumab, nesitumumab, nivolumab, obinutuzumab, ofatumumab, olaratumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, rituximab, trastuzumab, etc.); human growth hormone (hGH); bovine growth hormone (bGH); follicle-stimulating hormone (FSH); interferon (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ, consensus interferon, etc.); insulin (e.g., Novolin, Humulin, Humalog, Lancrine). (e.g., TAS, Ultralente), insulin-like growth factors (e.g., IGF-I, IGF-II); blood factors (but not limited to these, e.g., ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human factor VIIa), factor VIIa, factor VIII (e.g., KOGENATE®), factor IX, β-globin, hemoglobin, etc., e.g., factor X, tissue plasminogen activator (TPA), etc.);Colony-stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neurasta (pegfilgrastim), granulocyte-monocyte colony-stimulating factor, megakaryocyte colony-stimulating factor, etc.), transforming growth factors (e.g., TGF-beta, TGF-alpha); interleukins (e.g., IL-1, IL-2 (e.g., Proleukin®), IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IE-12, etc.); growth factors (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factor (FGF, e.g., aFGF, bFGF, e.g., FIB) LAST® (Trafermin), glial cell line-derived growth factor (GDNF), nerve growth factor (NGF), stem cell factors (e.g., STEMGEN® (Ancestim)), keratinocyte growth factor, hepatocyte growth factor, etc.); soluble receptors (e.g., ENBREL® (Etanercept), soluble VEGF receptor, soluble interleukin receptor, soluble γ / δ T-cell receptors, such as TNF-α-binding soluble receptors; enzymes (e.g., α-glucosidase, CERAZYME® (imiglucarase, β-glucocerebrosidase, CEREDASE® (algluculose)); enzyme activators (e.g., tissue plasminogen activator); chemokines (e.g., IP-10, Mig, Groα / IL-8, expressed and secreted regulated normal T cells (RANTES), MIP-1α, MIP-1ρ, MCP-1, PF-4, etc.); angiogenic agents (e.g., vascular endothelial growth factor (VEGF); anti-angiogenic agents (e.g., soluble VEGF receptor); protein vaccines;Bradykinin, cholecystokinin, gustin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, galanin, growth hormone-releasing hormone, bombesin, warfarin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, and other neuroactive peptides; thrombolytic agents, atrial natriuretic peptide, bone morphogenetic protein, etc. Other proteins such as rhombopoietin, relaxin, glial fibrillary acidic protein, follicle-stimulating hormone, human alpha-1 antitrypsin, leukemia inhibitors, transforming growth factors, tissue factor, insulin-like growth factor, luteinizing hormone, follicle-stimulating hormone, macrophage activator, tumor necrosis factor, neutrophil chemotactic factors, nerve growth factor, and other metalloproteinase tissue inhibitors; vasoactive intestinal peptides, angiogenin, angiotropin, fibrin; hirudin; leukemia inhibitors; or payloads such as IL-1 receptor antagonists (e.g., Kineret® (Anakinra)).

[0258] In some embodiments, the conjugate is of formula X or a pharmaceutically acceptable salt thereof. [ka] During the ceremony, m is an integer between 1 and 150. In each occurrence, G is independently a trans-cyclooctene moiety that has been substituted by arbitrary choice. D is the payload, L in each appearance 1 It is an independent linker.

[0259] In some embodiments of the conjugates described herein, each trans-cyclooctene portion is independently, [ka] And in the formula, R in each appearance 1A C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Selected from the group consisting of alkoxys, q is 0, 1, or 2, q1 is either 0 or 1. R in each appearance 1B G 1 -OH, -NR 1c -C 1-4 Alkilen-G 1 , -NR 1c -C 1-4 Alkylene-N(R) 1d )2, -NR 1c -C 1-6 Alkylene-N(C) 1-4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R) 1c )-C 1-6 Alkylene-CO2H,-N(R) 1f )-C 2-4 Alkylene-(N(C) 1-4 Alkylene-CO2H)-C 2-4 Alkilen) n -N(C 1-4 Alkylene-CO2H)2,-N(R) 1c )CHR 1e C(O)OC 1-6 Alkyl, -N(R 1c )-C 1-6 Alkylene-C(O)OC 1-6 Alkyl, -N(R 1f )-C 2-4 Alkylene-(N(C) 1-4 Alkylene C(O)OC 1-6 Alkyl)-C 2-4 Alkilen) n -N(C 1-4 Alkylene C(O)OC 1-6 Alkyl)2,-N(R 1c )-C 1-6 Alkylene-SO3H,-N(R) 1c )-(CH2CH2O)1-3 -CH2CH2N((CH2CH2O) 1-3 -C 1-6 Alkylene-CO2H)2 and -N(R 1c )-CH(CH2O-(CH2CH2O) 0-2 -C 1-6 Selected from the group consisting of alkylene-CO2H)2, R in each appearance 1c and R 1d These are independently hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, R in each appearance 1f Hydrogen and C are independent of each other. 1-6 Alkyl, or C 1-4 It is alkylene-CO2H. In each occurrence, n is independently 0, 1, 2, or 3. L in each appearance 2 These are independently -C(O)- and C 1-3 Selected from the group consisting of alkylenes, G in each appearance 1 These are independently, arbitrarily substituted heterocyclines.

[0260] In some embodiments, the conjugate is of formula X or a pharmaceutically acceptable salt thereof. [ka] During the ceremony, G is the trans-cyclooctene moiety, and in each occurrence, G is independent. [ka] And, L in each appearance 1 It is independently a linker, m is an integer between 1 and 150. D is the payload, R in each appearance 1A C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Selected from the group consisting of alkoxys, q is 0, 1, or 2. q1 is either 0 or 1. R in each appearance 1B G 1 , OH, -NR 1c -C 1-4 Alkilen-G 1 , -NR 1c -C 1-4 Alkylene-N(R) 1d )2, -NR 1c -C 1-6 Alkylene-N(C) 1-4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R) 1c )-C 1-6 Alkylene-CO2H,-N(R) 1c )CHR 1e C(O)OC 1-6 Alkyl, -N(R 1f )-C 2-4 Alkylene-(N(C) 1-4 Alkylene-CO2H)-C 2-4 Alkilen) n -N(C 1-4 Alkylene-CO2H)2,-N(R) 1c )-C 1-6 Alkylene-C(O)OC 1-6 Alkyl, -N(R 1f )-C 2-4 Alkylene-(N(C) 1-4 Alkylene C(O)OC 1-6 Alkyl)-C 2-4 Alkilen) n -N(C 1-4 Alkylene C(O)OC 1-6 Alkyl)2,-N(R 1c )-C 1-6 Alkylene-SO3H,-N(R) 1c)-(CH2CH2O) 1-3 -CH2CH2N((CH2CH2O) 1-3 -C 1-6 Alkylene-CO2H)2 and -N(R 1c )-CH(CH2O-(CH2CH2O) 0-2 -C 1-6 Selected from the group consisting of alkylene-CO2H)2, R in each appearance 1c and R 1d These are independently hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, R in each appearance 1f Hydrogen and C are independent of each other. 1-6 Alkyl, or C 1-4 It is alkylene-CO2H. In each occurrence, n is independently 0, 1, 2, or 3. L in each appearance 2 These are independently -C(O)- and C 1-3 Selected from the group consisting of alkylenes, G in each appearance 1 These are independently, arbitrarily substituted heterocyclines.

[0261] In some embodiments, q1 is 1.

[0262] In some embodiments, the payload is an immunomodulatory agent payload.

[0263] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, or immune checkpoint inhibitor payload, or a pharmaceutically acceptable salt thereof.

[0264] In some embodiments, the payload is a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, carmustine, ixabépirone, patupirone (epoterone class), platinum-based drugs, exatecan, orlistin (drastatin 10, MMAE, MMAD, MMAF), duocalmycin, pyrrolobenzodiazapene dimer, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin), immunosuppressants. The following are selected from disease control agents (e.g., cyclosporine A, rapamycin, etc.), antifungal agents (e.g., amphotericin, etc.), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, etc.), matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

[0265] In some embodiments, the payload may include therapeutic agents for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepyrone, patupyrone (epoteron class), platinum-based drugs, exatecan, orlistin (drastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, etc.), immunosuppressants (e.g., cyclosporine A, rapamycin, etc.), antifungal agents (e.g., The following are selected from amphotericin, antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin beta, flucytosine, emtricitabine, gentamicin, colistin, etc.), lurubinectedin, gardiquimod, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

[0266] Reference to a payload means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original unmodified payload are replaced by covalent bonds to one or more linkers. The payload is modified to originate from a known nuclear payload and to covalently bond to trans-cyclooctene, which is substituted by at least one optional linker. Even after being modified to reach the compounds described herein, the payload maintains equivalent biological activity to that observed in the original unmodified payload. In certain embodiments, the payload exhibits binding activity or inhibition that is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of that observed in the original unmodified payload.

[0267] In certain embodiments, hydrogen atoms bonded to heteroatoms (e.g., N, O, or S) in the original unmodified payload are replaced by covalent bonds to the linker. In certain embodiments, halogen atoms on the payload are replaced for bonding to the rest of the compound. In certain embodiments, hydrogen atoms on the payload are replaced for bonding to the rest of the compound. In certain embodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on nitrogen. In certain embodiments, the hydrogen atom is on oxygen. In certain embodiments, the hydrogen atom is on carbon.

[0268] In some embodiments, G in each occurrence is independent, [ka] That is the case.

[0269] In some embodiments, G in each occurrence is independent, [ka] That is the case.

[0270] In some embodiments, the payload is a monoclonal antibody payload. The monoclonal antibody used herein as the payload may be the entire monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the antibody is an immune cell engager and will therefore induce or trigger an immune response. In some embodiments, the monoclonal antibody or fragment thereof is CD3 (NCBI gene ID 916), CD28 (NCBI gene ID 940), CD137 (4-1BB) (NCBI gene ID 3604), CD16 (NCBI gene ID 2214), NKG2D (NCBI gene ID 22914), CD64 (NCBI gene ID 2209), GITR / TNFRSF18 (NCBI gene ID 8487), CD25 (NCBI gene ID 3559), CD40 (NCBI gene ID 958), CD4 (NCBI gene ID 920), CXCR4 (NCBI gene ID 7852), G-CSFR (NCBI gene ID 1441), GM-CSFR (NCBI gene ID 1438), CD122 (NCBI gene ID 920). The target is one or more of the following genes: gene ID 3560, PD1 (NCBI gene ID 5133), CTLA4 (NCBI gene ID 1493), LAG3 (NCBI gene ID 3902), TIGIT (NCBI gene ID 201633), NCR1 (NCBI gene ID 9437), TIM3 (NCBI gene ID 84868), VISTA (NCBI gene ID 64115), CD134 (NCBI gene ID 7293), CD27 (NCBI gene ID 939), CD40L (NCBI gene ID 959), ICOS (NCBI gene ID 29851), BAFFR (NCBI gene ID 115650), LFA-1 (NCBI gene ID 3689), or BTLA (NCBI gene ID 151888).

[0271] In certain embodiments, the payload is an antibody or antibody fragment that targets CD3, such as OKT3, SP34, UCHT1, teplizumab, otelixizumab, bicilizumab, or foralumab, or an antibody fragment derived therefrom.

[0272] In certain embodiments, the payload is a CD28-targeting antibody or antibody fragment, such as teralizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0273] In certain embodiments, the payload is an antibody or antibody fragment that targets CD137(4-1BB), such as utomirumab, urerumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0274] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD16, such as AFM13, or an antibody fragment derived therefrom.

[0275] In a particular embodiment, the payload is an antibody or antibody fragment that targets NKG2D, such as NNC0152-0002 or JNJ-64304500, or an antibody fragment derived therefrom.

[0276] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD64, such as H22, or an antibody fragment derived therefrom.

[0277] In certain embodiments, the payload is an antibody or antibody fragment that targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0278] In certain embodiments, the payload is a CD25-targeting antibody or antibody fragment, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0279] In certain embodiments, the payload is a CD40-targeting antibody or antibody fragment, or an antibody fragment derived therefrom, such as iscarimab, ABBV-323, preserumab (ASKP-1240), BI-655064, FFP-104, BMS986090, dacetuzumab, or lucatumumab.

[0280] In certain embodiments, the payload is a CD4-targeting antibody or antibody fragment, or an antibody fragment derived therefrom, such as MAX.16H5, IT1208, zanorimumab (HuMax-CD4), UB-421, or MTRX1011A.

[0281] In a particular embodiment, the payload is an antibody or antibody fragment that targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0282] In a particular embodiment, the payload is an antibody or antibody fragment that targets a G-CSFR such as CSL324, or an antibody fragment derived therefrom.

[0283] In a particular embodiment, the payload is a GM-CSFR-targeting antibody or antibody fragment, such as maprilimumab, or an antibody fragment derived therefrom.

[0284] In a particular embodiment, the payload is an antibody or antibody fragment that targets CD122, such as Hu-Mik(beta)1, or an antibody fragment derived therefrom.

[0285] In certain embodiments, the payload is a PD-1-targeting antibody or antibody fragment, such as CC-90006, semiprimab, camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0286] In certain embodiments, the payload is an antibody or antibody fragment that targets CTLA4, such as tremelimumab or ipilimumab, or an antibody fragment derived therefrom.

[0287] In certain embodiments, the payload is an antibody or antibody fragment that targets LAG3, such as relatrimab (BMS-986016), GSK2831781, semiprimab (REGN3767), fabezerimab, ieramilimab, or mavezelimab, or an antibody fragment derived therefrom.

[0288] In certain embodiments, the payload is an antibody or antibody fragment that targets TIGIT, such as BMS-986207, tilagolumab, vivostrimab, etigirimab, dombanarimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0289] In a particular embodiment, the payload is an antibody or antibody fragment that targets NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0290] In certain embodiments, the payload is an antibody or antibody fragment that targets TIM3, such as covolimab, Sym023, LY3321367, BMS-986258, SHR-1702, sabatrimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0291] In certain embodiments, the payload is an antibody or antibody fragment that targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0292] In certain embodiments, the payload is an antibody or antibody fragment that targets CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0293] In certain embodiments, the payload is an antibody or antibody fragment that targets CD27, such as varylumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0294] In a particular embodiment, the payload is a CD40L-targeting antibody or antibody fragment, such as dapyrolizumab, or an antibody fragment derived therefrom.

[0295] In certain embodiments, the payload is an antibody or antibody fragment that targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0296] In a particular embodiment, the payload is an antibody or antibody fragment that targets BAFFR, such as ianarumab, or an antibody fragment derived therefrom.

[0297] In a particular embodiment, the payload is an antibody or antibody fragment that targets LFA-1, such as efalizumab, or an antibody fragment derived therefrom.

[0298] In certain embodiments, the payload is an antibody or antibody fragment that targets BTLA, such as icatrimab, or an antibody fragment derived therefrom.

[0299] In some embodiments, the payload is an anti-CD3(αCD3) monoclonal antibody, or a derivative or analog thereof. In some embodiments, the anti-CD3(αCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative or analog thereof.

[0300] In some embodiments, at least one payload is selected from poly(ADP-ribose) polymerase (PARP), duocalmycin, pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, anti-CD3 (αCD3) monoclonal antibody, lurubinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, and inhibitors of MMAE, or derivatives or analogs thereof.

[0301] In some embodiments, D is a payload selected from poly(ADP-ribose) polymerase (PARP), duocalmycin, pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, and inhibitors of anti-CD3 (αCD3) monoclonal antibodies, or derivatives or analogs thereof.

[0302] In some embodiments, at least one payload is selected from lurubinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, seco-duocalmycin SA, and MMAE, or derivatives or analogs thereof.

[0303] In some embodiments, the payload is a poly(ADP-ribose) polymerase (PARP) inhibitor, derivative thereof, or analogue. In some embodiments, the poly(ADP-ribose) polymerase inhibitor (PARP inhibitor) is niraparib, talazoparib, olaparib, pamiparib, lucaparib, veliparib, iniparib, 3-aminobenzamide, CEP-9722, E7016, or derivative thereof or analogue.

[0304] In some embodiments, the payload is [ka] That is the case.

[0305] In some embodiments, the payload is duocalmycin, or a derivative or analog thereof. In some embodiments, duocalmycin is duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, CC-1065, adzeresin, karzeresin, bizeresin, or a derivative or analog thereof.

[0306] In some embodiments, the payload is [ka] [ka] Includes.

[0307] In some embodiments, the payload comprises pyrrolobenzodiazepine (PBD), or a derivative or analog thereof. In some embodiments, pyrrolobenzodiazepine (PBD) is [1,2]diazepino[3,4-e]indole, or a derivative or analog thereof.

[0308] In some embodiments, the payload is [ka] Includes.

[0309] In some embodiments, the payload is a tubulin polymerization inhibitor. In some embodiments, the payload is hemiasterlin, HTI-286, or a derivative or analog thereof.

[0310] In some embodiments, the payload is [ka] Includes.

[0311] In some embodiments, the payload is [ka] Includes.

[0312] In some embodiments, the payload is a topoisomerase inhibitor. In some embodiments, the payload includes camptothecin, or its derivatives or analogs. In some embodiments, the payload includes topotecan, irinotecan, siratecan, cocitecan, exatecan, lurtotecan, gimatecan, berotecan, or rubitecan.

[0313] In some embodiments, the payload is [ka] Includes.

[0314] In some embodiments, the payload is [ka] Includes.

[0315] In some embodiments, the payload is [ka] Includes.

[0316] In some embodiments, the payload is [ka] Includes.

[0317] In some embodiments, the payload is [ka] Includes.

[0318] In some embodiments, the payload is [ka] Includes.

[0319] In some embodiments, the payload is [ka] Includes.

[0320] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the linker L 1 It is covalently bonded to a lysine, serine, threonine, or tyrosine residue present on the payload. In some embodiments, the polypeptide contains one or more lysine residues. In some embodiments, the linker L 1 It is covalently bonded to lysine residues present on the payload.

[0321] In some embodiments, the payload includes an N-terminal amino acid and a linker L 1 It is covalently bonded to the N-terminal amino acid.

[0322] In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1 to 5.

[0323] In some embodiments, the payload is an immunomodulatory agent payload.

[0324] In some embodiments, the immunomodulatory agent payload is an antibody payload.

[0325] In some embodiments, the immunomodulatory agent payload is an immune checkpoint inhibitor payload. In some embodiments, the immune checkpoint inhibitor payload is a payload of pizilizumab, cintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, TIGIT inhibitors (e.g., LAG-3 such as an anti-LAG-3 antibody, TIM-3 such as an anti-TIM-3 antibody), B7 molecules, or BTLA pathway antagonists.

[0326] In some embodiments, the immune checkpoint inhibitor payload is an immune checkpoint inhibitor antibody payload. In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is nivolumab, pembrolizumab, pizilizumab, cintilimab, or AMP-224 payload.

[0327] In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload. In some embodiments, the PD-L1 inhibitor payload is atezolizumab, avelumab, durvalumab, or BMS-936559 payload.

[0328] In some embodiments, the immune checkpoint inhibitor antibody payload is a CTLA4 inhibitor payload. In some embodiments, the CTLA4 inhibitor payload is an ipilimumab or tremelimumab payload.

[0329] In some embodiments, the immune checkpoint inhibitor payload is an indoleamine 2,3-dioxygenase (IDO) inhibitor payload. In some embodiments, the IDO inhibitor payload is indoximod or epacadostat payload.

[0330] In some embodiments, the immunomodulatory payload is a cytokine payload.

[0331] In some embodiments, the cytokine payload is interferon, interleukin, tumor necrosis factor, erythropoietin, MIP3a, ICAM, macrophage colony-stimulating factor, erythropoietin (EPO), granulocyte colony-stimulating factor (GCSF), or granulocyte-macrophage colony-stimulating factor payload.

