Tetrazine-based targeting agents for in vivo delivery of a payload

Abstract

The present disclosure relates generally to tetrazine-based targeting agents for bioorthogonal delivery of a payload to a targeted location in a subject, which tetrazine-based targeting agents have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy.

Description

TETRAZINE-BASED TARGETING AGENTS FOR IN VIVO DELIVERY OF A PAYLOADCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Numbers 63 / 500,246, filed May 4, 2024, and 63 / 536,292, filed September 1, 2023, each of which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates generally to tetrazine-based targeting agents for bioorthogonal delivery of a payload to a targeted location in a subject, which tetrazine-based targeting agents have applications, e.g., in the treatment of cancer, tumor growth, and immunotherapy.BACKGROUND

[0003] Bioorthogonal conjugation or click reactions are selective and orthogonal (non-interacting with) functionalities found in biological systems, and have found use in various applications in the fields of chemistry, chemical biology, molecular diagnostics, and medicine, where they can be used to facilitate the selective manipulation of molecules, cells, particles and surfaces, and the tagging and tracking of biomolecules in vitro and in vivo. These reactions include the Staudinger ligation, the azide-cyclooctyne cycloaddition, and the inverse-electron-demand Diels-Alder reaction.SUMMARY

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

[0005] . The tetrazine-based targeting agents described herein are designed to, once administered to a subject, localize at a target site within the subject. The tetrazine-based targeting agent can be administered locally or systemically. Once administered, a prodrug comprising a payload or therapeutic agent and one or more complimentary bioorthogonal components (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine -based targeting agent in vivo, allows for targeted delivery of the payload or therapeutic agent. In some embodiments, the tetrazine-based targeting agent comprises is a therapeutic targeting agent.

[0006] In some embodiments, the tetrazine-based targeting agents described herein comprise a diagnostic agent such that the tetrazine-based targeting agents described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.

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

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

[0009] In some embodiments, the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a lymphoma or leukemia. In some embodiments, the cancer is a hematologic malignancy.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A and Figure IB show Compound 111 binds to both human (Fig. 1 A) and mouse (Fig. IB) isoforms of recombinant Nectin-4 in a ligand binding assay, while Compound 111-C does not bind in either case.

[0011] Figure ! shows Compounds B and F convert to MMAE and exatecan, respectively, after contact with Compound 111.

[0012] Figure 3 shows the tumor response of Compound 111 + Compound B in NCI-H292 tumors in BALB / c nude mice.DETAILED DESCRIPTION

[0013] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.1. Definitions

[0014] Certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that suchcombinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.A. Definitions

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0016] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as 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 plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0017] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9- 1.1. Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0018] The conjunctive term “or” includes any and all combinations of one or more listed elements associated by the conjunctive term. For example, the phrase “an apparatus comprising A or B” may refer to an apparatus including A where B is not present, an apparatus including B where A is not present, or an apparatus where both A and B are present. The phrases “at least one of A, B, . . . and N” or “at least one of A, B, . . . N, or combinations thereof’ are defined in the broadest sense to mean one or more elements selected from the group comprising A, B, . . . and N, that is to say, any combination of one or more of the elements A, B, . . . or N including any one element alone or in combination with one or more of the other elements which may also include, in combination, additional elements not listed.

[0019] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0020] The term “alkyl” as used herein, means a straight or branched, saturated hydrocarbon chain containing from 1 to 30 carbon atoms. The term “lower alkyl” or “C1-C6-alkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “C1-C3- alkyl” means a straight or branched chain hydrocarbon containing from 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, 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.

[0021] The term “alkoxy” as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0022] The term “alkenyl” as used herein, means a hydrocarbon chain containing from 2 to 30 carbon atoms with 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 a phenyl.

[0023] The term “alkynyl,” as used herein, refers to straight or branched monovalent hydrocarbyl groups having from 2 to 30 carbon atoms, such as 2 to 20, or 2 to 10 carbon atoms and having at least 1 site of triple bond unsaturation. The term “alkyne” also includes non-aromatic cycloalkyl groups of from 5 to 20 carbon atoms, such as from 5 to 10 carbon atoms, having single or multiple rings and having 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, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0024] The term “alkoxyalkyl” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0025] The term “alkylene” as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 30 carbon atoms, for example, of 2 to 10 carbon atoms. Representative examples of alkylene 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-.

[0026] The term “amino acid” refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the 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) and pyrrolidine and selenocysteine. Non-natural amino acids refer to amino acid analogs having the same basic chemical structure as a naturally occurring amino acid, i.e., by way of example only, an a- carbon attached to a hydrogen, carboxyl group, amino group, and R group. Such analogs can have a modified R group (e.g., norleucine as an example) or retain a modified peptide backbone while retaining the same basic chemical structure as a natural amino acid. Non-limiting examples of non-natural amino acids or amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formyl glycine, phenyl glycine, para-azidophenyl glycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(l,2,4,5-tetrazine))- phenylglyine, and 4-(3-methyl-(l,2,4,5-tetrazine))-phenylalanine.

[0027] 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 fused systems. Representative examples of aryls include, but are not limited to, phenyl, naphthyl, and anthracenyl. The monocyclic, bicyclic, and tricyclic aryls are connected to the parent molecular moiety through any carbon atom contained within the rings, and can be unsubstituted or substituted. The aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings. Thus, if a bicyclic ring system or tricyclic ring system contains a non-aromatic ring, the ring system is a cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to the remainder of the molecule.

[0028] In some embodiments, the term “aryl” as used herein, refers to a phenyl group, or bicyclic aryl or tricyclic aryl fused ring systems. Bicyclic fused ring systems are exemplified by a phenyl group appended to the parent molecular moiety and fused to a phenyl group. Tricyclic fused ring systems are exemplified by a phenyl group appended to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl. The monocyclic, bicyclic, and tricyclic aryls are connected to the parent molecular moiety through any carbon atom contained within the rings, and can be unsubstituted or substituted.

[0029] The term “azide” as used herein, refers to the functional group -N3.

[0030] The term “cycloalkyl” as used herein, 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. Cycloalkyl ring systems may contain one or more double bonds, so long as the ring is not aromatic; and thus, the termcycloalkyl includes cycloalkenyl ring systems. Representative examples of cycloalkyl 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 a cycloalkyl group is fused to an aryl or heteroaryl as defined herein, regardless of the point of attachment to the remainder of the molecule.

[0031] In some embodiments, the term “cycloalkyl” as used herein, refers to a carbocyclic ring system containing three to ten carbon atoms, zero heteroatoms and zero double bonds. Representative examples of cycloalkyl 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 appended to the parent molecular moiety and is fused to an aryl group as defined herein, a heteroaryl group as defined herein, or a heterocycle as defined herein.

[0032] The term “cycloalkenyl” as used herein, means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.

[0033] The term “cyclooctene” as used herein, refers to a substituted or unsubstituted non-aromatic cyclic alkyl group of 8 carbon atoms, 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).

[0034] The term “fluoroalkyl” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromcthyl, difluoromcthyl, pcntafluorocthyl, and trifluoropropyl such as 3,3,3-trifluoropropyl.

[0035] The term “alkoxyfluoroalkyl” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through a fluoroalkyl group, as defined herein.

[0036] The term “fluoroalkoxy” as used herein, means at least one fluoroalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkyloxy include, but are not limited to, difluoromethoxy, trifluoromethoxy and 2,2,2- trifluoroethoxy.

[0037] The term “halogen” or “halo” as used herein, means Cl, Br, I, or F.

[0038] The term “haloalkyl” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms arc replaced by a halogen.

[0039] The term “haloalkoxy” as used herein, means at least one haloalkyl group, as defined herein, is appended to the parent molecular moiety through an oxygen atom.

[0040] The term “heteroalkyl” as used herein, means an alkyl group, as defined herein, in which one or more of the carbon atoms has been replaced by a heteroatom selected from S, Si, O, P and N. The heteroatom may be oxidized. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.

[0041] The term “heteroaryl” as used herein, refers to an aromatic group having a single ring, multiple rings or multiple fused rings, with one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. In some embodiments, the term “heteroaryl” as used herein, refers to an aromatic monocyclic ring or an aromatic bicyclic ring system or an aromatic tricyclic ring system. The aromatic monocyclic rings are five or six membered rings 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). The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-l,2- dihydropyridinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indolyl, quinolinyl, imidazopyridine, benzooxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. The monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings, and can be unsubstituted or substituted. In some embodiments, the aromatic bicyclic ring system or aromatic tricyclic ring system does not contain non-aromatic rings. Thus, if a bicyclic ring system or tricyclic ring system contains a non-aromatic ring, the ring system is a cycloalkyl or heterocyclyl, depending on whether a heteroatom is present in the non-aromatic ring, regardless of the point of attachment to the remainder of the molecule.

[0042] In some embodiments, the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. In some embodiments, exemplary bicyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended to the parent molecular moiety and fused to a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocycle, as defined herein. In some embodiments, the tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended to the parent molecular moiety and fused to two of a monocyclic cycloalkyl group, as defined herein, a monocyclic aryl group, as defined herein, a monocyclic heteroaryl group, as defined herein, or a monocyclic heterocycle, as defined herein.

[0043] The terms “heterocyclyl,” “heterocycle,” or “heterocyclic” as used herein, refers to a non- aromatic ring system containing 3 to 10, or 3 to 8, or 3 to 6, or 5 to 10, carbon atoms and at least one (e.g., 1-5, 1-4, 1-3, 1-2, or 1) heteroatom, and optionally one or more oxo and / or double bonds. Theterms “heterocyclyl”, “heterocycle” or “heterocyclic” include monocyclic, bicyclic, tricyclic, fused, spirocyclic, or bridged ring systems, provided that at least one non-aromatic ring system containing at least one heteroatom is present. In some embodiments, the monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. In some embodiments, the three- or four-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 five-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 six-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 seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azctidinyl, azcpanyl, aziridinyl, diazcpanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, l,3-dimethylpyrimidine-2,4(lH,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring 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 two, three, or four 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]hept-2-yl), 2,3-dihydro-l W-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non- adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2, 5-epoxypentalene, hexahydro-2 / / -2,5-methanocyclopenta[b]furan, hexahydro- l / / -l,4-methanocyclopenta[c]furan, aza-adamantane (l-azatricyclo[3.3.1.13,7]decane), and oxa- adamantane (2-oxatricyclo[3.3.1.13,7]dccanc). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings, and can be unsubstituted or substituted.

[0044] The term “hydroxyl” as used herein, means an -OH group.

[0045] The term “hydroxyalkyl” as used herein, 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.

[0046] The term “substituted” refers to a group that may be further substituted with one or more nonhydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0047] The term “tetrazine” refers to a substituted or unsubstituted aromatic cyclic group of 2 carbon atoms and 4 nitrogen atoms, having a single ring with 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 a “Tz” group. The term “tetrazine-based” generally refers to a tetrazine as defined herein or a moiety which can be converted to a tetrazine, such as an optionally substituted dihydro tetrazine.

[0048] The term “selectively delivering” refers to delivering an agent (e.g., a payload) to an organ or tissue (or portion thereof) in need of treatment or diagnosis, without significant binding to other nontarget organs or tissues (or portions thereof). In some embodiments, the tetrazine-based targeting agents described herein do not themselves have a therapeutic effect, but rather are designed to allow the selective or targeted delivery of a therapeutic agent. However, it may be that the tetrazine-based targeting agent does have a therapeutic effect, and thus, such constructs are not excluded by the present disclosure.

[0049] The term “payload” refers to an agent for delivery to a target site in a subject. Tn some embodiments, payloads include therapeutic or diagnostic agents.

[0050] The term “therapeutic agent” refers to an agent capable of treating and / or ameliorating a condition or disease, or one or more symptoms thereof, in a subject. Therapeutic agents of the present disclosure also include prodrug forms of therapeutic agents.

[0051] The term “diagnostic agent” refers to agents that assist in diagnosing conditions or diseases. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are imaging agents that are magnetic under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles including iron nanoparticles and iron microparticles. Optical probes are fluorescent compounds that can be detected by excitation at one wavelength of radiation and detection at a second, different, wavelength of radiation. Optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (l,r-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate) and DiR (1,1’- dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots.Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to,3H,HC,13N,18F,19F,60Co,64Cu,67Cu,58Ga,82Rb,89Zr,90Sr,90Y, "Tc,99mTc,n iIn,123I,124I,125I,129I,131I,137Cs,177Lu,186Re,188Re,211At, Rn, Ra, Th, U, Pu, and241Am.

[0052] The term “targeting agent” refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and the specific target. “Stable association” means that a compound is bound to, e.g., covalently or non- covalently, or otherwise associated with, another moiety or structure under standard physiological conditions. Stable associations, or binding, may include covalent bonds and non-covalent interactions, such as, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. Targeting agents include ligands that specifically bind (or substantially specifically bind) a particular clinically-relevant target receptor or cell surface target. Specific targeting agents are described herein.

[0053] The term “targeted organ or tissue” refers to an organ or tissue that is being targeted for delivery of the payload. Representative organs and tissues for targeting include those that can be targeted by chemical or biological targeting agents, as well as those organs and tissues that cannot be targeted by chemical or biological targeting agents.

[0054] The term “implanting” refers to surgical implantation into a subject’s body.

[0055] The term “contacting” or “contact” refers to the process of bringing into contact at least two distinct species such that they can interact with each other, such as in a non-covalent or covalent binding interaction or binding reaction. It should be appreciated, however, the resulting complex or reaction product can be produced directly from an interaction or a reaction between the added reagents or from an intermediate from one or more of the added reagents or moictics, which can be produced in the contacting mixture.

[0056] The term “binding agent” refers to an agent having a functional group capable of forming a covalent bond to a complementary functional group of another binding agent in a biological environment. Binding between binding agents in a biological environment may also be referred to as bioconjugation. Binding agents include bioorthogonal binding agents, which are binding agents having bioorthogonal functional groups. Bioorthogonal functional groups of bioorthogonal binding agents selectively react with a complementary bioorthogonal functional group of another bioorthogonal binding partner.Selective reaction between bioorthogonal binding partners can minimize side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non- complementary bioorthogonal functional groups. Bioorthogonal moieties or functional groups of bioorthogonal binding agents include, but are not limited to, an azide and alkyne for formation of a triazole via Click-chemistry reactions, trans-cyclooctene (TCO) and tetrazine (Tz) (e.g., 1, 2,4,5- tetrazine), and others. The binding agents useful in the present disclosure may have a high reactivity with the corresponding binding agent so that the reaction is rapid.

[0057] The term “functionalized” refers to a moiety having a functional group attached to the moiety, such as for example a moiety having a binding agent functional group (e.g., a bioorthogonal functional group) attached thereto.

[0058] The term “administering” refers to any suitable route of administration to a subject, such as, but not limited to, oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.

[0059] The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0060] The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.

[0061] The term “pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0062] As used herein, the term “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which an agent(s) of the present disclosure may be administered may include mammals, particularly primates, especially humans. For veterinary applications, suitable subjects may include, for example, livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals, such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.

[0063] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition or symptom(s) thereof in a patient, such as a mammal (particularly a human) that includes: (a) ameliorating the disease or medical condition or symptom(s) thereof, such as, eliminating or causing regression of the disease or medical condition or symptom(s) thereof in a patient; (b) suppressing the disease or medical condition or symptom(s) thereof, for example by, slowing or arresting the development of the disease or medical condition or symptom(s) thereof in a patient; or (c) alleviating a symptom of the disease or medical condition or symptom(s) thereof in a patient.

[0064] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[0065] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0066] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0067] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 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 contemplated.

[0068] The compounds may exist as stereoisomers wherein asymmetric or chiral centers are present. The stereoisomers are “R” or “.S'” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “.S'” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof, and these arc specifically included within the scope of this disclosure. Stereoisomers include enantiomers and diastereomers and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared synthetically from commercially available starting materials, which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by methods of resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography, and optional liberation of the optically pure product from the auxiliary as described in Furniss, Hannaford, Smith, and Tatchell, “Vogel’ s Textbook of Practical Organic Chemistry,” 5thedition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns, or (3) fractional recrystallization methods.

[0069] It should be understood that the compounds may possess tautomeric forms as well as geometric isomers, and that these also constitute an aspect of the disclosure.

[0070] The present disclosure also includes isotopically-labeled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the disclosure are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to,2H,3H,13C,14C,15N,18O,17O,31P,32P,35S,18F, and36C1, respectively. Substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements, and, hence, may be preferred in some circumstances. The compound may incorporate positron-emitting isotopes for medical imaging and positron-emitting tomography (PET) studies for determining the distribution of receptors. Suitable positron-emitting isotopes that can be incorporated arenC,13N,15O, and18F. Isotopically-labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labeled reagent in place of non-isotopically-labeled reagent.B. Tetrazine-Based Targeting Agents

[0071] The tetrazine-based targeting agents described herein are designed to, once administered to a subject, localize at a target site within the subject. The tetrazine-based targeting agents can be administered locally or systemically. Once administered, a prodrug comprising a complimentary bioorthogonal component (e.g., a payload or therapeutic agent with a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine-based targeting agent in vivo, allows for targeted drug delivery of a payload or therapeutic agent. In some embodiments, the tetrazine -based targeting agents described herein comprise a diagnostic agent such that the tetrazine-based targeting agent as described herein can be used in diagnosing conditions or diseases, with or without administering a payload or therapeutic agent.

[0072] Provided herein are tetrazine-based targeting agents which comprise one or more targeting moieties covalently conjugated to one or more tetrazine moieties optionally via a linker, provided that at least one targeting moiety is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent. In some embodiments, at least one targeting moiety is selected from Table 1.

[0073] In some embodiments, each tetrazine moiety is independently of Formula I:wherein:R1, at each occurrence, is independently selected from the group consisting of 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'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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa;Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or hetero arylene, is independently optionally substituted with one to four R21;Ra, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;R21, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andR"', at each occurrence, is independently selected from alkyl and aryl.

[0074] In some embodiments, R3and R4are both absent, and the dotted lines are optional double bonds to form a tetrazine.

[0075] In some embodiments, R3and R4are each independently hydrogen or a group capable of being removed after a triggering event, and the dotted lines are optional double bonds to form a dihydrotetrazine.

[0076] In some embodiments, R2, at each occurrence, is independently cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to three Z1.

[0077] In some embodiments, each tetrazine moiety is independently of Formula II:wherein:R1, at each occurrence, is independently selected from the group consisting of 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'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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, hetcroalkyl, heterocyclyl, cycloalkyl, aryl, or hctcroaryl, is independently optionally substituted with one to three Z1;R21, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalky 1, 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;Ring A, at each occurrence, is independently cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to three Z1; t, at each occurrence, is independently 0, 1, 2, 3, or 4; each Z1is independently selected from 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", NRR ", NR'C(=O)R", NR’C(=S)R", NRC(=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' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andRT", at each occurrence, is independently selected from alkyl and aryl.

[0078] In some embodiments, the linker is branched.

[0079] In some embodiments, the tetrazine-based targeting agent comprises more than one targeting moieties.

[0080] In some embodiments, the tetrazine-based targeting agent comprises more than one tetrazine moiety.

[0081] In some embodiments, the linker is linear.

[0082] It is to be understood that the linker can be branched or linear and independently one or more (e.g., 1-16) linkers covalently attached to a targeting moiety. Further, more than one targeting moiety can be covalently attached via a branched linker.

[0083] In some embodiments, provided is a tetrazine -based targeting agent of Formula IX:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; each L is independently a linker; each p is independently 1-8;R1, at each occurrence, is independently selected from the group consisting of 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'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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa;Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R21;Rl, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;R21, at each occurrence, is 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, -Cf=C))-heterocyclyl, or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from 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 ";R" and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andR"', at each occurrence, is independently selected from alkyl and aryl.

[0084] In some embodiments, provided is a tetrazine-based targeting agent of Formula IX, wherein X is a targeting agent selected from Table 1.

[0085] In some embodiments, provided is a tetrazine-based targeting agent of Formula III:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent:L is a linker: p is 1-16;R1, at each occurrence, is independently selected from the group consisting of 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'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)RR", SC(=S)R'R", NR'C(=O)NR"R", and NR'C(=S)NR "R"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa;Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or hetero arylene, is independently optionally substituted with one to four R21;Ra, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;R21, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andR'", at each occurrence, is independently selected from alkyl and aryl.

[0086] In some embodiments, provided is a tetrazine-based targeting agent of Formula III:wherein:X is a targeting agent selected from Table 1;L is a linker; p is 1-16;R1, at each occurrence, is independently selected from the group consisting of 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'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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa;Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R21;Rl, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl;R21, at each occurrence, is 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, -Cf=C))-heterocyclyl, or -C(=O)-cycloalkyl; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from 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 ";R" and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andR"', at each occurrence, is independently selected from alkyl and aryl.

[0087] In some embodiments, provided is a tetrazine-based targeting agent of Formula IA, Formula IIA, or Formula VA:wherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an intcgrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA)targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;L, at each occurrence, is independently a linker;R1, at each occurrence, is independently selected from the group consisting of 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", 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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R2, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event;R20, at each occurrence, is independently selected from the group consisting of 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', 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 ";R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31a, and R31bare each independently hydrogen, Ci-Ce-alkyl, or Ci-Ce-haloalkyl; each Z1is independently selected from 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(=SjNR"R";R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R"', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently 0, 1, 2, 3, or 4.

[0089] In some embodiments, tetrazine moietyrepresented by:

[0090] In some embodiments, R1, at each occurrence, is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1.

[0091] In some embodiments, R1, at each occurrence, is independently hydrogen or alkyl optionally substituted with one to three Z1.

[0092] In some embodiments, Z1, at each occurrence, is independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0093] In some embodiments, R21, at each occurrence, is independently halo, alkyl, or haloalkyl.

[0094] In some embodiments, p is 1-5, or 1-4, or 1-3, or 1-2, or 1.

[0095] In some embodiments, t, at each occurrence, is 0.

[0096] In some embodiments, R2or Ring A is pyridyl or phenyl.

[0097] In some embodiments, R2or Ring A is other than pyridyl or phenyl.

[0098] In some embodiments of Formula II, the moiety:

[0099] In some embodiments of Formula II, the moiety:

[0100] In some embodiments, provided is a tetrazine-based targeting agent of Formula IID:wherein X is a targeting agent as disclosed herein. In some embodiments, X is a targeting agent selected from Table 1.

[0101] In some embodiments, provided is a tetrazine-based targeting agent of Formula HE:wherein X is a targeting agent as disclosed herein. In some embodiments, X is a targeting agent selected from Table 1.

[0102] In some embodiments, provided is a tetrazine-based targeting agent of Formula I-A, Formula II- A, or Formula V-A:wherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;L, at each occurrence, is independently a linker;R1, at each occurrence, is independently selected from the group consisting of 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", 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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, -Cl=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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R3and R1are both absent; or R3and R1are each independently hydrogen or a group capable of being removed after a triggering event;R20, at each occurrence, is independently selected from the group consisting of 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', 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", NRC(=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";R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Rl, R31aand R31bare each independently hydrogen, Ci-Cf, -alkyl, or Ci-Ce-haloalkyl; each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R'", at each occurrence, is independently selected from aryl and alkyl; andt, at each occurrence, is independently 0, 1, 2, 3, or 4.

[0103] In certain embodiments, when ring A is aryl, then R3and R4are both present.

[0104] In one embodiment, provided is a tetrazine-based targeting agent of Formula I-A:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent: p is 1-16;L, at each occurrence, is independently a linker;R20, at each occurrence, is independently selected from the group consisting of 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', 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 ";R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;RTand R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; andR'" at each occurrence is independently selected from aryl and alkyl.

[0105] In one embodiment, provided is a tetrazine-based targeting agent of Formula IIA-a:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;L, at each occurrence, is independently a linker;R20, at each occurrence, is independently selected from the group consisting of 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', 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";R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalky 1, alkenyl, alkynyl, alkoxy, haloalkoxy, hctcroalkyl, aryl, hctcroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;Ra, R31aand R31bare each independently hydrogen, Ci-Ce-alkyl, or Ci-Ce-haloalkyl;RTand R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R'", at each occurrence, is independently selected from aryl and alkyl; and t is independently is 0, 1, 2, 3, or 4. roio6i In one embodiment, R22, at each occurrence, is independently a linker of 1 to 100 linking atoms, and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups.

[0107] In one embodiment, provided is a tetrazine-based targeting agent of Formula IIA-b:wherein L, p, X, and R20are each independently as defined herein.

[0108] In one embodiment, provided is a tetrazine-based targeting agent of Formula IIB-a:wherein L, p, and X are each independently as defined herein.

[0109] In one embodiment, provided is a tetrazine-based targeting agent of Formula IlC-a:wherein L, p, and X are each independently as defined herein.