[0332] In some embodiments, the interleukin payload is selected from IL-1 to IL-40. In some embodiments, the interleukin payload is IL-2, IL-7, IL-12, IL-15, IL-18, or IL-21.

[0333] In some embodiments, the immunomodulatory payload is type 1 cytokines (IL-2, IL-12, TNF-B, IFN-γ).

[0334] In some embodiments, the cytokine payload includes IFN-alpha, IFN-beta, IFN-gamma, PEGylated IFN-α, and apolipoprotein AI fusion proteins having IFN-α, interleukin, IL-2, IL-2 covalently bound to immunoglobulin (e.g., sergutuzumabu amnaleukin, RO6874281), IL-2 covalently bound to PEG molecules (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegyrodecakin), IL-7, IL-12, IL-15, recombinant aglycosylated IL-15, IL-15 fusion proteins having an IL-15Rα binding domain (e.g., RLI), a triple fusion protein containing human IL-15, an IL-15Rα binding domain, and apolipoprotein AI, ALT-803 (IgG1 The group selected consists of IL-15, IL-18, IL-21, tumor necrosis factor, TNF-alpha, TNF-beta, erythropoietin (EPO), MIP3a, ICAM, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), GM-CSF, and talimogene laherparepvec.

[0335] In some embodiments, the immunomodulatory agent payload is a chemokine payload.

[0336] In some embodiments, the chemokine payload is a CCL27, CCL28, CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, or CXCL14 payload.

[0337] In some embodiments, the immunomodulatory payload is a chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0338] In some embodiments, the immunomodulator is a monoclonal antibody specific to a cytokine or cytokine receptor.

[0339] In some embodiments, the immunomodulatory payload includes a polypeptide.

[0340] In some embodiments, the polypeptide comprises one or more lysine residues.

[0341] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0342] In some embodiments, trans-cyclooctene is ligated to one or more lysine residues.

[0343] In some embodiments, trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0344] In some embodiments, the polypeptide contains an N-terminal amino acid, and the appearance of the bioorthogonal moiety is linked to the N-terminal amino acid.

[0345] In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1 to 5. In some embodiments, m is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, m is 1.

[0346] In some embodiments, the trans-cyclooctene functionalized prodrug of formula XI, [ka] or a pharmaceutically acceptable salt thereof, in the formula, R in each appearance 1a Hydrogen and C are independent of each other.1-4 Alkyl, and C 1-4 Selected from the group consisting of haloalkyls, R in each appearance 1b Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C(O)OH, C(O)OC 1-4 Alkyl, C(O)N(R) 1c )CHR 1e CO2H, C(O)N(R) 1c )CHR 1e C(O)OC 1-4 Alkyl, C(O)N(R) 1c )-C 1-6 Alkylene-CO2H and C(O)N(R) 1c )-C 1-6 Alkylene-C(O)OC 1-4 Selected from the group consisting of alkyl groups, R in each appearance 1c These are independently hydrogen or C 1-4 It is alkyl, R in each appearance 1e -C 1-4 Alkylene-CO2H, -C 1-4 Alkylene-CONH2, or -C 1-4 It is alkylene-OH, In each occurrence, D is independently a payload. L in each appearance 1 It is independently a linker, In each occurrence, p' is independently 0, 1, or 2. In each occurrence, p'' is independently 1, 2, or 3.

[0347] In some embodiments, D in each occurrence is independently selected from the group consisting of anticancer drug payloads, Toll-like receptor (TLR) agonist payloads, and interferon gene stimulator (STING) agonist payloads.

[0348] In some embodiments, R 1a It is hydrogen.

[0349] In some embodiments, R 1a C 1-4 It is alkyl.

[0350] In some embodiments, R 1a This is CH3.

[0351] In some embodiments, R 1b These are C(O)OH and C(O)OC 1-4 Alkyl, C(O)N(R) 1c )CHR 1e CO2H, C(O)N(R) 1c )CHR 1e C(O)OC 1-4 Alkyl, C(O)N(R) 1c )-C 1-6 Alkylene-CO2H and C(O)N(R) 1c )-C 1-6 Alkylene-C(O)OC 1-4 Selected from the group consisting of alkyl groups.

[0352] In some embodiments, R 1b C(O)OH, C(O)N(R) 1c )CHR 1e CO2H and C(O)N(R) 1c Selected from the group consisting of CH2CO2H.

[0353] In some embodiments, R 1b -NR 1c -CH2CH2-N(CH3)3 + , -N(R 1c )-CH2CH2-SO3H,-N(R 1c )-(CH2CH2O)3-CH2CH2N((CH2CH2O)3-CH2CH2-CO2H)2, and -N(R 1c It is selected from the group consisting of )-CH(CH2O-CH2CH2-CO2H)2.

[0354] In some embodiments, the trans-cyclooctene portion (G) is [ka] That is the case.

[0355] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0356] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0357] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0358] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0359] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0360] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0361] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0362] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0363] In some embodiments, the trans-cyclooctene portion is [ka] That is the case.

[0364] In some embodiments, the trans-cyclooctene portion is [ka] And R 2 R is -OH, 2-aminoethanesulfonic acid, N-linked natural or unnatural amino acids, or optionally substituted ethylenediamines, 2 This can optionally be further substituted with a polyether.

[0365] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0366] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0367] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0368] In some embodiments, the trans-cyclooctene portion is [ka] Includes.

[0369] In some embodiments, R 1e These are -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0370] In some embodiments, R 1e is -C 1-4 It is alkylene-CO2H.

[0371] In some embodiments, R 1e It is -CH2CO2H.

[0372] In some embodiments, R 1b is -C(O)N(R 1c )-C 1-6 It is alkylene-CO2H.

[0373] In some embodiments, R 1b is -C(O)N(R 1c It is CH2CO2H.

[0374] In some embodiments, R 1c It is hydrogen.

[0375] In some embodiments, R 1b It is hydrogen.

[0376] In some embodiments, R 1b It is C(O)OH.

[0377] In some embodiments, linker L 1It may have 1 to 100 linked atoms and may contain ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, a linker may have 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0378] In some embodiments, linker L 1 It may contain one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety independently and optionally oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

[0379] In some embodiments, linker L 1 This may also be the following: -Y 10 -(CH2) n’ -Y 20 -(CH2) m” -Y 30 - During the ceremony, Y 10 , Y 20 , and Y 30 Each of them is independent, combined, -NR 110 -, -O-, -S(O) 0-2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene can be independently and optionally oxo, halo, or C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. n' and m'' are independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0380] In a particular embodiment, the linker is a coupling.

[0381] In certain embodiments, the linker is not a link. In certain embodiments, each R 110 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 Haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, each R 120 Hydrogen and C are independent of each other. 1-4 Alkyl, C 1-4 They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

[0382] Representative linkers, though not limited to these, include the following: [ka]

[0383] Representative linkers, though not limited to these, include the following: [ka]

[0384] In some embodiments, linker L 1 This may include one or more of the following: polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diyrbis(methylcarbamate), arylene (ee, phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, linker L 1 teeth, [ka] It may include.

[0385] In some embodiments, linker L 1 This may contain one or more natural or non-natural amino acids, which may be called peptide linkers. If drug (D) contains an amino moiety, the linker may be attached to it using peptide linkers consisting of a carboxylacyl unit and one or more amino acids that constitute a protein or peptide sequence. In some embodiments, linker L 1 It may also contain self-destructing spacers that provide spacing between drug and protein peptide sequences.

[0386] In some embodiments, linker L 1The linker may be a peptide linker represented as "AYZQW", where "A" is a carboxylacyl unit, "Y" and "Z" are one or more natural or non-natural amino acids, together forming a peptide sequence, and "Q" and "W" are optional additional linkers having 1 to 50 linking atoms, or 5 to 10 linking atoms, or 1 to 10 linking atoms, spacing the peptide and drug, D, or bioorthogonal portion. In certain embodiments, one or more of the amino acids in the peptide linker are N-methylated.

[0387] In some embodiments, Y may be at least one amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In some embodiments, Y may be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0388] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, acetyl or formyl-protected lysine, arginine, tosyl or nitro-protected arginine, histidine, ornithine, acetyl or formyl-protected ornithine, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0389] In some embodiments, exemplary YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0390] In a particular embodiment, A is -OC(O)-.

[0391] In a particular embodiment, Q is -OC(O)-.

[0392] In a particular embodiment, W is -OC(O)-. In a particular embodiment, Q is absent, and W is -OC(O)-.

[0393] In a particular embodiment, -QW is, [ka] That is the case.

[0394] In a particular embodiment, -QW is, [ka] That is the case.

[0395] In certain embodiments, the peptide linker is specifically modified to be selectively cleaved (e.g., enzymatically cleaved) to release a drug, such as by one or more tumor-associated proteases.

[0396] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., dipeptide, tripeptide, or tetrapeptide). However, it will be understood that peptide linkers with up to 5, 6, 7, or 8 amino acid residues can also be suitably used.

[0397] In a particular embodiment, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly[sequence number], Ala-Leu-Ala-Leu[sequence number], Phe-N 9 -Tosyl-Arg, or Phe-N 9It is -nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Val-Ala, or Ala-Ala.

[0398] In a particular embodiment, linker L 1 teeth, [ka] That is the case.

[0399] The aforementioned linker is the amino acid side chain of D, such as lysine or cysteine ​​(for example, [ka] It can be attached to the right side.

[0400] In some embodiments, the payload is covalently bonded to the linker via an amide bond, and for example, the payload may be an amine-containing payload for bonding the payload to the carbonyl group of the linker, or in other cases, the payload may be a carboxyl-containing payload for bonding the payload to the amine group of the linker. In some examples, the payload and the linker together form a carbamate group, and for example, the payload may be an amine-containing payload for bonding the payload to the acyloxy group of the linker. In some examples, the payload and the linker together form a carbonate group, and for example, the payload may be a hydroxyl-containing payload for bonding the payload to the acyloxy group of the linker.

[0401] In some embodiments, L 1 teeth, [ka] or -O-, L 3a is a combination or C 1-6 It is alkylene, L 4a is a combination, -NHN:, -N(R 10 )-C 2-6 Alkylene-N(R) 11 )-,-N(R 12 )-C 2-3 Alkylene-N(R) 13 )C(O)-, -N(R 10 )-C 1-6 Alkylene-C(O)NHN:,-NHNHC(O)C 1-6 Alkylene-C(O)NHN:,-CH(NHC(O)R 14 )C 1-4 Alkylene-SSC 1-4 Alkylene-OC(O)-,-NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1-6 Alkylene-CH(G x )OC(O)-, [ka] And, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 19 Each of them independently consists of hydrogen or C 1-4 It is alkyl, R 16 is hydrogen, C 1-4 Alkyl, -C 1-4 Alkylene-OH,-C 1-4 Alkylene-OC 1-4 Alkyl, -C 1-4 Alkylene-CO2H, or -C 1-4 It is alkylene-CONH2, R in each appearance 17 These are independently hydrogen or -CH2OC(O)-, G x The following are optional choices: halogen, C 1-4 Alkyl, C 1-4 Haloalkyl, C1-4 This is a phenyl compound substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0402] In a particular embodiment, linker L 1 It is -OC(O)-.

[0403] In some embodiments, L 1 teeth, [ka] And, L 3a It is a combination, L 4a teeth, [ka] And, R 12 and R 13 Each of them independently consists of hydrogen or C 1-4 It is alkyl.

[0404] In some embodiments, p'' is 1. In some embodiments, p' is 1.

[0405] In some embodiments, [ka] [ka] And, R in each appearance 18 These are independently hydrogen or -CH2OC(O)NHD', R D This refers to hydrogen or C on the nitrogen atom of the payload. 1-4 It is alkyl, D and D' are independently payload portions.

[0406] In some embodiments, D or D' is a cyclic dinucleotide payload portion, an imidazo[4,5-c]quinoline-4-amine payload portion, a TLR agonist payload portion, a STING agonist payload portion, or an anticancer drug payload portion.

[0407] In some embodiments, [ka] And, R 12 and R 13 Each of them independently consists of hydrogen or C 1-4 It is alkyl, D and D' are independently payload portions (e.g., anticancer drug payload portions).

[0408] In some embodiments, p' is 0.

[0409] In some embodiments, p'' is 2 or 3.

[0410] In some embodiments, p is 2, [ka] That is the case.

[0411] Those skilled in the art will recognize that the payload (D or D') bound to the linker does not refer to the payload molecule itself, but rather to a portion of the payload molecule bound to the linker. The release of the payload (D or D') from the prodrug is the release of the payload itself.

[0412] The payload (D or D') may be any of the anticancer agents described herein.

[0413] In some embodiments, the payload includes a TLR7 / 8 agonist. In some embodiments, the payload includes garzikimod.

[0414] In some embodiments, the payload includes camptothecin or a derivative thereof. In some embodiments, the payload includes exatecan.

[0415] In some embodiments, the payload includes MMAE or a derivative thereof.

[0416] In some embodiments, the payload includes paclitaxel or a derivative thereof.

[0417] In some embodiments, the payload includes docetaxel or a derivative thereof.

[0418] In some embodiments, the payload has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It holds.

[0419] In a particular embodiment, the trans-cyclooctene functionalized prodrug is as follows: [ka] [ka] Selected from.

[0420] E. Treatment method Aspects of this disclosure include methods for delivering a payload to a target location in an object. In certain embodiments, the method includes selectively delivering a payload to a target location in an object. Selective delivery of a payload includes delivering the payload to a target location (e.g., an organ or tissue or part thereof) without targeting other locations in the object (e.g., other organs or tissues or parts thereof) that do not require the administration of the payload. Selective delivery of a payload can be achieved by using tetrazine-based targeting agents and functionalized payloads described herein.

[0421] In some examples, the tetrazine-based targeting agents of this disclosure may be localized to a desired target site in a subject. For example, the method of this disclosure may include administering a tetrazine-based targeting agent described herein to a subject. The tetrazine-based targeting agent may be administered to the subject at a desired target site in the subject. In some examples, the tetrazine-based targeting agent may be injected topically into the subject at a desired target site in the subject. In some embodiments, the tetrazine-based targeting agent is administered systemically. In these embodiments, the tetrazine-based targeting agent may be localized to a desired target site in the subject by specific binding of the tetrazine-based targeting agent to its target (e.g., antibody-antigen interaction), or it may be localized to the surface of a desired target (e.g., a cell surface) by specific binding of the tetrazine-based targeting agent to its target (e.g., antibody-antigen interaction).

[0422] As described herein, selective binding can occur between bioorthogonal binding partners (e.g., between tetrazine in a tetrazine-based targeting agent and its complementary trans-cyclooctene in a prodrug). Upon administration of the tetrazine-based targeting agent and the resulting localization to a desired location in the target, the selective binding of tetrazine and its complementary binder to trans-cyclooctene on the prodrug localizes the payload to the desired target location.

[0423] A method for treating cancer is provided herein, comprising administering a therapeutically effective amount of a tetrazine-based targeting agent described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctempordrug to a subject in need of cancer treatment.

[0424] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal cancer / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

[0425] In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0426] In some embodiments, cancer is a solid tumor.

[0427] In some embodiments, the cancer is a soft tissue sarcoma.

[0428] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0429] In some embodiments, the method also includes enhancing or inducing an immune response. In some embodiments, the immune response is an increase in one or more of the following: leukocytes, lymphocytes, monocytes, and eosinophils.

[0430] In some embodiments, the method further comprises administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctenprodrugs thereof. Anticancer agents, immunomodulators, and trans-cyclooctenprodrugs thereof are known in the art.

[0431] The indications for this approach include both hematological and solid tumors. In certain embodiments, this approach can be used for the treatment and / or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all different subtypes of soft tissue sarcomas and osteosarcomas. The composition may be for the treatment and / or diagnosis of pigmented villonodular synovitis.

[0432] In certain embodiments, the approach can be used to treat and / or diagnose hematological malignancies such as myelodysplastic syndrome, acute myeloid leukemia, chronic myelodisplastic syndrome, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammaglobulinemia, plasmacytomyeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin lymphoma, and others.

[0433] The compositions of this disclosure find use in the treatment and / or diagnosis of a condition or disease in a subject who may be treated or diagnosed by administration of a payload (e.g., a parent drug (i.e., a drug before conjugation into the composition)). "Treatment" means that at least improvement of the symptoms associated with the condition afflicting the subject is achieved, where improvement is used in a broad sense to mean at least a reduction of a parameter associated with the condition being treated, e.g., the severity of the symptoms. Thus, treatment includes situations in which a pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) so that the subject no longer suffers from that condition, or at least the symptoms that characterize that condition. Treatment may include inhibition, i.e., prevention of the onset or further onset of clinical symptoms, e.g., alleviation or complete inhibition of an active disease. Treatment may include alleviation, i.e., causing a regression of clinical symptoms. For example, in the context of cancer, the term “treating” includes any or all of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing overall tumor volume, extending survival time, and improving one or more cancer-related symptoms.

[0434] The subject to be treated may be any subject in need of therapy, and hereby the subject to be treated is any subject that can be treated with the parent drug. Thus, a variety of subjects may be treated using the compositions disclosed herein. Generally, such subjects are “mammals,” and humans are among the subjects. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., animal models of diseases), and non-human primates (e.g., chimpanzees and monkeys).

[0435] In certain embodiments, additional therapeutic agents and methods, though not limited to these, can be used for the treatment, prevention, and / or diagnosis of solid tumors including, but not limited to, melanoma (e.g., unresectable metastatic melanoma), kidney cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), endometrial cancer, breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal cancer / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid cancer, pancreatic neuroendocrine tumors, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma. The disclosed approach is suitable for use as an adjuvant / neoadjuvant system. For example, the particles disclosed herein can be placed during a biopsy, and upon return of the study results, the physician can deliver the appropriate cocktail to the desired site in the body. This would minimize tumor size, particularly in the case of surgically resectable tumors. Then, at the end of surgery, the surgeon can administer additional tetrazine-based targeted agents to target the surgical cavity and treat the patient with further doses of therapy (e.g., chemotherapy using the disclosed approach) to minimize the risk of any cancer cells that might have been missed at the resection margin.

[0436] In certain embodiments, the tetrazine-based targeted agents disclosed herein can be administered, allowing a physician to deliver the appropriate cocktail to a desired site in the body. This would minimize tumor size, particularly in the case of surgically resectable tumors. Then, at the end of surgery, the surgeon can administer additional tetrazine-based targeted agents to target the surgical cavity and treat the patient with further doses of therapy (e.g., chemotherapy using the disclosed approach) to minimize the risk of any cancer cells that might have been missed at the resection margin.

[0437] In certain embodiments, the disclosed method provides the ability to position particles as disclosed herein at the time of biopsy. Upon return of the results, the physician can deliver the immunomodulator to the biopsy site.

[0438] In certain embodiments, the disclosed method provides a physician's ability to deliver immunomodulatory agents such as TLR agonists, STING agonists, chemokines (agents that attract cancer cells and / or immune cells), and adjuvants, as well as chemotherapeutic agents in combination with immunotherapeutic agents, to enhance the immune system with fewer side effects. This combination approach would be beneficial to the patient. The chemotherapeutic agent would treat a solid tumor or a specific site, while the enhanced response of the immunotherapy would help distant metastatic sites. For example, in certain embodiments, the disclosed composition and method can enhance the efficacy of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer) using anthracyclines, taxanes, gemcitabine, and other agents.

[0439] cancer The methods disclosed may be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include persistent growth signaling, evasion of growth inhibitors, resistance to cell death, activation of replication immortality, induction of angiogenesis, and activation of invasion and metastasis. The methods disclosed may enhance or induce an immune response against cancer in a subject. The immune response may result in an increase in one or more of the following: leukocytes, lymphocytes, monocytes, and eosinophils.

[0440] Cancers that can be treated by the disclosed methods include, but are not limited to, astrocytoma, adrenocortical carcinoma, appendiceal cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, brainstem cancer, brainstem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse endogenous pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, and gastric cancer. Cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, mesothelioma, oral cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, solid tumor, squamous cell carcinoma, stomach cancer This includes cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, non-small cell lung cancer (NSCLC), diffuse large B-cell lymphoma (DLBCL), or oral-lingual squamous cell carcinoma (OTSCC).

[0441] In some embodiments, the cancers that can be treated by the disclosed method are melanoma, kidney cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the cancer is a non-fibrosarcoma. In some embodiments, the cancer is a diffuse endogenous pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0442] In some embodiments, cancers that can be treated by the disclosed methods include hematological malignancies such as myelodysplastic syndrome, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammaglobulinemia, plasmacytomyeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin lymphoma, and others.

[0443] While not constrained by any particular theory, local release of certain anticancer agents using the compounds and methods of this disclosure may induce or contribute to immunogenic cell death (ICD). For example, certain anticancer agents (e.g., anthracyclines, cyclophosphamide, oxaliplatin) have been reported to induce ICD. Kroemer et al. Annu. Rev. Immunol. 2013(31), 51-72. Immunogenic apoptosis of cancer cells can induce an effective antitumor immune response by activating dendritic cells (DCs) and, consequently, certain T cell responses. ICD is characterized by the secretion of damage-associated molecular patterns (DAMPs). Three key DAMPs are exposed on the cell surface during ICD. Calreticulin (CRT), one of the DAMP molecules normally located in the lumen of the endoplasmic reticulum (ER), translocates after inducing immunogenic apoptosis on the surface of dead cells, where it functions as an "eat me" signal to professional phagocytic cells. Other important surface-exposed DAMPs are heat shock proteins (HSPs), namely HSP70 and HSP90, which are also under stress conditions that translocate to the cell membrane. On the cell surface, they have immunostimulatory effects based on their interaction with several antigen-presenting cell (APC) surface receptors such as CD91 and CD40, and also promote the cross-presentation of antigens derived from tumor cells on MHC class I molecules, which then leads to a CD8+ T cell response. Other important DAMPs characteristic of ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered a late apoptosis marker, and its release into the extracellular space appears to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) 2 and 4, which are expressed on APCs. The most recently discovered DAMP released during immunogenic cell death is ATP, which, when secreted, acts as a "find-me" signal for monocytes, inducing their attraction to apoptotic sites. Kroemer et.al.Curr.Op.Immunol.2008(20),504-511.

[0444] Therefore, the local release of ICD-inducing substances using the compounds and methods of this disclosure can be beneficially combined with one or more immunomodulators.

[0445] In certain embodiments, tetrazine-based targeting agents can be used for the treatment, prevention, and / or diagnosis of solid tumors including, but are not limited to, melanoma (e.g., unresectable metastatic melanoma), kidney cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer such as metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, and pancreatic cancer.

[0446] The disclosed approach is suitable for use as an adjuvant / neoadjuvant system. For example, the targeted portion disclosed herein can be placed during a biopsy, and upon return of the study results, the physician can administer an appropriate cocktail to deliver the treatment to the desired site in the body (the compounds disclosed herein and any additional therapeutic agents(s) of their choice). The biopsy results may indicate the amount and type of treatment to be delivered to the site of the tumor. For example, chemokines (agents that attract cancer cells and / or immune cells) and adjuvants to enhance the immune system with fewer side effects, as well as chemotherapy agents, can be delivered and combined with immunotherapeutic agents.

[0447] The disclosed method may include one or more systemic doses of a targeted portion focusing on one or more locations. The disclosed method may be used to deliver a functionalized payload to these locations by systemic or topical administration. In some embodiments, the tetrazine-based targeting agent is delivered systemically. In some embodiments, both the tetrazine-based targeting agent and the payload (i.e., the TCO-labeled payload) are delivered systemically. In some embodiments, the tetrazine-based targeting agent is delivered topically.

[0448] The disclosed compounds and compositions may be administered before surgical resection. The disclosed methods may minimize tumor size before surgical resection. This will minimize tumor size, particularly in the case of surgically resectable tumors. The disclosed conjugates, compounds, and compositions may be administered during surgical resection. The disclosed conjugates, compounds, and compositions may be administered after surgical resection. A tetrazine-based targeting agent may be placed around the surgical cavity at the end of surgical resection, and the target may then be treated with additional doses to minimize the risk of any cancer cells that may have been missed at the resection margin.

[0449] The disclosed method may include multiple systemic doses of a functionalized payload focusing on a single site. The disclosed method may be used to deliver a second payload. If the tumor is resistant to the first payload, the disclosed method may be used to administer the second functionalized payload. The second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by a tetrazine-based targeting agent used in the first prodrug.

[0450] The functionalized payloads disclosed herein may function as adjuvants. This combination approach may be beneficial to patients. While chemotherapeutic agents may treat solid tumors or specific sites and enhance or induce immune responses, the enhanced response of immunotherapy with functionalized payloads and / or separate agents may aid distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods may enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C; Pfizer) using anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogs, and other agents.

[0451] The disclosed method may be used to treat diffuse endogenous pontine glioma. Diffuse endogenous pontine glioma (DIPG) is a pediatric brainstem tumor that can be highly malignant and difficult to treat. There is no known treatment for DIPG, and survival rates have remained bleak for the past 40 years. Patients with DIPG have a median overall survival of only 11 months, and a 2-year survival rate of less than 10%. DIPG accounts for 75–80% of pediatric brainstem tumors, affecting an estimated 200–300 children each year in the United States. The rarity of this devastating disease and the lack of experimental model systems to date have hindered research, and survival rates have remained the same for the past 40 years. Diagnosis of DIPG may begin with clinical symptoms and may be confirmed by MRI. The disease may begin with several months of systemic symptoms, including behavioral changes and school difficulties, diplopia, abnormal or limited eye movements, asymmetrical smiles, loss of balance, and weakness. Alternatively, severe neurological deterioration may occur more rapidly, with symptoms present for less than a month before diagnosis. Clinical examination may reveal polyneuropathy, cord signs such as hyperreflexia and clonus, and the triad of ataxia. Dilatation of the pontine portion of the brainstem can lead to obstructive hydrocephalus and increased intracranial pressure.

[0452] The nuclei essential for life-sustaining functions such as respiration and heart rate are located in the pons, and without treatment, DIPG can impair respiration and heart rate.

[0453] The disclosed method may include multiple systemic doses of a functionalized payload focusing on a single site. The disclosed method may be used to deliver a second payload. If the tumor is resistant to the first payload, the disclosed method may be used to administer the second functionalized payload. The second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by a tetrazine-based targeting agent used in the first prodrug.

[0454] Mode of administration The treatment method may include any number of forms of administration of the disclosed conjugate, compound, or composition. The forms of administration may include tablets, pills, sugar-coated tablets, hard and soft gel capsules, granules, pellets, skin patches, skin creams, skin gels, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixirs, solid emulsions, solid dispersions, or dispersible powders. In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein may also be dispersed in microparticles, such as nanoparticle compositions.

[0455] For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent such as water, a buffer, oil with or without a solubilizer, a surfactant, a dispersant, or an emulsifier. Suitable oils include, for example, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil, and sesame oil. For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be administered in the form of aqueous, lipid, oily, or other types of solutions or suspensions, or in the form of liposomes or nanosuspensions.

[0456] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injections and infusions.

[0457] The amount of composition administered to a subject can be initially determined based on the dose of the parent drug and / or the guidance of the administration regimen. Generally, the composition can provide targeted delivery and / or an enhanced serum half-life of the conjugated drug, and therefore can provide at least one of a reduced dose or reduced administration in the administration regimen. Thus, the composition can result in a reduced dose and / or reduced administration in the administration regimen compared to the parent drug before conjugation in the composition of this disclosure.

[0458] Pharmaceutical formulations may be provided in unit dosage forms. In such forms, the pharmaceutical formulation may be subdivided into unit doses containing an appropriate amount of the composition of the present disclosure. The unit dosage forms may be packaged formulations, and the packaging may contain discrete amounts of formulations such as packetized tablets, capsules, and powder in pouches, vials, or ampoules.

[0459] In some embodiments, a kit is provided comprising a tetrazine-based targeting agent described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, and instructions for use thereof.

[0460] In some embodiments, the kit further includes a prodrug.

[0461] The compositions of this disclosure may be present in any preferred amount and may depend on a variety of factors, including but not limited to the subject's weight and age, disease state, etc. Preferred dosage ranges for the compositions of this disclosure include 0.1 mg to 10,000 mg, or 1 mg to 1,000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, preferred dosages of the compositions of this disclosure include 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg.

[0462] In some embodiments, multiple doses of the composition are administered. The frequency of administration of the composition can vary depending on any of several factors, such as the severity of the symptoms or the condition of the subject. For example, in some embodiments, the composition is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0463] The compositions of the Disclosure may be administered at any preferred frequency, interval, and duration. For example, the compositions of the Disclosure may be administered once per hour, or twice, three times, or more times per hour, once per day, or two, three times, or more times per day, or once every two, three, four, five, six, or seven days, in order to provide a subject with a desired dose level. When the compositions of the Disclosure are administered more than once per day, typical intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes, as well as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours. The compositions of this disclosure may be administered once, twice, three times, or more times, over intervals of 1 hour, 1 to 6 hours, 1 to 12 hours, 1 to 24 hours, 6 to 12 hours, 12 to 24 hours, 1 day, 1 to 7 days, 1 week, 1 to 4 weeks, 1 month, 1 to 12 months, more than 1 year, or indefinitely.

[0464] The compositions of this disclosure may be co-administered with other activators. Co-administration includes administering the compositions of this disclosure and the activators within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours from each other. Co-administration also includes administering the compositions of this disclosure and the activators simultaneously or nearly simultaneously (for example, within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes from each other), or sequentially in any order. Furthermore, each of the compositions of this disclosure and the activators may be administered once daily, or two, three, or more times daily, to provide a desired daily dose level.

[0465] Co-administration can be achieved through co-transplantation or co-injection.

[0466] In some embodiments, co-administration can be achieved by co-formulation, for example, by preparing a single pharmaceutical formulation containing both the composition and the activator of the Disclosure. In other embodiments, the composition and the activator of the Disclosure can be formulated separately and co-administered to a subject.

[0467] The compositions and activators of this disclosure may be present in formulations in any suitable weight ratio, for example, 1:100 to 100:1 (w / w), or 1:50 to 50:1, or 1:25 to 25:1, or 1:10 to 10:1, or 1:5 to 5:1 (w / w). The compositions and other activators of this disclosure may be present in any suitable weight ratio, such as 1:100 (w / w), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (w / w). Other dosages and dosage ratios of the compositions and activators of this disclosure are preferred in formulations and methods described herein.

[0468] Combination therapy In one embodiment, a method is provided for treating cancer or enhancing or inducing an immune response, the method comprising administering to a subject in need a therapeutically effective amount of a tetrazine-based targeting agent of the present disclosure or a pharmaceutically acceptable salt or composition thereof; and a prodrug such as those described herein; and optionally, a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctene prodrugs thereof.

[0469] This disclosure also provides pharmaceutically acceptable combinations comprising tetrazine-based targeting agents described herein, or pharmaceutically acceptable salts thereof, or compositions thereof, for use in the treatment or prevention of cancer, or for use in enhancing or inducing an immune response; prodrugs described herein; and optionally, additional therapeutic agents selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctene prodrugs thereof.

[0470] This disclosure also provides the use of tetradine-based targeting agents described herein, or pharmaceutically acceptable salts or compositions thereof; prodrugs such as those described herein; and optionally, a therapeutically effective amount of additional therapeutic agents selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctene prodrugs thereof, for use in the treatment or prevention of cancer, or in enhancing or inducing an immune response, in a pharmaceutically effective amount.

[0471] In the methods and uses described herein, the components of a pharmaceutically acceptable combination may be administered / used simultaneously, separately, sequentially, and in any order, and the components may be administered separately or as a fixed combination. For example, disease progression or delay of treatment according to this disclosure may include the administration of a first active ingredient in the form of a free salt or pharmaceutically acceptable salt, simultaneously or sequentially in any order, in a joint therapeutic effective dose or effective dose, e.g., a daily dose corresponding to the amount described herein. The individual active ingredients of a combination may be administered separately at different times during the course of treatment, or simultaneously in divided or single dosage forms. Accordingly, this disclosure should be understood to encompass all such regimens of simultaneous or alternating treatment, and the term “administer” should be interpreted accordingly. Accordingly, as used herein, a pharmaceutically acceptable combination defines either a fixed combination of one unit dosage form or separate dosage forms for concomitant administration, and concomitant administration may be independently, simultaneously, or at different times. As a further example, tetrazine-based targeting agents (or therapeutic tetrazine-based targeting agents) and prodrugs may be administered simultaneously (e.g., via co-injection or co-transplantation), separately, or sequentially, followed by the administration of additional therapeutic agents selected from the group consisting of anticancer agents, their immunomodulators, or trans-cyclooctemin prodrugs.

[0472] Methods and uses in the treatment of cancer include the administration / localization of tetrazine-based targeting agents to tumors. In the methods and uses disclosed herein, the administration of prodrugs, or pharmaceutically acceptable salts or compositions thereof; tetrazine-based targeting agents; and optionally additional therapeutic agents may inhibit tumor growth.

[0473] Additional therapeutic agents may be administered simultaneously with or sequentially to the disclosed conjugates and compositions. Sequential administration includes administration before or after the disclosed conjugates and compositions. Additional therapeutic agents may be administered before the disclosed conjugates and compositions. Additional therapeutic agents may be administered after the disclosed conjugates and compositions. Additional therapeutic agents may be administered simultaneously with the disclosed conjugates and compositions. In some embodiments, the additional therapeutic agent may be administered in the same composition as the disclosed conjugates. In other embodiments, there may be a time interval between the administration of the additional therapeutic agent and the administration of the disclosed conjugate or composition. In some embodiments, administration of the additional therapeutic agent with the disclosed conjugate or composition may allow for lower doses and / or less frequent administration of the other therapeutic agents. When used in combination with one or more other active ingredients, the conjugates or compositions and other active ingredients of this disclosure may be used in lower doses than when each is used alone. Accordingly, the pharmaceutically acceptable compositions of this disclosure include those containing one or more other active ingredients in addition to the conjugates of this disclosure.

[0474] anticancer drugs Examples of anticancer drugs, though not limited to these, include abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Aldara (imiquimod), and Aldeslo Ikin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), asparaginase, erwinia chrysanthemum, Avastin (bevacizumab), axitinib, azacitidine, BEA COPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and I131 iodine tositumomab), bicalutamide, bleomycin, bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, Campath (aremtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfil Zomib, Casodex (bicalutamide), CeeNU (lomustine), Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib,CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin difutitox, denosumab, DepoCyt (liposomal cytarabine) Bin), DepoFoam (liposomalcitarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxa tin (oxaliplatin), eltrombopagolamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (bismodegib), erlotinib hydrochloride, Erwinaze (asparaginase erwinia chrysanthemum), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet ( Doxorubicin hydrochloride (liposome), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), Folfiri, Folfiri-bevacizumab, Folfiri-cetuximab, Folfirinox, Folfox (leucovorin, fluorouracil, oxaliplatin), Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV tetravalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin,Gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV tetravalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hyper-CV AD, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), iodine-131 tositumomab and tositumoma Mab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepyrone, Ixempra (ixabepyrone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Kypro lis (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), liposomalcitarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3 Month (leuprolide acetate), Lupron Depot-4 Month (leuprolide acetate), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechloretamine hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide),Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Prelixafor), Mustargen (Mechloretamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin Stabilized Nano (Particle formulation), Navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omasetaxin mepesuccinate, Oncaspar (peguaspar gauze), Ontak (deniroikin difutitox), OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin Stabilized nanoparticle formulations, Palifermin, Palonosetron hydrochloride, Pamidronate disodium, Panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), Pazopanib hydrochloride, Pegaspargaze, Peginterferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), Pemetrexed disodium, Perjeta (pertuzumab), Pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Prelixafor, Pomalidomide, Po malyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopagolamine), Provenge (ciproisel-T), Purinethol (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV tetravalent vaccine, recombinant interferon alpha-2b, regorafenib,Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol (talc), ciproisel-T, sorafenib tosylate, Sprycel (dasatinib), Stanford V, Sterilized talc powder (talc), Steritalc (talc), Stivarga (regorafenib), Sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alpha-2b), Synovir (thalidomide), Synribo (omacetaxin mepesuccinate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tocitumomab and 1 131 Iodine tocitumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomes, vinorelbine tartrate, bismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine),Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexla, Examples include zoxane hydrochloride, Ziv-aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0475] The anticancer drugs may be PBD dimer, calicheamicin, speromycin, tubulisin B, rhizoxin, drastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothyron B, taxol, cryptophycin, SN38, Velcade, bruseantine, DAVLBH, DM1, filantoside, alimta, T2 toxin, MMC, vantaranib, vinorelbine, breferdin, sunitinib, daunomycin, semacanib, tarceva, irressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustard, etoposide, or 5-FU.

[0476] In certain embodiments, the anticancer agent is an anthracycline. In certain embodiments, the anticancer agent is a taxane. In certain embodiments, the anticancer agent is gemcitabine. In certain embodiments, the anticancer agent is doxorubicin. In certain embodiments, the anticancer agent is docetaxel. In certain embodiments, the anticancer agent is SN38. In certain embodiments, the anticancer agent is monomethyl auristatin E.

[0477] Compound synthesis Targeted moieties may be prepared using the methods disclosed herein and modifications thereof, as will be apparent from the consideration of the methods disclosed herein and methods well known in the art. In addition to the teachings herein, conventional and well known synthetic methods may be used. The synthesis of typical targeted moieties disclosed herein can be achieved as disclosed in the following examples. Where available, reagents and starting materials may be purchased commercially from, for example, Sigma Aldrich or other chemical suppliers.

[0478] Given typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), it will be understood that other process conditions may also be used unless otherwise specified. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through routine optimization procedures.

[0479] Furthermore, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. Suitable protecting groups for various functional groups, as well as preferred conditions for protecting and deprotecting specific functional groups, are well known in the art. For example, numerous protecting groups are described in Wuts, PGM, Greene, TW, & Greene, TW (2006). Greene's protective groups in organic synthesis. Hoboken, NJ, Wiley-Interscience, and the references cited therein.

[0480] The compounds provided herein (e.g., compounds of formula I) can be prepared by the methods described in the following examples or by methods adapted from the literature (see, for example, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126).

[0481] Exemplary payloads may be prepared according to methods adapted from the literature (see, for example, WO2022 / 032191, WO2021 / 007160, WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, these methods, in whole, are incorporated herein). [Examples]

[0482] The following embodiments are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that function well in the implementation of the Disclosure and can therefore be considered to constitute a particular form for implementation. However, those skilled in the art will understand that many modifications can be made in light of the Disclosure to the specific embodiments disclosed, and that similar or equivalent results can still be obtained without departing from the spirit and scope of the Disclosure.

[0483] LC-MS analysis method: Test specimens were subjected to PNGaseF(IgG) and DTT or DTT alone (Fab) in RapiGest according to the manufacturer's protocol. Samples were diluted to 100 μg / mL with water and centrifuged at 4°C at 16.1 k RCF for 10 minutes. The samples were then analyzed by LC-MS (LC-Q-TOF), and the mass spectrum was reconstructed from the charge ladder.