[0110] In one embodiment, provided is a tetrazine-based targeting agent of Formula Ill-a:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;L, at each occurrence, is independently a linker;R20, at each occurrence, is independently selected from the group consisting of 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', 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";R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalky 1, alkenyl, alkynyl, alkoxy, haloalkoxy, hctcroalkyl, aryl, hctcroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;RT", at each occurrence, is independently selected from aryl and alkyl; and t is independently is 0, 1, 2, 3, or 4.

[0111] In some embodiments, provided is a tetrazine-based targeting agent represented by Formula IID-a:wherein X and R20arc each independently as defined herein. In some embodiments, R20is methyl.

[0112] Also provided is a tetrazine-based targeting agent of Formula IIE-a:wherein p and X are each independently as defined herein.L0113J Also provided is a tetrazine-based targeting agent of Formula HF-a:wherein p and X are each independently as defined herein.

[0114] Also provided is a tetrazine -based targeting agent of Formula IIG-a:wherein p and X are each independently as defined herein.

[0115] Also provided is a tetrazine-based targeting agent of Formula IIH:wherein X is as defined herein.

[0116] In some embodiments, provided is a tetrazine-based targeting agent of Formula IIH:wherein X is a targeting agent selected from Table 1.

[0117] In some embodiments, provided is a tetrazine-based targeting agent of Formula IIH-a:wherein X is a targeting agent selected from Table 1.

[0118] In some embodiments, X is a nectin-4 targeting agent.

[0119] In some embodiments of Formula IIA-a, at least one of:

[0120] In some embodiments of Formula IIA-a, at least one of:defined herein.

[0121] In some embodiments of Formula IIA-a, at least one of:

[0122] In some embodiments of Formula IIA-a, at least one of:defined herein.

[0123] In some embodiments of Formula IIA-a, at least one of:

[0124] In one embodiment, provided herein is a tetrazine-based targeting agent of Formula V-A:ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present; provided that when ring A is aryl, then R3and R4are both present;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;L, at each occurrence, is independently a linker;R1, at each occurrence, is independently selected from the group consisting of 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", 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"; wherein each alkyl,alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R2, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;RT", at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently 0, 1, 2, 3, or 4.

[0125] In one embodiment, provided is a tetrazine-based targeting agent of Formula V-A:ring A is cycloalkyl, heterocyclyl, or heteroaryl; the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA)targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent: p is 1-16;L, at each occurrence, is independently a linker;R1, at each occurrence, is independently selected from the group consisting of 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", 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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R2, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R3and R4are both absent; orR3is a group capable of being removed after a triggering event;R4is hydrogen or R3; each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R'", at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently 0, 1, 2, 3, or 4.

[0126] In one embodiment, provided is a tetrazine-based targeting agent of Formula VI-A:wherein each of R1, R2, R3, R4, ring A, L, p, t, and X are independently as defined herein.

[0127] In some embodiments, R4is hydrogen.

[0128] In some embodiments of the tetrazine-based targeting agents described herein, R3is a group capable of being removed after a triggering event. In some embodiments, the triggering event occurs in vivo. Once the triggering event occurs and R3is removed, the dihydrotetrazine moiety is oxidized to provide a tetrazine as in Formula VII-A:wherein each of R1, R2, ring A, L, p, t, and X are independently as defined herein.

[0129] The triggering event is initiated after administration of the tetrazine-based targeting agent to the subject, and can be initiated by any means, such as internal means (e.g., via enzymatic cleavage of a functional group, optionally followed by a decomposition) or by external means (e.g., photocleavable linkers).

[0130] In some embodiments, R3comprises an amino acid sequence specific for cleavage by a protease or esterase.

[0131] In some embodiments, R3comprises an amino acid sequence specific for cleavage by a protease as shown in Table 1A.Table 1A

[0132] In some embodiments, R3comprises an amino acid sequence specific for cleavage by a cathepsin, matrix metalloprotease (MMP), or PSMA. For example, in some embodiments, R3comprises Vai- Ala, Val-Cit, Ala-Ala, Phe-Lys, Lys-Lys, Phe-Arg, or Gly-Gly-Gly for cleavage by cathepsins. In some embodiments, R3comprises Ac-yE-PLG-S(OBn)YL, or Ac-PLG-HofOrnL, where Hof is homophenylalanine and Orn is ornithine for cleavage by MMPs. In some embodiments, R3comprises an amino acid sequence as shown Table IB.Table IBI indicates cleavage siteSpecial amino acid abbreviation:Cit: Citrulline; Cha: P-cyclohexylalanine; Hof: homophenylalanine; Nva: aminosuberic acid; Dpa: D- phenylalanine; Nle: Norleucine; Smc: S-methylcysteine* the listing of multiple amino acids before, between, or after a slash indicate alternative amino acids that can be substituted at the position; indicates that any amino acid may be substituted for the corresponding amino acid indicated in the middle column** x is any L-amino acid other than prolineHy is any hydrophobic L-amino acid y indicates that bond is a gamma carboxy linkage

[0133] Additional cleavable groups are described in Choi, et al., Theranostics. 2012; 2(2): 156-178, in which Table 2 is hereby incorporated by reference.

[0134] In some embodiments, R3is photolabile. In some embodiments, the photolabile group is labile, or decomposes, with exposure to light at a wavelength matched to the absorbance profile of the photolabile group.

[0135] In some embodiments,I? is a direct bond or linker; andX1is -NO2, an optionally substituted sugar moiety, or an optionally substituted peptide unit comprising one or more natural or unnatural amino acids.

[0136] In some embodiments, at least one of the moiety:is represented by a formula selected from:independently as defined herein, and optionally the ring A portion may be substituted with one or more R2moieties.

[0137] In some embodiments, at least one of the moiety:is represented by a formula selected from:substituted with a PEG, an amino acid, ester, amide, amine, -C(O)OH, -SO2, -SO3, -PO3, -PO4, or other solubility enhancing substituent; and each of L, ring A, R1, R2, t, p, and X are independently as defined herein.F0138] In some embodiments, ring A is cycloalkyl. In some embodiments, ring A is heterocyclyl. In some embodiments, ring A is heteroaryl. In some embodiments, ring A is aryl.

[0139] 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.

[0140] In some embodiments, ring A is phenyl.

[0141] In some embodiments, at least one of the moiety:is represented by a formula selected from:are independently as defined herein.

[0142] In some embodiments, R1, at each occurrence, is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1.

[0143] In some embodiments, R1, at each occurrence, is independently hydrogen or alkyl optionally substituted with one to three Z1.

[0144] In some embodiments, Z1, at each occurrence, is independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

[0145] In some embodiments, R2, at each occurrence, is independently halo, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl. In some embodiments, R2, at each occurrence, is independently halo, alkyl, or haloalkyl. In some embodiments, R2, at each occurrence, is independently halo or alkyl.

[0146] In some embodiments, t at each occurrence, is 0.

[0147] Also provided is a tetrazine-based targeting agent of Formula VA-a:wherein p and X are each independently as defined herein. Also provided is a tetrazine -based targeting agent of Formula VB-a:wherein p and X are each independently as defined herein.

[0148] In some embodiments, ring A is other than pyridyl. In some embodiments, ring A is other than aryl. In some embodiments, ring A is other than phenyl.B. Targeting Moieties

[0149] Provided herein are tetrazine-based targeting agents which comprise one or more targeting moieties covalently bonded to one or more tetrazine moieties. The targeting moieties described herein are designed to, once administered to a subject, localize at a target site within the subject. Once the tetrazine-based targeting agent is administered, a prodrug comprising a complimentary bioorthogonal component (i.e., a trans-cyclooctene moiety) can be administered, which when in contact with the tetrazine-based targeting agent in vivo, allows for targeted drug delivery of a payload or therapeutic agent.

[0150] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent.

[0151] The targeting moieties as used herein are derived from known compounds (i.e., targeting agents) which target (or bind) to a desired target. The parent or known targeting agent (e.g., compounds or peptides) are modified to be conjugated to a tetrazine moiety, optionally via a linking moiety as defined herein. The targeting moieties, even after modification to arrive at the compounds described herein, maintain biological activity, which is comparable to that observed in the original, unmodified targeting agent. In certain embodiments, the targeting moieties exhibit a binding activity which 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 targeting agent.

[0152] In certain embodiments, the terms “modified” and “derived from” as used in reference to a targeting agent, means that one or more atoms of the original, unmodified targeting agent (i.e., a known targeting agent) is replaced by a covalent bond to the tetrazine moiety, optionally via a linking moiety. In certain embodiments, one hydrogen atom bound to a heteroatom (e.g., N, O, or S) or one reactive functional group (e.g., -OH) of the original, unmodified targeting agent (i.e., a known targeting agent) is replaced by a covalent bond to a tetrazine moiety, optionally via a linking moiety. In certain embodiments, particularly when the targeting agent comprises amino acids, including an additional amino acid or a conservative substitution provides a synthetic handle (e.g., introducing a lysine or cysteine),

[0153] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment NECTIN-4 peptide targeting agent. See, e.g., Table 1, entry 1.

[0154] 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).

[0155] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment PSMA peptide targeting agent.

[0156] 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).

[0157] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment cholecystokinin-2 receptor (CCK2R) peptide targeting agent.

[0158] The tetrazine-based targeting agents disclosed herein can be tested for CCK2R-binding using assays known in the art (e.g., Hbrmann 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, tetrazine-based targeting agents disclosed herein can be used in methods for treating cancer or gastrointestinal disorders.

[0159] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment PD-L1 peptide targeting agent.

[0160] 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 / sl3045-017-0479-y).

[0161] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 3 targeting agent.

[0162] 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).

[0163] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 6 targeting agent.

[0164] 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 av06 or av[38 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(CAN)2(NAN)]+ complexes by trifluoromethylation of the NAN ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039 / C9CC07666E).

[0165] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment integrin alpha V beta 8 targeting agent.

[0166] The tetrazine -based targeting agents disclosed herein can be tested for integrin alpha V 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 av06 or av08 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(CAN)2(NAN)]+ complexes by trifluoromethylation of the NAN ligand. Chemical Communications, 55(98), 14777-14780. doi: 10.1039 / C9CC07666E).

[0167] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment FAP targeting agent.

[0168] 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.).

[0169] In some embodiments, the targeting moiety, or X, is a non-antibody or non-antibody fragment carbonic anhydrase IX (CAIX) targeting agent.

[0170] 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).

[0171] As used herein, the phrase “non-antibody or non-antibody fragment” is intended to refer to targeting agents that are generally considered antibodies or antibody fragments, such as compounds, including peptides, less than 15 kDa, or less that 10k Da, or less than 9 kDa, or less that 8 kDa, or less that 7 kDa, or less that 6 kDa, or less that 5 kDa.

[0172] In some embodiments, the targeting moiety, or X, is selected from Table 1. For representative procedures for the preparation of various Targeting moieties, see, e.g., 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., Mm- / . Chem., 2021, 45, 5291; WO2019 / 243832; and Zana, et al., Bioconjugate Chem. 2023, 34, 7, 1205-1211.Table 1Linkers

[0173] In the tetrazine-based targeting agents disclosed herein, the linkers can be linear or branched (including, but not limited to, bivalent, trivalent, tetravalent branching). Thus, in one tetrazine-basedtargeting agent, there may be one or more targeting moiety covalently bonded to one or more tetrazine moiety. In the Formulas disclosed herein, the “L” may be inside or outside of the “p” designation, however, it is intended that the linker can be linear with one bond to the tetrazine moiety and one bond to the targeting moiety, wherein there are more than one [tetrazine-L]- moieties bonded to a single targeting moiety; or branched such that the linker has a bond to two tetrazine moieties and one bond to the targeting moiety.

[0174] In some embodiments, L, or the linker, comprises one or more of a hydrazone, a hydrazide, a disulfide, a N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), a N-succinimidyl-4-(2- pyridyldithio)butyrate (SPDB), a 4-(4’-acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acid, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

[0175] In some embodiments, L, or the linker, comprises 1 to 100 linking atoms, from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.

[0176] In some embodiments, L, or the linker, comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and CM haloalkyl.

[0177] In some embodiments, L, or the linker, is bonded to X via a cysteine or lysine residue on X.

[0178] In some embodiments, L, or the linker, is a non-cleavable linker.

[0179] In some embodiments, L, or the linker, is a cleavable linker.

[0180] In some embodiments, L, or the linker, comprises one or more amino acids.

[0181] In some embodiments, L, or the linker, comprises a polypeptide.

[0182] In some embodiments, L, or the linker, is an alkylene linker optionally comprising one or more - O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

[0183] In some embodiments, L, or the linker, is of the formula:-Y10-(CHR130)n'-Y20-(CHR140)n'-Y30-(CHR150)ni"-Y40- wherein: each of Y10, Y20, Y30, and Y40are independently a bond, -NR110-, -O-, -S(0)o-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OCIO)-, -OC(O)O-, -('CHiCHiOj s-, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene;wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and Ci-4 haloalkyl; each R110is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, hctcroaryl, cycloalkyl, or heterocyclyl; each R130is independently hydrogen, CIM alkyl, CIM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; each R140is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; each R150is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or an amino acid side chain; and n', n", and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0184] In some embodiments, L, or the linker, is of the formula:-Y10-(CH2)n-Y20-(CH2)m.-Y30- whcrcin: each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(0)o-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, CM alkyl, CM alkoxy, and CM haloalkyl; each R110is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and n' and m" are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0185] In certain embodiments, each R110is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120is independently hydrogen, CM alkyl, CM haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl. In certain embodiments, the linker is not a bond.

[0186] The L, or the linker, may comprise one or more of polyethylene glycol (e.g., PEG having an average molecular weight of from 300 g / mol to 10,000 g / rnol), ethylene- l,2-diylbis(methylcarbamate, anarylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate.

[0187] In some embodiments, the linker comprises one or more of:

[0188] In some embodiments, the linker comprises one or more of:

[0189] In some embodiments, the linker comprises one or more of:

[0190] In some embodiments, the linker comprises one or more H . In some embodiments, the linker comprises one or more

[0191] In some embodiments, the linker is, or comprises one or more:

[0192] In some embodiments, the linker is, or comprises one or more:

[0193] In some embodiments, the linker comprises one or more natural or unnatural amino acids, which may be referred to as a peptide linker. The linker may be a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence. The linker may also contain a self-immolating spacer which spaces the drug and the protein peptide sequence.

[0194] In some embodiments, the linker may be a peptide containing linker represented by “A — Y — Z — X2— W” in which “A” is the carboxylic acyl unit, “Y” and “Z” are each one or more natural or unnatural amino acids and together form a peptide sequence, and “X2” and “W” are optional additional linkers having from 1 to 50 linking atoms, or from 5 to 10 linking atoms, or from 1 to 10 linking atoms which spaces the peptide and the payload, D, or the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.

[0195] 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.

[0196] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline. In some embodiments Z may be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0197] Exemplary Y-Z combinations include Valine-Citrulline; Valine- Alanine; and Alanine-Alanine.

[0198] In some embodiments, A is -OC(O)-.

[0199] In some embodiments, X2is -OC(O)-.

[0200] In some embodiments, W is -OC(O)-. In some embodiments, X2is absent and W is -OC(O)-.

[0201] In some embodiments, the moiety — X2— W comprises H . In someembodiments, the moiety — X2is H

[0202] In some embodiments,

[0203] In some embodiments,

[0204] In some embodiments, the peptide linker is specifically tailored so that it will be selectively cleaved (e.g., enzymatically cleaved) releasing the drug, such as by one or more of the tumor-associated proteases.

[0205] In some embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a di-, tri-, or tetra-peptide). It will be understood, however, that peptide linkers up to five, six, seven, or eight amino acid residues may also suitably be employed.

[0206] In some embodiments, the peptide linker is Phe-Lys, Val-Lys, Vai- 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-N9-tosyl-Arg, or Phe-N9-Nitro-Arg. In certain embodiments, the peptide linker is Phe-Lys, Val-Lys, Vai-Ala, Ala-Ala, Val-Val, Val-Cit, or D-Phe-L-Phe-Lys. In certain embodiments, the peptide linker is Val-Cit, Vai-Ala, or Ala-Ala.

[0207] In some embodiments, L, or the linker, is or comprises one or more of:L0208J In some embodiments, the linker L comprises one or more of:

[0209] In some embodiments, the linker L comprises one or more of:

[0210] The foregoing linkers may bond to an amino acid side chain present on X, such as a lysine or

[0211] In some embodiments, L, or the linker, is -C(O)L4- or -C(O)Ci4>alkyleneC(O)L4-;R12, R13, R14, R15, and R19are each independently hydrogen or Cmalkyl;R16is hydrogen, Cwalkyl, -Ci-4alkylene-OH, -CMalkylene-OCwalkyl, -Ci-.alkylcnc-CXhH, or -Ci-4alkylene-CONH2; andGxis phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, Cwalkyl, Cwhaloalkyl, Cwalkoxy, cyano, and nitro.

[0212] In some embodiments, L, or the linker, comprises a carbonyl moiety for conjugating the tetrazine moiety to the linker or X. For example, the linker may comprise a polypeptide moiety (PPM) having the lysine residue and lysine side chain and the PPM may also have additional lysines, or other amino acid side chains conjugated to the carbonyl moiety. In some embodiments, the linker L may comprise polypeptidelysine side chain

[0213] In some embodiments, L, or the linker, is or comprises one or more of:

[0214] In some embodiments, L, or the linker, is or comprises one or more of:

[0215] In some embodiments, L, or the linker, is or comprises one or more of:

[0216] In some embodiments, L, or the linker, is or comprises one or more of:

[0217] In some embodiments, L, or the linker, is:

[0218] In some embodiments, L, or the linker, is:

[0220] In some embodiments, L, or the linker, is or comprises one or more

[0221] In some embodiments, L, or the linker, is or comprises one or more

[0222] In some embodiments, L, or the linker, is or comprises one or more

[0223] In some embodiments, L, or the linker, is or comprises one or more

[0224] In some embodiments, L, or the linker, is or comprises one or moreL, or the linker, is or comprises one or more0 0

[0227] In some embodiments, L, or the linker, is or comprises one or moreO O

[0228] In some embodiments, L, or the linker, is or comprises one or moreO O

[0229] In some embodiments, the linker L is, or comprises one or more

[0230] In one embodiment, provided is a tetrazine-based targeting agent of Formula I:wherein: p is 1-16;R20, at each occurrence, is independently selected from the group consisting of 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', 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”;R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R"" at each occurrence is independently selected from aryl and alkyl;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent;L, at each occurrence, is independently a linker selected from the group consisting of:

[0231] In one embodiment, provided is a tetrazine-based targeting agent of Formula II:wherein:X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CAIX) targeting agent; p is 1-16;R20, at each occurrence, is independently selected from the group consisting of 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’, 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", NRC(=O)NR"R ", and NR’C(=S)NR"R";R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl;R1, R31aand R31bare each independently hydrogen, Ci-Cs-alkyl, or Ci-Ce-haloalkyl;R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R'", at each occurrence, is independently selected from aryl and alkyl; t is independently is 0, 1, 2, 3, or 4;L, at each occurrence, is independently a linker selected from the group consisting of:

[0232] In one embodiment, provided is a tetrazine-based targeting agent of Formula V :wherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl;the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present; provided that when ring A is aryl, then R3and R4are both present;X is a non-antibody or non-antibody fragment targeting agent selected from a NECTIN-4 peptide targeting agent, a PSMA peptide targeting agent, a CCK2R peptide targeting agent, a PD-L1 peptide targeting agent, an integrin alpha V beta 3 targeting agent, an integrin alpha V beta 6 targeting agent, an integrin alpha V beta 8 targeting agent, a FAP targeting agent, a CEACAM5 (also known as CEA) targeting agent, and a carbonic anhydrase IX (CA1X) targeting agent; p is 1-16;R1, at each occurrence, is independently selected from the group consisting of 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", 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"; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R2, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1;R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; each Z1is independently selected from 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";R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl;R'", at each occurrence, is independently selected from aryl and alkyl; t, at each occurrence, is independently 0, 1, 2, 3, or 4;L, at each occurrence, is independently a linker selected from the group consisting of:

[0233] 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; wherein each may be optionally substituted.

[0234] In some embodiments, p is 1-5, or 1-4, or 1-3, or 1-2.

[0235] In certain embodiments, p is dependent on the size and / or number of available binding sites on X for forming a covalent bond to L.

[0236] In one embodiment, the tetrazine-based targeting agent is of Formula:wherein n is 1-10.

[0237] In one embodiment, the tetrazine -based targeting agent is as shown in Table 2.Table 2C. Support Compositions

[0238] The support composition comprises a support. In certain embodiments, the support composition is a therapeutic support composition. Supports may be biocompatible supports compositions, i.e., compatible with the subject’s body. In some instances, a support is non-toxic to the subject and does not substantially react with tissue or biological compounds in the subject. For example, the support can be a hydrogel, among others. A support is capable of implantation into a subject’s body and supporting binding agents (e.g., a TCO-containing group), as well as payloads after the binding agents conjugate. Representative supports include, but are not limited to polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports also include particles, such as nanoparticles, microparticles, and the like.

[0239] Hydrogels may be polysaccharide hydrogels, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, and the like. Other suitable sugar-based biomaterials include those described in Polymer Advanced Technology., 2014, 25, 448-460. Polymers that may be used as the support can include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(ortho esters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, and blends / composites / co-polymers thereof. Representative polyethers include, but are not limited to, poly(ethylene glycol) (PEG), polypropylene glycol) (PPG), triblock Pluronic ([PEG]n-[PPG]m-[PEG]n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). The support can 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), and the like.

[0240] 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 a hyaluronic acid (e.g., a non-hydrogel hyaluronic acid substantially without crosslinks). In some embodiments, the support is chitosin.

[0241] In certain embodiments, the support is a particle. Particles of the present disclosure can have a diameter that is 2 cm or less, such as 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles can be nanoparticles or microparticles. Nanoparticles include particles having average dimensions in the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having average dimensions in the micrometer scale (e.g., 1000 pm or less). By “average” is meant the arithmetic mean. In some embodiments, the nanoparticles have a diameter ranging from 1 nm to 1 pm, such as from 10 nm to 1 pm, or 25 nm to 1 pm, or 50 nm to 1 pm, or 75 nm to 1 pm, or 100 nm to 1 pm, or 150 nm to 1 pm, or 200 nm to 1 pm, or 250 nm to 1 pm, or 300 nm to 1 pm, or 350 nm to 1 pm, or 400 nm to 1 pm, or 450 nm to 1 pm, or 500 nm to 1 pm. In other embodiments, the microparticles have a diameter ranging from 1 pm to 1 mm, such as from 10 pm to 1 mm, or 25 pm to 1 mm, or 50 pm to 1 mm, or 75 pm to 1 mm, or 100 pm to 1 mm, or 150 pm to 1 mm, or 200 pm to 1 mm, or 250 pm to 1 mm, or 300 pm to 1 mm, or 350 pm to 1 mm, or 400 pm to 1 mm, or 450 pm to 1 mm, or 500 pm to 1 mm. In further embodiments, small particles on the order of 10-100 nm in diameter may be assembled to form larger complexes, such as clusters or assemblies on the order of 1-10 pm. Particles of the present disclosure may be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes may also be used, such as, but not limited to, ellipsoid, cubic, cylindrical, conical, needle, or other irregular shapes.

[0242] A “particle” may take the form of any fabricated material, a molecule, cryptophan, a virus, a phage, etc. The particle may be composed of a material, such as, but not limited to, a metal, a ceramic, a plastic, a glass, a composite, a polymer, a hydrogel, and the like. For example, the particles may be made of an inert material, such as alginate or iron oxide. In some examples, the particles may be magnetic and can be formed from a paramagnetic, super-paramagnetic or ferromagnetic material, or other material thatresponds to a magnetic field. Further, a particle may be of any shape, for example, spheres, rods, non- symmetrical shapes, etc. The particles, or a group of several particles in a complex, may be functionalized with a receptor that has a specific affinity to bind to or interact with a clinically relevant substrate. The receptor may be inherent to the particle itself. For example, the particle itself may be a virus or a phage with an inherent affinity for certain substrates. Additionally or alternatively, the particles can be functionalized by covalently or otherwise attaching or associating a receptor that specifically binds or otherwise recognizes a particular clinically relevant substrate. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule with a defined affinity for a target substrate. Examples of material that may be used for the “particles” and / or “carrier” 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, poly anhydrides, polyphosphazines, poly(vinyl alcohols), poly(alkylcnc oxides), poly(allylamincs)(PAM), poly(acrylatcs), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers or graft copolymers of any of the above. These examples do not limit their concentration, their cross-linking with different agents, their method of administration, their tailored degradation profiles and other characteristics known to those skilled in the art.

[0243] The particles, or a group of several particles in a complex, may be functionalized with a tetrazine -based targeting agent (e.g., a ligand or antibody) that specifically binds (or substantially specifically binds) to a target (e.g., a target receptor or a cell surface target, such as a clinically relevant receptor or cell surface target (e.g., antigen)). The tctrazinc-bascd targeting agent may be attached directly to the particle itself. The tetrazine-based targeting agent can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule with a specific affinity for a target receptor or cell surface target. In some instances, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, or other cellular receptors or cell surface targets. Other compounds or molecules, such as fluorophores or autofluorescent or luminescent markers, which may assist in detecting the particles (e.g., in vivo detection), may also be attached to the particles. The ligands and / or detectable labels may be attached directly to the particle or attached to the particle through bioorthogonal functional groups as described herein.