[0484] Example 1: Exemplary synthesis of a tetrazine-based targeting agent The targeting agent in PBS is mixed with tetrazine-NHS (e.g., 20 equivalents). The reactants are thoroughly mixed and aged at room temperature for 1 hour, at which point the reactants are quenched by adding 1 volume of 0.1 M Tris buffer. The resulting solution is buffer-changed with 0.01 M PBS to remove excess reagent and buffer salts. The resulting solution of the targeting agent is analyzed by SDS-Page and LC-MS to confirm the formation of the tetrazine-based targeting agent.

[0485] Example 2: Synthesis of tetrazine-based targeting agents 1-4 [ka] [ka] [ka] [ka] [ka]

[0486] Procedure for preparing Peptide 1 [ka]

[0487] Peptide synthesis: The peptides were synthesized using standard Fmoc chemistry.

[0488] DCM is added to a container containing CTC resin (5 mmol, 0.83 mmol / g, 6.02 g) and Fmoc-Lys(Alloc)-OH (2.34 g, 5 mmol, 1.0 equivalent) while bubbling with N2.

[0489] Add DIEA (6.0 equivalents) dropwise and mix for 2 hours.

[0490] Add MeOH (7.0 mL) and mix for 0.5 hours.

[0491] Drain and wash 5 times with DMF.

[0492] Add 20% piperidine / DMF and allow to react for 0.5 hours.

[0493] Drain and wash 5 times with DMF.

[0494] Add the Fmoc-amino acid solution and mix for 30 seconds, then add the activation solution. Continue the reaction with N2 bubbling for 1 hour.

[0495] Repeat steps 4-7 for the next amino acid coupling. TIFF2026518126000203.tif71170

[0496] 20% piperidine in DMF was used for 30 minutes to deprotect Fmoc. The coupling reaction was monitored by the ninhydrin test, and the resin was washed five times with DMF.

[0497] Urea formation: To a mixture of NH2-Glu(OtBu)-OtBu (17.50 g, 17.50 mmol, 4.00 equivalents) and TEA (8.4 g, 35.00 mmol, 8.00 equivalents) in DMF (20 mL), CDI (11.40 g, 17.50 mmol, 4.00 equivalents) was added at 0°C. The reaction mixture was stirred at 20°C for 1 hour. After filtration, the filtrate was added to the resin, and subsequently, DMAP (2.13 g, 17.50 mmol, 1.00 equivalent) was added while bubbling with N2 at 20°C for 16 hours. The coupling reaction was monitored by the ninhydrin test and showed no color.

[0498] Peptide cleavage and purification: Add the cleavage buffer (5% DTT / 2.5% TIS / 2.5% H2O / 90% TFA) to a flask containing the side-chain protected peptide at room temperature and stir for 1 hour.

[0499] The peptide is precipitated with cold isopropyl ether and then centrifuged (at 3000 rpm for 3 minutes).

[0500] Wash with isopropyl ether two more times.

[0501] The solvent was removed under vacuum to obtain the crude product. The crude product was purified by preparative HPLC (A: 0.075% TFA in H2O, B: CH3CN) to obtain compound 3 (0.76 g, purity 99.8%, yield 47%) as a white solid. LCMS[M+H]+ calculated value 656.8; measured value 656.3

[0502] Purification conditions: TIFF2026518126000204.tif114170

[0503] Procedure for preparing Peptide 2 [ka]

[0504] Peptide synthesis: The peptides were synthesized using standard Fmoc chemistry.

[0505] DCM is added to a container containing CTC resin (5 mmol, 0.83 mmol / g, 6.02 g) and Fmoc-Ahx-OH (1.76 g, 5 mmol, 1.0 equivalent) while bubbling with N2.

[0506] Add DIEA (6.0 equivalents) dropwise and mix for 2 hours.

[0507] Add MeOH (7.5 mL) and mix for 0.5 hours.

[0508] Drain and wash 5 times with DMF.

[0509] Add 20% piperidine / DMF and allow to react for 0.5 hours.

[0510] Drain and wash 5 times with DMF.

[0511] Add the Fmoc-amino acid solution and mix for 30 seconds, then add the activation solution. Continue the reaction with N2 bubbling for 1 hour.

[0512] Repeat steps 4-7 for the next amino acid coupling. TIFF2026518126000206.tif34170

[0513] 20% piperidine in DMF was used for deprotection for 30 minutes. The coupling reaction was monitored by a ninhydrin test, and the resin was washed five times with DMF.

[0514] Peptide cleavage and purification: Add the cleavage solution (50% TFA / DCM) to a flask containing the side-chain protected peptide at room temperature and stir for 1 hour.

[0515] After filtration, the filtrate was collected.

[0516] The filtrate was concentrated under reduced pressure to obtain compound 4 (1.0 g, purity 90.0%, yield 68.4%) as a white solid. LCMS[M+H] + Calculated value: 468.6; Measured value: 468.1; LCMS[M+Na] + Calculated value: 490.6; Measured value: 490.1

[0517] Procedure for the preparation of compound 2 [ka] A solution of compound 1 (800 mg, 3.47 mmol) and HOSu (600 mg, 5.21 mmol) in THF (16.0 mL) was mixed with DIC (646 μL, 4.17 mmol). The mixture was stirred at 25°C for 10 hours. LC-MS showed that compound 1 (RT=0.843 min) was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was quenched at 25°C with an additional H2O (50.0 mL) and then extracted with DCM (100 mL × 3). The combined organic layers were washed with saturated brine (100 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain the residue. The crude product, compound 2 (1.67 g), was obtained as a purple solid. LCMS[M+H] + Calculated value: 328.3; Measured value: 328.0

[0518] Procedure for preparing target 1 [ka] DIEA (319 μL, 1.83 mmol) was added to a solution of compound 3 (400 mg, 610 μmol) and compound 2 (299 mg, 915 μmol) in DMF (8.00 mL). The reaction mixture was stirred at 25 °C for 2 hours. LC-MS showed that compound 3 (RT=0.865 min) was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was filtered to remove the solid and obtain a filtrate. The filtrate was then purified by preparative HPLC (TFA conditions) to obtain target 1 (144 mg, 166 μmol, yield 27.2%) as a purple solid. LC-MS[M+H] + Calculated value: 869.0; Measured value: 868.6

[0519] Procedure for preparing target 3 [ka] DIEA (234 μL, 1.34 mmol) was added to a solution of compound 4 (314 mg, 672 μmol) and compound 2 (330 mg, 1.01 mmol) in DMF (8.00 mL). The reaction mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 4 (RT=0.897 min) was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was filtered to remove the solid and obtain a filtrate. The filtrate was then purified by preparative HPLC (TFA conditions) to obtain target 3 (212 mg, 312 μmol, yield 46.4%) as a purple solid. LC-MS[M+H] + Calculated value: 680.8; Measured value: 680.6

[0520] Procedure for the preparation of compound 3 [ka] HATU (2.48 g, 6.53 mmol) and DIEA (1.69 g, 13.1 mmol) were slowly added to a solution of compound 2 (1.30 g, 2.97 mmol) in DMF (15 mL). The mixture was stirred for 10 minutes before adding compound 2A (1.25 g, 6.24 mmol). LC-MS showed that compound 2 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was filtered to remove undissolved residue. The residue was purified by preparative HPLC (TFA conditions). Compound 3 (1.60 g, 55.6% yield) was obtained as a red oil. LCMS[M+H] + Calculated value 804.9; Measured value 804.7.LCMS[M+Na] + Calculated value: 826.9; Measured value: 826.5

[0521] Procedure for the preparation of compound 4 [ka] To a solution of compound 3 (1.60 g, 1.99 mmol) in DCM (20 mL), TFA (7.60 g, 67.6 mmol) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 3 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure, the DCM was removed, and the residue was obtained. Compound 4 (2.10 g, crude) was obtained as a red oil. LCMS[M+H]+ Calculated value 704.8; Measured value 704.5

[0522] Procedure for the preparation of compound 5 [ka] To a solution of tetrahydrofuran-2,5-dione (390 mg, 3.89 mmol) and compound 4 (2.10 g, 2.99 mmol) in DCM (20 mL), DIEA (773 mg, 5.98 mmol) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 4 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was quenched at 0°C by adding 1N HCl (50 mL), then diluted with DCM (20 mL), and extracted with DCM (30 mL x 3). The combined organic layer was washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound 5 (1.70 g, crude) was obtained as a red oil. LCMS[M+H]+ Calculated value 804.9; Measured value 804.6

[0523] Procedure for the preparation of compound 6 [ka] To a solution of compound 5 (1.70 g, 2.11 mmol) in DCM (20 mL), EDCI (810 mg, 4.23 mmol) and 1-hydroxypyrrolidine-2,5-dione (730 mg, 6.34 mmol) were added. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 5 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was added to 1N HCl (100 mL). The mixture was extracted with DCM (150 mL x 3). The combined organic layer was washed with NaHCO3 (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by preparative HPLC (TFA conditions). Compound 6 (2.20 g, crude) was obtained as a red solid. LCMS[M+H]+ Calculated value 902.0; Measured value 901.6

[0524] Procedure for preparing target 2 [ka] To a solution of compound 6 (700 mg, 777 μmol) in DMF (7 mL), DIEA (301 mg, 2.33 mmol) and compound 1 (611 mg, 932 μmol) were added. The mixture was stirred at 25°C for 3 hours. LC-MS showed that compound 6 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was filtered to remove undissolved residue. The residue was purified by preparative HPLC (TFA conditions). Target 2 (220 mg, yield 19.6%) was obtained as a red solid. LCMS[M+H]+ Calculated value 1442.6; Measured value 1442.9

[0525] Procedure for preparing target 4 [ka] To a solution of compound 6 (700 mg, 777 μmol) in DMF (7 mL), DIEA (301 mg, 2.33 mmol) and compound 7 (436 mg, 932 μmol) were added. The mixture was stirred at 25°C for 3 hours. LC-MS showed that compound 6 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was filtered to remove undissolved residue. The residue was purified by preparative HPLC (TFA conditions). Target 4 (210 mg, yield 21.5%) was obtained as a red solid. LCMS[M+H]+ Calculated value 1254.5; Measured value 1253.3

[0526] Example 3: Synthesis of compounds 111, 253, 254, 257, and 259 Procedure for the preparation of compound 2 [ka] To a solution of compound 1 (150 mg, 745 μmol) and compound 1A (170 mg, 1.49 mmol) in DMF (2.0 mL), DIEA (193 mg, 1.49 mmol, 260 μL) was added. The mixture was stirred at 25°C for 2 hours. LC-MS showed that compound 1 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 2 (164 mg, 520 μmol, yield 69.8%) as a red solid. LCMS[M+H]+ Calculated value 316.3; Measured value 316.1

[0527] Procedure for the preparation of compound 3 [ka] To a solution of compound 2 (100 mg, 317 μmol) and HOSu (54.8 mg, 476 μmol) in DMF (1.0 mL), DCC (131 mg, 634 μmol, 128 μL) was added at 0°C. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 2 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was filtered, and the filtrate was added to isopropyl ether (10 mL x 2) to form a red solid. This was filtered and dried to obtain compound 3 (90.0 mg, 212 μmol, yield 66.7%, purity 97.0%) as a red solid, which was used in the next step without further purification. LCMS[M+H]+ Calculated value 413.4; Measured value 413.1

[0528] Procedure for the preparation of compound 4 [ka]

[0529] Peptide synthesis: The peptides were synthesized using standard Fmoc chemistry.

[0530] Add DMF (200 mL) to a container containing Rink amide MBHA resin (5.00 mmol, 8.50 g, degree of substitution: 0.60 mmol / g) and allow to expand for 2 hours.

[0531] Drain the fluid, then perform a DMF wash three times for 30 seconds each.

[0532] Add 20% piperidine / DMF and mix for 30 minutes.

[0533] Drain the fluid, then run a DMF wash five times for 30 seconds each.

[0534] Add the Fmoc-amino acid solution and mix for 30 seconds, then add the coupling reagent and bubble with N2 for about 1 hour.

[0535] Repeat steps 2-5 for the next amino acid coupling. TIFF2026518126000219.tif158170

[0536] 20% piperidine in DMF was used for deprotection for 30 minutes. The coupling reaction was monitored by a ninhydrin test, and the resin was washed five times with DMF.

[0537] Peptide cleavage and purification: Add the cleavage buffer (90% TFA / 2.5% TIS / 2.5% H2O / 5.0% DTT) to a flask containing the side-chain protected peptide at room temperature and stir for 2 hours.

[0538] The peptide is precipitated with cold isopropyl ether and then centrifuged (at 3000 rpm for 3 minutes).

[0539] Wash with isopropyl ether two more times.

[0540] The crude peptide was dried under vacuum to obtain compound 4 (12.0 g, crude) as a white solid.

[0541] LCMS[M / 3]+Calculated value 878.0; Measured value 878.6; LCMS[M / 2]+Calculated value 1317.0; Measured value 1317.5

[0542] Procedure for the preparation of compound 5 [ka] Crude peptide compound 4 (12.0 g, 4.56 mmol) was dissolved in 20% MeCN / H2O (5000 mL), and then TATA (1.48 g, 5.92 mmol) was added to the stirred peptide solution at 25°C for 30 minutes. After the addition, the mixture was stirred at 25°C for 30 minutes, and then NH4HCO3 was added to pH 8. The resulting mixture was stirred at 25°C for 16 hours. LC-MS indicated that the reaction was complete. The combined five batches were purified by preparative HPLC (A: 0.1% TFA in H2O, B: MeCN), and then re-purified by preparative HPLC (A: 0.1% TFA in H2O, B: MeCN) to obtain compound 5 (1.35 g, purity 98.6%) as a white solid. LCMS[M / 3+H]+Calculated value 962.1; Measured value 962.0; LCMS[M / 2+H]+Calculated value 1442.6; Measured value 1442.3

[0543] Purification conditions: TIFF2026518126000221.tif65170

[0544] Procedure for the preparation of Nectin-4 peptide, compound 111 [ka] To a solution of compound 5 (400 mg, 139 μmol) in DMF (4.0 mL), DIEA (53.8 mg, 416 μmol, 72.5 μL) and compound 3 (59.0 mg, 139 μmol) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 5 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (A: 0.1% TFA in H2O, B: MeCN) to obtain compound 111 (260 mg, 81.8 μmol, yield 57.9%, purity 96.9%, TFA salt) as a pink solid. Next, the pink solid was purified by preparative HPLC (A: 0.05% HCl in H2O, B: MeCN) to obtain compound 111 (208 mg, 65.4 μmol, yield 46.3%, purity 98.1%, HCl salt) as a pink solid. LCMS[M / 3+H] + Calculated value: 1061.2; Measured value: 1061.0

[0545] Note: TFA conditions: The reaction mixture was dissolved in DMF and purified by preparative HPLC (Gilson GX-281; ​​Gemini® 250×30 mm, C18, 5 μm, 110 Å; elution of 28%-48% of the solution (A: 0.1% TFA in H2O, B: MeCN) for 40 minutes at room temperature; observed under wavelengths (220 / 254 nm), flow rate 20 mL / min, retention time 23 minutes).

[0546] HCl conditions: The TFA salt was dissolved in ACN / H2O, and 10% of the solution (A: 0.5% NH4Cl in H2O, B: MeCN) was eluted for 20 minutes at room temperature, followed by 10% of the solution (A: 0.05% HCl in H2O, B: MeCN) being eluted for 10 minutes at room temperature, and then preparative HPLC (AUNO LC2000; Gemini-C18, 150×30mm, 5um, 110Å; 20%-40% of the solution (A: 0.05% HCl in H2O, B: MeCN) was eluted for 40 minutes at room temperature; observed under wavelengths (220 / 254nm), with a flow rate of 20 mL / min and a retention time of 27 minutes).

[0547] Purification conditions: TIFF2026518126000223.tif226170

[0548] Procedure for the preparation of Nectin-4 Peptide-TCO, Compound 253 [ka] [ka]

[0549] General procedure for the preparation of compound 3 [ka] Compound 1 (200 mg, 994 μmol, 1.00 equivalent) and DIEA (257 mg, 1.99 mmol, 346 μL, 2.00 equivalent) were added to a solution of Compound 2 (1.62 g, 4.97 mmol, 5.00 equivalents) in DMF (15.0 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed that Compound 1 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain Compound 3 (210 mg, 392 μmol, yield 39.5%, purity 98.3%, TFA) as a pink solid, which was confirmed by LC-MS and HPLC. LC-MS:R t = 0.816 minutes, MS calculated value: 412.41, MS measured value: [M+H] + =413.1.HPLC:R t = 7.945 minutes, purity: 98.3%.

[0550] General procedure for the preparation of compound 4A Peptide synthesis: Peptides were synthesized using standard Fmoc chemistry. 1) Resin preparation: Add DMF to a container containing Rink Amide MBHA resin (0.50 mmol, 1.00 equivalent, degree of substitution: 0.33 mmol / g) and allow to expand for 2 hours. 2) Deprotection: 20% piperidine in DMF (30.0 mL) was added, and the resin was stirred with N2 at 22°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) and filtered to obtain the resin. 3) Coupling: A solution of HBTU (2.85 equivalents) and Fmoc-Cys(Trt)-OH (3.00 equivalents) in DMF (3.00 mL) was added to the resin, then DIEA (6.00 equivalents) was added, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was washed with DMF (20.0 mL x 5). 4) Repeat steps 2-3 above for the coupling of the following amino acids: (1-25) TIFF2026518126000227.tif192170

[0551] Peptide cleavage and purification: 1) The cutting solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) was added to a flask containing resin at 22°C and stirred for 2.5 hours. 2) The peptide was precipitated with cold isopropyl ether (500 mL). The solution was filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (150 mL x 2). The crude peptide was dried under vacuum for 2 hours to obtain crude peptide compound 4A (1.30 g), and the crude peptide was identified by LC-MS.

[0552] LCMS:R t = 0.803 minutes, MS calculated value: 2633.08, MS measured value: [M / 3+H] + =878.8, [M / 2+H] + =1317.5.

[0553] General procedure for the preparation of compound 5A To a solution of crude linear peptide compound 4A (1.30 g, 1.00 equivalent) in H2O (300 mL) and ACN (200 mL), 1,3,5-triacryloylhexahydro-1,3,5-triazine (0.15 g, 1.20 equivalent) and NH4HCO3 in H2O (1.00 M) were added to slowly adjust the reaction mixture pH to 8-9. The mixture was stirred at 20°C for 4.0 hours. LC-MS showed the detection of one major peak with the desired mass. Subsequently, HCl (1.00 M) in water was added to bring the pH down to 6-7. The mixture was lyophilized to obtain the crude bicyclic peptide. The crude bicyclic peptide was purified by preparative HPLC (condition A: 0.075% TFA in H2O, B: ACN) to obtain compound 5A (240 mg, yield 16.6%, purity 84.5%, TFA salt) as a white solid, which was confirmed by LC-MS and HPLC.

[0554] LCMS:R t = 0.786 minutes, MS calculated value: 2882.35, MS measured value: [M / 3+H] + =961.9, [M / 2+H] + =1442.2.

[0555] HPLC:R t = 7.554 minutes, purity: 84.5%.

[0556] Purification conditions: TIFF2026518126000228.tif75170

[0557] General procedure for preparing target 253 To a solution of compound 6A (170 mg, 59.0 μmol, 1.00 equivalent) in DMF (1.80 mL), DIEA (22.9 mg, 177 μmol, 30.8 μL, 3.00 equivalent) and compound 3 (34.2 mg, 64.9 μmol, 1.1 equivalent, TFA) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 6A was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA and AcOH conditions) to obtain target 253 (54.0 mg, 16.1 μmol, yield 27.2%, purity 96.3%, HOAC) as a pink solid, which was confirmed by LC-MS and HPLC.