[0244] In certain embodiments, the support is a bone graft material, such as a bone graft substitute material. A bone graft substitute material is a material structurally similar to bone. In some instances, a bone graft substitute material is bioresorbable such that the bone graft substitute material can dissolve or be absorbed in the body over time. A bone graft substitute material can be osteoconductive, such that it facilitates blood vessel and new bone formation into the bone graft substitute material. In some instances, the bone graft substitute material is osteoinductive, such that facilitates the formation of new bone through active recruitment of mesenchymal stem cells from the surrounding tissue. For example, growth factors, such as bone morphogenetic proteins, may be included in the bone graft substituteI llmaterial. Bone graft substitute materials include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and the like, and combinations thereof.

[0245] In certain embodiments, the support compositions comprise substituted alginate having units of formula:wherein the dashed line represents a bond to L.

[0246] In some embodiments, the support compositions comprise substituted hyaluronic acid having units of formula:wherein the dashed line represents a bond to L.

[0247] The hyaluronic acid derivative includes a hyaluronic acid having a plurality of glucuronic acid units and a tetrazine -containing group linked or directly bonded to a glucuronic acid unit of the hyaluronic acid. The hyaluronic acid may also have a plurality of N-acetylglucosamine units. In certain embodiments, the N-acetylglucosamine units of the hyaluronic acid are not linked or conjugated to the tetrazine-containing group.

[0248] The tetrazine-containing group can be linked or directly bonded through a carboxylic acid of a glucuronic acid unit. The tetrazine-containing group can be incorporated into the hyaluronic acid from about 0.1% to about 80% as measured by the % of carboxylic acids being linked or conjugated to the tetrazine-containing group, such as about 1 % to about 75%, about 5% to about 75%, about 10% to about 50%, or about 40% to about 75% as measured by the % of carboxylic acids being linked or conjugated to L of the tetrazine-containing group.

[0249] Additional support compositions are exemplified in WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, the entire contents of each of which is incorporated herein by reference in their entirety.D. Trans-Cyclooctene Functionalized Prodrugs

[0250] Trans-cyclooctene functionalized prodrugs are known in the art, including prodrugs of anticancer agents, 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 follow.

[0251] In some embodiments, the trans-cyclooctene functionalized prodrugs is a conjugate comprised of a payload linked to one or more trans-cyclooctene moieties.

[0252] In some embodiments, the conjugate (or trans-cyclooctene functionalized prodrug) comprises an immunomodulatory agent payload, such as for example, an immunomodulatory agent payload selected from the group consisting of a cytokine, chemokine, chemokine antagonist, therapeutic monoclonal antibody, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

[0253] In some embodiments, the immunomodulatory agent payload is an inhibitor of a cytokine payload, or a pharmaceutically acceptable salt thereof.

[0254] In some embodiments, the inhibitor of a cytokine payload is an inhibitor of TNF-a, infliximab, certolizumab, TGF-0, galunisertib, fresolimumab, M7824, CSF-1, pexidartinib, or cabiralizumab.

[0255] In some embodiments, the conjugate comprises a monoclonal antibody, or a pharmaceutically acceptable salt thereof.

[0256] In some embodiments, the conjugate comprises a therapeutic protein payload, or a pharmaceutically acceptable salt thereof.

[0257] In some embodiments, the therapeutic protein payload is an antibody -based drug, Fc fusion protein, anticoagulant, blood factor, bone morphogenetic protein, engineered protein scaffold, enzyme, growth factor, hormone, interferon, interleukin, or thrombolytic.

[0258] In some embodiments, the therapeutic protein payload is a cytokine, chemokine, growth factor, hormone, antibody, or antigen.

[0259] In some embodiments, the therapeutic protein payload is a payload of erythropoietin (EPO, e.g., native EPO or synthetic EPO (see, e.g., US 2003 / 0191291), such as, but not limited to, e.g., PROCRIT®, EPREX®, or EPOGEN® (epoetin-a), ARANESP® (darbepoietin-a), NEORECORMON®, EPOGIN® (epoetin-P), and the like); a growth hormone (e.g., a somatotropin, e.g., GENOTROPIN®, NUTROPIN®, NORDITROPIN®, SAIZEN®, SEROSTIM®, HUMATROPE®, etc.); theraputic monoclonal antibody (e.g Atezolizumab, Avelumab, Bevacizumab, Cemiplimab, Cetuximab, Daratumumab, Dinutuximab, Durvalumab, Elotuzumab, Ipilimumab, Isatuximab, Mogamulizumab, Necitumumab, 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-y, IFN-a, IFN-P, IFN- co; IFN-r, consensus interferon, and the like); insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.), insulin-like growth factor (e.g., IGF-I, IGF-II); blood factors (e.g., Factor X, tissue plasminogen activator (TP A), and the like, such as, but not limited to, e.g., ACTIVASE® (alteplase) tissue plasminogen activator, NOVOSEVEN® (recombinant human factor Vila), Factor Vila, FactorVIII (e.g., KOGENATE®), Factor IX, 0-globin, hemoglobin, and the like); colony stimulating factors (e.g., granulocyte-CSF (G-CSF, e.g., NEUPOGEN® (filgrastim)), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), Neulasta (pegfilgrastim), granulocyte-monocyte colony stimulating factor, megakaryocyte colony stimulating factor, and the like), 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, and the like); a growth factor (e.g., epidermal growth factor (EGF), platelet-derived growth factor (PDGF, e.g., REGRANEX® (beclapermin)), fibroblast growth factors (FGFs, e.g., aFGF, bFGF, such as FIB LAST® (trafermin)), glial cell line -derived growth factor (GDNF), nerve growth factor (NGF), stem cell factor (e.g., STEMGEN® (ancestim)), keratinocyte growth factor, a hepatocyte growth factor, and the like); a soluble receptor (e.g., a TNF-a-binding soluble receptor such as ENBREL® (etanercept), a soluble VEGF receptor, a soluble interleukin receptor, a soluble y / 3 T cell receptor, and the like); an enzyme (e.g., a-glucosidase, CERAZYME® (imiglucarase, f>- glucoccrcbrosidasc, CEREDASE® (algluccrasc); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP- 10, Mig, Groa / IL-8, regulated and normal T cell expressed and secreted (RANTES), MIP-la, MIP-lp, MCP-1, PF-4, and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropinreleasing 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, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha- 1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, a tissue factor, an insulin-like growth factor, a luteinizing hormone, a follicle stimulating hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor, a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; or an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); and the like

[0260] In some embodiments, the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof:wherein m is an integer from 1-150;G, at each occurrence, is independently an optionally substituted trans-cyclooctene moiety;D is a payload;L1, at each occurrence, is independently a linker.

[0261] In some embodiments of the conjugates described herein, each trans-cyclooctene moiety is independently:wherein:RIA, at each occurrence, is independently selected from the group consisting of Cwalkyl, Ci- dialoalkyl, and Ci.4alkoxy; q is 0, 1, or 2; ql is 0 or 1;RIB, at each occurrence, is independently selected from the group consisting of G1, -OH, -NR1L-C walkylene-G1, -NR1C-C i.4alkylene-N(Rld)2, -NR1C-C ^alkylene-NIC i _4alky 1)3+, -N(R1C)CHR1CCO2H, -N(R1C)-Ci.6alkylene-CO2H, -N(Rlf)-C2.4alkylene-(N(Ci.4alkylene-CO2H)-C2.4alkylene)„-N(Ci.4alkylene-CO2H)2, -N(Rlc)CHRleC(O)OCi-6alkyl, -N(Rlc)-Ci-6alkylene-C(O)OCi-6alkyl, -N(Rlf)-C2.4alkylene-(N(Ci.4alkyleneC(O)OCi.6alkyl)-C2.4alkylene)„-N(Ci.4alkyleneC(O)OCi.6alkyl)2, -N(R1C)-C i.6alkylene-SO3H, -N(R1C)-(CH2CH2O) I.3-CH2CH2N((CH2CH2O) i.3-C i.6alkylene-CO2H)2, and -N(Rlc)-CH(CH2O-(CH2CH2O)0-2-Ci.6alkylene-CO2H)2;Rlcand Rld, at each occurrence, are independently hydrogen or Ci^alkyl;Rle, at each occurrence, is independently -Ci-4alkylene-CO2H, -Ci-4alkylene-CONH2, or -C i-4alkylene-OH ;Rlf, at each occurrence, is independently hydrogen, Ci-ealkyl, or Ci-4alkylene-CO2H; n, at each occurrence, is independently 0, 1, 2, or 3;L2, at each occurrence, is independently selected from the group consisting of -C(O)- and Ci.3alkylene; andG1, at each occurrence, is independently an optionally substituted heterocyclyl.

[0262] In some embodiments, the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof,whereinG is the trans-cyclooctene moiety, and G, at each occurrence, is independentlyL1, at each occurrence, is independently a linker; m is an integer from 1-150;D is a payload;RIA, at each occurrence, is independently selected from the group consisting of Ci^alkyl, Ci-Jialoalkyl, and Ci-4alkoxy; q is 0, 1 or 2; ql is 0 or 1;RIB, at each occurrence, is independently selected from the group consisting of G1, OH, -NRlc-Ci.4alkylene-G1, -NRlc-Ci-4alkylene-N(Rld)2, -NRlc-Ci-6alkylene-N(Ci-4alkyl)j+, -N(Rlc)CHRleCO2H, -N(R1C)-Ci.6alkylene-CO2H, -N(Rlc)CHRleC(O)OCi.6alkyl, -N(Rlf)-C2-4alkylene -(N(Ci-4alkylene-CO2H)-C2.4alkylene)11-N(Ci-4alkylene-CO2H)2, -N(Rlc)-Ci-6alkylene-C(O)OCi.6alkyl,-N(Rlf)-C2.4alkylene -(N(Ci.4alkyleneC(O)OCi.6alkyl)-C2-4alkylene)11-N(Ci.4alkyleneC(O)OCi-6alkyl)2,Rlcand Rld, at each occurrence, are independently hydrogen or Ci-4alkyl;Rle, at each occurrence, is independently -Ci-4alkylene-CO2H, -Ci-4alkylene-CONH2, or -C i .4alkylene-OH ;Rlf, at each occurrence, is independently hydrogen, Ci-ealkyl, or Ci.4alkylene-CO2H; n, at each occurrence, is independently 0, 1, 2, or 3;L2, at each occurrence, is independently selected from the group consisting of -C(O)- and Ci.3alkylene; andG1, at each occurrence, is independently an optionally substituted heterocyclyl.

[0263] In some embodiments, ql is 1.

[0264] In some embodiments, the payload is an immunomodulatory agent payload.

[0265] In some embodiments, the payload is a therapeutic monoclonal antibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

[0266] In some embodiments, the payload is selected from a therapeutic agent for treating cancer (e.g., doxorubicin, daunorubicin, PNU-159682, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, baccatin III, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, auristatin (dolastatin 10, MMAE, MMAD, MMAF), duocarmycin, pyrrolobenzodiazapene dimer, mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, and the like), a matrix metalloproteinase (MMP) inhibitor, L-dopa, oseltamivir, cefalexin, 5-amino levulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic- adenosine monophosphatidyl (c-AMP).

[0267] In some embodiments, the payload is selected from a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, auristatin (dolastatin 10, MMAE, MMAD, MMAF) mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, and the like), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, and the like), lurbinectedin, gardiquimod, a matrix metalloproteinase (MMP) inhibitor, L-dopa, oseltamivir, cefalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic-adenosine monophosphatidyl (c-AMP).

[0268] Reference to a payload, means that one or more atoms, including hydrogen or non-hydrogen atoms, of the original, unmodified payload is replaced by a covalent bond to one or more linker. The payloads are derived from the known nuclear payload and are modified to be covalently bonded to at least one optionally substituted trans-cyclooctene via a linker. The payloads, even after modification to arrive at the compounds described herein, maintain biological activity which is comparable to that observed in the original, unmodified payload. In certain embodiments, the payloads exhibit a binding activity or inhibition which 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.

[0269] In certain embodiments, a hydrogen atom bound to a heteroatom (e.g., N, O, or S) of the original, unmodified payload is replaced by a covalent bond to a linker. In certain embodiments, a halogen atom on a payload is replaced for attachment to the remainder of the compound. In certain embodiments, a hydrogen atom on a payload is replaced for attachment to the remainder of the compound. In certainembodiments, the hydrogen atom is on a heteroatom. In certain embodiments, the hydrogen atom is on a nitrogen. In certain embodiments, the hydrogen atom is on an oxygen. In certain embodiments, the hydrogen atom is on a carbon.

[0270] In some embodiments, G, at each occurrence, is independently

[0271] In some embodiments, G, at each occurrence, is independently

[0272] In some embodiments, the payload is a monoclonal antibody payload. A monoclonal antibody for use herein as a payload can be an entire monoclonal antibody, or a fragment thereof (c.g., antigenbinding fragment (Fab)). In some embodiments, the antibody is an immune cell engager, and as such would induce or elicit an immune response. In some embodiments, the monoclonal antibody, or fragment thereof, targets one or more of 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 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).

[0273] In certain embodiments, the payload is an antibody or antibody fragment which targets CD3, such as OKT3, SP34, UCHT1, Teplizumab, Otelixizumab, Visilizumab, or Foralumab, or an antibody fragment derived therefrom.

[0274] In certain embodiments, the payload is an antibody or antibody fragment which targets CD28, such as Thcralizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0275] In certain embodiments, the payload is an antibody or antibody fragment which targets CD 137 (4-1BB), such as Utomilumab, Urelumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0276] In certain embodiments, the pay load is an antibody or antibody fragment which targets CD 16, such as AFM13, or an antibody fragment derived therefrom.

[0277] In certain embodiments, the payload is an antibody or antibody fragment which targets NKG2D, such as NNC0152-0002 orJNJ-64304500, or an antibody fragment derived therefrom.

[0278] In certain embodiments, the payload is an antibody or antibody fragment which targets CD64, such as H22, or an antibody fragment derived therefrom.

[0279] In certain embodiments, the payload is an antibody or antibody fragment which targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0280] In certain embodiments, the payload is an antibody or antibody fragment which targets CD25, such as Daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0281] In certain embodiments, the payload is an antibody or antibody fragment which targets CD40, such as Iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, Dacetuzumab, or Lucatumumab, or an antibody fragment derived therefrom.

[0282] In certain embodiments, the payload is an antibody or antibody fragment which targets CD4, such as MAX.16H5, IT1208, Zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.

[0283] In certain embodiments, the payload is an antibody or antibody fragment which targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0284] In certain embodiments, the payload is an antibody or antibody fragment which targets G-CSFR, such as CSL324, or an antibody fragment derived therefrom.

[0285] In certain embodiments, the payload is an antibody or antibody fragment which targets GM- CSFR, such as Mavrilimumab, or an antibody fragment derived therefrom.

[0286] In certain embodiments, the pay load is an antibody or antibody fragment which targets CD 122, such as Hu-Mik(beta)!, or an antibody fragment derived therefrom.

[0287] In certain embodiments, the pay load is an antibody or antibody fragment which targets PD-1, such as CC-90006, Ccmiplimab, Camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0288] In certain embodiments, the payload is an antibody or antibody fragment which targets CTLA4, such as Tremelimumab or ipilimumab, or an antibody fragment derived therefrom.

[0289] In certain embodiments, the payload is an antibody or antibody fragment which targets LAG3, such as Relatlimab (BMS-986016), GSK2831781, Cemiplimab (REGN3767), Favezelimab, leramilimab, or Mavezelimab, or an antibody fragment derived therefrom.

[0290] In certain embodiments, the payload is an antibody or antibody fragment which targets TIGIT, such as BMS-986207, Tiragolumab, Vibostolimab, Etigilimab, Domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0291] In certain embodiments, the payload is an antibody or antibody fragment which targets NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0292] In certain embodiments, the payload is an antibody or antibody fragment which targets TIM3, such as Cobolimab, Sym023, LY3321367, BMS-986258, SHR-1702, Sabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0293] In certain embodiments, the payload is an antibody or antibody fragment which targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0294] In certain embodiments, the pay load is an antibody or antibody fragment which targets CD 134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0295] In certain embodiments, the payload is an antibody or antibody fragment which targets CD27, such as Varlilumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0296] In certain embodiments, the payload is an antibody or antibody fragment which targets CD40L, such as Dapirolizumab, or an antibody fragment derived therefrom.

[0297] In certain embodiments, the payload is an antibody or antibody fragment which targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0298] In certain embodiments, the payload is an antibody or antibody fragment which targets BAFFR, such as lanalumab, or an antibody fragment derived therefrom.

[0299] In certain embodiments, the pay load is an antibody or antibody fragment which targets LFA-1, such as Efalizumab, or an antibody fragment therefrom.

[0300] In certain embodiments, the payload is an antibody or antibody fragment which targets BTLA, such as Icatolimab, or an antibody fragment derived therefrom.

[0301] In some embodiments, the payload is an anti-CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof. In some embodiments, the anti-CD3 (aCD3) monoclonal antibody is SP34, UCHT1, or OKT3, or a derivative, or analog thereof.

[0302] In some embodiments, at least one payload is selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, an anti- CD3 (aCD3) monoclonal antibody, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, and MMAE, or a derivative, or analog thereof.

[0303] In some embodiments, D is a payload selected from an inhibitor of poly (ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterlin, HTI-286, and an anti- CD3 (aCD3) monoclonal antibody, or a derivative, or analog thereof.

[0304] In some embodiments, at least one payload is selected from lurbinectedin, MSA- 2, gardiquimod, ciprofloxacin, Paclitaxel, Gemcitabine, Mitomycin C, Etoposide, exatecan, Seco-Duocarmycin SA, and MMAE, or a derivative, or analog thereof.

[0305] In some embodiments, a payload is an inhibitor of poly (ADP-ribose) polymerase (PARP), or a derivative, or analog thereof. In some embodiments, the inhibitor of poly (ADP-ribose) polymerase (PARP inhibitor) is niraparib, talazoparib, olaparib, pamiparib, rucaparib, veliparib, iniparib, 3- aminobenzamide, CEP-9722, E7016, or a derivative, or analog thereof.

[0306] In some embodiments, a payload is:

[0307] In some embodiments, a payload is a duocarmycin, or a derivative, or analog thereof. In some embodiments, the duocarmycin is Duocarmycin A, Duocarmycin Bl, Duocarmycin B2, DuocarmycinCl, Duocarmycin C2, Duocarmycin D, Duocarmycin SA, CC-1065, adozelesin, carzelesin, bizelesin, or a derivative, or analog thereof.

[0308] In some embodiments, a pay load comprises:

[0309] In some embodiments, a payload comprises a pyrrolobenzodiazepine (PBD), or a derivative, or analog thereof. In some embodiments, the pyrrolobenzodiazepine (PBD) is [l,2]diazepino[3,4-e]indole, or a derivative, or analog thereof.

[0310] In some embodiments, a payload comprises:

[0311] In some embodiments, a payload is an inhibitor of tubulin polymerization. In some embodiments, a payload is hemiasterlin, HTI-286, or a derivative, or analog thereof.

[0312] In some embodiments, a payload comprises:

[0313] In some embodiments, a payload comprises:

[0314] In some embodiments, the payload is a topoisomerase inhibitor. In some embodiments, the payload comprises camptothecin, or a derivative, or analog thereof. In some embodiments, the payload comprises topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, or rubitecan.

[0315] In some embodiments, the pay load comprises:

[0316] In some embodiments, the payload comprises:

[0317] In some embodiments, the payload comprises:[ nts, the pay load comprises:

[0319] In some embodiments, the pay load comprises:

[0321] In some embodiments, the pay load comprises:

[0322] 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 L1is covalently bonded to a lysine, serine, threonine, or tyrosine residue present on the payload. In some embodiments, the polypeptide comprises one or more lysine residues. In some embodiments, the linker L1is covalently bonded to a lysine residue present on the payload.

[0323] In some embodiments, the payload comprises an N-terminal amino acid, wherein the linker L1is covalently bonded to a N-terminal amino acid.

[0324] In some embodiments, m is 1-20. In some embodiments, m is 1-10. In some embodiments, m is 1-5.

[0325] In some embodiments, the payload is an immunomodulatory agent payload.

[0326] In some embodiments, the immunomodulatory agent payload is an antibody payload.

[0327] In some embodiments, the immunomodulatory agent payload is the immune checkpoint inhibitor payload. In some embodiments, the immune checkpoint inhibitor payload is a payload of pidilizumab, sintilimab, AMP-224, atezolizumab, durvalumab, BMS-936559, tremelimumab, indoximod, epacadostat, a TIGIT inhibitor (e.g., LAG-3, such as an anti-LAG-3 antibody; TIM-3, such as an anti-TIM-3 antibody), a B7 molecule, or a BTLA pathway antagonist.

[0328] 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 aPD-1 inhibitor payload. In some embodiments, the PD-1 inhibitor payload is a nivolumab, pembrolizumab, pidilizumab, sintilimab, or AMP-224 payload.

[0329] In some embodiments, the immune checkpoint inhibitor antibody payload is a PD-L1 inhibitor payload. In some embodiments, the PD-L1 inhibitor payload is an atezolizumab, avelumab, durvalumab, or BMS-936559 payload.

[0330] 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.

[0331] 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 an indoximod or epacadostat payload.

[0332] In some embodiments, the immunomodulatory agent payload is a cytokine payload.

[0333] In some embodiments, the cytokine payload is an 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.

[0334] In some embodiments, the interleukin pay load is chosen 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.

[0335] In some embodiments, the immunomodulatory agent payload is a type 1 cytokine (IL-2, IL- 12, TNF-B, IFN-g).

[0336] In some embodiments, the cytokine payload is selected from the group consisting of IFN-alpha, IFN-beta, IFN-gamma, pegylated IFN-a, and apolipoprotein A-I fusion protein with IFN-a, interleukin, IL-2, IL-2 covalently bound to immunoglobulins (e.g., cergutuzumab amunaleukin, RO6874281), IL-2 covalently bound to PEG molecules (e.g., NKTR-214), IL-10, PEGylated IL-10 (e.g., pegilodecakin), IL- 7, IL- 12, IL-15, recombinant aglycosylated IL- 15, fusion protein of IL- 15 with the binding domain of IL- 15Ra (e.g., RLI), triple fusion protein comprising human IL-15, the binding domain of IL-15Ra and apolipoprotein A-I, ALT-803 (IL-15 fused to IgGl Fc domain), 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.

[0337] In some embodiments, the immunomodulatory agent payload is the chemokine payload.

[0338] 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.

[0339] In some embodiments, the immunomodulatory agent payload is the chemokine antagonist payload. In some embodiments, the chemokine antagonist payload is a plerixafor payload.

[0340] In some embodiments, the immunomodulatory agent is a monoclonal antibody specific to a cytokine or a cytokine receptor.

[0341] In some embodiments, the immunomodulatory agent payload comprises a polypeptide.

[0342] In some embodiments, the polypeptide comprises one or more lysine residues.

[0343] In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues.

[0344] In some embodiments, the trans-cyclooctene is linked to one of the one or more lysine residues.

[0345] In some embodiments, the trans-cyclooctene is independently linked to one or more lysine, serine, threonine, or tyrosine residues.

[0346] In some embodiments, the polypeptide comprises an N-terminal amino acid, wherein an occurrence of the bioorthogonal moiety is linked to the N-terminal amino acid.

[0347] In some embodiments, m is 1-20. In some embodiments, m is 1-10. In some embodiments, m is 1-5. 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.

[0348] In some embodiments, the trans-cyclooctene functionalized prodrug of Formula XI:or a pharmaceutically acceptable salt thereof, wherein:Rla, at each occurrence, is independently selected from the group consisting of hydrogen, Ci- 4alkyl, and Ci-ihaloalkyl;Rlb, at each occurrence, is independently selected from the group consisting of hydrogen, Ci-4alkyl, CiJialoalkyl, C(O)OH, C(O)OCMalkyl, C(O)N(Rlc)CHRleCO2H, C(O)N(Rlc)CHRleC(O)OCi.4alkyl, C(O)N(R1C)-Ci.6alkylene-CO2H, and C(O)N(R1C)-Ci.6alky lene-C(O)OCwalkyl;Rlc, at each occurrence, is independently hydrogen or C i al ky I :Rle, at each occurrence, is independently -Ci.4alkylene-CO2H, -Ci.4alkylene-CONH2, or -C i .4alkylene-OH ;D, at each occurrence, is independently a payload;L1, at each occurrence, is independently a linker;p', at each occurrence, is independently 0, 1, or 2; and p'', at each occurrence, is independently 1, 2, or 3.

[0349] In some embodiments, D, at each occurrence, is independently selected from the group consisting of an anticancer agent payload, a toll-like receptor (TLR) agonist payload and a stimulator of interferon genes (STING) agonist payload.

[0350] In some embodiments, R1ais hydrogen.

[0351] In some embodiments, R1ais C1-4alkyl.

[0352] In some embodiments, R1ais CH3.

[0353] In some embodiments, R1bis selected from the group consisting of C(O)OH, C(O)OC1-4alkyl, C(O)N(R1c)CHR1eCO2H, C(O)N(R1c)CHR1eC(O)OC1-4alkyl, C(O)N(R1c)–C1-6alkylene–CO2H, and C(O)N(R1c)–C1-6alkylene–C(O)OC1-4alkyl.

[0354] In some embodiments, R1bis selected from the group consisting of C(O)OH, C(O)N(R1c)CHR1eCO2H, and C(O)N(R1c)CH2CO2H.

[0355] In some embodiments, R1bis selected from the group consisting of –NR1c–CH2CH2–N(CH3)3+, –N(R1c)–CH2CH2–SO3H, –N(R1c)–(CH2CH2O)3–CH2CH2N((CH2CH2O)3–CH2CH2–CO2H)2, and – N(R1c)–CH(CH2O–CH2CH2–CO2H)2.

[0356] In some embodiments, the trans-cyclooctene moiety (G) is: OH

[0358] In some embodiments, the trans-cyclooctene moiety is

[0359] In some embodiments, the trans-cyclooctene moiety

[0360] In some embodiments, the trans-cyclooctene moiety is

[0361] In some embodiments, the trans-cyclooctene moiety

[0362] In some embodiments, the trans-cyclooctene moiety

[0363] In some embodiments, the trans-cyclooctene moiety

[0364] In some embodiments, the trans-cyclooctene moiety is

[0365] In some embodiments, the trans-cyclooctene moiety is

[0366] In some embodiments, the trans-cyclooctene moiety-OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine; wherein R2may be optionally further substituted with a polyether.

[0367] In some embodiments, the trans-cyclooctene moiety comprises

[0368] In some embodiments, the trans-cyclooctene moiety comprises

[0369] In some embodiments, the trans-cyclooctene moiety of comprises

[0370] In some embodiments, the trans-cyclooctene moiety of comprises

[0371] In some embodiments, Rleis -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH.

[0372] In some embodiments, Rleis -Chalky lene-CO2H.

[0373] In some embodiments, Rleis -CH2CO2H.

[0374] In some embodiments, Rlbis -C(O)N(Rlc)-Ci-6alkylene-CO2H.

[0375] In some embodiments, Rlbis -C(O)N(R1C)CH2CO2H.

[0376] In some embodiments, Rlcis hydrogen.

[0377] In some embodiments, Rlbis hydrogen.

[0378] In some embodiments, R1bis C(O)OH.

[0379] In some embodiments, linker L1may have 1 to 100 linking atoms, and may include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, linkers may have from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.

[0380] In some embodiments, linker L1may comprise one or more (e.g., 1-10 or 1-5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1-10 or 1-5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4alkyl, C1-4alkoxy, and C1-4haloalkyl.

[0381] In some embodiments, linker L1may be of the formula: -Y10-(CH2)n’-Y20-(CH2)m''-Y30- wherein: each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(O)0-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, - OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl; each R110is independently hydrogen, C1-4alkyl, C1-4haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; each R120is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0382] In certain embodiments, the linker is a bond.

[0383] In certain embodiments, the linker is not a bond. In certain embodiments, each R110is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and each R120is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl.

[0384] Representative linkers include, but are not limited to, those shown below:

[0385] Representative linkn below:

[0386] In some embodim, yethylene glycol (e.g., PEG having an average molecular weight of from 300 g / mol to 10,000 g / mol), ethylene-1,2- diylbis(methylcarbamate, an arylene (e.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. In some embodiments, linker L1may comprise .

[0387] In some embodiments, linker L1may comprise one or more natural or unnatural amino acids, which may be referred to as a peptide linker. Where the drug (D) comprises an amino moiety, the linker may be bound thereto using a peptide linker made up of a carboxylic acyl unit, and one or more amino acids making up a protein or peptide sequence. In some embodiments, linker L1may also contain a self- immolating spacer which spaces the drug and the protein peptide sequence.

[0388] In some embodiments, linker L1may be a peptide linker represented by “A—Y—Z—Q—W” in which “A” is the carboxylic acyl unit, “Y” and “Z” are each one or more natural or unnatural amino acids and together form a peptide sequence, and “Q” and “W” are optional additional linkers having from 1 to 50 linking atoms, or from 5 to 10 linking atoms, or from 1 to 10 linking atoms which spaces the peptideand the drug, D, or the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.

[0389] 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.

[0390] In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline. In some embodiments, Z may be at least one amino acid selected from the group consisting of alanine, lysine and citrulline.

[0391] In some embodiments, exemplary Y-Z combinations include Valine-Citrulline; Valine-Alanine; and Alanine-Alanine.

[0392] In certain embodiments, A is -OC(O)-.

[0393] In certain embodiments, Q is -OC(O)-.

[0394] In certain embodiments, W is -OC(O)-. In certain embodiments, Q is absent and W is -OC(O)-.

[0395] In certain embodiments .

[0396] In certain embodiments .

[0397] In certain embodiments, ored so that it will be selectively cleaved (e.g., enzymatically cleaved) releasing the drug, such as by one or more of the tumor-associated proteases.

[0398] In certain embodiments, the peptide linker has a chain length of two to four amino acid residues (i.e., a di-, tri-, or tetra-peptide). It will be understood, however, that peptide linkers up to five, six, seven, or eight amino acid residues may also suitably be employed.

[0399] In certain 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: ], Ala-Leu-Ala-Leu [SEQ ID NO: ], Phe-N9-tosyl-Arg, or Phe-N9-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.,h ,).e.g., the payload may be an amine-containing payload for attachment of the payload to a carbonyl group of the linker, or, in other cases, the payload may be a carboxyl-containing payload for attachment of the payload to an amine group of the linker. In some instances, the payload and linker, together form a carbamate group; e.g., the payload may be an amine-containing payload for attachment of the payload to an acyloxy group of the linker. In some instances, the payload and linker, together form a carbonate group; e.g., the payload may be a hydroxy-containing payload for attachment of the payload to an acyloxy group of the linker. O O

[0403] In some embodiments1 L3aL4aO–; L3ais a bond or C1-6alL4ais a bond, –NHN:, –N(R10)–C2-6alkylene–N(R11)–, –N(R12)–C2-3alkylene–N(R13)C(O)–, –N(R10)–C1-6alkylene–C(O)NHN: –NHNHC(O)C1-6alkylene–C(O)NHN: ,R , R , R , R , R , R , and R are each independently hydrogen or C1-4alkyl; R16is hydrogen, C1-4alkyl, –C1-4alkylene–OH, –C1-4alkylene–OC1-4alkyl, –C1-4alkylene–CO2H, or –C1-4alkylene–CONH2;R17, at each occurrence, is independently hydrogen or –CH2OC(O)–; and Gxis phenyl optionally substituted with 1-5 substituents independently selected from the group consisting of halogen, C1-4alkyl, C1-4haloalkyl, C1-4alkoxy, cyano, and nitro.

[0404] In certain embodiments, linker L1is -OC(O)-. O O 1 L3aL4a

[0405] In some embodiments L is ; L3ais a bond;and

[0406] In some embodiments, p” is 1. In some embodiments, p’ is 1. ,,, at eac occurrence, s n epenenty y rogen or – 2 ( )N ; RDis hydrogen or C1-4alkyl on a nitrogen atom of a payload; and D and D’ are independently a payload moiety.

[0408] In some embodiments, D or D’ is a cyclic dinucleotide payload moiety, imidazo[4,5-c]quinolin- 4-amine payload moiety, TLR agonist payload moiety, STING agonist payload moiety, or anticancer agent payload moiety. orD and D’ are independently a payload moiety (e.g., anticancer agent payload moiety).

[0410] In some embodiments, p’ is 0.

[0411] In some embodiments, p” is 2 or 3.

[0412] In some embodiments .

[0413] The person skilled ined to a linker does not refer to a payload molecule per se, but refers to the portion of the payload molecule bonded to the linker. Release of the payload (D or D’) from a prodrug, releases the payload per se.

[0414] A payload (D or D’) may be an anticancer agent payload of any of the anticancer agents described herein.

[0415] In some embodiments, the payload comprises a TLR7 / 8 agonist. In some embodiments, the payload comprises gardiquimod.

[0416] In some embodiments, the payload comprises camptothecin, or derivative thereof. In some embodiments, the payload comprises exatecan.

[0417] In some embodiments, the payload comprises MMAE, or derivative thereof.

[0418] In some embodiments, the payload comprises paclitaxel, or derivative thereof.

[0419] In some embodiments, the payload comprises docetaxel, or derivative thereof.

[0420] In some embodiments, the payload has the structure:,,,, ,[ ] spects o t e present scosure ncue met os or e verng a payoa to a target ocaton in a subject. In certain embodiments, the method includes selectively delivering a payload to the target location in a subject. Selective delivery of the payload includes delivering the payload to the target location (e.g., an organ or tissue, or portion thereof), without targeting other locations in the subject (e.g., other organs or tissues, or portions thereof) that do not need administration of the payload. Selective delivery of the payload may be achieved through use of the tetrazine-based targeting agent and the functionalized payloads described herein.

[0423] In some instances, a tetrazine-based targeting agent of the present disclosure may be localized to a desired target location in a subject. For example, methods of the present disclosure may include administering to a subject a tetrazine-based targeting agent as described herein. The tetrazine-based targeting agent may be administered to the subject at a desired target location in the subject. In some instances, the tetrazine-based targeting agent may be injected locally into the subject at the desired target location in the subject. In some embodiments, the tetrazine-based targeting agent is administered systemically. In these embodiments, the tetrazine-based targeting agent may localize at a desired target location in the subject through specific binding of the tetrazine-based targeting agent to its target (e.g., antibody-antigen interaction, and the like), or may localize on the surface of a desired target (e.g., a cell surface) through specific binding of the tetrazine-based targeting agent to its target (e.g., antibody- antigen interaction, and the like).

[0424] As described herein, selective binding between bioorthogonal binding partners (e.g., between a tetrazine of the tetrazine-based targeting agent and its complementary trans-cyclooctene of a prodrug may occur. Due to the administration of the tetrazine-based targeting agent and resulting localization to a desired location in the subject, the selective binding between the tetrazine and its complementary binding agent of the trans-cyclooctene on the prodrug will localize the payload to the desired target location.

[0425] Provided herein is a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a tetrazine-based targeting agent as described herein, or a pharmaceutically acceptable salt thereof, and a trans-cyclooctene prodrug.

[0426] In some embodiments, the cancer is metastatic. In some embodiments the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilm’s tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0427] In some embodiments, the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue carcinoma, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0428] In some embodiments, the cancer is a solid tumor.

[0429] In some embodiments, the cancer is a soft tissue sarcoma.

[0430] In some embodiments, the soft tissue sarcoma is a fibrosarcoma, rhabdomyosarcoma, or Ewing’s sarcoma.

[0431] In some embodiments, the method also comprises enhancing or eliciting an immune response. In some embodiments the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0432] In some embodiments, the method further comprising administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof. Anticancer agents, immunomodulatory agents, and their trans-cyclooctene prodrugs are known in the art.

[0433] Indications for this approach include cancer, both hematological and solid cancers. In certain embodiments, the approach can be used for the treatment and / or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing’s sarcoma, and all the different subtypes of soft tissue sarcoma as well as osteosarcoma. The compositions can be for the treatment and / or diagnosis of pigmented vilonodular synovitis.

[0434] In certain embodiments, the approach can be used for the treatment and / or diagnosis of hematological malignancies such as myelodysplastic syndromes, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s 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’s lymphoma, as well as others.

[0435] The compositions of the present disclosure find use in treatment and / or diagnosis of a condition or disease in a subject that is amenable to treatment or diagnosis by administration of the payload (e.g., the parent drug (i.e., the drug prior to conjugation to the composition)). By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. Treatment may include inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease. Treatment may include relief, that is, causing the regression of clinical symptoms. For example, in the context of cancer, the term “treating” includes any or all of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, prolonged survival and ameliorating one or more symptoms associated with a cancer.

[0436] The subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable totreatment using the compositions disclosed herein. Generally, such subjects are “mammals,” with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys).

[0437] In certain embodiments, additional therapeutic agents, and methods can be used for the treatment, prevention, and / or diagnosis of solid tumors, including but not limited to, melanoma (e.g., unresectable, metastatic melanoma), renal 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 carcinoma, 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 carcinoma, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilm's tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostatic adenocarcinoma, nasopharyngeal carcinoma, cutaneous T-cell lymphoma, among others. The disclosed approach lends itself well as an adjuvant / neoadjuvant system. For example, particles as disclosed herein could be placed during the biopsy, once the results from the study come back, the practitioner could deliver the appropriate cocktail to the desired site in the body. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. Then at the end of the surgery, the surgeon could administer additional tetrazine-based targeting agent to the subject to target the surgical cavity and treat the patient with further doses of treatment (e.g. chemotherapy through the disclosed approach) to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0438] In certain embodiments, a tetrazine -based targeting agent as disclosed herein could be administered and the practitioner could deliver the appropriate cocktail to the desired site in the body. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. Then at the end of the surgery, the surgeon could administer additional tetrazine -based targeting agent to the subject to target the surgical cavity and treat the patient with further doses of treatment (e.g. chemotherapy through the disclosed approach) to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0439] In certain embodiments, the disclosed methods provide the ability to place particles as disclosed herein at the time of the biopsy. When the results return, the practitioner can deliver through to the biopsy site immunomodulatory agents.

[0440] In certain embodiments, the disclosed methods provide the ability for a practitioner to deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents combined with immunotherapy agents. This combinationapproach would be beneficial to patients. The chemotherapy agent would treat the solid tumor or specific location, while the enhanced response of the immunotherapy would help with distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods could employ or be used with anthracyclines, taxanes, gemcitabine and other agents to enhance the efficacy of one or more immunomodulatory agents such as ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).Cancer

[0441] The disclosed methods may be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may include sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, enablement of replicative immortality, induction of angiogenesis, and the activation of invasion and metastasis. The disclosed methods may enhance or elicits an immune response against a cancer in the subject. The immune response may lead to an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0442] Cancer that may be treated by the disclosed methods, includes, but is not limited to, astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brain stem cancer, brain stem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse intrinsic pontine glioma, ductal cancer, endometrial cancer, ependymoma, Ewing’s sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular cancer, histiocytosis, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, nonHodgkin’s lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue carcinoma, soft tissue sarcoma, solid tumor, squamous cell carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, 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 tongue squamous cell carcinoma (OTSCC).

[0443] In some embodiments, the cancer that may be treated by the disclosed methods is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue carcinoma, 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 carcinoma. In some embodiments, the cancer is afibrosarcoma. In some embodiments, the cancer is diffuse intrinsic pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0444] In some embodiments, the cancer that may be treated by the disclosed methods is a hematological malignancy, such as myelodysplastic syndromes, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s 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’s lymphoma, as well as others.

[0445] Without being bound by a particular theory, local release of certain anti-cancer agents using the compounds and methods of the disclosure may produce or contribute to immunogenic cell death (ICD). For example, certain anti-cancer 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 through activation of dendritic cells (DCs) and consequent activation of specific T cell response. ICD is characterized by secretion of damage-associated molecular patterns (DAMPs). Three important DAMPs which are exposed to the cell surface during ICD. Calreticulin (CRT), one of the DAMP molecules, which is normally in the lumen of endoplasmic reticulum (ER), is translocated after the induction of immunogenic apoptosis to the surface of dying cell where it functions as an "eat me" signal for professional phagocytes. Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which are under stress condition also translocated to the plasma membrane. On the cell surface they have an immunostimulatory effect, based on their interaction with number of antigen-presenting cell (APC) surface receptors like CD91 and CD40 and also facilitate cross presentation of antigens derived from tumor cells on MHC class I molecule, which than leads to the CD8+ T cell response. Other important DAMPs, characteristic for ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered to be late apoptotic marker and its release to the extracellular space seems 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 receptor (TLR) 2 and 4, which are expressed on APCs. The most recently found DAMP released during immunogenic cell death is ATP, which functions as a "find-me" signal for monocytes when secreted and induces their attraction to the site of apoptosis. Kroemer et. al. Curr. Op. Immunol. 2008 (20), 504-511.

[0446] Thus, local release of ICD inducers using the compounds and methods of the disclosure may be beneficially combined with one or more immunomodulatory agents.

[0447] In certain embodiments, the tetrazine -based targeting agent can be used for the treatment, prevention, and / or diagnosis of solid tumors, including but not limited to, melanoma (e.g. , unresectable, metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, such as metastaticepithelial 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, pancreatic cancer, among others.

[0448] The disclosed approach lends itself well as an adjuvant / neoadjuvant system. For example, targeting moieties as disclosed herein could be placed during the biopsy, once the results from the study come back, the practitioner could administer the appropriate cocktail to deliver treatment to the desired site in the body (compounds as disclosed herein and optional additional therapeutic agent(s)). The results of the biopsy may indicate the amount and type of treatment to deliver to the site of a tumor. For example, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants to enhance the immune system with fewer side effects as well as the chemotherapeutics agents could be delivered and combined with immunotherapy agents.

[0449] The disclosed methods may include one or multiple systemic doses of targeting moieties that focus at one location or more locations. The disclosed methods may be used to deliver a functionalized payload to these location through systemic or local administration. In some embodiments, the tetrazinebased targeting agent is delivered systemically. In some embodiments, the tetrazine-based targeting agent and the payload (i.e., a TCO-labeled payload) are both delivered systemically. In some embodiments, the tetrazine-based targeting agent is delivered locally.

[0450] The disclosed compounds and compositions may be administered prior to surgical resection. The disclosed methods may minimize the size of the tumor prior to surgical resection. This would minimize the size of the tumor particularly in the context of a surgically resectable tumor. The disclosed conjugates, compounds and compositions may be administered during surgical resection. The disclosed conjugates, compounds and compositions may be administered after surgical resection. The tetrazinebased targeting agent may be placed around the surgical cavity at the end of surgical resection and the subject may then be treated with further doses of a treatment to minimize the risk of any cancer cells that may have been missed in the surgical margins.

[0451] The disclosed methods may include multiple systemic doses of functionalized payload that focus at one location. The disclosed methods may be used to deliver a second payload. The disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload. A 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 the tetrazine-based targeting agent used for the first prodrug.

[0452] The functionalized payloads disclosed herein may function as adjuvants. This combination approach would be beneficial to patients. The chemotherapy agent would treat the solid tumor or specific location and may enhance or elicit an immune response, while the enhanced response of the immunotherapy of the functionalized payload and / or separate agent may help with distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods could employ or beused with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, camptothecin analogues and other agents to enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).

[0453] The disclosed methods may be used to treat diffuse intrinsic pontine gliomas. Diffuse intrinsic pontine gliomas (DIPG) are pediatric brainstem tumors that may be highly malignant and may be difficult to treat. There is no known curative treatment for DIPG, and survival odds have remained dismal over the past four decades. DIPG patients have a median overall survival of just 11 months, with a two-year survival rate below 10%. DIPG account for 75-80% of brainstem tumors in children, affecting an estimated 200-300 children in the U.S. each year. The rarity of this devastating disease and previous lack of experimental model systems has impeded research, and over the past four decades survival odds have remained the same. Diagnosis of DIPG may begin with clinical symptoms and may be confirmed by MRI. The disease may begin with several months of generalized symptoms, including behavioral changes and difficulties in school, double vision, abnormal or limited eye movements, an asymmetric smile, loss of balance, and weakness. Alternately, severe neurologic deterioration may happen more quickly, with symptoms present for less than a month prior to diagnosis. Clinical examination may reveal the triad of multiple cranial neuropathies, long tract signs such as hyperreflexia and clonus, as well as ataxia. Expansion of the pons section of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure.

[0454] Nuclei critical for life-sustaining function such as breathing and heartbeat in are located in the pons and without treatment, breathing and heartbeat may be damaged by DIPG.

[0455] The disclosed methods may include multiple systemic doses of functionalized payload that focus at one location. The disclosed methods may be used to deliver a second payload. The disclosed methods may be used to administer a second functionalized payload if the tumor is resistant to the first payload. A 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 the tetrazine-based targeting agent used for the first prodrug.Modes of Administration

[0456] Methods of treatment may include any number of modes of administering a disclosed conjugate, compound or composition. Modes of administration may include tablets, pills, dragees, 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 the pharmaceutical composition, the conjugate, compound or compositions disclosed herein may also be dispersed in a microparticle, e.g. a nanoparticulate composition.

[0457] For parenteral administration, the conjugates, compounds or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent, such as water, buffer, oils with orwithout solubilizers, surface-active agents, dispersants or emulsifiers. Suitable oils may 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 an aqueous, lipid, oily or other kind of solution or suspension, or even administered in the form of liposomes or nano-suspensions.

[0458] The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0459] The amount of composition administered to a subject can be initially determined based on guidance of a dose and / or dosage regimen of the parent drug. In general, the compositions can provide for targeted delivery and / or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the compositions can provide for reduced dose and / or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in a composition of the present disclosure.

[0460] The pharmaceutical formulation may be provided in unit dosage form. In such form the pharmaceutical formulation may be subdivided into unit doses containing appropriate quantities of the compositions of the present disclosure. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, such as packeted tablets, capsules, and powders in pouches, vials or ampoules.

[0461] In some embodiments, provided is a kit comprising a tetrazine -based targeting agent, or a pharmaceutically acceptable salt thereof, as described herein, or the pharmaceutical composition comprising the same, and instructions for use thereof.

[0462] In some embodiments, the kit further comprising a prodrug.

[0463] Compositions of the present disclosure can be present in any suitable amount, and can depend on various factors including, but not limited to, weight and age of the subject, state of the disease, etc.Suitable dosage ranges for the composition of the present disclosure include from 0.1 mg to 10,000 mg, or 1 mg to 1000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For instance, suitable dosages for the composition of the present 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.

[0464] In some embodiments, multiple doses of a composition are administered. The frequency of administration of a composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, a composition is administered once per month, twice per month, three times per month, every other week (qow), once perweek (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0465] The compositions of the present disclosure can be administered at any suitable frequency, interval and duration. For example, the composition of the present disclosure can be administered once an hour, or two, three or more times an hour, once a day, or two, three, or more times per day, or once every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, so as to provide the desired dosage level to the subject. When the composition of the present disclosure is administered more than once a day, representative intervals include 5 min, 10 min, 15 min, 20 min, 30 min, 45 min and 60 minutes, as well as 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, and 24 hours. The composition of the present disclosure can be administered once, twice, or three or more times, for an hour, for 1 to 6 hours, for 1 to 12 hours, for 1 to 24 hours, for 6 to 12 hours, for 12 to 24 hours, for a single day, for 1 to 7 days, for a single week, for 1 to 4 weeks, for a month, for 1 to 12 months, for a year or more, or even indefinitely.

[0466] The compositions of the present disclosure can be co- administered with another active agent. Co-administration includes administering the composition of the present disclosure and active agent within 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, or 24 hours of each other. Coadministration also includes administering the composition of the present disclosure and active agent simultaneously or approximately simultaneously (e.g., within about 1 min, 5 min, 10 min, 15 min, 20 min, or 30 minutes of each other), or sequentially in any order. In addition, the composition of the present disclosure and the active agent can each be administered once a day, or two, three, or more times per day so as to provide the desired dosage level per day.

[0467] Co-administration can be accomplished by coimplantation or coinjection.

[0468] In some embodiments, co-administration can be accomplished by co-formulation, e.g., preparing a single pharmaceutical formulation including both the composition of the present disclosure and the active agent. In other embodiments, the composition of the present disclosure and the active agent can be formulated separately and co-administered to the subject.

[0469] The composition of the present disclosure and the active agent can be present in a formulation in any suitable weight ratio, such as from 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 composition of the present disclosure and the other active agent can be present in any suitable weight ratio, such as l :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 composition of the present disclosure and the active agent are suitable in the formulations and methods described herein.Combination Therapies

[0470] In one aspect, provided is a method of treating cancer or enhancing or eliciting an immune response comprising administering to a subject in need thereof: a therapeutically effective amount of a tetrazine-based targeting agent of the disclosure, or a pharmaceutically acceptable salt or compositionthereof; and a prodrug, such as those as described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0471] The disclosure also provides a pharmaceutical combination comprising a tetrazine-based targeting agent described herein, or a pharmaceutically acceptable salt, or composition thereof; a prodrug as described herein; and optionally an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for use in the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response.

[0472] The disclosure also provides the use of a pharmaceutical combination comprising a tetrazinebased targeting agent as described herein, or a pharmaceutically acceptable salt, or composition thereof; a prodrug, such as those described herein; and optionally a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof for the treatment or prevention of a cancer or for use in enhancing or eliciting an immune response.