[0558] LCMS:R t = 1.363 minutes, MS calculated value: 3179.67, MS measured value: [M / 3+H] + =1060.5, [M+H] + =1590.6.

[0559] HPLC:R t = 5.914 minutes, purity: 96.3%.

[0560] Purification conditions: TIFF2026518126000229.tif75170TIFF2026518126000230.tif75170

[0561] Procedure for the preparation of Nectin-4 Peptide-TCO, Compound 254 [ka] [ka]

[0562] General procedure for the preparation of compound 4B Peptide synthesis: Peptides were synthesized using standard Fmoc chemistry. 1) Resin preparation: Add DMF to a container containing Rink Amide MBHA resin (0.50 mmol, 1.00 equivalent, degree of substitution: 0.33 mmol / g) and allow to expand for 2 hours. 2) Deprotection: 20% piperidine in DMF (30.0 mL) was added, and the resin was stirred with N2 at 22°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) and filtered to obtain the resin. 3) Coupling: A solution of HBTU (2.85 equivalents) and Fmoc-Cys(Trt)-OH (3.00 equivalents) in DMF (3.00 mL) was added to the resin, then DIEA (6.00 equivalents) was added, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was washed with DMF (20.0 mL x 5). 4) Repeat steps 2-3 above for the coupling of the following amino acids: (1-25) TIFF2026518126000233.tif192170

[0563] Peptide cleavage and purification: 1) The cutting solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) was added to a flask containing resin at 22°C and stirred for 2.5 hours. 2) The peptide was precipitated with cold isopropyl ether (500 mL). The solution was filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (150 mL x 2). The crude peptide was dried under vacuum for 2 hours to obtain crude peptide compound 4B (1.30 g), and the crude peptide was identified by LC-MS.

[0564] LCMS:R t = 0.760 minutes, MS calculated value: 2697.18, MS measured value: [M / 4+H] + =675.4, [M / 3+H] + =900.1, [M / 2+H] + =1349.6.

[0565] General procedure for the preparation of compound 5B To a solution of crude linear peptide compound 4B (1.30 g, 1.00 equivalent) in H2O (300 mL) and ACN (200 mL), 1,3,5-triacryloylhexahydro-1,3,5-triazine (0.15 g, 1.20 equivalents) and NH4HCO3 in H2O (1.00 M) were added to slowly adjust the reaction mixture pH to 8-9. The mixture was stirred at 20°C for 4.0 hours. LC-MS showed the detection of one major peak with the desired mass. Subsequently, HCl (1.00 M) in water was added to pH 6-7. The mixture was lyophilized to obtain the crude bicyclic peptide. The crude bicyclic peptide was purified by preparative HPLC (condition A: 0.075% TFA in H2O, B: ACN) to obtain compound 5B (237 mg, yield 16.1%, purity 99.2%, TFA salt) as a white solid, which was confirmed by LC-MS and HPLC.

[0566] LCMS:R t = 0.745 minutes, MS calculated value: 2946.45, MS measured value: [M / 4+H] + =737.7, [M / 3+H] + =983.3, [M / 2+H] + =1474.2.

[0567] HPLC:R t = 6.786 minutes, purity: 99.2%.

[0568] Purification conditions: TIFF2026518126000234.tif75170

[0569] General procedure for preparing target 254 To a solution of compound 5B (160 mg, 54.3 μmol, 1.00 equivalent) in DMF (1.60 mL), DIEA (21.1 mg, 163 μmol, 28.4 μL, 3.00 equivalent) and compound 3 (31.5 mg, 59.7 μmol, 1.10 equivalent, TFA) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 5B was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain target 254 (43.0 mg, 12.8 μmol, yield 23.6%, purity 96.6%, HOAC) as a pink solid, which was confirmed by LC-MS and HPLC.

[0570] LCMS:R t = 1.282 minutes, MS calculated value: 3243.76, MS measured value: [M / 4+H] + =811.8, [M / 3+H] + =1082.2, [M / 2+H] + =1622.8.

[0571] HPLC:R t = 4.318 minutes, purity: 96.6%.

[0572] Purification conditions: TIFF2026518126000235.tif75170TIFF2026518126000236.tif75170

[0573] Procedure for the preparation of Nectin-4 Peptide-TCO, Compound 257 [ka] [ka]

[0574] General procedure for the preparation of compound 4C Peptide synthesis: Peptides were synthesized using standard Fmoc chemistry. 1) Resin preparation: Add DMF to a container containing Rink Amide MBHA resin (0.50 mmol, 1.00 equivalent, degree of substitution: 0.33 mmol / g) and allow to expand for 2 hours. 2) Deprotection: 20% piperidine in DMF (30.0 mL) was added, and the resin was stirred with N2 at 22°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) and filtered to obtain the resin. 3) Coupling: A solution of HBTU (2.85 equivalents) and Fmoc-Cys(Trt)-OH (3.00 equivalents) in DMF (3.00 mL) was added to the resin, then DIEA (6.00 equivalents) was added, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was washed with DMF (20.0 mL x 5). 4) Repeat steps 2-3 above for the coupling of the following amino acids: (1-25) TIFF2026518126000239.tif191170

[0575] Peptide cleavage and purification: 1) The cutting solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) was added to a flask containing resin at 22°C and stirred for 2.5 hours. 2) The peptide was precipitated with cold isopropyl ether (500 mL). The solution was filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (150 mL x 2). The crude peptide was dried under vacuum for 2 hours to obtain crude peptide compound 4C (1.30 g), and the crude peptide was identified by LC-MS.

[0576] LCMS:R t = 0.774 minutes, MS calculated value: 2693.17, MS measured value: [M / 4+H] + =674.5, [M / 3+H] + =898.8, [M / 2+H] + =1347.5.

[0577] General procedure for the preparation of compound 5C To a solution of crude linear peptide compound 4C (1.30 g, 1.00 equivalent) in H2O (300 mL) and ACN (200 mL), 1,3,5-triacryloylhexahydro-1,3,5-triazine (0.15 g, 1.20 equivalents) and NH4HCO3 in H2O (1.00 M) were added to adjust the reaction mixture pH to 8-9. The mixture was stirred at 20°C for 4.0 hours. LC-MS showed the detection of one major peak with the desired mass. Subsequently, HCl (1.00 M) in water was added to pH 6-7. The mixture was lyophilized to obtain the crude bicyclic peptide. The crude bicyclic peptide was purified by preparative HPLC (condition A: 0.075% TFA in H2O, B: ACN) to obtain compound 5C (272 mg, yield 18.5%, purity 99.1%, TFA salt) as a white solid, which was confirmed by LC-MS and HPLC. LC-MS:R t = 0.774 minutes, MS calculated value: 2942.44, MS measured value: [M / 4+H] + =736.6, [M / 3+H] + =981.8, [M / 3+H] + =1472.2.HPLC:R t = 7.041 minutes, purity: 99.1%.

[0578] Purification conditions: TIFF2026518126000240.tif75170

[0579] General procedure for preparing target 257 To a solution of compound 5C (150 mg, 51.0 μmol, 1.00 equivalent) in DMF (1.50 mL), DIEA (19.8 mg, 153 μmol, 26.6 μL, 3.00 equivalent) and compound 3 (32.2 mg, 61.2 μmol, 1.20 equivalent, TFA) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 5C was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain target 257 (40.0 mg, 11.8 μmol, yield 23.1%, purity 95.4%, HOAC) as a pink solid, which was confirmed by LC-MS and HPLC. LC-MS:Rt = 1,300 minutes, MS calculated value: 3243.76, MS measured value: [M / 4+H] + =810.9, [M / 3+H] + =1080.6, [M / 2+H] + =1620.6.HPLC:R t = 4.822 minutes, purity: 95.4%.

[0580] Purification conditions: TIFF2026518126000241.tif75170TIFF2026518126000242.tif75170

[0581] Procedure for the preparation of Nectin-4 Peptide-TCO, Compound 259 [ka] [ka]

[0582] General procedure for the preparation of compound 4D Peptide synthesis: Peptides were synthesized using standard Fmoc chemistry. 1) Resin preparation: Add DMF to a container containing Rink Amide MBHA resin (0.50 mmol, 1.00 equivalent, degree of substitution: 0.33 mmol / g) and allow to expand for 2 hours. 2) Deprotection: 20% piperidine in DMF (30.0 mL) was added, and the resin was stirred with N2 at 22°C for 30 minutes. The resin was washed with DMF (30.0 mL x 5) and filtered to obtain the resin. 3) Coupling: A solution of HBTU (2.85 equivalents) and Fmoc-Cys(Trt)-OH (3.00 equivalents) in DMF (3.00 mL) was added to the resin, then DIEA (6.00 equivalents) was added, and the mixture was stirred with N2 at 20°C for 30 minutes. The resin was washed with DMF (20.0 mL x 5). 4) Repeat steps 2-3 above for the coupling of the following amino acids: (1-28) TIFF2026518126000245.tif213170

[0583] Peptide cleavage and purification: 1) The cutting solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) was added to a flask containing resin at 22°C and stirred for 2.5 hours. 2) The peptide was precipitated with cold isopropyl ether (500 mL). The solution was filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (150 mL x 2). The crude peptide was dried under vacuum for 2 hours to obtain crude peptide compound 4D (1.50 g), and the crude peptide was identified by LC-MS. LCMS:R t = 0.857 minutes, MS calculated value: 2869.35, MS measured value: [M / 2 + H] + =1435.2.

[0584] General procedure for the preparation of compound 5D To a solution of crude linear peptide compound 4D (1.50 g, 1.00 equivalent) in H2O (300 mL) and ACN (200 mL), 1,3,5-triacryloylhexahydro-1,3,5-triazine (0.15 g, 1.20 equivalents) and NH4HCO3 in H2O (1.00 M) were slowly added to adjust the reaction mixture pH to 8-9. The mixture was stirred at 20°C for 4.0 hours. LC-MS showed the detection of one major peak with the desired mass. Subsequently, HCl (1.00 M) in water was added to pH 6-7. The mixture was lyophilized to obtain the crude bicyclic peptide. The crude bicyclic peptide was purified by preparative HPLC (condition A: 0.075% TFA in H2O, B: ACN) to obtain compound 5D (200 mg, yield 14.2%, purity 99.8%, TFA salt) as a white solid, which was confirmed by HPLC. LCMS:R t = 0.844 minutes, MS calculated value: 3118.62, MS measured value: [M / 3+H] + =1040.4.HPLC:R t = 8.768 minutes, purity: 99.8%.

[0585] Purification conditions: TIFF2026518126000246.tif75170

[0586] General procedure for the preparation of compound 6D To a solution of compound 5D (170 mg, 54.5 μmol, 1.00 equivalent) in DMF (2.00 mL), DIEA (21.1 mg, 164 μmol, 28.5 μL, 3.00 equivalent) and compound 3 (28.7 mg, 54.5 μmol, 1.00 equivalent, TFA) were added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 5D was completely consumed and one major peak with the desired mass was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to obtain compound 6D (111 mg, 29.8 μmol, yield 54.8%, purity 91.9%) as a pink solid, which was confirmed by LC-MS and HPLC. LC-MS:R t = 0.915 minutes, MS calculated value: 3415.94, MS measured value: [M / 4+H] + =854.8, [M / 3+H] + =1139.4, [M / 2+H] + =1708.6.HPLC:R t = 9.397 minutes, purity: 91.9%.

[0587] General procedure for preparing target 259 To a solution of compound 6D (120 mg, 35.2 μmol, 1.00 equivalent) in DMF (1.00 mL), Et3N (182 mg, 1.80 mmol, 0.25 mL, 51.1 equivalents) was added. The mixture was stirred at 25°C for 5 hours. LC-MS showed that compound 6D was completely consumed and one major peak with the desired mass was detected. The reaction mixture was added to isopropyl ether (12.0 mL), forming a pink solid, which was filtered and dried to obtain the residue. The residue was purified by preparative HPLC (TFA and AcOH conditions) to obtain target 259 (42.0 mg, 12.6 μmol, yield 35.9%, purity 96.0%) as a pink solid, which was confirmed by LC-MS. LC-MS:R t= 1.321 minutes, MS calculated value: 3193.70, MS measured value: [M / 4+H] + =799.4, [M / 3+H] + =1065.4, [M / 2+H] + =1597.1.HPLC:R t = 5.051 minutes, purity: 96.0%.

[0588] Purification conditions: TIFF2026518126000247.tif75170TIFF2026518126000248.tif76170

[0589] Example 4: HCC1954 Xenograft Model Animal studies will be conducted according to AAALAC guidance and the IACUC protocol. Female Balb / c nude mice were transplanted into the right flank with HCC1954 (5e6 cells + Matrigel) cells grown in the exponential growth phase in 0.2 mL of PBS. The animals were examined until the tumor volume reached approximately 200 mm². 3 Randomization occurs when the target is reached. Animals are administered intravenously with either saline (days 1-4), a tetrazine-based targeting agent (day 0, 5 mg / kg) + saline (days 1-4), or a tetrazine-based targeting agent (day 0, 5 mg / kg) + TCO prodrug (days 1, 2, 3, and 4, 120 mg / kg).

[0590] Tumor volume was measured in two dimensions twice a week using calipers, and the volume was expressed in mm using the following formula. 3 Expressed in units: V = 0.5a × b 2 In the formula, a and b are the long and short diameters of the tumor, respectively. The reduction in tumor growth due to the administration of the prodrug demonstrates the effectiveness of the system described herein.

[0591] Suitable prodrugs for use in the methods described herein can be prepared and administered as described in WO2020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126.

[0592] Example 5: General procedure for preparing compound A [ka] Compound 2 (800 mg, 1.79 mmol) in DMF (4.00 mL) was added at 0°C to a solution of MMAE (1.40 g, 1.95 mmol) and DIEA (690 mg, 5.34 mmol) in DMF (4.00 mL), and the mixture was stirred at 25°C for 16 hours. Then, HOBt (480 mg, 3.56 mmol) in DMF (0.50 mL) was added to the reaction mixture at 0°C, and the reaction mixture was stirred at 25°C for 1.0 hour. After the reaction mixture was cooled to 0°C, TBAF (1 M, 4.45 mL in THF) was added. After stirring the mixture at 25°C for 2.0 hours, another batch of TBAF (1 M, 4.45 mL in THF) was added at 0°C, and the reaction mixture was continued to stir at 25°C for 12.0 hours. LC-MS showed that one major peak was at the desired mass. The resulting reaction mixture was purified by preparative HPLC (column: Welch XB-C18 7μm 110 A 250×50mm; mobile phase: [water (0.1% TFA)-ACN]; B%: 50-70% - 40 minutes; number of injections: 2, retention time: 37 minutes, flow rate: 60 mL / min) to obtain compound A (450 mg, purity 99.0%; 64.6 mg, 99.2%, yield 31.2%).

[0593] LCMS(m / z):928.6[M+H] +

[0594] 1HNMR:(400MHz,DMSO-d6):δ 8.46-8.28(m,1H),8.03-7.84(m,1H),7.64(d,J=8.8Hz,1H),7.35-7.23(m,4H),7.21-7.13(m,1 H),6.05-5.57(m,2H),5.10(s,1H),4.81-4.39(m,3H),4.35-4.19(m,1H),4.05-3.92(m,2H),3. 40-3.09(m,11H),3.08-2.83(m,5H),2.48-2.37(m,2H),2.31-2.09(m,5H),2.07-1.89(m,3H),1 .88-1.64(m,6H),1.63-1.33(m,4H),1.32-1.16(m,1H),1.08-0.97(m,9H),0.90-0.68(m,18H).

[0595] Example 6: General procedure for preparing compound B [ka]

[0596] General procedure for the preparation of compound 4 [ka] To a solution of compound 2 (1.00 g, 5.43 mmol) in DCM (10 mL), DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol), DMAP (1.33 g, 10.9 mmol), and compound 3 (1.61 g, 8.14 mmol) were added. The mixture was stirred at 25°C for 16 hours. TLC showed that compound 2 was completely consumed and one new spot was formed. The reaction mixture was partitioned into DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with saturated citric acid aqueous solution (3 mL) and brine (20 mL), then dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1-1 / 1) to obtain compound 4 (700 mg, yield 39.4%).

[0597] 1 HNMR(400MHz,CDCl3):δ ppm 1.12(s,3H),1.60(dd,J=15.45,6.19Hz,1H),1.79-1.87(m,2H),1.92(br d,J=5.88Hz,1H),1.95(s,1H),1.98-2.00(m,1H),2.02(br d,J=4.13Hz,1H),2.26(dd,J=11.63,3.88Hz,1H),2.30-2.36(m,1H),2.77-2.89(m,1H),2.88-2.88(m,1H),3.00(dd,J=16.95,4 .57Hz,1H),3.70(s,4H),3.75(s,3H),4.80(dt,J=8.00,4.50Hz,1H),5.66(dd,J=16.63,2.38Hz,1H),6.02-6.12(m,1H),6.54(br d,J=7.88Hz,2H).

[0598] General procedure for the preparation of compound 6 [ka] To a solution of compound 4 (700 mg, 2.14 mmol) in DCM (5 mL), Py (846 mg, 10.7 mmol) and compound 5 (1.72 g, 8.55 mmol) in DCM (5 mL) were added. The mixture was stirred at 25°C for 1 hour. TLC showed that compound 4 was completely consumed and a new spot was formed. The reaction mixture was partitioned into DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with saturated citric acid aqueous solution (3 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1-1 / 1) to obtain compound 6 (490 mg, yield 46.5%).

[0599] 1HNMR(400MHz,CDCl3):δ ppm 1.17(s,3H),1.55-1.61(m,1H),1.58(br s,1H),1.76(dd,J=14.76,6.25Hz,1H),1.87-2.03(m,3H),2.06-2.15(m,1H),2.19-2.41(m,3H),2.82(dd,J=17.13,4.50Hz,1H),3.0 3(dd,J=17.07,4.44Hz,1H),3.72(s,3H),3.77(s,3H),4.78-4.86(m,1H),5.67(dd,J=16.70,2.44Hz,1H),6.03-6.14(m,1H),6.58(br d,J=7.88Hz,1H),7.40-7.45(m,2H),8.27-8.33(m,2H).

[0600] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (490 mg, 995 μmol) and MMAE (714 mg, 995 μmol) in DMF (4 mL), DIEA (64.3 mg, 497 μmol) and HOBt (202 mg, 1.49 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 6 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (under 0.1% TFA conditions) to obtain compound 7 (500 mg, 46.9% yield).

[0601] 1HNMR(400MHz,CDCl3):δ ppm 0.84(br d,J=6.75Hz,4H),0.89(br d,J=4.50Hz,5H),0.92(br d,J=6.63Hz,4H),0.98(br d,J=6.25Hz,3H),1.04(br d,J=6.88Hz,3H),1.16(s,3H),1.25-1.27(m,3H),1.59-1.74(m,3H),1.88(br d,J=9.38Hz,4H),2.07(br d,J=8.38Hz,5H),2.27(br s,4H),2.36-2.43(m,2H),2.45-2.53(m,1H),2.89(br s,6H),2.95-3.01(m,4H),3.04(br s,2H),3.29-3.34(m,3H),3.36-3.47(m,5H),3.67-3.73(m,4H),3.76(s,3H),3.82-3.89(m,1H),4.05-4.19(m,3H),4.28(br s,1H),4.63-4.86(m,3H),4.96(d,J=2.50Hz,1H),5.24(br s,1H),5.63(br d,J=18.14Hz,1H),5.82(br s,1H),6.53-6.74(m,3H),7.30-7.41(m,5H).

[0602] General procedure for the preparation of compound B [ka] To a solution of compound 7 (500 mg, 467 μmol) in MeOH (5 mL), LiOH·H2O (196 mg, 4.67 mmol) in H2O (2 mL) was added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 7 was completely consumed and a single major peak with the desired mass was detected. The residue was adjusted to pH approximately 2 with saturated citric acid aqueous solution and then purified by preparative HPLC (0.1% TFA conditions) to obtain compound B (265 mg, yield 53.4%).

[0603] 1HNMR(400MHz,CDCl3):δ ppm 0.80-1.04(m,25H),1.09(s,3H),1.24(d,J=6.88Hz,3H),1.69-1.82(m,2H),1.88-1.94(m,3H),2.02-2.11(m,4H),2.16(br d,J=18.64Hz,1H),2.11-2.24(m,2H),2.32(br d,J=5.25Hz,2H),2.40-2.45(m,1H),2.52(br d,J=5.50Hz,2H),2.81(br dd,J=14.01,4.88Hz,1H),2.94-3.04(m,2H),3.08(s,2H),3.14-3.27(m,6H),3.33(s,1H),3.39(s,3H),3.48-3.57(m,2H),3.94(br d,J=1.25Hz,1H),4.05-4.18(m,4H),4.30(br dd,J=6.19,4.82Hz,2H),4.54-4.67(m,4H),4.91(br d,J=2.00Hz,2H),5.30(br s,1H),5.64-5.73(m,1H),5.79-5.89(m,1H),6.61(br d,J=7.38Hz,1H),7.30-7.42(m,5H),7.55-7.64(m,1H).

[0604] Example 7: Alternative route to compound B and synthesis of compound C. [ka]

[0605] General procedure for the preparation of compound 2 [ka] To a solution of compound 1 (20.0 g, 83.2 mmol) in MeOH (80 mL), KOH (8.19 g, 124 mmol) in H2O (80 mL) was added. The mixture was stirred at 25°C for 24 hours. The reaction was monitored by TLC (compound 1, PE / SiO=5 / 1, R f(=0.5). The reaction mixture was extracted with MTBE (3 × 400 mL). The combined organic layers were washed with water (100 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain the undesirable ester. The aqueous layer was acidified with 1 M HCl to pH=4 while cooling in an ice bath (T < 7°C). The aqueous layer was extracted with MTBE (3 × 400 mL). The combined MTBE layers were dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain compound 2 (5.50 g, yield 35.9%). The crude product was used in the next step without further purification.

[0606] 1 H NMR:(400MHz,DMSO-d6):δ ppm 11.9(br s,1H),5.81-5.94(m,1H),5.58(dd,J=16.45,2.31Hz,1H),4.65(br s,1H),4.24(br s,1H),2.04-2.24(m,2H),1.87-2.03(m,1H),1.61-1.86(m,4H),1.36-1.46(m,1H),0.97(s,3H).

[0607] General procedure for the preparation of compound 3 [ka] To a solution of compound 2 (8.00 g, 43.4 mmol) in MeCN (160 mL), DIEA (39.3 g, 304 mmol) and DSC (47.8 g, 186.4 mmol) were added. The mixture was stirred at 40°C for 14 hours. Completion of the reaction was confirmed by TLC (compound 2, DCM / MeOH = 10 / 1, R). f(=0.5). The reaction mixture was poured into water (400 mL), and the temperature was then raised from 20°C to 27°C. After 15 minutes, the mixture was cooled to 17°C in an ice bath and stirred for 15 minutes. The solid was filtered, washed with water (3 × 20 mL), and dried under vacuum at 35°C for 4 hours to obtain crude compound 3 (9.60 g). Acetonitrile (20 mL) was added to the crude product, and the mixture was heated at 40°C for 1 hour using mechanical stirring. Heating was stopped, and the mixture was cooled to 8°C in an ice bath for 15 minutes. The solid was filtered, washed with acetonitrile (2 × 10 mL), and dried under vacuum at 35°C for 3 hours to obtain compound 3 (6.65 g, yield 36.3%).

[0608] 1 H NMR:(400MHz,CDCl3):δ 6.03-6.14(m,1H),5.60-5.67(m,1H),5.29(br s, 1H), 2.80-2.88 (m, 8H), 2.25-2.47 (m, 4H), 1.94-2.18 (m, 4H), 1.29 (s, 3H).

[0609] General procedure for the preparation of compound 4 [ka] To a solution of compound 3 (100 mg, 0.24 mmol) in DMF (20 mL), MMAE (136 mg, 0.19 mmol) and DIEA (61.2 mg, 0.47 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed the detection of one major peak with the desired mass. The residue was purified by preparative HPLC (water (0.1% FA)-ACN) to obtain compound 4 (41.0 mg, yield 16.9%).

[0610] LCMS(m / z):1025.6(M+H) + .

[0611] General procedure for the preparation of compound B [ka] To a solution of compound 4 (500 mg, 0.49 mmol) and compound 4-1 (519 mg, 3.90 mmol) in DMF (10 mL), DIEA (378 mg, 2.93 mmol) and DMAP (119 mg, 0.97 mmol) were added. The mixture was stirred at 25°C for 12 hours. LC-MS showed the detection of one major peak with the desired mass. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound B (161 mg, yield 31.6%).

[0612] 1 H NMR:(400MHz,MeOD):δ 7.73-8.00(m,1H),7.20-7.39(m,4H),5.78-5.96(m,1H),5.73(br s,1H),5.20-5.28(m,1H),5.13-5.20(m,1H),4.49-4.74(m,3H),4.17-4.28(m, 2H),4.04-4.10(m,1H),3.85-3.90(m,1H),3.50-3.80(m,2H),3.31-3.50(m,9H ),3.28-3.30(m,3H),2.77-3.12(m,6H),2.44-2.58(m,2H),1.78-2.36(m,13H) ,1.56-1.72(m,2H),1.22-1.48(m,3H),1.08-1.23(m,9H),0.80-1.07(m,18H).

[0613] LCMS(m / z):1043.62(M+H) + ; 1065.61(M+Na) + .

[0614] General procedure for the preparation of compound C [ka] To a solution of compound 4 (320 mg, 0.31 mmol) and compound 4-2 (187 mg, 2.50 mmol) in DMF (3.2 mL), DIEA (242 mg, 1.87 mmol) and DMAP (76.3 mg, 0.62 mmol) were added. The mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 4 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound C (92.0 mg, yield 29.9%).

[0615] 1 H NMR(400MHz,MeOD):δ 7.86-8.00(m,1H),7.15-7.45(m,5H),5.67-5.98(m,2H),5.17(br s,1H),4.50-4.74(m,2H),4.03-4.29(m,3H),3.81-3.89(m,2H),3.51-3.77(m,2H),3.46-3.50(m,1H),3.33-3.45(m,5H),3.30(br s,4H),3.20(dt,J=11.57,7.47Hz,1H),3.02-3.15(m,3H),2.90-3.01(m,1H),2.41-2.57(m,2H), 1.65-2.39(m,15H),1.52-1.64(m,1H),1.26-1.51(m,2H),1.08-1.23(m,9H),0.82-1.07(m,18H).

[0616] LCMS(m / z):985.6(M+H) + .

[0617] Example 8: Synthesis of Compound D [ka] [ka]

[0618] General procedure for the preparation of compound 6 [ka] To a solution of compound 5 (150 g, 689 mmol, HCl) in NaOH (1 M, 1.38 L) and NaHCO3 (1 M, 1.38 L), (2,5-dioxopyrrolidine-1-yl)2,2,2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L) was added. The mixture was stirred at 25°C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the dioxane. The residue was extracted with MTBE (5 L), and then the aqueous phase was adjusted to pH approximately 4 with saturated KHSO4 aqueous solution and extracted with RINKAN (5 L). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. To a solution of the crude product described above in MeOH (2 L), SOCl2 (90.2 g, 758 mmol) was added, and the mixture was stirred at 25°C for 2 hours. LC-MS indicated that the reaction was complete and that a single major peak with the desired mass was detected. The reaction mixture was adjusted to a pH of approximately 9-10 with saturated NaHCO3 aqueous solution and then extracted with siRNA (5 L). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was precipitated with PE (10 vol) to obtain compound 6 (190 g, yield 74.4%).

[0619] 1 H NMR:(400MHz,CDCl3):δ 3.25(br s,1H)3.85(s,3H)4.64-4.83(m,2H)5.30(dd,J=9.51,1.13Hz,1H)5.92(br d,J=9.38Hz,1H)7.30-7.45(m,5H).

[0620] LCMS(m / z):391.9 / 393.9(M+H) + .

[0621] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (185 g, 499 mmol) in toluene (1.9 L), 4-methylbenzenesulfonate pyridine (3.90 g, 15.4 mmol) and 4-methoxybenzaldehyde dimethyl acetal (121 g, 666 mmol) were added. The mixture was stirred at 110°C for 4 hours. LC-MS showed the detection of one major peak with the desired mass. The reaction mixture was then cooled to 25°C, and the reaction mixture was concentrated under reduced pressure to remove toluene. The residue was diluted with H2O (500 mL) and then extracted with siRNA (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 7 (285 g, crude), which was carried over as is.

[0622] General procedure for the preparation of compound 8 [ka] To a solution of compound 7 (285 g, crude) in MeOH (2000 mL), KOH (42.5 g, 758 mmol) in H2O (1000 mL) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 7 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was extracted with MTBE (5 L). The aqueous layer was diluted with saturated KHSO4 (1 L) aqueous solution and extracted with siRNA (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was precipitated with PE (10 vol) to obtain compound 8 (95.0 g, yield 34.3%).

[0623] 1 H NMR(400MHz,MeOD):δ 3.82(s,3H)4.41-4.47(m,1H)4.50-4.56(m,1H)4.60(d,J=4.88Hz,1H)5.47(d,J=4.75Hz,1H)6.46(s,1H)6.86-6.94(m,2H)7.34-7.46(m,7H).

[0624] LCMS (m / z): 495.9 (M + Na)+ .

[0625] General procedure for the preparation of 7-Troc-Baccatin III [ka] To a solution of baccatin III (30.0 g, 51.1 mmol) in DCM (300 mL), DMAP (625 mg, 5.11 mmol), pyridine (14.2 g, 179 mmol), and 2,2,2-trichloroethyl carbonochloride (15.2 g, 71.6 mmol) were added. The mixture was stirred at 25°C for 0.5 hours. LC-MS showed that baccatin III was completely consumed and a single major peak with the desired mass was detected. The residue was diluted with water (300 mL), extracted with DCM (300 mL), washed with water (200 mL) and brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 7-Troc-baccatin III (45.0 g, yield 34.3%).

[0626] LCMS (m / z): 761.5 / 763.5 (M+Na) + .

[0627] General procedure for the preparation of compound 9 [ka] To a solution of 7-Troc-baccatin III (26.0 g, 34.1 mmol) and compound 8 (32.4 g, 68.2 mmol) in DCM (1000 mL), DMAP (4.20 g, 34.1 mmol) and DCC (21.1 g, 102 mmol) were added. The mixture was stirred at 0°C for 1 hour. LC-MS showed that compound 8 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was filtered. The crude product was washed with saturated NH4Cl aqueous solution (100 mL) and water (1000 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 9 (35.0 g, crude).

[0628] LCMS(m / z):1240.0 / 1242.0(M+Na) + .

[0629] General procedure for the preparation of compound 10 [ka] To a solution of compound 9 (80.0 g, 65.6 mmol) in MeOH (350 mL), 4-methylbenzenesulfonic acid; hydrate (24.9 g, 131 mmol) was added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that approximately 50% of compound 9 remained, and one major peak with the desired mass was detected. The reaction mixture was filtered and concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluate was concentrated under reduced pressure to remove the solvent, and then extracted with siRNA (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 10 (13.0 g, yield 17.9%).

[0630] LCMS(m / z):1120.2(M+Na) + .

[0631] General procedure for the preparation of compound 11 [ka] Compound 10 (13.0 g, 11.8 mmol) was mixed with a solution of DMAP (722 mg, 5.90 mmol), EDCI (2.70 g, 14.2 mmol), and benzoic acid (1.70 g, 14.2 mmol) in DCM (260 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 10 was completely consumed and a single major peak with the desired mass was detected. The reaction mixture was washed with saturated citric acid aqueous solution (100 mL), saturated NaHCO3 aqueous solution (100 mL), and water (200 mL), dried over NaSO4, filtered, and concentrated under reduced pressure to obtain compound 11 (11.0 g, yield 77.3%).

[0632] LCMS(m / z):1204.1(M+H)+ .

[0633] General procedure for the preparation of compound 12 [ka] To a solution of compound 11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL), Zn dust (21.6 g, 331 mmol) was added. The mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 11 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was filtered, diluted with H2O (500 mL), and then extracted with siRNA (100 mL × 3). The combined organic layer was washed with saturated NaHCO3 aqueous solution (200 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound 12 (5.0 g, yield 21%).

[0634] LCMS(m / z):854.3(M+H) + .

[0635] General procedure for the preparation of compound 13 [ka] Compound 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and Compound 3 (3.90 g, 8.80 mmol) were dissolved in DMF (50 mL). The mixture was stirred at 25°C for 16 hours. LC-MS showed that approximately 50% of Compound 12 remained, and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain Compound 13 (505 mg, yield 7.4%).

[0636] LCMS(m / z):1161.4(M+H) + .

[0637] General procedure for the preparation of compound D [ka] To a solution of compound 13 (150 mg, 0.13 mmol) in DMF (1.50 mL), DMAP (94.7 mg, 0.78 mmol), compound 13-1 (66.7 mg, 0.65 mmol), and DIEA (100 mg, 0.78 mmol) were added. The mixture was stirred at 25°C for 16 hours. LC-MS showed that compound 13 was completely consumed and a single major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain compound D (75.0 mg, yield 50.5%).

[0638] LCMS(m / z):1148.5(M) + .

[0639] Example 9: General procedure for the preparation of compound E [ka] Compound 13 (350 mg, 0.30 μmol), DMAP (221 mg, 1.81 mmol), and Compound 14 (249 mg, 0.39 mmol, HCl) were dissolved in DMF (0.3 mL). The mixture was stirred at 25°C for 16 hours. LC-MS showed that Compound 13 was completely consumed and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to obtain Compound E (205 mg, yield 41.3%).

[0640] LCMS(m / z):1646.5(M+H) + .

[0641] Example 10: 3-(5-aminomethylpyrimidine)-6-methyl-1,2,4,5-tetrazine [ka] To a solution of N-Boc-2-cyano-5-aminomethylpyrimidine(1) in dry acetonitrile, hydrazine and nickel(II) triflate are added. The reaction mixture is then heated overnight and the starting materials are consumed by TLC. Sodium nitrite (dissolved in water), followed by 1 M hydrochloric acid, is added to the reaction mixture. The reaction mixture is then stirred at ambient temperature until the reaction is determined to be complete by HPLC. The reaction mixture is then partitioned between ethyl acetate and water. The organic layer is washed with water (three times), followed by brine (once), and then dried over sodium sulfate. The solution is filtered, and the filtrate is concentrated under reduced pressure to obtain the product, which can be carried over without further purification.

[0642] To solution 2 in 3-(5-aminomethylpyrimidine)-6-methyl-1,2,4,5-tetrazine(3)dioxane, hydrochloric acid (4M in dioxane) was added. The reaction mixture was then stirred at ambient temperature until the starting materials were consumed. The product was isolated by filtration, and the precipitate was washed with diethyl ether to obtain the product optionally as an HCl salt.

[0643] 6-(6-methyl-1,2,4,5-tetrazin-3-yl)-3-pyridinemethanamine may also be used in the compounds and methods described herein, which may be prepared in accordance with the art or purchased from a commercial source (e.g., Enamine US Inc., New Jersey, USA). [ka]

[0644] Example 11: Val-Cit-PABC-dihydrotetrazine [ka] N-Boc-3-(5-aminomethylpyrimidine)-6-methyl-1,2,4,5-dihydrotetrazine (4) In a sealed flask, thiourea dioxide is added to a solution of tetrazine 2 in DMF / H2O (v / v=10 / 1) at ambient temperature. The reaction mixture is then heated with stirring until the solution changes color from pink to colorless. The reaction mixture is concentrated under reduced pressure, and the resulting residue is dried under vacuum to obtain dihydrotetrazine 4, which can be used directly without further purification.

[0645] A solution of nitrophenyl carbonate 5 in toluene is added to a solution of dihydrotetrazine 4. The reaction mixture is then stirred at ambient temperature. Upon completion, the reaction mixture is concentrated under reduced pressure, and the resulting residue is purified by flash chromatography to obtain compound 6.

[0646] To solution 6 of 1-(N-acyl-(Val-Cit)-PABC)-3-(5-aminomethylpyrimidine)-6-methyl-1,2,4,5-dihydrotetrazine(7) in dioxane, hydrochloric acid (4 M in dioxane) was added. The reaction mixture was then stirred at ambient temperature until the starting materials were consumed. The product was isolated by filtration, and the precipitate was washed with diethyl ether to obtain the product optionally as an HCl salt.

[0647] Example 12: Dihydrotetrazine (Target 5) [ka] Compound 1 (5.00 g, 26.9 mmol, 1.00 equivalent, HCl) and Compound 2 (2.55 g, 26.9 mmol, 1.00 equivalent, HCl) were added at 20°C to a solution of N2H4.H2O (9.74 g, 190 mmol, 9.44 mL, 98% purity, 7.08 equivalents) in EtOH (35.0 mL). The mixture was stirred at 78°C for 3 hours. LC-MS analysis of the reaction mixture showed that Compound 1 was completely consumed. H2O (100 mL) was added to the reaction mixture at 20°C, and the resulting solution was concentrated under reduced pressure at 40°C to remove EtOH. The reaction mixture was extracted with 150 mL of ethyl acetate (50.0 mL x 3), and the combined organic layer was washed with 20.0 mL of brine (20.0 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, n-heptane / ethyl acetate = 100 / 1-1 / 1), and then further purified by preparative HPLC (HCl conditions) to obtain target 5 (100 mg, 2.87 mmol) as a white solid.

[0648] 1 ¹H NMR (400 MHz, methanol-d4): δ ppm 7.94-8.13 (m, 2H), 7.53-7.75 (m, 3H), 2.64 (s, 3H). LCMS:M+H + =175.1

[0649] Example 13: 3-methyl-6-(1-methyl-1H-imidazole-4-yl)-1,2,4,5-tetrazine (Target 4) [ka] 3-methyl-6-(1-methyl-1H-imidazole-4-yl)-1,2,4,5-tetrazine (Target 4): A solution of 1-methylimidazole-4-carbonitride (200 mg, 1.87 mmol), MeCN (268 mg, 6.54 mmol), and zinc; trifluoromethanesulfonate (68 mg, 0.19 mmol) in 1 mL of dioxane was mixed with NH2NH2.H2O (2.34 g, 46.68 mmol) at 25°C, and the mixture was stirred under N2 at 65°C for 16 hours. The mixture was then cooled to 25°C, and a solution of NaNO2 (387 mg, 5.60 mmol) in H2O (3 mL) was added dropwise at 25°C. The mixture was stirred at 25°C for 3 hours. The mixture was cooled to room temperature, adjusted to pH=3 with 1 M hydrochloric acid aqueous solution. The aqueous phase was extracted with DCM (3 × 5 mL). The combined organic matter was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (SiO2, DCM:MeOH = 10:1) to obtain 3-methyl-6-(1-methyl-1H-imidazole-4-yl)-1,2,4,5-tetrazine (Target 4) (20.1 mg, 6.1%).