[0473] In the methods and uses described herein, the components of the pharmaceutical combinations may be administered / used simultaneously, separately, or sequentially, and in any order, and the components may be administered separately or as a fixed combination. For example, the delay of progression or treatment of diseases according to the disclosure may comprise administration of the first active ingredient in free or pharmaceutically acceptable salt form and administration of the second active ingredient in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts or effective amounts, e.g. in daily dosages corresponding to the amounts described herein. The individual active ingredients of the combination can be administered separately at different times during the course of therapy or concurrently in divided or single dosage forms. The instant disclosure is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. Thus, a pharmaceutical combination, as used herein, defines either a fixed combination in one dosage unit form or separate dosages forms for the combined administration where the combined administration may be independently at the same time or at different times. As a further example, the tetrazine -based targeting agent (or therapeutic tetrazine-based targeting agent) and prodrug may be administered / used simultaneously (e.g., through coinjection or coimplantation), separately, or sequentially, followed by administration of the additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0474] The methods and uses in treating cancer include administering / localizing the tetrazine-based targeting agent at a tumor. In the methods and uses disclosed herein, the administration of the prodrug, or a pharmaceutically acceptable salt, or composition thereof; the tetrazine-based targeting agent; and optionally an additional therapeutic agent may inhibit the growth of the tumor.

[0475] Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed conjugates and compositions. Sequential administration includes administration before or after the disclosed conjugates and compositions. An additional therapeutic agent may be administered before the disclosed conjugates and compositions. An additional therapeutic agent may be administered after the disclosed conjugates and compositions. An additional therapeutic agent may be administered at the same time as the disclosed conjugates and compositions. In some embodiments, the additional therapeutic agent or agents may be administered in the same composition as the disclosed conjugates. In other embodiments, there may be an interval of time between administration of the additional therapeutic agent and the disclosed conjugates or compositions. In some embodiments, administration of an additional therapeutic agent with a disclosed conjugate or composition may allow lower doses of the other therapeutic agents and / or administration at less frequent intervals. When used in combination with one or more other active ingredients, the conjugates or compositions of the present disclosure and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions of the present disclosure include those that contain one or more other active ingredients, in addition to a conjugates of the present disclosure.Anticancer agents

[0476] Exemplary anti-cancer agents include, but are not limited to, 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), Aldesleukin, 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 chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and 1 131 Iodine Tositumomab), Bicalutamide, Bleomycin, Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib- S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carboplatin, Carboplatin-Taxol, Carfilzomib, 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 liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine), 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), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), 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 Quadrivalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gcmtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Ifex (Ifosfamide), Ifosf amide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa- 2b), Iodine 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Kyprolis (Carfilzomib), Lapatinib Ditosylate, Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Liposomal Cytarabine, 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), Mechlorethamine 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 (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin- stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylatc), Nilotinib, Nolvadex (Tamoxifen Citrate), Nplatc (Romiplostim), Obinutuzumab, Ofatumumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), OEPA, OPP A, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation,Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Rasburicase, R-CHOP, R-CVP, Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadrivalent Vaccine, Recombinant Interferon Alfa- 2b, Regorafenib, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Sipuleucel-T, Sorafenib Tosylate, Sprycel (Dasatinib), Stanford V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa- 2b), Synovir (Thalidomide), Synribo (Omacctaxinc Mcpcsuccinatc), 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), Tositumomab and 1 131 Iodine Tositumomab, 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 Liposome, Vinorelbine Tartrate, Vismodegib, 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 (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), and Zytiga (Abiraterone Acetate).

[0477] The anticancer agent may be a PBD dimer, calicheamicin, speromycin, tubulysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothilone B, taxol, cryptophycin, SN38, velcade, bruceantin, DAVLBH, DM1, Phyllanthoside, Alimta, T2 Toxin, MMC, vantalanib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, iressa, irinotecan, LY - 541503, geldanomycin, gemcitabine, methotrexate, gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustards, etoposide, or 5-FU.

[0478] In certain embodiments, an anticancer agent is an anthracycline. In certain embodiments, anticancer agent is a taxane. In certain embodiments, anticancer agent is gemcitabine. In certain embodiments, anticancer agent is doxorubicin. In certain embodiments, anticancer agent is docetaxel. Incertain embodiments, anticancer agent is SN38. In certain embodiments, anticancer agent is monomethyl auristatin E.Synthesis of the Compounds

[0479] The targeting moieties may be prepared using the methods disclosed herein and routine modifications thereof, which will be apparent given the disclosure herein and methods well known in the art. Conventional and well-known synthetic methods may be used in addition to the teachings herein. The synthesis of typical targeting moieties described herein may be accomplished as described in the following examples. If available, reagents and starting materials may be purchased commercially, e.g., from Sigma Aldrich or other chemical suppliers.

[0480] It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0481] Additionally, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, numerous protecting groups are described in Wuts, P. G. M., Greene, T. W., & Greene, T. W. (2006). Greene's protective groups in organic synthesis. Hoboken, NJ., Wiley- Interscience, and references cited therein.

[0482] Compounds provided herein (e.g., compounds of Formula I, etc.) can be prepared by methods described in the Examples below or adapted from the literature (see, e.g., W02020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126).

[0483] Exemplary payloads can be prepared can be prepared according to methods adapted from the literature (see, e.g., WO2022 / 032191, WO2021 / 007160, W02020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126, which methods are incorporated herein in their entirety).EXAMPLES

[0484] The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques to function well in the practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0485] LCMS Analysis Method: Test articles were subjected to PNGaseF (IgG) and DTT or DTT alone (Fab) in RapiGest according to the manufacturer’s protocol. Samples were diluted to 100 pg / mL with water and centrifuged at 16.1k RCF for 10 min at 4 °C. The samples were then analyzed by LCMS (LC-Q-TOF) and the mass spectra reconstructed from the charge ladder.Example 1: Exemplary Synthesis of Tetrazine-Based Targeting Agent

[0486] Targeting agent in PBS is mixed with of tetrazine-NHS (e.g., 20 equivalents). The reaction is mixed thoroughly and aged at room temperature for 1 hour, at which time the reaction is quenched by the addition of 1 volume of 0.1 M Tris buffer. The resulting solution is buffer exchanged to 0.01 M PBS to remove excess reagent and buffer salts. The resulting solution of targeting agent is analyzed by SDS- Page and LCMS confirming the formation of the tetrazine-based targeting agent.Example 2: Synthesis of Tetrazine-Based Targeting Agents 1-4Target 3Target 4

[0487] Procedure for preparation of peptide 13

[0488] Peptide Synthesis:

[0489] The peptide was synthesized using standard Fmoc chemistry.

[0490] Add DCM to the vessel containing CTC Resin (5 mmol, 0.83 mmol / g, 6.02 g) and Fmoc- Lys(Alloc)-OH (2.34 g, 5 mmol, 1.0 eq) with N2 bubbling.

[0491] Add DIEA (6.0 eq) dropwise and mix for 2 hrs.

[0492] Add MeOH (7.0 mL) and mix for 0.5 hr.

[0493] Drain and wash with DMF for 5 times.

[0494] Add 20% piperidine / DMF and react for 0.5 hr.

[0495] Drain and wash with DMF for 5 times.

[0496] Add Fmoc-amino acid solution and mix 30 secs, and then add activation solution. The reaction lasts for 1 hr with N3bubbling.

[0497] Repeat step 4 to 7 for next amino acid coupling.

[0498] 20% piperidine in DMF was used for Fmoc deprotection for 30 mins. The coupling reaction was monitored by ninhydrin test, and the resin was washed with DMF for 5 times.

[0499] Forming Urea: To a mixture of NH2-Glu(OtBu)-OtBu (17.50 g, 17.50 mmol, 4.00 equiv.), TEA (8.4 g, 35.00 mmol, 8.00 equiv.) in DMF (20 mL) was added CDI (11.40 g, 17.5.00 mmol, 4.00 equiv.) at 0 °C. The reaction mixture was stirred at 20 °C for 1 h. After filtration, the filtrate was added to the resin,followed by DMAP (2.13 g, 17.50 mmol, 1.00 equiv.) with N2 bubbling for 16 h at 20 °C. The coupling reaction was monitored by ninhydrin test and showed colorless.

[0500] Peptide Cleavage and Purification:

[0501] Add cleavage buffer (5% DTT I 2.5% TIS / 2.5% H2O / 90% TFA) to the flask containing the side chain protected peptide at room temperature and stir for 1 hr.

[0502] The peptide is precipitated with cold isopropyl ether and centrifuged (3 mins at 3000 rpms)

[0503] Isopropyl ether washes two additional times.

[0504] Remove the solvent under vacuum to get the crude product. The crude was purified by Prep- HPLC (A: 0.075% TFA in H2O, B: CH3CN) to give compound 3 (0.76 g, 99.8% purity, 47% yield) as a white solid. LCMS [M + H]+ calcd 656.8; observed 656.3

[0505] Purification conditions:

[0506] Procedure for preparation of peptide 2

[0507] Peptide Synthesis:

[0508] The peptide was synthesized using standard Fmoc chemistry.

[0509] Add DCM to the vessel containing CTC Resin (5 mmol, 0.83 mmol / g, 6.02 g) and Fmoc-Ahx- OH (1.76 g, 5 mmol, 1.0 eq) with N2 bubbling.

[0510] Add DIEA (6.0 eq) dropwise and mix for 2 hrs.

[0511] Add MeOH (7.5 mL) and mix for 0.5 hr.

[0512] Drain and wash with DMF for 5 times.

[0513] Add 20% piperidine / DMF and react for 0.5 hr.

[0514] Drain and wash with DMF for 5 times.

[0515] Add Fmoc-amino acid solution and mix 30 secs, and then add activation solution. The reaction lasts for 1 hr with N2 bubbling.

[0516] Repeat step 4 to 7 for next amino acid coupling.

[0517] 20% piperidine in DMF was used for Fmoc deprotection for 30 mins. The coupling reaction was monitored by ninhydrin test, and the resin was washed with DMF for 5 times.

[0518] Peptide Cleavage and Purification:

[0519] Add cleavage solution (50%TFA / DCM) to the flask containing the side chain protected peptide at room temperature and stir for 1 hr.

[0520] After filtration, the filtrate was collected.

[0521] The filtrate was concentrated under reduced pressure to give compound 4 (1.0 g, 90.0% purity, 68.4% yield) as a white solid. LCMS [M + H]+calcd 468.6; observed 468.1; LCMS [M + Na]+calcd 490.6; observed 490.1

[0522] Procedure for preparation of Compound 2

[0523] To a solution of Compound 1 (800 mg, 3.47 mmol) and HOSu (600 mg, 5.21 mmol) in THF (16.0 mL) was added DIC (646 uL, 4.17 mmol). The mixture was stirred at 25 °C for 10 hrs. LC-MS showed Compound 1 (RT=0.843 min) was consumed completely and one main peak with desired mass was detected. The reaction mixture was quenched by additional HzO (50.0 mL) at 25 °C, and then extracted by DCM (100 mL * 3). The combined organic layer was washed by saturated brine (100 mL) and dried over anhydrous NazSO i, concentrated under reduced pressure to give a residue. The crude product Compound 2 (1.67 g) was obtained as a purple solid. LCMS [M + H]+ealed 328.3; observed 328.0

[0524] Procedure for preparation of Target 1

[0525] To a solution of Compound 3 (400 mg, 610 umol) and Compound 2 (299 mg, 915 umol) in DMF (8.00 mL) was added D1EA (319 uL, 1.83 mmol). The reaction mixture was stirred at 25 °C for 2 hrs. LC-MS showed that Compound 3 (RT=0.865 min) was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove solid and get a filtrate. Then the filtrate was purified by prep-HPLC (TFA condition) to give Target 1 (144 mg, 166 umol, 27.2% yield) as a purple solid. LCMS [M + H]+ealed 869.0; observed 868.6

[0526] Procedure for preparation of Target 3

[0527] To a solution of Compound 4 (314 mg, 672 umol) and Compound 2 (330 mg, 1.01 mmol) in DMF (8.00 mL) was added DIEA (234 uL, 1.34 mmol). The reaction mixture was stirred at 25 °C for 1 hr. LC-MS showed that Compound 4 (RT=0.897 min) was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove solid and get a filtrate. Then the filtrate was purified by prep-HPLC (TFA condition) to give Target 3 (212 mg, 312 umol, 46.4% yield) as a purple solid. LCMS [M + H]+calcd 680.8; observed 680.6

[0528] Procedure for preparation of compound 3

[0529] HATU (2.48 g, 6.53 mmol) and DIEA (1.69 g, 13.1 mmol) were added slowly to a solution of compound 2 (1.30 g, 2.97 mmol) in DMF (15 mL). The mixture was stirred for 10 min before adding compound 2A (1.25 g, 6.24 mmol). LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition). Compound 3 (1.60 g, 55.6% yield) was obtained as a red oil. LCMS [M + H]+calcd 804.9; observed 804.7. LCMS [M + Na]+calcd 826.9; observed 826.5

[0530] Procedure for preparation of Compound 4

[0531] To a solution of compound 3 (1.60 g, 1.99 mmol) in DCM (20 mL) was added TFA (7.60 g, 67.6 mmol). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 3 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove DCM and give a residue. Compound 4 (2.10 g, crude) was obtained as a red oil. LCMS [M + H]+ ealed 704.8; observed 704.5

[0532] Procedure for preparation of Compound 5

[0533] 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) was added DIEA (773 mg, 5.98 mmol). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 4 was consumed completely and one main peak with desired mass was detected. The reaction mixture was quenched by addition 1 N HC1 (50 mL) at 0 °C, and then diluted with DCM (20 mL) and extracted with DCM (30 mL * 3). The combined organic layers were washed with Brine (30 mL), dried over NazSCL, filtered and concentrated under reduced pressure to give a residue. Compound 5 (1.70 g, crude) was obtained as a red oil. LCMS [M + H]+ ealed 804.9; observed 804.6

[0534] Procedure for preparation of Compound 6

[0535] To a solution of compound 5 (1.70 g, 2.11 mmol) in DCM (20 mL) was added EDCI (810 mg, 4.23 mmol) and 1 -hydroxypyrrolidine-2, 5-dione (730 mg, 6.34 mmol). The mixture was stirred at 25 °C for 2 hr. LC-MS showed compound 5 was consumed completely and one main peak with desired mass was detected. The reaction mixture was added into 1 N HC1 (100 mL). The mixture was extracted with DCM (150 mL * 3). The combined organic layers were washed with NaHCOj (200 mL), dried over NazSO i. filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition). Compound 6 (2.20 g, crude) was obtained as a red solid. LCMS [M + H]+ calcd 902.0; observed 901.6

[0536] Procedure for preparation of Target 2

[0537] To a solution of compound 6 (700 mg, 777 pmol) in DMF (7 mL) was added DIEA (301 mg, 2.33 mmol) and compound 1 (611 mg, 932 pmol). The mixture was stirred at 25 °C for 3 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition). Target 2 (220 mg, 19.6% yield) was obtained as a red solid. LCMS [M + H]+ calcd 1442.6; observed 1442.9

[0538] Procedure for preparation of Target 4

[0539] To a solution of compound 6 (700 mg, 777 pmol) in DMF (7 mL) was added DIEA (301 mg, 2.33 mmol) and compound 7 (436 mg, 932 pmol). The mixture was stirred at 25 °C for 3 hr. LC-MS showed compound 6 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition). Target 4 (210 mg, 21.5% yield) was obtained as a red solid. LCMS [M + H]+ calcd 1254.5; observed 1253.3Example 3: Synthesis of Compounds 111, 253, 254, 257, and 259

[0540] Procedure for preparation of compound 2

[0541] To a solution of compound 1 (150 mg, 745 pmol), compound 1A (170 mg, 1.49 mmol) in DMF (2.0 mL) was added DIEA (193 mg, 1.49 mmol, 260 pL). The mixture was stirred at 25 °C for 2 hrs. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give compound 2 (164 mg, 520 pmol, 69.8% yield) as a red solid. LCMS [M + H]+ calcd 316.3; observed 316.1

[0542] Procedure for preparation of compound 3

[0543] To a solution of compound 2 (100 mg, 317 pmol), HOSu (54.8 mg, 476 pmol) in DMF (1.0 mL) was added DCC (131 mg, 634 pmol, 128 pL) at 0 °C. The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered, filtrate was added to isopropyl ether (10 mL x 2), red solid formed, filtered and dried to give compound 3 (90.0 mg, 212 pmol, 66.7% yield, 97.0% purity) as a red solid and used into the next step without further purification. LCMS [M + H]+ calcd 413.4; observed 413.1

[0544] Procedure for preparation of compound 4Peptide Synthesis:

[0545] The peptide was synthesized using standard Fmoc chemistry.

[0546] Add DMF (200 mL) to the vessel containing Rink amide MBHA Resin (5.00 mmol, 8.50 g, sub: 0.60 mmol / g) and swell for 2 hrs.

[0547] Drain and then DMF wash 30 secs with 3 times.

[0548] Add 20% piperidine / DMF and mix for 30 mins.

[0549] Drain and then DMF wash 30 secs with 5 times.

[0550] Add Fmoc-amino acid solution and mix 30 secs, then add coupling regents, N? bubbling for about 1 hr.

[0551] Repeat step 2 to 5 for next amino acid coupling.

[0552] 20% piperidine in DMF was used for Fmoc deprotection for 30 mins. The coupling reaction was monitored by ninhydrin test, and the resin was washed with DMF for 5 times.

[0553] Peptide Cleavage and Purification:

[0554] Add cleavage buffer (90% TFA / 2.5% TIS 72.5% H2O 75.0% DTT) to the flask containing the side chain protected peptide at room temperature and stir for 2 hrs.

[0555] The peptide is precipitated with cold isopropyl ether and centrifuged (3 mins at 3000 rpms).

[0556] Isopropyl ether washes two additional times.

[0557] Dry the crude peptide under vacuum to give compound 4 (12.0 g, crude) as a white solid.

[0558] LCMS [M / 3]+ calcd 878.0: observed 878.6; LCMS [M / 2]+ calcd 1317.0; observed 1317.5

[0559] Procedure for preparation of compound 5L0560J Crude peptide compound 4 (12.0 g, 4.56 mmol) was dissolved in 20% McCN / HiO (5000 mL), then to the stirred solution of the peptide was added TATA (1.48 g, 5.92 mmol) at 25 °C over 30 mins. After addition, the mixture was stirred at 25 °C for 30 mins, and then NH4HCO3 was added for pH to 8. The resulting mixture was stirred at 25 °C for 16 hrs. LCMS showed the reaction was complete. The combined 5 batches was purified by Prep-HPLC (A: 0.1% TFA in H2O, B: MeCN) and re-purified byPrep-HPLC (A: 0.1% TFA in H2O, B: MeCN) to give compound 5 (1.35 g, 98.6% purity) as a white solid. LCMS [M / 3 + H]+ calcd 962.1; observed 962.0; LCMS [M / 2 + H]+ calcd 1442.6; observed 1442.3

[0561] Purification conditions:

[0562] Procedure for preparation of nectin-4 peptide, Compound 111

[0563] To a solution of compound 5 (400 mg, 139 pmol) in DMF (4.0 mL) was added DIEA (53.8 mg, 416 pmol, 72.5 pL) and compound 3 (59.0 mg, 139 pmol). The mixture was stirred at 25 °C for 1 hr.LC-MS showed compound 5 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (A: 0.1% TFA in H2O, B: MeCN) to give Compound 111 (260 mg, 81.8 jtmol, 57.9% yield, 96.9% purity, TFA salt) as a pink solid. Then the pink solid was purified by prep-HPLC (A: 0.05 % HC1 in H2O, B: MeCN) to give Compound 111 (208 mg, 65.4 pmol, 46.3% yield, 98.1% purity, HC1 salt) as a pink solid. LCMS [M / 3 + H]+calcd 1061.2; observed 1061.0

[0564] Note: TFA condition: The reaction mixture was dissolved in DMF, purified by prep-HPLC (Gilson GX-281; Gemini® 250*30 mm, C18, 5 um, 110A; Eluent of 28%-48% of solution (A: 0.1% TFA in H2O, B: MeCN) for 40 mins at room temperature; observed under wavelength (220 / 254 nm), flow rate was 20 mL / Min, retention time was 23 mins).

[0565] HC1 condition: The TFA salt was dissolved in ACN / H2O, eluent of 10% of solution (A: 0.5 % NH4CI in H2O, B: MeCN) for 20 mins at room temperature, then eluent of 10% of solution (A: 0.05 % HC1 in H2O, B: MeCN) for 10 mins at room temperature, followed by prep-HPLC (AUNO LC2000; Gemini-C18, 150*30 mm, 5 um, 110A; Eluent of 20%-40% of solution (A: 0.05 % HC1 in H2O, B: MeCN) for 40 mins at room temperature; observed under wavelength (220 / 254 nm), flow rate was 20 mL / Min, retention time was 27 mins).

[0566] Purification conditions:Procedure for preparation of nectin-4 peptide-TCO, Compound 253General procedure for preparation of compound 3

[0567] To a solution of compound 2 (1.62 g, 4.97 mmol, 5.00 equiv.) in DMF (15.0 mL) was added compound 1 (200 mg, 994 pmol, 1.00 equiv.) and DIEA (257 mg, 1.99 mmol, 346 pL, 2.00 equiv.). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give compound 3 (210 mg, 392 pmol, 39.5% yield, 98.3% purity, TFA) as a pink solid which was confirmed via LCMS and HPLC. LCMS: Rt= 0.816 min, MS cal.: 412.41, MS observed: [M+H]+= 413.1. HPLC: Rt = 7.945 min, purity: 98.3%.General procedure for preparation of compound 4A

[0568] Peptide Synthesis: The peptide was synthesized using standard Fmoc chemistry.) Resin preparation: Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol / g) and swell for 2 hrs. ) Deprotection: 20% piperidine in DMF (30.0 mL) was added and agitated the resin with Ni at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin. ) Coupling: A solution of HBTU (2.85 equiv.) and Fmoc-Cys(Trt)-OH (3.00 equiv.) in DMF (3.00 mL) was added to the resin, then the DIEA (6.00 equiv.) was added, the mixture was agitated with N2at 20 °C for 30 mins. The resin was washed with DMF (20.0 mL x 5). ) Repeat above step 2 to 3 for the coupling of following amino acids: (1-25)

[0569] Peptide Cleavage and Purification:1) Add cleavage solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) to the flask containing resin at 22 °C and stirred for 2.5 hrs.2) Precipitated the peptide with cold isopropyl ether (500 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (150 mL x 2). Dry the crude peptide under vacuum 2 hrs to get the crude peptide compound 4A (1.30 g), the crude peptide was confirmed via LCMS.

[0570] LCMS: Rt= 0.803 min, MS cal.: 2633.08, MS observed: [M / 3+H]+= 878.8, [M / 2+H]+= 1317.5.General procedure for preparation of compound 5A

[0571] To a solution of crude linear peptide compound 4A (1.30 g, 1.00 equiv.) in H2O (300 mL) and ACN (200 mL) was added l,3,5-triacryloylhexahtdro-l,3,5-triazine (0.15 g, 1.20 equiv.) and NH4HCO3 in H2O (1.00 M) slowly to adjust the reaction mixture pH to 8 ~ 9. The mixture was stirred at 20 °C for 4.0 hrs. LCMS showed one main peak with desired mass was detected. Then HO in water (1.00 M) was added until the pH to 6 ~ 7. The mixture was lyophilized to give the crude bicyclic peptide. The crude bicyclic peptide was purified by prep-HPLC (condition A: 0.075% TFA in H2O, B: ACN) to give compound 5A (240 mg, 16.6% yield, 84.5% purity, TFA salt) as a white solid which was confirmed via LCMS and HPLC.

[0572] LCMS: Rt= 0.786 min, MS cal.: 2882.35, MS observed: [M / 3+H]+= 961.9, [M / 2+H]+= 1442.2.

[0573] HPLC: Rt= 7.554 min, purity: 84.5%.Purification conditions:General procedure for preparation of Target 253

[0574] To a solution of compound 6A (170 mg, 59.0 prnol, 1.00 equiv.) in DMF (1.80 mL) was added DIEA (22.9 mg, 177 jrmol, 30.8 pL, 3.00 equiv.) and compound 3 (34.2 mg, 64.9 jrmol, 1.1 equiv., TFA). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 6A was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified byprep-HPLC (TFA condition and AcOH condition) to give Target 253 (54.0 mg, 16.1 pmol, 27.2% yield, 96.3% purity, HO AC) as a pink solid which was confirmed via LCMS and HPLC.

[0575] LCMS: Rt= 1.363 min, MS cal.: 3179.67, MS observed: [M / 3+H]+= 1060.5, [M+H]+= 1590.6.