[0650] LCMS(ESI+): m / z = 177.2[M+H] +

[0651] 1 H NMR(400MHz,CDCl3)(ET60578-12-P1M):δ=8.00(s,1H),7.68(s,1H),3.85(s,3H),3.06(s,3H).

[0652] Example 14: 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8) [ka] Isoxazole-3-carboxamide(2): To a solution of isoxazole-3-carboxylic acid (10 g, 88.44 mmol) in DMF (646 mg, 8.84 mmol) and DCM (100 mL), oxalyl dichloride (13.47 g, 106.13 mmol) was added under N2 at 0°C. The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in THF (50 ml) and adjusted to pH=9 with NH3.H2O at 0°C. The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain isoxazole-3-carboxamide(2) (3.3 g, 33.3%).

[0653] 1 H NMR(400MHz,DMSO):δ=9.05(d,J=1.6Hz,1H),8.12(s,1H),7.82(s,1H),6.85(d,J=1.6Hz,1H)

[0654] Isoxazole-3-Carbonitrile(3): To a solution of isoxazole-3-carboxamide (500 mg, 4.46 mmol) in pyridine (18 mL), POCl3 (1.03 g, 6.69 mmol) was added under N2 at 20°C. The mixture was stirred at 20°C for 2 hours. After stirring for 2 hours, the mixture was cooled in an ice bath and water (10 mL) was added. The aqueous phase was adjusted to pH=4 by adding 3 M HC1 aqueous solution and extracted with MTBE (3 × 10 mL). The combined organic matter was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain isoxazole-3-Carbonitrile(3) (280 mg, 66.7%).

[0655] 1 H NMR(400MHz,MeOD):δ 9.06(d,J=1.6Hz,1H),7.02(d,J=1.2Hz,1H)

[0656] 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8): To a solution of isoxazole-3-carbonitride (220 mg, 2.34 mmol) in EtOH (2 mL), NH2NH2.H2O (1.87 g, 37.42 mmol) was added at 20°C for 0.5 hours. Then, MeCN (384 mg, 9.35 mmol) and 3-sulfanylpropanoic acid (248 mg, 2.34 mmol) were added to the mixture under N2 at 20°C. The mixture was stirred at 45°C for 12 hours. After that, NaNO2 (500 mg) was added, and the reaction mixture was stirred at 20°C for 0.5 hours. 3M hydrochloric acid was added dropwise to pH=1, and the reaction mixture was stirred at 20°C for 0.5 hours. The aqueous phase was extracted with RINKAN (3 × 10 mL). The combined organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative HPLC (FA) under the following conditions: column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; B%: 5%-45%, for 8 minutes, to obtain 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (12.45 mg, 3.26%). LCMS (ESI+): m / z = 164.1 [M+H] +

[0657] 1H NMR (400MHz, MeOD): δ 9.01(d,J=1.6Hz,1H),7.34(d,J=1.6Hz,1H),3.11(s,3H).

[0658] Example 15: 3-methyl-6-(1H-pyrazole-1-yl)-1,2,4,5-tetrazine (Target 13) [ka] Methyl(E)-hydrazine carbohydrazonothioate (2): To a mixture of 1,3-diaminothiourea (100 g, 942.06 mmol) in MeOH (500 mL), MeI (160.46 g, 1.13 mol) was added at 25 °C, and the mixture was stirred under N2 at 80 °C for 1.5 hours. ¹H NMR indicated that the reaction was complete. The resulting pale yellow solution was cooled to room temperature until the solid precipitated. It was then diluted with MTBE (500 mL). The mixture was cooled on ice for 2 hours and filtered. The collected solid was washed with MTBE and dried under reduced pressure to obtain 1,3-diamino-2-methyl-isothiourea; hydroiodide (compound 2) (132 g, yield 56.48%, HI).

[0659] 1 H NMR(400MHz,DMSO):δ=10.96-9.08(m,1H),5.80-4.81(m,2H),2.37(s,3H)

[0660] To a solution of 1,3-diamino-2-methylisothiourea (30 g, 249.63 mmol) in 3-methyl-6-(methylthio)-1,2,4,5-tetrazine(4):DMF (750 mL), 1,1,1-triethoxyethane (44.55 g, 274.60 mmol) was added at 25°C, and the mixture was stirred under N2 at 25°C for 5 minutes. Then, TEA (25.26 g, 249.63 mmol) was added to the mixture at 25°C, and the mixture was stirred at 50°C for 3 hours. TLC showed that the reaction was complete. The mixture was poured into water (1000 mL) and extracted with ethyl acetate (3 × 500 mL). The combined organic layers were washed with brine (500 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1-5 / 1) to obtain 3-methyl-6-methylsulfanyl-1,2,4,5-tetrazine (compound 4) (5.8 g, 16.3%).

[0661] 1 H NMR (400MHz, CDCl3): δ=2.99(s,3H),2.74(s,3H).

[0662] 3-methyl-6-(1H-pyrazole-1-yl)-1,2,4,5-tetrazine (Target 13): To a mixture of 1H-pyrazole (144 mg, 2.11 mmol) in THF (2 mL), NaH (84 mg, 2.11 mmol, 60% purity) was added at 0°C, and the mixture was stirred under N2 at 0°C for 15 minutes. Then, 3-methyl-6-methylsulfanyl-1,2,4,5-tetrazine (200 mg, 1.41 mmol) in THF (1 mL) was added to the mixture at 0°C, and the mixture was stirred under N2 at 25°C for 2 hours. TLC showed that the reaction was complete. The mixture was poured into saturated NH4Cl (5 mL) and extracted with ELISA (3 × 3 mL). The combined organic layers were washed with brine (3 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative TLC (SiO2, PE:Â1 = 3:1) to obtain 3-methyl-6-pyrazole-1-yl-1,2,4,5-tetrazine (Target 13) (20 mg, 8.8%). LC-MS (ESI+): m / z = 163.1 [M+H] +

[0663] 1 H NMR (400MHz, CDCl3): δ=8.74(d,J=2.8Hz,1H),8.02(d,J=1.2Hz,1H),6.68(dd,J=1.6,2.8Hz,1H),3.14(s,3H).

[0664] Example 16: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15) [ka] To a solution of 4-(aminomethyl)-3-(trifluoromethyl)benzonitrile (300 mg, 1.50 mmol) in tert-butyl(4-cyano-2-(trifluoromethyl)benzyl)carbamate (2): Boc2O (360 mg, 1.65 mmol) and TEA (227 mg, 2.25 mmol) were added under N2 at 20°C. The mixture was stirred at 20°C for 2 hours. The mixture was cooled and poured into H2O (10 mL), and the aqueous phase was extracted with DCM (3 × 10 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (PE:SiO=1:0-3:1) to obtain tert-butyl(4-cyano-2-(trifluoromethyl)benzyl)carbamate (2) (400 mg, 88.9%).

[0665] 1 H NMR (400MHz, CDCl3): δ 7.94(s,1H),7.87-7.81(m,1H),7.79-7.73(m,1H),5.00(s,1H),4.56(d,J=6.0Hz,2H),1.47(s,9H).

[0666] To a solution of tert-butyl N-[[4-cyano-2-(trifluoromethyl)phenyl]methyl]carbamate (3): tert-butyl N-[[4-cyano-2-(trifluoromethyl)phenyl]methyl]carbamate (150 mg, 0.50 mmol) in EtOH (0.5 mL), NH2NH2.H2O (400 mg, 7.99 mmol) was added at 20°C for 0.5 hours. Then, MeCN (82 mg, 2.00 mmol) and 3-sulfanylpropanoic acid (53 mg, 0.50 mmol) were added to the mixture under N2 at 20°C. Subsequently, NaNO2 (500 mg) was added, and the reaction mixture was stirred at 20°C for 0.5 hours. 3M hydrochloric acid was added to pH=1, and the reaction mixture was stirred at 20°C for 0.5 hours. The aqueous phase was extracted with SiO2 (3 × 10 mL). The combined organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by preparative TLC (SiO2, PE:SiO2 = 3:1) to obtain tert-butyl N-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methyl)carbamate (3) (50 mg, 14.1%).

[0667] 1 H NMR(400MHz,CDCl3):δ 8.91(s,1H),8.78(d,J=8.4Hz,1H),7.84(d,J=8.4Hz,1H),5.11-4.96(m,1H),4.63(d,J=6.8Hz,2H),3.14(s,3H),1.49(s,9H).

[0668] (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methaneamine (Target 15): To a solution of tert-butyl N-((4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methyl)carbamate (50 mg, 0.14 mmol) in MeOH (2 mL), HCl / MeOH (2 mL, 4 M) was added under N2 at 20°C. The mixture was stirred at 20°C for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (FA) under the following conditions: Phenomenex Luna 80×30mm×3um, phase: [water (FA)-ACN]; B%: 1%-25%, for 8 minutes, to obtain (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methaneamine (4.8 mg, 15.9%).

[0669] LCMS(ESI+): m / z = 270.0[M+H] +

[0670] 1 H NMR (400MHz, MeOD): δ 8.93(s,1H),8.89(d,J=8.4Hz,1H),8.49(s,1H),7.96(d,J=8.0Hz,1H),4.37(s,2H),3.09(s,3H).

[0671] Example 17: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 17) [ka] 3-((1,3-dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile(2): To a solution of 3-(hydroxymethyl)-4-(trifluoromethyl)benzonitrile (4.5 g, 22.38 mmol) in THF (50 mL), isoindolin-1,3-dione (3.3 g, 22.38 mmol), PPh3 (11.7 g, 44.76 mmol), and DIAD (6.79 g, 33.57 mmol) were added under N2 at 0°C. The resulting mixture was then stirred at 20°C for 16 hours. The reaction mixture was quenched by adding water (15 mL) and extracted with ELISA (3 × 10 mL). The combined organic phase was washed with brine (15 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the residue, which was then purified by column chromatography to obtain 3-((1,3-dioxoisoindorin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (2) (5.1 g, 69%).

[0672] 1 H NMR(400MHz, CDCl3)δ=7.91-8.03(m,2H),7.84(dd,J=5.6,2.8Hz,3H),7.70(d,J=8.0Hz,1H),7.46(s,1H),5.13(s,2H).

[0673] 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (3): To a solution of 3-((1,3-dioxoisoindorin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (1 g, 3.03 mmol) in EtOH (20 mL), NH2NH2.H2O (3.79 g, 60.56 mmol, 80% purity) was added at 20°C, and the mixture was stirred at 20°C for 12 hours. 6M hydrochloric acid was added to bring the pH down to 1, and the reaction mixture was stirred at 20°C for 2 hours. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was poured into water (50 mL), and the pH was adjusted to 11 with 6M NaOH aqueous solution (20 mL). Next, the mixture was extracted with toluene (3 × 50 mL), the organic layer was washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (3) (600 mg, 99.0%).

[0674] 1 H NMR (400MHz, CDCl3): δ=8.09(s,1H),7.72-7.80(m,1H),7.62-7.71(m,1H),4.12ppm(s,2H).

[0675] To a solution of tert-butyl(5-cyano-2-(trifluoromethyl)benzyl)carbamate(4):dioxane (6 mL) and 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (0.3 g, 1.50 mmol) and NaOH (180 mg, 4.50 mmol) in H2O (3 mL), Boc2O (654 mg, 3.00 mmol) was added at 20°C, and the mixture was stirred at 20°C for 2 hours. The reaction mixture was quenched with H2O (10 mL) and extracted with RINKAN (3 × 10 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (PE:Â=1:0-0:1) to obtain tert-butyl(5-cyano-2-(trifluoromethyl)benzyl)carbamate (4) (300 mg, 66.6%).

[0676] 1 H NMR (400MHz, CDCl3): δ=7.89(s,1H),7.75(s,1H),7.65-7.71(m,1H),4.88-5.19(m,1H),4.54(br d,J=6.0Hz,2H),1.48ppm(s,9H).

[0677] To a solution of tert-butyl(5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5): tert-butyl(5-cyano-2-(trifluoromethyl)benzyl)carbamate (500 mg, 1.67 mmol), MeCN (273 mg, 6.66 mmol), and 3-sulfanylpropanoic acid (177 mg, 1.67 mmol) in EtOH (3 mL), N2H4.H2O (1.33 g, 26.64 mmol) was added at 25°C, and the mixture was stirred under N2 at 65°C for 16 hours. The mixture was then cooled to room temperature, and a solution of NaNO2 (345 mg, 5.00 mmol) in H2O (2.5 mL) was added dropwise at 25°C. The mixture was stirred at 25°C for 3 hours. The mixture was cooled to room temperature and adjusted to pH=3 with 1M hydrochloric acid aqueous solution. The mixture was extracted with DCM (20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: siRNA = 20:1-1:1) to obtain tert-butyl (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5) (140 mg, 22.8%).

[0678] 1 H NMR (400MHz, CDCl3): δ=8.78(s,1H),8.60(d,J=8.0Hz,1H),7.88(d,J=8.0Hz,1H),5.08(s,1H),4.67(s,1H),3.15(s,3H),1.49(s,9H).

[0679] (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15): To a solution of tert-butyl N-[[5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl]methyl]carbamate (5) (140 mg, 0.38 mmol) in MeOH (2 mL), HCl / MeOH (2 mL, 4 M) was added under N2 at 20 °C. The mixture was stirred at 20 °C for 2 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative TLC (SiO2, DCM:MeOH = 10:1) to obtain [5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl]methanamine (18.0 mg, 17.7%).

[0680] LCMS(ESI+): m / z = 270.0[M+H] +

[0681] 1 H NMR(400MHz,MeOD):δ 8.88(s,1H),8.60(d,J=8.4Hz,1H),7.94(d,J=8.4Hz,1H),4.11(s,2H),3.08(s,3H)

[0682] Example 18: 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (Target 6) [ka] To a mixture of tert-butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl) carbamate:EtOH (140 mL) and 4-iodobenzonitrile (15 g, 65.50 mmol), tert-butyl N-(2-cyanoethyl) carbamate (44.59 g, 261.99 mmol), 3-sulfanylpropanoic acid (6.95 g, 65.50 mmol), and NH2NH2.H2O (59.02 g, 1.18 mol) were added under N2 at 0°C. The mixture was stirred at 45°C for 16 hours. The mixture was then cooled to 20°C, and a solution of NaNO2 in H2O (40 mL) was added dropwise at 20°C. The mixture was stirred at 20°C for 1 hour. Under ice cooling, the pH was adjusted to 3 with a 1M hydrochloride aqueous solution, and then extracted with DCM (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: siRNA = 10:1-1:1) to obtain tert-butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl) carbamate (14 g, 50%).

[0683] LCMS: m / z = 372.0 [M - BuOH + H] + .

[0684] A mixture of tert-butyl(2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethane-1-amine:HCl / HCl (4M, 100 mL) and tert-butyl(2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl) carbamate (6.5 g, 5.85 mmol) was stirred at 25°C for 1 hour. The reaction product was concentrated under reduced pressure to obtain 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethane-1-amine hydrochloride (5.8 g, 80%).

[0685] LCMS: m / z = 328.2[M+H] + .

[0686] To a mixture of 1-(2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea:DCM (45 mL) and CDI (3.35 g, 20.63 mmol), TEA (4.18 g, 41.27 mmol) and 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethane-1-amine (5.8 g, 13.76 mmol) were added under N2 at -40°C. The mixture was stirred at -40°C for 1 hour. Then, MeNH2 (2 M, 17 mL) and TEA (4.17 g, 41.24 mmol) were added under N2 at 0°C. The mixture was stirred at 25°C for 12 hours. The reaction product was concentrated under reduced pressure to obtain a residue. The residue was ground in DCM. The obtained solid was collected by filtration, washed with DCM (40 mL), and dried under reduced pressure to obtain 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methylurea (3 g, 57%). LCMS: m / z = 385.0 [M + H] + .

[0687] 1H NMR (400MHz, DMSO): δ=8.24(d,J=8.4Hz,2H)8.06(d,J=8.4Hz,2H)6.09(br t,J=5.6Hz,1H)5.77(br d,J=4.8Hz,1H)3.53-3.57(m,2H)3.36-3.40(m,2H)2.45(d,J=4.4Hz,3H).

[0688] To a mixture of tert-butyl(4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate:2-methyl-2-butanol (4 mL) and H2O (1 mL), 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methylurea (1 g, 2.60 mmol) and (tert-butoxycarbonylamino)methyltrifluoroboron;potassium hydride (926 mg, 3.90 mmol) were added under N2 at 25°C. The mixture was stirred at 80°C for 16 hours. The mixture was diluted with H2O (15 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic matter was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:ethyl = 5:1-0:1) to obtain tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (200 mg, 20%).

[0689] LCMS: m / z = 388.3[M+H] + .

[0690] 1H NMR (400MHz, DMSO): δ=8.44(br d,J=8.0Hz,2H),7.52(br d,J=7.6Hz,3H),6.08(br t,J=5.6Hz,1H),5.78(br s,1H),4.26(br d,J=5.6Hz,2H),3.54-3.58(m,2H),3.38(br d,J=6.4Hz,2H),2.45(d,J=4.4Hz,3H),1.41(s,9H).

[0691] A mixture of 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (target 6):tert-butyl(4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (150 mg, 0.39 mmol) was mixed with HCl / ELISA (4 M, 3 mL) under N2 at 25°C. The mixture was stirred at 25°C for 1 hour. The reaction product was concentrated under reduced pressure. The crude product was purified by preparative HPLC under the following conditions: column: Phenomenex C18 100×30mm×5μm; mobile phase: A: 10mM NH4HCO3 in water, B: MeCN; B% in A: 20%-50%, for 10 minutes, to obtain 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (63 mg, 29%). LCMS: m / z = 288.2 [M + H] + .

[0692] 1H NMR (400MHz, DMSO): δ=8.52(d,J=8.0Hz,2H),8.45(br s,2H),7.77(d,J=8.2Hz,2H),6.14(br t,J=5.6Hz,1H),5.76-5.83(m,1H),4.18(br s,2H),3.56(q,J=6.4Hz,2H),3.38-3.41(m,2H),2.44(d,J=4.4Hz,3H).

[0693] Example 19: 4-((S)-2-((S)-2-acetamido-3-methylbutanamide)-5-ureidopentanamide)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b) [ka] To a solution of benzonitrile (10 g, 96.97 mmol), ACN (31.85 g, 775.79 mmol), and 3-mercaptopropanoic acid (10.29 g, 96.97 mmol) in 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine(2):EtOH (100 mL), NH2NH2.H2O (79.26 g, 1.55 mol) was added dropwise at 0°C under N2. The mixture was stirred at 40°C for 16 hours. The mixture was quenched with water (100 mL), the pH was adjusted to 4 by adding HCl (1 M), and the mixture was extracted with ELISA (3 × 100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The mixture was slurryed with MTBE:siRNA (10:1, 100 mL) and filtered, and the solid was as desired. The solid was purified by p-HPLC (FA) under the following conditions: column: Phenomenex luna C18 (250 × 70 mm, 15 μm); mobile phase: [water (FA)-ACN]; B%: 8%-35%, for 22 minutes to obtain 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (10 g, 29%).

[0694] LCMS, m / z = 175.1 [M + H] +

[0695] 1 H NMR (400MHz, DMSO): δ 8.50(br s,1H),8.29(br s,1H),7.74(dd,J=1.6,7.6Hz,2H),7.47-7.35(m,3H),1.78(s,3H).

[0696] 4-Nitrophenyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate(4): To a solution of 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (3 g, 17.22 mmol) in DCM (30 mL), DIEA (6.68 g, 51.66 mmol) and then (4-nitrophenyl)carbonochloride (3.64 g, 18.08 mmol) were added under N2 at 0°C...