[0576] HPLC: Rt= 5.914 min, purity: 96.3%.Purification conditions:Procedure for preparation of nectin-4 peptide-TCO, Compound 254General procedure for preparation of compound 4B

[0577] Peptide Synthesis: The peptide was synthesized using standard Fmoc chemistry.1) Resin preparation: Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol / g) and swell for 2 hrs.2) Deprotection: 20% piperidine in DMF (30.0 mL) was added and agitated the resin with N2 at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin.3) Coupling: A solution of HBTU (2.85 equiv.) and Fmoc-Cys(Trt)-OH (3.00 equiv.) in DMF (3.00 mL) was added to the resin, then the DIEA (6.00 equiv.) was added, the mixture was agitated with N2 at 20 °C for 30 mins. The resin was washed with DMF (20.0 mL x 5).4) Repeat above step 2 to 3 for the coupling of following amino acids: (1-25)[05781 Peptide Cleavage and Purification:1) Add cleavage solution (150 mL, 90.0% TFA / 5.00% DTT72.50% TIS / 2.50% H2O) to the flask containing resin at 22 °C and stirred for 2.5 hrs.2) Precipitated the peptide with cold isopropyl ether (500 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (150 mL x 2). Dry the crude peptide under vacuum 2 hrs to get the crude peptide compound 4B (1.30 g), the crude peptide was confirmed via LCMS.

[0579] LCMS: Rt= 0.760 min, MS cal.: 2697.18, MS observed: [M / 4+H]+= 675.4, [M / 3+H]+= 900.1 , [M / 2+H]+= 1349.6.General procedure for preparation of compound 5B[0580J To a solution of crude linear peptide compound 4B (1.30 g, 1.00 equiv.) in H2O (300 mL) and ACN (200 mL) was added l,3,5-triacryloylhexahtdro-l,3,5-triazine (0.15 g, 1.20 equiv.) and NH4HCO3 in H2O (1.00 M) slowly to adjust the reaction mixture pH to 8 ~ 9. The mixture was stirred at 20 °C for 4.0 hrs. LCMS showed one main peak with desired mass was detected. Then HO in water (1.00 M) wasadded until the pH = 6 ~ 7. The mixture was lyophilized to give the crude bicyclic peptide. The crude bicyclic peptide was purified by prep-HPLC (condition A: 0.075% TFA in H2O, B: ACN) to give compound 5B (237 mg, 16.1% yield, 99.2% purity, TFA salt) as a white solid which was confirmed via LCMS and HPLC.

[0581] LCMS: Rt= 0.745 min, MS cal.: 2946.45, MS observed: [M / 4+H]+= 737.7, [M / 3+H]+= 983.3, [M / 2+H]+= 1474.2.

[0582] HPLC: Rt= 6.786 min, purity: 99.2%.Purification conditions:General procedure for preparation of Target 254

[0583] To a solution of compound 5B (160 mg, 54.3 pmol, 1.00 equiv.) in DMF (1.60 mL) was added DIEA (21.1 mg, 163 pmol, 28.4 pL, 3.00 equiv.) and compound 3 (31.5 mg, 59.7 |imol, 1.10 equiv., TFA). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 5B was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give Target 254 (43.0 mg, 12.8 pmol, 23.6% yield, 96.6% purity, HOAC) as a pink solid which was confirmed via LCMS and HPLC.

[0584] LCMS: Rt = 1.282 min, MS cal.: 3243.76, MS observed: [M / 4+H]+= 811.8, [M / 3+H]+= 1082.2, [M / 2+H]+= 1622.8.

[0585] HPLC: Rt= 4.318 min, purity: 96.6%.Purification conditions:Procedure for preparation of nectin-4 peptide-TCO, Compound 257General procedure for preparation of compound 4C

[0586] Peptide Synthesis: The peptide was synthesized using standard Fmoc chemistry.1) Resin preparation: Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol / g) and swell for 2 hrs.2) Deprotection: 20% piperidine in DMF (30.0 mL) was added and agitated the resin with Ni at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin.3) Coupling: A solution of HBTU (2.85 equiv.) and Fmoc-Cys(Trt)-OH (3.00 equiv.) in DMF (3.00 mL) was added to the resin, then the DIEA (6.00 equiv.) was added, the mixture was agitated with N? at 20 °C for 30 mins. The resin was washed with DMF (20.0 mL x 5).4) Repeat above step 2 to 3 for the coupling of following amino acids: (1-25)[05871 Peptide Cleavage and Purification:1) Add cleavage solution (150 mL, 90.0% TFA / 5.00% DTT / 2.50% TIS / 2.50% H2O) to the flask containing resin at 22 °C and stirred for 2.5 hrs.2) Precipitated the peptide with cold isopropyl ether (500 niLi. Filter and collect the filter cake. The filter cake was washed with isopropyl ether (150 mL x 2). Dry the crude peptide under vacuum 2 hrs to get the crude peptide compound 4C (1.30 g), the crude peptide was confirmed via LCMS.

[0588] LCMS: Rt= 0.774 min, MS cal.: 2693.17, MS observed: [M / 4+H? = 674.5, [M / 3+H]+=898.8, [M / 2+H]+= 1347.5.General procedure for preparation of compound 5C

[0589] To a solution of crude linear peptide compound 4C (1.30 g, 1.00 equiv.) in H2O (300 mL) and ACN (200 mL) was added l,3,5-triacryloylhexahtdro-l,3,5-triazine (0.15 g, 1.20 equiv.) and NH4HCO3 in H2O (1.00 M) slowly to adjust the reaction mixture pH to 8 ~ 9. The mixture was stirred at 20 °C for 4.0 hrs. LCMS showed one main peak with desired mass was detected. Then HO in water (1.00 M) was added until the pH = 6 ~ 7. The mixture was lyophilized to give the crude bicyclic peptide. The crude bicyclic peptide was purified by prep-HPLC (condition A: 0.075% TFA in H2O, B: ACN) to give compound 5C (272 mg, 18.5% yield, 99.1% purity, TFA salt) as a white solid which was confirmed via LCMS and HPLC. LCMS: Rt= 0.774 min, MS cal.: 2942.44, MS observed: [M / 4+H]+= 736.6, [M / 3+H]+= 981.8, [M / 3+H]+= 1472.2. HPLC: Rt= 7.041 min, purity: 99.1%.Purification conditions:General procedure for preparation of Target 257

[0590] To a solution of compound 5C (150 mg, 51.0 pmol, 1.00 equiv.) in DMF (1.50 mL) was added DIEA (19.8 mg, 153 pmol, 26.6 pL, 3.00 equiv.) and compound 3 (32.2 mg, 61.2 pmol, 1.20 equiv., TFA). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 5C was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give Target 257 (40.0 mg, 11.8 pmol, 23.1% yield, 95.4% purity, HO AC) as a pink solid which was confirmed via LCMS and HPLC. LCMS: Rt= 1.300 min, MS cal.: 3243.76, MS observed: [M / 4+H]+= 810.9, [M / 3+H]+= 1080.6, [M / 2+H]+= 1620.6. HPLC: Rt= 4.822 min, purity: 95.4%.Purification conditions:Procedure for preparation of nectin-4 peptide-TCO, Compound 2595D

[0591] Peptide Synthesis: The peptide was synthesized using standard Fmoc chemistry.1) Resin preparation: Add DMF to the vessel containing Rink Amide MBHA Resin (0.50 mmol, 1.00 equiv., sub: 0.33 mmol / g) and swell for 2 hrs.2) Deprotection: 20% piperidine in DMF (30.0 mL) was added and agitated the resin with N2 at 22 °C for 30 mins. The resin was washed with DMF (30.0 mL x 5) and filtered to get the resin.3) Coupling: A solution of HBTU (2.85 equiv.) and Fmoc-Cys(Trt)-OH (3.00 equiv.) in DMF (3.00 mL) was added to the resin, then the DIEA (6.00 equiv.) was added, the mixture was agitated with N2 at 20 °C for 30 mins. The resin was washed with DMF (20.0 mL x 5).4) Repeat above step 2 to 3 for the coupling of following amino acids: (1-28)Peptide Cleavage and Purification:1) Add cleavage solution (150 mL, 90.0% TFA / 5.00% DTT72.50% TIS / 2.50% H2O) to the flask containing resin at 22 °C and stirred for 2.5 hrs.2) Precipitated the peptide with cold isopropyl ether (500 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (150 mL x 2). Dry the crude peptide under vacuum 2 hrs to get the crude peptide compound 4D (1.50 g), the crude peptide was confirmed via LCMS.

[0592] LCMS: Rt= 0.857 min, MS cal.: 2869.35, MS observed: [M / 2+H? = 1435.2.General procedure for preparation of compound 5D

[0593] To a solution of crude linear peptide compound 4D (1.50 g, 1.00 equiv.) in H2O (300 mL) and ACN (200 mL) was added l,3,5-triacryloylhexahtdro-l,3,5-triazine (0.15 g, 1.20 equiv.) and NH4HCO3 in H2O (1.00 M) slowly to adjust the reaction mixture pH to 8 ~ 9. The mixture was stirred at 20 °C for 4.0 hrs. LCMS showed one main peak with desired mass was detected. Then HC1 in water (1.00 M) was added until the pH = 6 ~ 7. The mixture was lyophilized to give the crude bicyclic peptide. The crude bicyclic peptide was purified by prep-HPLC (condition A: 0.075% TFA in H2O, B: ACN) to give compound 5D (200 mg, 14.2% yield, 99.8% purity, TFA salt) as a white solid which was confirmed via HPLC. LCMS: Rt= 0.844 min, MS cal.: 3118.62, MS observed: [M / 3+H? = 1040.4. HPLC: R, = 8.768 min, purity: 99.8%.Purification conditions:General procedure for preparation of compound 6D

[0594] To a solution of compound 5D (170 mg, 54.5 pmol, 1.00 equiv.) in DMF (2.00 mL) was added DIEA (21.1 mg, 164 pmol, 28.5 pL, 3.00 equiv.) and compound 3 (28.7 mg, 54.5 pmol, 1.00 equiv., TFA). The mixture was stirred at 25 °C for 1 hr. LC-MS showed compound 5D was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified byprep-HPLC (TFA condition) to give compound 6D (111 mg, 29.8 prnol, 54.8% yield, 91.9% purity) as a pink solid which was confirmed via LCMS and HPLC. LCMS: Rt= 0.915 min, MS cal.: 3415.94, MS observed: [M / 4+H? = 854.8, [M / 3+H? = 1139.4, [M / 2+H]+= 1708.6. HPLC: Rt = 9.397 min, purity: 91.9%.General procedure for preparation of Target 259

[0595] To a solution of compound 6D (120 mg, 35.2 pmol, 1.00 equiv.) in DMF (1.00 mL) was added EhN (182 mg, 1.80 mmol, 0.25 mL, 51.1 equiv.). The mixture was stirred at 25 °C for 5 hrs. LC-MS showed compound 6D was consumed completely and one main peak with desired mass was detected. The reaction mixture was added to isopropyl ether (12.0 mL), pink solid formed, filtered and dried to give a residue. The residue was purified by prep-HPLC (TFA condition and AcOH condition) to give Target 259 (42.0 mg, 12.6 jrmol, 35.9% yield, 96.0% purity) as a pink solid which was confirmed via LCMS. LCMS: Rt= 1.321 min, MS cal.: 3193.70, MS observed: [M / 4+H]+= 799.4, [M / 3+H? = 1065.4, [M / 2+H]+= 1597.1. HPLC: Rt= 5.051 min, purity: 96.0%.Purification conditions:Example 4: HCC1954 Xenograft Model

[0596] Animal studies are conducted in accordance with IACUC protocols following the guidance of the AAALAC. Female Balb / c nude mice are implanted with HCC1954 grown in exponential phase in right flank (5e6 cells + Matrigel) in 0.2 mL PBS. The animals are randomized when the tumor volume reached -200 mm3. Animals are dosed IV with saline (days 1-4), the tetrazine -based targeting agent (day 0, 5 mg / kg) + saline (days 1-4), or tetrazine-based targeting agent (day 0, 5 mg / kg) + TCO prodrug (days 1, 2, 3 & 4, 120 mg / kg).

[0597] Tumor volume is measured twice weekly in two dimensions using a caliper, and the volume is expressed in mm3using the formula: V - 0.5 a x b2where a and b are the long and short diameters of the tumor, respectively. A decrease in tumor growth with administration of the prodrug shows efficacy of the system described therein.

[0598] Suitable prodrugs for use in the methods disclosed herein can be prepared and administered as described in W02020 / 077140, WO2018 / 187740, WO2017 / 044983, WO2015 / 139025, and WO2014 / 205126.Example 5: General procedure for preparation of Compound ACompound A

[0599] To a solution of MMAE (1.40 g, 1.95 mmol) and DIEA (690 mg, 5.34 mmol) in DMF (4.00 mL) was added compound 2 (800 mg, 1.79 mmol) in DMF (4.00 mL) at 0 °C, the mixture was stirred at 25 °C for 16 hrs. Then HOBt (480 mg, 3.56 mmol) in DMF (0.50 mL) was added to the above reaction mixture at 0 °C, after the reaction mixture was stirred at 25 °C for 1.0 hr, the reaction mixture was cooled to 0 °C, and TBAF ( 1 M in THF, 4.45 mL) was added. After the mixture was stirred for 2.0 hrs at 25 °C, another batch of TBAF (1 M in THF, 4.45 mL) was added at 0 °C, the reaction mixture was continued to stirring for 12.0 hrs at 25 °C. LC-MS showed one main peak was desired mass. The resulting reaction mixture was purified by Prep- HPLC (column: Welch XB-C18 7 pm 110 A 250*50 mm; mobile phase: [water (0.1% TFA)-ACN]; B%: 50-70%-40 min. number of injections: 2, Retention time: 37 min, flow rate: 60 mL / min) to give Compound A (450 mg, 99.0% purity; 64.6 mg, 99.2%, 31.2% yield).

[0600] LCMS (m / z): 928.6 [M+H]+

[0601] 1HNMR: (400 MHz, DMSO-d6): δ 8.46 - 8.28 (m, 1H), 8.03 - 7.84 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.35 - 7.23 (m, 4H), 7.21 - 7.13 (m, 1H), 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). Example 6: General procedure for preparation of Compound B O O NH2O NO2O HCl O O Cl O O H

[0602] To a solutiono compoun ( . g, 5. mmo) n ( m ) was added DIEA (2.10 g, 16.3 mmol), EDCI (2.08 g, 10.9 mmol) and DMAP (1.33 g, 10.9 mmol) and compound 3 (1.61 g, 8.14 mmol). The mixture was stirred at 25 °C for 16 hrs. TLC indicated compound 2 was consumed completely and one new spot formed. The reaction mixture was partitioned between DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with sat. citric acid aq. (3 mL) and brine (20 mL), then dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Petroleum ether / Ethyl acetate = 3 / 1 to 1 / 1) to give compound 4 (700 mg, 39.4% yield).

[0603] 1HNMR (400MHz, CDCl3): δ ppm 1.12 (s, 3 H), 1.60 (dd, J =15.45, 6.19 Hz, 1 H), 1.79 - 1.87 (m, 2 H), 1.92 (br d, J = 5.88 Hz, 1 H), 1.95 (s, 1 H), 1.98 - 2.00 (m, 1 H), 2.02 (br d, J = 4.13 Hz, 1 H),2.26 (dd, J = 11.63, 3.88 Hz, 1 H), 2.30 - 2.36 (m, 1 H), 2.77 - 2.89 (m, 1 H), 2.88 - 2.88 (m, 1 H), 3.00 (dd, J = 16.95, 4.57 Hz, 1 H), 3.70 (s, 4 H), 3.75 (s, 3 H), 4.80 (dt, J = 8.00, 4.50 Hz, 1 H), 5.66 (dd, J = 16.63, 2.38 Hz, 1 H), 6.02 - 6.12 (m, 1 H), 6.54 (br d, J = 7.88 Hz, 2 H). General procedure for preparation of Compound 6

[0604] T(846 mg, 10.7 mmol) and compound 5 (1.72 g, 8.55 mmol) in DCM (5 mL). The mixture was stirred at 25 °C for 1 hrs. TLC indicated compound 4 was consumed completely and one new spot formed. The reaction mixture was partitioned between DCM (20 mL) and H2O (10 mL). The organic phase was separated, washed with sat. citric acid aq. (3 mL) and brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Petroleum ether / Ethyl acetate = 3 / 1 to 1 / 1) to give compound 6 (490 mg, 46.5% yield).

[0605] 1HNMR (400MHz, CDCl3): δ ppm 1.17 (s, 3 H), 1.55 - 1.61 (m, 1 H), 1.58 (br s, 1 H), 1.76 (dd, J = 14.76, 6.25 Hz, 1 H), 1.87 - 2.03 (m, 3 H), 2.06 - 2.15 (m, 1 H), 2.19 - 2.41 (m, 3 H), 2.82 (dd, J = 17.13, 4.50 Hz, 1 H), 3.03 (dd, J = 17.07, 4.44 Hz, 1 H), 3.72 (s, 3 H), 3.77 (s, 3 H), 4.78 - 4.86 (m, 1 H), 5.67 (dd, J = 16.70, 2.44 Hz, 1 H), 6.03 - 6.14 (m, 1 H), 6.58 (br d, J = 7.88 Hz, 1 H), 7.40 - 7.45 (m, 2 H), 8.27 - 8.33 (m, 2 H). General procedure for preparation of compound 7

[0606] To a solution of compound 6 (490 mg, 995 µmol) and MMAE (714 mg, 995 µmol) in DMF (4 mL) was added DIEA (64.3 mg, 497 µmol) and HOBt (202 mg, 1.49 mmol). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed compound 6 was consumed completely and one main peak withdesired mass was detected. The residue was purified by prep-HPLC (0.1% TFA conditions) to give compound 7 (500 mg, 46.9% yield).

[0607] 1HNMR (400MHz, CDCl3): δ ppm 0.84 (br d, J = 6.75 Hz, 4 H), 0.89 (br d, J = 4.50 Hz, 5 H), 0.92 (br d, J = 6.63 Hz, 4 H), 0.98 (br d, J = 6.25 Hz, 3 H), 1.04 (br d, J = 6.88 Hz, 3 H), 1.16 (s, 3 H), 1.25 - 1.27 (m, 3 H), 1.59 - 1.74 (m, 3 H), 1.88 (br d, J = 9.38 Hz, 4 H), 2.07 (br d, J = 8.38 Hz, 5 H), 2.27 (br s, 4 H), 2.36 - 2.43 (m, 2 H), 2.45 - 2.53 (m, 1 H), 2.89 (br s, 6 H), 2.95 - 3.01 (m, 4 H), 3.04 (br s, 2 H), 3.29 - 3.34 (m, 3 H), 3.36 - 3.47 (m, 5 H), 3.67 - 3.73 (m, 4 H), 3.76 (s, 3 H), 3.82 - 3.89 (m, 1 H), 4.05 - 4.19 (m, 3 H), 4.28 (br s, 1 H), 4.63 - 4.86 (m, 3 H), 4.96 (d, J = 2.50 Hz, 1 H), 5.24 (br s, 1 H), 5.63 (br d, J = 18.14 Hz, 1 H), 5.82 (br s, 1 H), 6.53 - 6.74 (m, 3 H), 7.30 - 7.41 (m, 5 H). General procedure for preparation of Compound B O H O N O O H O OH LiOH

[0608] To a solution of compound 7 (500 mg, 467 µmol) in MeOH (5 mL) was added LiOH·H2O (196 mg, 4.67 mmol) in H2O (2 mL). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed compound 7 was consumed completely and one main peak with desired mass was detected. The residue was adjusted pH ~ 2 with sat. citric acid aq.,then purified by prep-HPLC (0.1% TFA condition) to give Compound B (265 mg 53.4% yield).

[0609] 1HNMR (400MHz, CDCl3): δ ppm 0.80 - 1.04 (m, 25 H), 1.09 (s, 3 H), 1.24 (d, J = 6.88 Hz, 3 H), 1.69 - 1.82 (m, 2 H), 1.88 - 1.94 (m, 3 H), 2.02 - 2.11 (m, 4 H), 2.16 (br d, J = 18.64 Hz, 1 H), 2.11 - 2.24 (m, 2 H), 2.32 (br d, J = 5.25 Hz, 2 H), 2.40 - 2.45 (m, 1 H), 2.52 (br d, J = 5.50 Hz, 2 H), 2.81 (br dd, J = 14.01, 4.88 Hz, 1 H), 2.94 - 3.04 (m, 2 H), 3.08 (s, 2 H), 3.14 - 3.27 (m, 6 H), 3.33 (s, 1 H), 3.39 (s, 3 H), 3.48 - 3.57 (m, 2 H), 3.94 (br d, J = 1.25 Hz, 1 H), 4.05 - 4.18 (m, 4 H), 4.30 (br dd, J = 6.19, 4.82 Hz, 2 H), 4.54 - 4.67 (m, 4 H), 4.91 (br d, J = 2.00 Hz, 2 H), 5.30 (br s, 1 H), 5.64 - 5.73 (m, 1 H), 5.79 - 5.89 (m, 1 H), 6.61 (br d, J = 7.38 Hz, 1 H), 7.30 - 7.42 (m, 5 H), 7.55 - 7.64 (m, 1 H).Example 7: Alternative route to Compound B and Synthesis of Compound C. `General procedure for preparation of compound 2

[0610] To a solution of compound 1 (20.0 g, 83.2 mmol) in MeOH (80 mL) was added KOH (8.19 g, 124 mmol) in H2O (80 mL). The mixture was stirred at 25 °C for 24 hrs. The reaction was monitored by TLC (compound 1, PE / EtOAc = 5 / 1, Rf = 0.5). The reaction mixture was extracted with MTBE (3 × 400 mL). The combined organic layers were washed with water (100 mL), dried with Na2SO4, filtered and concentrated in vacuo to provide the undesired ester. The aqueous layer was acidified with 1 M HCl until pH = 4 while cooling in an ice-water bath (T < 7 °C). The aqueous layer was extracted with MTBE (3 × 400 mL). The combined MTBE layers were was dried with Na2SO4, filtered and concentrated invacuo to provide the compound 2 (5.50 g, 35.9% yield). The crude product was used into the next step without further purification.

[0611] 1H NMR: (400 MHz, DMSO-d6): δ ppm 11.9 (br s, 1 H), 5.81 - 5.94 (m, 1 H), 5.58 (dd, J = 16.45, 2.31 Hz, 1 H), 4.65 (br s, 1 H), 4.24 (br s, 1 H), 2.04 - 2.24 (m, 2 H), 1.87 - 2.03 (m, 1 H), 1.61 - 1.86 (m, 4 H), 1.36 - 1.46 (m, 1 H), 0.97 (s, 3 H). General procedure for preparation of compound 3

[0612] To a solded DIEA (39.3 g, 304 mmol) and DSC (47.8 g, 186.4 mmol). The mixture was stirred at 40 °C for 14 hrs. The completion of the reaction was confirmed by TLC (compound 2, DCM / MeOH = 10 / 1, Rf = 0.5). The reaction mixture was poured in water (400 mL), then the temperature rose from 20 °C to 27 °C. After 15 mins, the mixture was cooled to 17 °C in an ice-water bath and stirred for 15 mins. The solid was filtered, washed with water (3 × 20 mL) and was dried under vacuum at 35 °C for 4 hrs to give crude compound 3 (9.60 g). Acetonitrile (20 mL) was added to the crude and the mixture was heated at 40 °C for 1 hrs using mechanical stirring. The heating was stopped and the mixture was cooled to 8 °C in an ice-water bath for 15 mins. The solid was filtered, washed with acetonitrile (2 ×10 mL) and dried under vacuum at 35 °C for 3 hrs to give compound 3 (6.65 g, 36.3% yield).

[0613] 1H NMR: (400 MHz, CDCl3): δ 6.03 - 6.14 (m, 1 H), 5.60 - 5.67 (m, 1 H), 5.29 (br s, 1 H), 2.80 - 2.88 (m, 8 H), 2.25 - 2.47 (m, 4 H), 1.94 - 2.18 (m, 4 H), 1.29 (s, 3 H). General procedure for preparation of compound 4

[0006] To a so ut on o compound 3 ( 00 mg, 0. mmo) n DMF ( 0 mL) was added MM E ( 36 mg, 0.19 mmol) and DIEA (61.2 mg, 0.47 mmol). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% FA)-ACN) to give compound 4 (41.0 mg, 16.9% yield).

[0615] LCMS (m / z): 1025.6 (M+H)+.General procedure for preparation of Compound Bn DMF (10 mL) was added DIEA (378 mg, 2.93 mmol) and DMAP (119 mg, 0.97 mmol). The mixture was stirred at 25 °C for 12 hrs. LC-MS showed one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give Compound B (161 mg, 31.6% yield).

[0617] 1H NMR: (400 MHz, MeOD): δ 7.73 - 8.00 (m, 1 H), 7.20 - 7.39 (m, 4 H), 5.78 - 5.96 (m, 1 H), 5.73 (br s, 1 H), 5.20 - 5.28 (m, 1 H), 5.13 - 5.20 (m, 1 H), 4.49 - 4.74 (m, 3 H), 4.17 - 4.28 (m, 2 H), 4.04 - 4.10 (m, 1 H), 3.85 - 3.90 (m, 1 H), 3.50 - 3.80 (m, 2 H), 3.31 - 3.50 (m, 9 H), 3.28 - 3.30 (m, 3 H), 2.77 - 3.12 (m, 6 H), 2.44 - 2.58 (m, 2 H), 1.78 - 2.36 (m, 13 H), 1.56 - 1.72 (m, 2 H), 1.22 - 1.48 (m, 3 H), 1.08 - 1.23 (m, 9 H), 0.80 - 1.07 (m, 18 H).

[0618] LCMS (m / z): 1043.62 (M+H)+; 1065.61 (M+Na)+. General procedure for preparation of Compound C

[0619] 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) was added DIEA (242 mg, 1.87 mmol) and DMAP (76.3 mg, 0.62 mmol). The mixturewas stirred at 25 °C for 12 hrs. LC-MS showed compound 4 was consumed completely and one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% TFA)- ACN) to give Compound C (92.0 mg, 29.9% yield).

[0620] 1H NMR (400 MHz, MeOD): δ 7.86 - 8.00 (m, 1 H), 7.15 - 7.45 (m, 5 H), 5.67 - 5.98 (m, 2 H), 5.17 (br s, 1 H), 4.50 - 4.74 (m, 2 H), 4.03 - 4.29 (m, 3 H), 3.81 - 3.89 (m, 2 H), 3.51 - 3.77 (m, 2 H), 3.46 - 3.50 (m, 1 H), 3.33 - 3.45 (m, 5 H), 3.30 (br s, 4 H), 3.20 (dt, J = 11.57, 7.47 Hz, 1 H), 3.02 - 3.15 (m, 3 H), 2.90 - 3.01 (m, 1 H), 2.41 - 2.57 (m, 2 H), 1.65 - 2.39 (m, 15 H), 1.52 - 1.64 (m, 1 H), 1.26 - 1.51 (m, 2 H), 1.08 - 1.23 (m, 9 H), 0.82 - 1.07 (m, 18 H).

[0621] LCMS (m / z): 985.6 (M+H)+. Example 8: Synthesis of Compound D

[0622] To a solup ( g, , ) ( , . ) and NaHCO3(1 M, 1.38 L) was added (2, 5-dioxopyrrolidin-1-yl) 2, 2, 2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L). The mixture was stirred at 25 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was extracted with MTBE (5 L), then the aqueous phase was adjusted pH~4 with Sat. KHSO4 aq. and extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. To a solution of above crude in MeOH (2 L) was added SOCl2 (90.2 g, 758 mmol) and the mixture was stirred at 25 °C for 2 hrs. LC-MS showed reaction was completed and one main peak with desired mass was detected. The reaction mixture was adjusted pH~9-10 with Sat. NaHCO3aq., then extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give crude. The crude was precipitated by PE (10 Vol) to give compound 6 (190 g, 74.4% yield).

[0623] 1H NMR: (400 MHz, CDCl3): δ 3.25 (br s, 1 H) 3.85 (s, 3 H) 4.64 - 4.83 (m, 2 H) 5.30 (dd, J = 9.51, 1.13 Hz, 1 H) 5.92 (br d, J = 9.38 Hz, 1 H) 7.30 - 7.45 (m, 5 H).

[0624] LCMS (m / z): 391.9 / 393.9 (M+H)+. General procedure for preparation of compound 7

[0625] To amethylbenzenesulfonic acid pyridine (3.90 g, 15.4 mmol) and 4-methoxybenzaldehyde dimethyl acetal (121 g, 666 mmol). The mixture was stirred at 110 °C for 4 hrs. LC-MS showed one main peak with desired mass was detected. Then reaction mixture was allowed to cool to 25 °C, The reaction mixture was concentrated under reduced pressure to remove toluene. The residue was diluted with H2O (500 mL), then extracted with EtOAc (500 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 7 (285 g, crude) which was carried forward as is. General procedure for preparation of compound 8

[0626] To a solution of compound 7 (285 g, crude) in MeOH (2000 mL) was added KOH (42.5 g, 758 mmol) in H2O (1000 mL). The mixture was stirred at 25 °C for 1 hrs. LC-MS showed compound 7 was consumed completely and one main peak with 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 phase layers were diluted with sat. KHSO4 (1L) aq. extracted with EtOAc (5 L), the combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give crude. The crude was precipitated by PE (10 Vol) to give compound 8 (95.0 g, 34.3% yield).

[0627] 1H NMR (400 MHz, MeOD): δ 3.82 (s, 3 H) 4.41 - 4.47 (m, 1 H) 4.50 - 4.56 (m, 1 H) 4.60 (d, J = 4.88 Hz, 1 H) 5.47 (d, J = 4.75 Hz, 1 H) 6.46 (s, 1 H) 6.86 - 6.94 (m, 2 H) 7.34 - 7.46 (m, 7 H).

[0628] LCMS (m / z): 495.9 (M+Na)+.General procedure for preparation of 7-Troc-baccatin Ⅲ

[0629] ToMAP (625 mg, 5.11 mmol) and pyridine (14.2 g, 179 mmol) and 2,2,2-trichloroethyl carbonochloridate (15.2 g, 71.6 mmol). The mixture was stirred at 25 °C for 0.5 hrs. LC-MS showed baccatin Ⅲ was consumed completely and one main peak with desired mass was detected. The residue was diluted with water (300 mL) and extracted with DCM (300 mL) and washed with water (200 mL ) and brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 7-Troc-baccatin III (45.0 g, 34.3% yield).

[0630] LCMS (m / z): 761.5 / 763.5 (M+Na)+. General procedure for preparation of compound 9[ ] o a so ut on o 7- roc- accat n ( . g, . mmo) an compoun ( . g, . mmo) in DCM (1000 mL) was added DMAP (4.20 g, 34.1 mmol) and DCC (21.1 g, 102 mmol). The mixture was stirred at 0 °C for 1 hrs. LC-MS showed compound 8 was consumed completely and one main peak with desired mass was detected. The reaction mixture filtered. The crude was washed by sat. NH4Cl aq. (100 mL) and water (1000 mL) dried over Na2SO4, filtered and concentrated under reduced pressure to give Compound 9 (35.0 g, crude).

[0632] LCMS (m / z): 1240.0 / 1242.0 (M+Na)+.General procedure for preparation of compound 10methylbenzenesulfonic acid; hydrate (24.9 g, 131 mmol). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed ~50% compound 9 was remained and one main peak with desired mass was detected. The reaction mixture filtered, concentrated and the residue was purified by prep-HPLC (Water (0.1% TFA)-ACN). The elution was concentrated under reduced pressure to remove solvent, then, extracted with EtOAc (500 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give compound 10 (13.0 g, 17.9% yield).

[0634] LCMS (m / z): 1120.2 (M+Na)+. General procedure for preparation of compound 11

[0635] To a solution of compound 10 (13.0 g, 11.8 mmol) and DMAP (722 mg, 5.90 mmol) and 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 hrs. LC-MS showed compound 10 was consumed completely and one main peak with desired mass was detected. The reaction mixture was washed with sat. citric acid aq. (100 mL), sat. NaHCO3 aq. (100 mL) and water (200 mL), dried over NaSO4, filtered and concentrated under reduced pressure to give compound 11 (11.0 g, 77.3% yield).

[0636] LCMS (m / z): 1204.1 (M+H)+.General procedure for preparation of compound 12was added Zn dust (21.6 g, 331 mmol). The mixture was stirred at 25 °C for 1 hrs. LC-MS showed compound 11 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered and diluted with H2O (500 mL), then extracted with EtOAc (100 mL * 3). The combined organic layers were washed with sat. NaHCO3 aq. (200 mL) and brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a crude product. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give Compound 12 (5.0 g, 21% yield).

[0638] LCMS (m / z): 854.3 (M+H)+. General procedure for preparation of compound 13 O N O H O O[ ] o a so u o o co pou . g, . o , . g, . o a co pou d 3 (3.90 g, 8.80 mmol) in DMF (50 mL). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed ~50% compound 12 was remained and one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give compound 13 (505 mg, 7.4% yield).

[0640] LCMS (m / z): 1161.4 (M+H)+.General procedure for preparation of Compound D.7 mg, 0.78 mmol) and compound 13-1 (66.7 mg, 0.65 mmol) and DIEA (100 mg, 0.78 mmol). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed compound 13 was consumed completely and one main peak with desired mass was detected. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give Compound D (75.0 mg, 50.5% yield).

[0642] LCMS (m / z): 1148.5 (M)+. Example 9: General procedure for preparation of Compound E

[0643] To a solution of compound 13 (350 mg, 0.30 µmol) and DMAP (221 mg, 1.81 mmol) and compound 14 (249 mg, 0.39 mmol, HCl) in DMF (0.3 mL). The mixture was stirred at 25 °C for 16 hrs. LC-MS showed compound 13 was consumed completely and one main peak with desired mass wasdetected. The residue was purified by prep-HPLC (Water (0.1% TFA)-ACN) to give Compound E (205 mg, 41.3% yield).

[0644] LCMS (m / z): 1646.5 (M+H)+. Example 10: 3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine [c-2- cyano-5-aminomethyl-pyrimidine (1) in dry acetonitrile is added hydrazine and nickel (II) triflate. The reaction mixture is then heated overnight; the starting material is consumed by TLC. To the reaction mixture is added sodium nitrite (dissolved in water), followed by 1 M hydrochloric acid. 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 (3x), followed by brine (1x), and then is dried over sodium sulfate. The solution is filtered and the filtrate is concentrated under reduced pressure to yield the product, which can be carried forward without further purification.

[0646] 3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-tetrazine (3). To a solution 2 in dioxane is added hydrochloric acid (4 M in dioxane). The reaction mixture is then stirred at ambient temperature until the starting material is consumed. The product is isolated by filtration and the precipitate is washed with diethyl ether to yield the product, optionally as the HCl salt.

[0647] 6-(6-Methyl-1,2,4,5-tetrazin-3-yl)-3-pyridinemethanamine can also be utilized in the compounds and methods described herein, which compound can be prepared according to the art or purchased from a commercial source (e.g., Enamine US Inc., New Jersey, USA).Example 11: Val-Cit-PABC-dihydrotetrazineflask, 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 color changes from pink to colorless. The reaction mixture is concentrated under reduced pressure and the resulting residue is dried under vacuum, providing dihydrotetrazine 4, which can be used directly without further purification.

[0649] To a solution of dihydrotetrazine 4 is added a solution of nitrophenyl carbonate 5 in toluene. 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 yield compound 6.

[0650] 1-(N-Acyl-(Val-Cit)-PABC)-3-(5-aminomethyl-pyrimidine)-6-methyl-1,2,4,5-dihydrotetrazine (7). To a solution 6 in dioxane is added hydrochloric acid (4 M in dioxane). The reaction mixture is then stirred at ambient temperature until the starting material is consumed. The product is isolated by filtration and the precipitate is washed with diethyl ether to yield the product, optionally as the HCl salt. Example 12: Dihydrotetrazine (Target 5)

[0651] To a solution of N2H4.H2O (9.74 g, 190 mmol, 9.44 mL, 98% purity, 7.08 eq) in EtOH (35.0 mL) was added compound 1 (5.00 g, 26.9 mmol, 1.00 eq, HCl) and compound 2 (2.55 g, 26.9 mmol, 1.00 eq, HCl) at 20 °C. The mixture was stirred at 78 °C for 3 hrs. LCMS analysis of the reaction mixture showed compound 1 was consumed completely. To the reaction mixture was added H2O (100 mL) at 20 °C and then the resulting solution was concentrated under reduced pressure at 40 °C to remove EtOH. The reaction mixture was extracted with ethyl acetate 150 mL (50.0 mL x 3) and the combined organic layers were washed with brine 20.0 mL (20.0 mL x 1), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, n- heptane / Ethyl acetate = 100 / 1 to 1 / 1) and then further purified by prep-HPLC (HCl condition) to give Target 5 (100 mg, 2.87 mmol) as a white solid.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 Example 13: 3-methyl-6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (Target 4)

[0652] 3-methyl-6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (Target 4) : To a solution of 1- methylimidazole-4-carbonitrile (200 mg, 1.87 mmol), MeCN (268 mg, 6.54 mmol) and zinc;trifluoromethanesulfonate (68 mg, 0.19 mmol) in dioxane (1 mL) was added NH2NH2.H2O (2.34 g, 46.68 mmol) at 25oC and the mixture was stirred at 65oC for 16 h under N2. Then the mixture was cooled to 25oC and added with a solution of NaNO2 (387 mg, 5.60 mmol) in H2O (3 mL) dropwise at 25oC. The mixture was stirred at 25oC for 3 h. The mixture was cooled to room temperature and adjusted to pH = 3 with 1 M aqueous hydrochloric acid. The aqueous phase was extracted with DCM (3 × 5 mL). The combined organics were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by prep-TLC (SiO2, DCM: MeOH = 10:1) to give 3-methyl- 6-(1-methyl-1H-imidazol-4-yl)-1,2,4,5-tetrazine (Target 4) (20.1 mg, 6.1%).

[0653] LCMS (ESI+): m / z = 177.2 [M+H]+

[0654] 1H NMR (400MHz, CDCl3) (ET60578-12-P1M): δ = 8.00 (s, 1H), 7.68 (s, 1H), 3.85 (s, 3H), 3.06 (s, 3H).Example 14: 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8)

[0655] 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) was added oxalyl dichloride (13.47 g, 106.13 mmol) at 0 °C under N2. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved THF (50 ml) and adjusted to pH = 9 with NH3.H2O at 0oC. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give isoxazole-3-carboxamide (2) (3.3 g, 33.3%).

[0656] 1H NMR (400MHz, DMSO): δ = 9.05 (d, J = 1.6 Hz, 1H), 8.12 (s, 1H), 7.82 (s, 1H), 6.85 (d, J = 1.6 Hz, 1H)

[0657] isoxazole-3-carbonitrile (3): To a solution of isoxazole-3-carboxamide (500 mg, 4.46 mmol) in pyridine (18 mL) was added POCl3 (1.03 g, 6.69 mmol) at 20°C under N2. The mixture was stirred at 20 °C for 2 h. After stirring for 2 h, the mixture was cooled by an ice bath and added with water (10 mL). The aqueous phase was adjusted to pH = 4 by addition of aqueous 3 M HC1 and extracted with MTBE (3 × 10 mL). The combined organics were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give isoxazole-3-carbonitrile (3) (280 mg, 66.7%).

[0658] 1H NMR (400MHz, MeOD): δ 9.06 (d, J = 1.6 Hz, 1H), 7.02 (d, J = 1.2 Hz, 1H)

[0659] 3-(6-methyl-1,2,4,5-tetrazin-3-yl)isoxazole (Target 8): To a solution of isoxazole-3- carbonitrile (220 mg, 2.34 mmol) in EtOH (2 mL) was added NH2NH2.H2O (1.87 g, 37.42 mmol) at 20 °C for 0.5 h. Then the mixture was added MeCN (384 mg, 9.35 mmol) and 3-sulfanylpropanoic acid (248 mg, 2.34 mmol) at 20 °C under N2. The mixture was stirred at 45 °C for 12 h. Thereafter, NaNO2 (500 mg) was added and the reaction mixture was stirred at 20 °C for 0.5 h.3 M hydrochloric acid was added dropwise until pH = 1, and the reaction mixture was stirred at 20 °C for 0.5 h. The aqueous phase was extracted with EtOAc (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 prep-HPLC (FA) with the following conditions: column: Phenomenex Luna C18100*30mm*5um; mobile phase: [water(FA)-ACN]; B%: 5%-45%, 8 min to give 3-(6-methyl-1,2,4,5- tetrazin-3-yl)isoxazole (12.45 mg, 3.26%). LCMS (ESI+): m / z = 164.1 [M+H]+

[0660] 1H NMR (400MHz, MeOD): δ 9.01 (d, J = 1.6 Hz, 1H), 7.34 (d, J = 1.6 Hz, 1H), 3.11 (s, 3H). Example 15: 3-methyl-6-(1H-pyrazol-1-yl)-1,2,4,5-tetrazine (Target 13)

[0661] methyl (E)-hydrazinecarbohydrazonothioate (2): To a mixture of 1,3-diaminothiourea (100 g, 942.06 mmol) in MeOH (500 mL) was added MeI (160.46 g, 1.13 mol) at 25oC and the mixture was stirred at 80oC for 1.5 h under N2. H NMR showed the reaction was completed. The resulting pale yellow solution was cooled to room temperature until solid precipitated. And then it was diluted with MTBE (500 mL). The mixture was cooled in ice for 2 h and filtered. The collected solid was washed with MTBE, dried under reduced pressure to give 1,3-diamino-2-methyl-isothiourea; hydroiodide (Compound 2) (132 g, 56.48% yield, HI).

[0662] 1H NMR (400MHz, DMSO): δ = 10.96 - 9.08 (m, 1H), 5.80 - 4.81 (m, 2H), 2.37 (s, 3H)

[0663] 3-methyl-6-(methylthio)-1,2,4,5-tetrazine (4): To a solution of 1,3-diamino-2-methyl- isothiourea (30 g, 249.63 mmol) in DMF (750 mL) was added 1,1,1-triethoxyethane (44.55 g, 274.60 mmol) at 25oC and the mixture was stirred at 25oC for 5 min under N2. Then the mixture was added TEA (25.26 g, 249.63 mmol) at 25oC and the mixture was stirred at 50oC for 3 h. TLC showed the reaction was completed. The mixture was poured into water (1000 mL) and extracted with EtOAc (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 to 5 / 1) to give 3-methyl-6-methylsulfanyl-1,2,4,5-tetrazine (Compound 4) (5.8 g, 16.3%).

[0664] 1H NMR (400MHz, CDCl3): δ = 2.99 (s, 3H), 2.74 (s, 3H).

[0665] 3-methyl-6-(1H-pyrazol-1-yl)-1,2,4,5-tetrazine (Target 13): To a mixture of 1H-pyrazole (144 mg, 2.11 mmol) in THF (2 mL) was added NaH (84 mg, 2.11 mmol, 60% purity) at 0oC and the mixturewas stirred at 0oC for 15 min under N2. Then the mixture was added 3-methyl-6-methylsulfanyl-1,2,4,5- tetrazine (200 mg, 1.41 mmol) in THF (1 mL) at 0oC and the mixture was stirred at 25oC for 2 h under N2. TLC showed the reaction was completed. The mixture was poured into sat.NH4Cl (5 mL) and extracted with EtOAc (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 prep-TLC (SiO2, PE: EtOAc = 3:1) to give 3-methyl-6-pyrazol-1-yl-1,2,4,5-tetrazine (Target 13) (20 mg, 8.8%). LCMS (ESI+): m / z = 163.1 [M+H]+

[0666] 1H NMR (400MHz, CDCl3): δ = 8.74 (d, J = 2.8 Hz, 1H), 8.02 (d, J = 1.2 Hz, 1H), 6.68 (dd, J = 1.6, 2.8 Hz, 1H), 3.14 (s, 3H). Example 16: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 15)

[0667] tert-butyl (4-cyano-2-(trifluoromethyl)benzyl)carbamate (2): To a solution of 4- (aminomethyl)-3-(trifluoromethyl)benzonitrile (300 mg, 1.50 mmol) in DCM (10 mL) was added Boc2O (360 mg, 1.65 mmol) and TEA (227 mg, 2.25 mmol) at 20 °C under N2. The mixture was stirred at 20 °C for 2 h. The mixture was cooled down and poured into H2O (10 mL), 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:EtOAc = 1:0 to 3:1) to give the tert-butyl (4-cyano-2- (trifluoromethyl)benzyl)carbamate (2) (400 mg, 88.9%).

[0668] 1H 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 = 6.0 Hz, 2H), 1.47 (s, 9H).

[0669] tert-butyl (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (3): To a solution of tert-butyl N-[[4-cyano-2-(trifluoromethyl)phenyl]methyl]carbamate (150 mg, 0.50 mmol) in EtOH (0.5 mL) was added NH2NH2.H2O (400 mg, 7.99 mmol) at 20 °C for 0.5 h. Then the mixture was added with MeCN (82 mg, 2.00 mmol) and 3-sulfanylpropanoic acid (53 mg, 0.50 mmol) at20 °C under N2. Thereafter, NaNO2 (500 mg) was added and the reaction mixture was stirred at 20 °C for 0.5 h.3 M hydrochloric acid was added until pH = 1, and the reaction mixture was stirred at 20 °C for 0.5 h. The aqueous phase was extracted with EtOAc (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 prep-TLC (SiO2, PE:EtOAc = 3:1) to give tert-butyl N-((4- (6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methyl)carbamate (3) (50 mg, 14.1%).

[0670] 1H NMR (400MHz, CDCl3): δ 8.91 (s, 1H), 8.78 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 5.11 - 4.96 (m, 1H), 4.63 (d, J = 6.8 Hz, 2H), 3.14 (s, 3H), 1.49 (s, 9H).

[0671] (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (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) was added HCl / MeOH (2 mL, 4 M) at 20 °C under N2. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by prep-HPLC (FA) with the following conditions: Phenomenex Luna 80*30mm*3um phase: [water(FA)-ACN]; B%: 1%-25%, 8 min to give (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (4.8 mg, 15.9%).

[0672] LCMS (ESI+): m / z = 270.0 [M+H]+

[0673] 1H NMR (400MHz, MeOD): δ 8.93 (s, 1H), 8.89 (d, J = 8.4 Hz, 1H), 8.49 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 4.37 (s, 2H), 3.09 (s, 3H). Example 17: (4-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)phenyl)methanamine (Target 17)

[0674] 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) was added isoindoline-1,3-dione (3.3 g, 22.38 mmol), PPh3 (11.7 g, 44.76 mmol) and DIAD (6.79 g, 33.57 mmol) at 0 °C under N2. Then the resulting mixture was stirred at 20 °C for 16 h. The reaction mixture wasquenched by addition of water (15 mL) and extracted with EtOAc (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 give a residue, which was purified by column to give 3-((1,3- dioxoisoindolin-2-yl)methyl)-4-(trifluoromethyl)benzonitrile (2) (5.1 g, 69%).

[0675] 1H NMR (400 MHz, CDCl3) δ = 7.91-8.03 (m, 2H), 7.84 (dd, J = 5.6, 2.8 Hz, 3H), 7.70 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 5.13 (s, 2H).

[0676] 3-(aminomethyl)-4-(trifluoromethyl)benzonitrile (3): To a solution of 3-((1,3-dioxoisoindolin- 2-yl)methyl)-4-(trifluoromethyl)benzonitrile (1 g, 3.03 mmol) in EtOH (20 mL) was added NH2NH2.H2O (3.79 g, 60.56 mmol, 80% purity) at 20 °C, then the mixture was stirred at 20 °C for 12 h.6 M hydrochloric acid was added until pH = 1, and the reaction mixture was stirred at 20 °C for 2 h. After the completion of the reaction, the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was poured into water (50 mL), adjusting pH to 11 with 6 M NaOH aqueous solution (20 mL). Then the mixture was extracted with EtOAc (3 × 50 mL) and organic layer was washed with brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 3- (aminomethyl)-4-(trifluoromethyl)benzonitrile (3) (600 mg, 99.0%).

[0677] 1H NMR (400 MHz, CDCl3): δ = 8.09 (s, 1H), 7.72-7.80 (m, 1H), 7.62-7.71 (m, 1H), 4.12 ppm (s, 2H).

[0678] tert-butyl (5-cyano-2-(trifluoromethyl)benzyl)carbamate (4): To a solution of 3- (aminomethyl)-4-(trifluoromethyl)benzonitrile (0.3 g, 1.50 mmol) and NaOH (180 mg, 4.50 mmol) in dioxane (6 mL) and H2O (3 mL) was added Boc2O (654 mg, 3.00 mmol) at 20 °C, then the mixture was stirred at 20 °C for 2 h. The reaction mixture was quenched with H2O (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (PE:EtOAc = 1:0 to 0:1) to give tert-butyl (5-cyano-2- (trifluoromethyl)benzyl)carbamate (4) (300 mg, 66.6%).

[0679] 1H NMR (400 MHz, 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 = 6.0 Hz, 2H), 1.48 ppm (s, 9H).

[0680] tert-butyl (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5): To a solution of 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) was added N2H4.H2O (1.33 g, 26.64 mmol) at 25oC and the mixture was stirred at 65oC for 16 h under N2. Then the mixture was cooled to r.t and added with a solution of NaNO2 (345 mg, 5.00 mmol) in H2O (2.5 mL) dropwise at 25oC. The mixture was stirred at 25oC for 3 h. The mixture was cooled to room temperature and adjusted to pH = 3 with 1 M aqueous hydrochloric acid. The mixture was extracted with DCM (20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc = 20:1 to 1:1) togive tert-butyl (5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2-(trifluoromethyl)benzyl)carbamate (5) (140 mg, 22.8%).