Claims

1. A tetrazine-based targeting agent comprising, optionally, one or more targeting moieties covalently conjugated to one or more tetrazine moieties via a linker, provided that at least one of the targeting moieties is a non-antibody or non-antibody fragment targeting agent selected from nectin-4 peptide targeting agents, PSMA peptide targeting agents, CCK2R peptide targeting agents, PD-L1 peptide targeting agents, integrin alpha-V beta-3 targeting agents, integrin alpha-V beta-6 targeting agents, integrin alpha-V beta-8 targeting agents, FAP targeting agents, and carbonic anhydrase IX (CAIX) targeting agents.

2. The tetrazine-based targeting agent according to claim 1, wherein at least one targeting portion is selected from Table 1.

3. Each tetrazine portion is independently of the one in formula I. 【Chemistry 1】 During the ceremony, R in each appearance １ These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ NR'R", C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O )R”, NR’C(=S)R”, NR’C(=O)OR”, NR’C(=S)OR”, NR’C(=O)SR”, NR’C(=S)SR”, OC(=O)NR’R”, SC Selected from the group consisting of (=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected to have 1 to 3 Z １ It has been replaced with, R ３ and R ４ Both are absent, or R ３ and R ４ Each of these is a group that can be removed independently after the hydrogen or trigger event. The dotted line represents R ３ and R ４ are both absent, a tetrazine, or when both R ３ and R ４ are both present, an optional double bond that forms dihydrotetrazine, Y in each appearance １ These are independently, directly bonded, O, S, NR ａ , or CR ３１ａ R ３１ｂ NR ａ Selected from the group consisting of, Y in each appearance ２ Each of these is independently directly bonded, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently and optionally bonded to 1 to 4 R ２１ It has been replaced with, R in each appearance ａ , R ３１ａ , and R ３１ｂ These are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl, R in each appearance ２１ Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has 1 to 3 Z of any choice. １ It has been replaced with, Each Z １ These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ Selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R'', In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. A tetrazine-based targeting agent according to claim 1 or 2, wherein R''' in each occurrence is independently selected from alkyl and aryl.

4. R ３ and R ４ The tetrazine-based targeting agent according to claim 3, wherein both are absent and the dotted line is an optional double bond forming tetrazine.

5. R ３ and R ４ The tetrazine-based targeting agent according to claim 3, wherein each of the groups is a hydrogen group that can be removed independently after a trigger event, and the dotted line is an optional double bond that forms dihydrotetrazine.

6. R in each appearance ２ Each is independently a cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each cycloalkylene, heterocyclylene, arylene, or heteroarylene independently has, at will, 1 to 3 Z １ A tetrazine-based targeting agent according to any one of claims 3 to 5, which is substituted with

7. Each tetrazine portion is independently of the one in Equation II. 【Chemistry 2】 During the ceremony, R in each appearance １ These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ NR'R", C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O )R”, NR’C(=S)R”, NR’C(=O)OR”, NR’C(=S)OR”, NR’C(=O)SR”, NR’C(=S)SR”, OC(=O)NR’R”, SC Selected from the group consisting of (=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected to have 1 to 3 Z １ It has been replaced with, R in each appearance ２１ Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has 1 to 3 Z of any choice. １ It has been replaced with, In each occurrence, ring A is independently a cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each cycloalkylene, heterocyclylene, arylene, or heteroarylene independently has 1 to 3 Z of any choice. １ It has been replaced with, In each occurrence, t is independently 0, 1, 2, 3, or 4. Each Z １ These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ Selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R'', In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. A tetrazine-based targeting agent according to any one of claims 1 to 6, wherein R''' in each occurrence is independently selected from alkyl and aryl.

8. The tetrazine-based targeting agent according to any one of claims 1 to 7, wherein the linker is branched.

9. The tetrazine-based targeting agent according to any one of claims 1 to 8, wherein the tetrazine-based targeting agent comprises two or more targeting moieties.

10. The tetrazine-based targeting agent according to any one of claims 1 to 9, wherein the tetrazine-based targeting agent comprises two or more tetrazine moieties.

11. The tetrazine-based targeting agent according to any one of claims 1 to 7, wherein the linker is a straight chain.

12. A tetrazine-based targeting agent of formula III, 【Transformation 3】 During the ceremony, X is a targeting agent selected from Table 1, L is the linker, p is between 1 and 16. R in each appearance １ These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, heteroaryl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ NR'R", C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O )R”, NR’C(=S)R”, NR’C(=O)OR”, NR’C(=S)OR”, NR’C(=O)SR”, NR’C(=S)SR”, OC(=O)NR’R”, SC Selected from the group consisting of (=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl can be independently and optionally selected to have 1 to 3 Z １ It has been replaced with, R ３ and R ４ Both are absent, or R ３ and R ４ Each of these is a group that can be removed independently after the hydrogen or trigger event. The dotted line is R ３ and R ４ If both are absent, then tetrazine, or R ３ and R ４ This represents an optional double bond that forms dihydrotetrazine when both are present. Y in each appearance １ These are independently, directly bonded, O, S, NR ａ , or CR ３１ａ R ３１ｂ NR ａ Selected from the group consisting of, Y in each appearance ２ Each of these is independently directly bonded, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene is independently and optionally bonded to 1 to 4 R ２１ It has been replaced with, R in each appearance ａ , R ３１ａ , and R ３１ｂ These are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl, R in each appearance ２１ Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has 1 to 3 Z of any choice. １ It has been replaced with, Each Z １ These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ Selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R'', In each occurrence, R' and R'' are independently selected from hydrogen, alkyl, and aryl. A tetrazine-based targeting agent of formula III, wherein R''' in each occurrence is independently selected from alkyl and aryl.

13. A tetrazine-based targeting agent of formula IA, formula IIA, or formula VA, 【Chemistry 4】 During the ceremony, Ring A is an aryl, cycloalkyl, heterocyclyl, or heteroaryl ring. The dotted line is R ３ and R ４ If both are absent, then tetrazine, or R ３ and R ４ This represents an additional bond that forms dihydrotetrazine when both are present. X is a targeting agent selected from Table 1, p is between 1 and 16. In each appearance, L is independently a linker. R in each appearance １ These independently include hydrogen, halo, cyano, nitro, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ NR'R", C(=O)OR', C(=O)SR', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(= O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", Selected from the group consisting of SC(=O)NR'R'', OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'', each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally associated with 1 to 3 Z １ It has been replaced with, R in each appearance ２ Each of these is independently a halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -C(=O)-alkyl, -C(=O)-haloalkyl, -C(=O)-alkenyl, -C(=O)-alkynyl, -C(=O)-alkoxy, -C(=O)-haloalkoxy, -C(=O)-heteroalkyl, -C(=O)-aryl, -C(=O)-heteroaryl, -C(=O)-heterocyclyl, or -C(=O)-cycloalkyl, and each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has 1 to 3 Z of any choice. １ It has been replaced with, R ３ and R ４ Both are absent, or R ３ and R ４ Each of these is a group that can be removed independently after the hydrogen or trigger event. R at each occurrence ２０ is independently hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, CF ３ CF ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'', NR'R'', NR'C(=O)R'', NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR'', NR'C(=S)SR'', OC(=O)NR'R'', SC(=O)NR'R'', OC(=S)R'R'''', SC(=S)R'R'', NR'C(=O)NR''R'', and NR'C(=S)NR''R'' and is selected from the group consisting of R in each appearance ２２ The linker is independently and optionally comprises one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional groups, and consists of 1 to 100 linked atoms. R in each appearance ３０ These are independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl. R ａ 、R ３１ａ 、and R ３１ｂ are each independently hydrogen, C １ -C ６ -alkyl, or C １ -C ６ -haloalkyl, and Each Z １ These independently include halo, oxo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''", OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S (=O) ２ Selected from NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R'', NR'R", NR'C(=O)R", NR'C(=S)R'', NR'C(=O)OR'', NR'C(=S)OR'', NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R'', NR'C(=O)NR"R", and NR'C(=S)NR"R'', In each occurrence, R' and R'' are independently selected from hydrogen, aryl, and alkyl. In each occurrence, R''' is independently selected from aryl and alkyl groups. A tetrazine-based targeting agent of formula IA, formula IIA, or formula VA, wherein t in each occurrence is independently 0, 1, 2, 3, or 4.

14. The aforementioned part: 【Transformation 5】 A tetrazine-based targeting agent according to claim 12, as represented by [the specified figure].

15. The aforementioned part: 【Transformation 6】 but, 【Chemistry 7-1】 【Chemistry 7-2】 A tetrazine-based targeting agent according to claim 3, as represented by [the specified figure].

16. R in each appearance １ Each element is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl, and each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl has, optionally, 1 to 3 Z １ A tetrazine-based targeting agent according to any one of claims 3 to 15, which is substituted with

17. R in each appearance １ Independently, hydrogen or optionally, 1 to 3 Z １ A tetrazine-based targeting agent according to any one of claims 3 to 16, wherein the alkyl is substituted with

18. Z in each appearance １ A tetrazine-based targeting agent according to any one of claims 3 to 17, wherein is independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

19. R in each appearance ２１ A tetrazine-based targeting agent according to any one of claims 3 to 18, wherein is independently a halo, alkyl, or haloalkyl.

20. A tetrazine-based targeting agent according to any one of claims 3 to 18, wherein t is 0 in each occurrence.

21. R ２ A tetrazine-based targeting agent according to any one of claims 3 to 20, wherein ring A is pyridyl or phenyl.

22. R ２ A tetrazine-based targeting agent according to any one of claims 3 to 20, wherein ring A is other than pyridyl or phenyl.

23. A tetrazine-based targeting agent according to any one of claims 1 to 22, wherein X further comprises an imaging contrast agent.

24. The tetrazine-based targeting agent according to claim 23, wherein the imaging contrast agent is a protein.

25. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker, comprises one or more amino acids, according to any one of claims 1 to 24.

26. L, -Y ２ -Y １ -L, -Y ２ A tetrazine-based targeting agent according to any one of claims 1 to 25, wherein -L, or the linker, comprises a polypeptide.

27. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker comprises one or more of the following: hydrazone, hydrazide, disulfide, N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), 4-(4'-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acids, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety, according to any one of claims 1 to 26.

28. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker comprises 1 to 100 linked atoms, 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, or 1 to 40 linked atoms, or 1 to 30 linked atoms, or 1 to 20 linked atoms, or 1 to 10 linked atoms, or 1 to 5 linked atoms, or 5 to 30 linked atoms, or 10 to 30 linked atoms, or 5 to 40 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms, according to any one of claims 1 to 27.

29. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety is independently and optionally oxo, halo, C １－４ Alkyl, C １－４ Alkoxy, and C １－４ A tetrazine-based targeting agent according to any one of claims 1 to 28, which may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

30. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, or heteroarylene moieties, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene moiety is independently and optionally oxo, halo, C １－４ Alkyl, C １－４ Alkoxy, and C １－４ A tetrazine-based targeting agent according to any one of claims 1 to 29, which may be substituted with 1 to 5 substituents independently selected from the haloalkyl group.

31. L, -Y ２ -Y １ -L, -Y ２ A tetrazine-based targeting agent according to any one of claims 1 to 30, wherein -L, or the linker, is an alkylene linker comprising, optionally, one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thiosuccinimide, or ketone functional groups.

32. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker having the following formula: -Y １０ —(CHR １３０ ( ｎ’ -Y ２０ —(CHR １４０ ( ｎ’’ -Y ３０ —(CHR １５０ ( ｍ’’ -Y ４０ — During the ceremony, Y １０ , Y ２０ , Y ３０ , and Y ４０ Each of them independently, combined, -NR １１０ -, -O-, -S(O) ０－２ -, -NR １１０ C(O)-, -C(O)NR １１０ -, -NR １１０ S(O) ２ -, -S(O) ２ NR １１０ -, -CR １２０ = N - NR １１０ -, -NR １１０ -N=CR １２０ -, -C(O)-, -OC(O)-, -OC(O)O-, -(CH ２ CH ２ O) １－５ -, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene, and each alkylene, alkenylene, alkynylene, arylene, or heteroarylene can be independently and optionally selected as oxo, halo, or C １－４ Alkyl, C １－４ Alkoxy, and C １－４ It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R １１０ Hydrogen, C １－４ Alkyl, C １－４ They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R １２０ Hydrogen, C １－４ Alkyl, C １－４ They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R １３０ Hydrogen, C １－４ Alkyl, C １－４ The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. Each R １４０ Hydrogen, C １－４ Alkyl, C １－４ The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. Each R １５０ Hydrogen, C １－４ Alkyl, C １－４ The side chains are haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or amino acid side chains. A tetrazine-based targeting agent according to any one of claims 1 to 31, wherein n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

33. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker having the following formula: -Y １０ —(CH ２ ( ｎ’ -Y ２０ —(CH ２ ( ｍ’’ -Y ３０ — During the ceremony, Y １０ , Y ２０ , and Y ３０ Each of them independently, combined, -NR １１０ -, -O-, -S(O) ０－２ -, -NR １１０ C(O)-, -C(O)NR １１０ -, -NR １１０ S(O) ２ -, -S(O) ２ NR １１０ -, -CR １２０ = N - NR １１０ -, -NR １１０ -N=CR １２０ -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, and each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene can be independently and optionally selected as oxo, halo, or C １－４ Alkyl, C １－４ Alkoxy, and C １－４ It is substituted with 1 to 5 substituents independently selected from the haloalkyl group. Each R １１０ Hydrogen, C １－４ Alkyl, C １－４ They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. Each R １２０ Hydrogen, C １－４ Alkyl, C １－４ They are haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl. A tetrazine-based targeting agent according to any one of claims 1 to 31, wherein n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

34. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker, 【Chemistry 8-1】 【Chemistry 8-2】 A tetrazine-based targeting agent according to any one of claims 1 to 33, which is or includes the same.

35. L, -Y ２ -Y １ -L, -Y ２ -L, or the linker, 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 A tetrazine-based targeting agent according to any one of claims 1 to 33.

36. A tetrazine-based targeting agent of formula IID, 【Chemistry 10】 In the formula, X is a tetrazine-based targeting agent of formula IID, selected from Table 1.

37. A tetrazine-based targeting agent of formula IIH, 【Chemistry 11】 In the formula, X is a tetrazine-based targeting agent of formula IIH, selected from Table 1.

38. Table 2 shows tetrazine-based targeting agents, or pharmaceutically acceptable salts thereof.

39. A pharmaceutical composition comprising a tetrazine-based targeting agent according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

40. A method for treating cancer or enhancing or inducing an immune response, comprising administering to a subject in need of such treatment an effective amount of a tetrazine-based targeting agent according to any one of claims 1 to 38, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, comprising a conjugate comprising a payload linked to one or more trans-cyclooctene moieties, or a pharmaceutically acceptable salt thereof.

41. The conjugate is of formula X or a pharmaceutically acceptable salt thereof. 【Chemistry 12】 During the ceremony, In each occurrence, G is independent. 【Chemistry 13】 And, L in each appearance １ It is independent and is a linker, m is an integer between 1 and 150. D is the payload, R in each appearance １Ａ C １－４ Alkyl, C １－４ Haloalkyl and C １－４ Selected from the group consisting of alkoxys, q is 0, 1, or 2, q1 is 0 or 1, R in each appearance １Ｂ G １ ,OH,-NR １ｃ -C １－４ Alkilen-G １ , -NR １ｃ -C １－４ Alkylene-N(R) １ｄ ) ２ , -NR １ｃ -C １－６ Alkylene-N(C) １－４ Alkyl) ３ ＋ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ )-C １－６ Alkylene-CO2 ２ H, -N(R １ｆ )-C ２－４ Alkylene-(N(C) １－４ Alkylene-CO2 ２ H)-C ２－４ Alkilen) ｎ -N(C) １－４ Alkylene-CO2 ２ H) ２ , -N(R １ｃ ) CHR １ｅ C(O)OC １－６ Alkyl, -N(R) １ｃ )-C １－６ Alkylene-C(O)OC １－６ Alkyl, -N(R) １ｆ )-C ２－４ Alkylene-(N(C) １－４ Alkylene-C(O)OC １－６ Alkyl)-C ２－４ Alkilen) ｎ -N(C) １－４ Alkylene-C(O)OC １－６ Alkyl) ２ , -N(R １ｃ )-C １－６ Alkylene-SO ３ H, -N(R １ｃ )-(CH ２ CH ２ O) １－３ -CH ２ CH ２ N((CH ２ CH ２ O) １－３ -C １－６ Alkylene-CO2 ２ H) ２ , and -N(R １ｃ )-CH(CH ２ O-(CH ２ CH ２ O) ０－２ -C １－６ Alkylene-CO2 ２ H) ２ Selected from the group consisting of, R in each appearance １ｃ and R １ｄ Independently, hydrogen or C １－４ It is alkyl, R in each appearance １ｅ -C １－４ Alkylene-CO2 ２ H, -C １－４ Alkilen-CONH ２ , or -C １－４ It is an alkylene-OH group. R in each appearance １ｆ Hydrogen, C １－６ Alkyl, or C １－４ Alkylene-CO2 ２ It is H, n in each occurrence is independently 0, 1, 2, or 3. L in each appearance ２ Independently, -C(O)- and C １－３ Selected from the group consisting of alkylenes, G in each appearance １ The method according to claim 40, wherein is an independently optionally substituted heterocyclyl.

42. The method according to claim 40 or 41, wherein the payload is an immunomodulatory agent payload.

43. The method according to any one of claims 40 to 42, wherein the payload is a therapeutic monoclonal antibody, a cytokine, a chemokine, a chemokine antagonist, or an immune checkpoint inhibitor payload, or a pharmaceutically acceptable salt thereof.

44. The payload may include therapeutic agents for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepylone, patupylone (epoterone class), platinum-based drugs, exatecan, orlistin (drastatin 10, MMAE, MMAD, MMAF), mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, etc.), immunosuppressants (e.g., cyclosporine A, rapamycin, etc.), antifungal agents (e.g., amphotericin, etc.), antibiotics ( The method according to any one of claims 40 to 43, for example, selected from vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin beta, flucytosine, emtricitabine, gentamicin, colistin, etc.), lurubinectedin, gardiquimod, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic adenosine monophosphatidyl (c-AMP).

45. The aforementioned conjugate, 【Chemistry 14-1】 【Chemistry 14-2】 The method according to claim 40, selected from the following.

46. The method according to any one of claims 40 to 45, wherein the method is a method for treating cancer.

47. The method according to claim 46, wherein the cancer is melanoma, kidney cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal cancer / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin lymphoma, Hodgkin lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal cancer, or cutaneous T-cell lymphoma.

48. The method according to claim 46 or 47, wherein the cancer is a solid tumor.

49. The method according to claim 46 or 47, wherein the cancer is a soft tissue sarcoma.

50. The method according to claim 49, wherein the cancer is a hematological malignancy such as myelodysplastic syndrome, acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammaglobulinemia, plasmacytomyeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, or nodular lymphocyte-predominant Hodgkin lymphoma.

51. The method according to any one of claims 40 to 50, wherein the method is a method for enhancing or inducing an immune response.

52. The method according to claim 51, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

53. The method according to any one of claims 40 to 52, further comprising administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of anticancer agents, immunomodulators, or trans-cyclooctenprodrugs thereof.

54. A kit comprising a tetrazine-based targeting agent according to any one of claims 1 to 39, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 40, and instructions for use thereof.

55. The kit according to claim 54, further comprising a conjugate containing a payload linked to one or more trans-cyclooctene portions, or a pharmaceutically acceptable salt thereof.

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