[0681] 1H NMR (400 MHz, CDCl3): δ = 8.78 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 5.08 (s, 1H), 4.67 (s, 1H), 3.15 (s, 3H), 1.49 (s, 9H).

[0682] (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) was added HCl / MeOH (2 mL, 4 M) at 20 °C under N2. The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by prep-TLC (SiO2, DCM:MeOH = 10:1) to give [5-(6-methyl-1,2,4,5-tetrazin-3-yl)-2- (trifluoromethyl)phenyl]methanamine (18.0 mg, 17.7%).

[0683] LCMS (ESI+): m / z = 270.0 [M+H]+

[0684] 1H NMR (400MHz, MeOD): δ 8.88 (s, 1H), 8.60 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 4.11 (s, 2H), 3.08 (s, 3H) Example 18: 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (Target 6)[ ] e - u y - - - o op e y - , , , - e a - -y e y ca a a e: o a u e o - iodobenzonitrile (15 g, 65.50 mmol) in EtOH (140 mL) was added 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) at 0 °C under N2. The mixture was stirred at 45 °C for 16 h. Then the mixture was cooled to 20oC and added with a solution of NaNO2 in H2O (40 mL) dropwise at 20oC. The mixture was stirred at 20oC for 1 h. Under ice-cooling, the pH was adjusted to 3 with 1 M aqueous hydrochloride 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:EtOAc = 10:1 to 1:1) to give tert-butyl (2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate (14 g, 50%).

[0686] LCMS: m / z = 372.0 [M-BuOH+H]+.

[0687] 2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethan-1-amine: A mixture of tert-butyl (2-(6-(4- iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)carbamate (6.5 g, 5.85 mmol) in HCl / EtOAc (4M, 100 mL) was stirred at 25 °C for 1 h. The reaction was concentrated under reduced pressure to give 2-(6-(4- iodophenyl)-1,2,4,5-tetrazin-3-yl)ethan-1-amine; hydrochloride (5.8 g, 80%).

[0688] LCMS: m / z = 328.2 [M+H]+.

[0689] 1-(2-(6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea: To a mixture of CDI (3.35 g, 20.63 mmol) in DCM (45 mL) was added TEA (4.18 g, 41.27 mmol) and 2-(6-(4-iodophenyl)-1,2,4,5- tetrazin-3-yl)ethan-1-amine (5.8 g, 13.76 mmol) at -40 °C under N2. The mixture was stirred at -40 °C for 1 h. Then MeNH2 (2 M, 17 mL) and TEA (4.17 g, 41.24 mmol) were added at 0 °C under N2. The mixture was stirred at 25 °C for 12 h. The reaction was concentrated under reduced pressure to give a residue. The residue was triturated with DCM. The resulting solid was collected by filtration, washed with DCM (40 mL) and dried under reduced pressure to give 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3- yl]ethyl]-3-methyl-urea (3 g, 57%). LCMS: m / z = 385.0 [M+H]+.

[0690] 1H NMR (400 MHz, DMSO): δ = 8.24 (d, J = 8.4 Hz, 2 H) 8.06 (d, J = 8.4 Hz, 2 H) 6.09 (br t, J = 5.6 Hz, 1 H) 5.77 (br d, J = 4.8 Hz, 1 H) 3.53 - 3.57 (m, 2 H) 3.36 - 3.40 (m, 2 H) 2.45 (d, J = 4.4 Hz, 3 H).

[0691] tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate: To a mixture of 1-[2-[6-(4-iodophenyl)-1,2,4,5-tetrazin-3-yl]ethyl]-3-methyl-urea (1 g, 2.60 mmol) and (tert- butoxycarbonylamino)methyl-trifluoro-boron;potassium hydride (926 mg, 3.90 mmol) in 2-METHYL-2- BUTANOL (4 mL) and H2O (1 mL) was added Cs2CO3 (1.70 g, 5.21 mmol) and ditert- butyl(cyclopentyl)phosphane;dichloropalladium;iron (170 mg, 0.26 mmol) at 25 °C under N2. The mixture was stirred at 80 °C for 16 h. The mixture was diluted with H2O (15 mL) and extracted with EtOAc (3 × 10 mL). The combined organics were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EtOAc = 5:1 to 0:1) to give tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3- yl)benzyl)carbamate (200 mg, 20%).

[0692] LCMS: m / z = 388.3 [M+H]+.

[0693] 1H NMR (400 MHz, DMSO): δ = 8.44 (br d, J = 8.0 Hz, 2 H), 7.52 (br d, J = 7.6 Hz, 3 H), 6.08 (br t, J = 5.6 Hz, 1 H), 5.78 (br s, 1 H), 4.26 (br d, J = 5.6 Hz, 2 H), 3.54 - 3.58 (m, 2 H), 3.38 (br d, J = 6.4 Hz, 2 H), 2.45 (d, J = 4.4 Hz, 3 H), 1.41 (s, 9 H).

[0694] 1-(2-(6-(4-(aminomethyl)phenyl)-1,2,4,5-tetrazin-3-yl)ethyl)-3-methylurea (Target 6): To a mixture of tert-butyl (4-(6-(2-(3-methylureido)ethyl)-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (150 mg,0.39 mmol) was added HCl / EtOAc (4 M, 3 mL) at 25°C under N2. The mixture was stirred at 25 °C for 1 h. The reaction was concentrated under reduced pressure. The crude product was purified by prep-HPLC with the following conditions: column: Phenomenex C18100 × 30 mm × 5 μm; mobile phase: A: 10 mM NH4HCO3in water, B: MeCN; B% in A: 20%-50%, 10 min to give 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]+.

[0695] 1H NMR (400 MHz, DMSO): δ = 8.52 (d, J = 8.0 Hz, 2 H), 8.45 (br s, 2 H), 7.77 (d, J = 8.2 Hz, 2 H), 6.14 (br t, J = 5.6 Hz, 1 H), 5.76 - 5.83 (m, 1 H), 4.18 (br s, 2 H), 3.56 (q, J = 6.4 Hz, 2 H), 3.38 - 3.41 (m, 2 H), 2.44 (d, J = 4.4 Hz, 3 H). Example 19: 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl-6- methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b)

[0696] 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (2): To a solution of benzonitrile (10 g, 96.97 mmol), ACN (31.85 g, 775.79 mmol), 3-mercaptopropanoic acid (10.29 g, 96.97 mmol) in EtOH (100 mL) was added dropwise NH2NH2.H2O (79.26 g, 1.55 mol) at 0 °C under N2. The mixture was stirred for 16 h at 40 °C. The mixture was quenched by water (100 mL), adjusted to pH = 4 by addition of HCl (1 M), extracted with EtOAc (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 slurried withMTBE:EtOAc (10:1, 100 mL), filtered and the solid was desired. The solid was purified by p-HPLC (FA) under the following condition: column: Phenomenex luna C18 (250 × 70 mm, 15 um); mobile phase: [water(FA)-ACN]; B%: 8%-35%, 22 min to give 3-methyl-6-phenyl-1,4-dihydro-1,2,4,5-tetrazine (10 g, 29%).

[0697] LCMS, m / z = 175.1 [M+H]+

[0698] 1H NMR (400MHz, DMSO): δ 8.50 (br s, 1H), 8.29 (br s, 1H), 7.74 (dd, J = 1.6, 7.6 Hz, 2H), 7.47-7.35 (m, 3H), 1.78 (s, 3H).

[0699] 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) was added DIEA (6.68 g, 51.66 mmol) and then (4-nitrophenyl) carbonochloridate (3.64 g, 18.08 mmol) at 0 °C under N2. The mixture was stirred for 2 h at 25 °C. The mixture was poured into water (45 mL), extracted with DCM (3 × 15 mL). The combined organic layers were washed with brine (15 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (PE:EtOAc = 1:0 to 0:1) to give 4-nitrophenyl 6-methyl-3-phenyl-1,2,4,5-tetrazine- 1(4H)-carboxylate (1 g, 17%).

[0700] LCMS, m / z = 340.0 [M+H]+

[0701] 1H NMR (400MHz, CDCl3): δ 8.36-8.28 (m, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.56-7.42 (m, 5H), 6.30-6.02 (m, 2H), 2.44 (s, 3H).

[0702] 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3- phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (6): To a solution of 4-nitrophenyl 6-methyl-3-phenyl- 1,2,4,5-tetrazine-1(4H)-carboxylate (705 mg, 1.66 mmol) and (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4- (hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1 g, 1.66 mmol) in DMF (10 mL) was added DIEA (644 mg, 4.99 mmol) at 25 °C under N2. The mixture was heated to 80 °C and stirred for 2 h. The crude was purified by p-HPLC (neutral) under the following condition: column: Phenomenex C18250 × 50 mm × 10 um; mobile phase: [water( NH4HCO3)-ACN]; B%: 14%-44%, 8 min to give 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5- ureidopentanamido)benzyl-6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (240 mg, 25%).

[0703] LCMS, m / z = 580.2 [M+H]+

[0704] 1H NMR (400MHz, DMSO): δ 9.97 (s, 1H), 9.84 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 7.88-7.79 (m, 2H), 7.60-7.47 (m, 5H), 7.23 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 5.98 (br t, J = 6.0 Hz, 1H), 5.47-5.35 (m, 2H), 5.12-5.04 (m, 1H), 4.43 (d, J = 5.6 Hz, 3H), 4.26 (dd, J = 5.6, 8.8 Hz, 1H), 3.07-2.94 (m, 2H), 2.18 (s, 3H), 2.14-2.04 (m, 1H), 1.75-1.55 (m, 2H), 1.51-1.30 (m, 2H), 0.92 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H)

[0705] 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl-6-methyl-3- phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (Target 1b): To a solution of 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (100 mg, 0.17 mmol) and DIEA (45 mg, 0.35 mmol) in THF (0.5 mL) was added DMAP (4 mg, 0.03 mmol) and then Ac2O (23 mg, 0.22 mmol) at 0 °C under N2. The mixture was stirred for 2 h at 25 °C. The mixture was concentrated under reduced pressure. The crude was purified by p-HPLC (neutral) under the following condition: column: Waters xbridge 150 × 25 mm × 10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 24%-54%, 8 min to give 4-((S)-2-((S)-2-acetamido-3-methylbutanamido)-5- ureidopentanamido)benzyl 6-methyl-3-phenyl-1,2,4,5-tetrazine-1(4H)-carboxylate (58.9 mg, 55%).

[0706] LCMS, m / z = 622.2 [M+H]+

[0707] 1H NMR (400MHz, DMSO): δ 10.09 (s, 1H), 9.85 (s, 1H), 8.44 (d, J = 7.2 Hz, 1H), 7.87-7.77 (m, 2H), 7.64-7.47 (m, 5H), 7.30 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 5.99 (br t, J = 5.6 Hz, 1H), 5.41 (s, 2H), 5.00 (s, 2H), 4.47-4.36 (m, 1H), 4.27 (dd, J = 5.6, 8.7 Hz, 1H), 3.10-2.88 (m, 2H), 2.18 (s, 3H), 2.14-2.06 (m, 1H), 2.04 (s, 3H), 1.77-1.57 (m, 2H), 1.52-1.32 (m, 2H), 0.92 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H).Example 20: Synthesis of Exatecan Prodrug Compound F[ ] roceure or prepara on o ompoun

[0709] To a solution of methyl compound 1 (25.0 g, 104 mmol, 1.0 eq.) in MeOH (125 mL) was added NaOMe (5.40 M, 105 mL, 5.5 eq.) in H2O (125 mL). The mixture was stirred at 25 °C for 24 hrs. Thereaction was monitored by TLC, TLC (PE: EA = 2:1, product Rf = 0.20) indicated the reactant (Rf = 0.60) was consumed. The reaction mixture was diluted with H2O (50 mL) and extracted with MTBE (4 × 500 mL). The aqueous layer was acidified with 1 M HCl until pH = 4 while cooling in an ice-water bath (T < 7 °C). The aqueous layer was extracted with MTBE (5 × 500 mL). The combined MTBE layers were dried with Na2SO4, filtered and concentrated under reduced pressure to give residue and evaporated with MeCN three times to give compound 2 (9.1 g, 49.3 mmol, 47.4% yield).

[0710] 1H NMR: 400 MHz, CDCl3 δ ppm 1.11 (s, 3 H), 1.65 (br dd, J = 15.82, 6.19 Hz, 1 H), 1.78 - 2.02 (m, 4 H), 2.05 - 2.42 (m, 4 H), 4.49 (br s, 1 H), 5.64 (dd, J = 16.63, 2.00 Hz, 1 H), 6.00 - 6.14 (m, 1 H).

[0711] Procedure for preparation of Compound 3. , . , . . (130 mL) was added DIEA (32.4 g, 250 mmol, 43.6 mL, 7.0 eq.) and DSC (39.4 g, 154 mmol, 4.3 eq.). The mixture was stirred at 25 °C for 16 hrs. The reaction was monitored by TLC, TLC (DCM: MeOH = 10:1, product Rf = 0.60) indicated the reactant (Rf = 0.20) was consumed. The reaction mixture was purified by re-crystallization from H2O (350 mL) at 25 °C, filtered and concentrated under reduced pressure to give a residue. The crude product was evaporated with MeCN for three times to give compound 3 (10.0 g, 21.6 mmol, 60.4% yield, 91.4% purity). HPLC: Rt= 2.14 min, purity: 91.4%.

[0713] 1H NMR: 400 MHz, CDCl3 δ ppm 1.28 (s, 3 H), 1.97 - 2.17 (m, 4 H), 2.26 - 2.49 (m, 4 H), 2.82 - 2.86 (m, 8 H), 5.29 (br s, 1 H), 5.63 (dd, J = 16.70, 2.19 Hz, 1 H), 6.02 - 6.15 (m, 1 H).

[0714] Procedure for preparation of Compound 5

[0715] To a solution of compound 3 (6.6 g, 15.6 mmol, 1.0 eq.) in DMF (70 mL) was added DIEA (4.04 g, 31.2 mmol, 5.44 mL, 2.0 eq.) and Exatecan (6.23 g, 11.7 mmol, 0.75 eq.). The mixture was stirred at25 °C for 1 hr. LC-MS (EC17183-7-P1A1) showed one main peak with desired mass (RT =0.44 min) was detected. The reaction mixture was filtered and purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water (FA)-ACN]; gradient: 40%-70% B over 15 min) to give compound 5 (6.0 g, 7.76 mmol, 49.6% yield, 96.1% purity).

[0716] LCMS (monitor): Rt = 0.44 min, MS cal.: 742.2, MS observed: [M+H]+= 743.1.

[0717] LCMS: Rt= 0.44 min, MS cal.: 742.2, MS observed: [M+H]+= 743.3.

[0718] HPLC: Rt = 3.04 min, purity: 96.1%.

[0719] Procedure for preparation of Compound F

[0070] To a sout on o compound 5- (9.95 g, 6.5 mmo , .5 eq.) n DMSO ( 0 mL) was added DIEA (2.85 g, 22.0 mmol, 3.85 mL, 2.0 eq.) and DMAP (2.70 g, 22.08 mmol, 2.0 eq.) and compound 5 (8.2 g, 11.04 mmol, 1 eq). The mixture was stirred at 25 °C for 16 hrs. LC-MS (EC17183-21-P1A11) showed one main peak with desired mass (Rt= 0.39 min) was detected. The reaction mixture was filtered, purified by prep-HPLC (FA condition) and exchange to AcOH salt to give Compound F (7.5 g, 5.89 mmol, 53.3% yield, 96.5% purity).

[0721] LCMS (monitor): Rt= 0.39 min, MS cal.: 1227.5, MS observed: [M+H]+= 1228.8.

[0722] LCMS: Rt = 0.38 min, MS cal.: 1227.5, MS observed: [M+H]+= 1228.7.

[0723] HPLC: Rt= 2.28 min, purity: 96.5%. Example 21: Synthesis of Exatecan Prodrug Compound G23.6 mmol) and DMAP (2.89 g, 23.6 mmol) and 2-aminoacetic acid (3.55 g, 47.3 mmol). The mixture was stirred at 25 °C for 120 hrs. LC-MS showed compound 4 was consumed completely and one main peak with desired mass (Rt =0.40 min) was detected. The reaction mixture was added TFA until pH= 6~7. The crude product was purified by reversed-phase HPLC (column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (TFA)-ACN]; gradient: 20%-50% B over 22 min) to give Compound G (1.02 g, 1.38 mmol, 41.1% yield, 95.3% purity). HRMS: MS calc. [M+H]+: 703.2774; MS observed = 703.2791. HPLC: Rt= 13.1 min, purity: 94.5%.Example 22: Synthesis of Exatecan Prodrug Compound H[ ] o a u e o p osge e g, . o was a e , , -6- hydroxy-1-methylcyclooct-4-ene-1-carboxylic acid (220 mg, 1.20 mmol) and DMAP (292 mg, 2.40 mmol). The mixture was stirred at room temperature for 30 min. The mixture was added to a mixture of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl -1,2,3,9,12,15-hexahydro-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate (700 mg, 1.32 mmol) and DIPEA (510 mg, 3.96 mmol) in DMF (10 mL). The resulting mixture was stirred at room temperature for 12 hr. The mixture was concentrated and purified by Prep-HPLC (CH3CN / H2O(FA)) 0% to 70%) to give Intermediate A (110 mg, yield 14%). LCMS: (m / z, C35H36FN3O8) = 646.3 [M+H]+

[0007] o a so ut on o ntermedate ( 5 mg, µmo) an U ( mg, 5 µmol) in DMF (5 mL) was added DIEA (38 mg, 300 µmol). The mixture was stirred at room temperature for 30 min and then 2-amino-N,N,N-trimethylethan-1-aminium(21 mg, 110 µmol) was added. The mixture was stirred at 25°C for additional 24 hr. The mixture was concentrated and purified by Prep-HPLC to give Compound H (21 mg, 29 %).

[0727] Mobile Phase: A: water (0.01%TFA) B: ACN (0.01%TFA)

[0728] Gradient: 5% increase to 95%B within 1.3 min, 95%B for 1.7 min.

[0729] Flow Rate: 2.0ml / min

[0730] Column:Sunfire C18,4.6*50mm,3.5um

[0731] Column Temperature: 45 C

[0732] Detection:UV(214,4nm) and MS(ESI,Pos mode, 110 to 1000 amu)

[0733] Pre-HPLC Method for Compound H:

[0734] The gradient was 5% B for 0.2 min, and to 95% B within 1.7 min then with a hold at 95% B for 1.3 min, back to 5% B within 0.01 min (2.3 mL / min flow rate).

[0735] Mobile phase A was 0.01% HCOOH in water, and mobile phase B was 0.01% HCOOH in HPLC grade CH3CN. The column used for the chromatography is a 4.6 x 50 mm XBridge C18 column (3.5µm particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive / negative electrospray ionization.Example 23: Synthesis of Exatecan Prodrug Compound J

[0736] DMF (5 mL) was added DIEA (38 mg, 300 µmol). The mixture was stirred at 25°C for 30 min. Then glycine (16 mg, 200 µmol) and NaHCO3(17 mg, 200 µmol) was added. The mixture was stirred at 25°C for 5 hr. The mixture was concentrated and purified by Prep-HPLC (CH3CN / H2O(FA)) 0% to 70%) to give Compound J (32 mg, 45.2 %).

[0737] Mobile Phase: A: water(0.01%TFA) B: ACN(0.01%TFA)

[0738] Gradient: 5% increase to 95%B within 1.3 min, 95%B for 1.7 min.

[0739] Flow Rate: 2.0ml / min

[0740] Column:Sunfire C18,4.6*50mm,3.5um

[0741] Column Temperature: 45 C

[0742] Detection:UV(214,4nm) and MS(ESI,Pos mode,110 to 1000 amu)

[0743] Pre-HPLC Method for Target 3

[0744] The gradient was 5% B for 0.2 min, and to 95% B within 1.7 min then with a hold at 95% B for 1.3 min, back to 5% B within 0.01 min (2.3 mL / min flow rate).

[0745] Mobile phase A was 0.01% HCOOH in water, and mobile phase B was 0.01% HCOOH in HPLC grade CH3CN. The column used for the chromatography is a 4.6 x 50 mm XBridge C18 column (3.5µm particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection as well as positive / negative electrospray ionization.Example 24: Synthesis of Compound K and Compound Lp . g, . mmol, 1.00 eq.) in EtOH (12.0 mL) was added AcOH (614 mg, 10.2 mmol, 585 μL, 0.89 eq.) and compound 2 (3.77 g, 17.1 mmol, 1.49 eq.). The mixture was stirred at 25 °C for 16 hrs. LCMS showed compound 1 was consumed completely and desired mass (MS cal.: 215.0, MS observed: [M+H]+= 216.0) was detected. The reaction mixture was concentrated under reduced pressure to compound 3 (5.67 g, crude) as yellow oil. LCMS-1: Rt= 0.36 min, MS cal.: 215.0, MS observed: [M+H]+= 216.0. Procedure for preparation of compound 4

[0747] To a solution of compound 3 (5.67 g, 26.3 mmol, 1.00 eq.) in DMF (25.0 mL) was added DIEA (10.2 g, 78.9 mmol, 13.7 mL, 3.00 eq.) and PNP (16.0 g, 52.6 mmol, 2.00 eq.). The mixture was stirred at 25 °C for 7 hrs. LCMS showed compound 3 was consumed completely and desired mass (MS cal.:380.05, MS observed: [M+H]+= 381.0) was detected. The reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (50 mL * 3). The combined organic layers were washed with brine (50 mL * 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (0.1% TFA condition) to give compound 4 (1.40 g, 3.66 mmol, 31.8% yield for 2 steps, 99.4% purity) as a yellow solid which was confirmed via LCMS, HPLC and HNMR. LCMS: Rt= 0.48 min, MS cal.: 380.0, MS observed: [M+H]+= 381.1. HPLC: Rt= 3.53 min, purity: 99.4%.1H NMR: 400 MHz, CDCl3 δ: 8.47 - 8.45 (m, 1H), 8.30 - 8.26 (m, 2H), 7.76 - 7.74 (m, 1H), 7.65 - 7.60 (m, 2H), 7.39 - 7.35 (m, 2H), 7.11 - 7.07 (m , 1H). Procedure for preparation of compound 5

[0748] To a solution of compound 4 (1.40 g, 3.66 mmol, 1.00 eq.) in DMF (14.0 mL) was added MMAE (2.63 g, 3.66 mmol, 1.00 eq.), HOBt (741 mg, 5.49 mmol, 1.50 eq.) and DIEA (945 mg, 7.32 mmol, 1.27 mL, 2.00 eq.). The mixture was stirred at 25 °C for 12 hrs. LCMS showed compound 4 was consumed completely and desired mass (MS cal.: 958.53, MS observed: [M+H]+= 959.5) was detected. The residue was purified by prep-HPLC (0.1% TFA condition) to give compound 5 (2.50 g, 2.54 mmol, 69.3% yield, 97.4% purity) as a white solid which was confirmed via LCMS and HPLC. LCMS: Rt= 0.55 min, MS cal.: 958.5, MS observed: [M+H]+= 959.7. HPLC: Rt= 3.93 min, purity: 97.4%.Procedure for preparation of compound 7 [07added AcOH (81.4 mg, 1.36 mmol, 77.6 μL, 0.89 eq.) and compound 5 (1.50 g, 1.52 mmol, 1.00 eq.). The mixture was stirred at 80 °C for 3 hrs. LCMS showed compound 5 was consumed completely and desired mass (MS cal.: 968.5, MS observed: [M+H]+= 969.5) was detected. The residue was purified by prep-HPLC (0.1% TFA condition) to give compound 7 (780 mg, 767 μmol, 50.4% yield, 99.0% purity, HCl) as a white solid which was confirmed via LCMS and HPLC. LCMS: Rt= 0.41 min, MS cal.: 968.5, MS observed: [M+H]+= 969.6. HPLC: Rt= 2.62 min, purity: 99.0%. Procedure for preparation of compound 8

[0750] To a solution of compound 8a (2.00 g, 10.8 mmol, 1.00 eq.) in ACN (10.0 mL) was added DIEA (4.21 g, 32.5 mmol, 5.67 mL, 3.00 eq.) and DSC (2.78 g, 10.8 mmol, 1.00 eq.) in ACN (10.0 mL). The mixture was stirred at 25 °C for 1 hr. LCMS showed desired mass (MS cal.: 281.1, MS observed: [M+Na]+= 304.1) was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether: EtOAc = 1: 1) to give compound 8 (1.82 g, 6.18 mmol, 56.9% yield, 95.5% purity) as a light yellow solid which was confirmed via LCMS and HNMR. LCMS: Rt= 0.44 min, MS cal.: 281.1, MS observed: [M+H]+= 304.0.HPLC: Rt = 1.77 min, purity: 95.5%.1H NMR: 400 MHz, CDCl3 δ: 6.13 - 6.06 (m, 1H), 5.68 - 5.64 (m, 2H), 4.51 (s, 1H), 2.82 - 2.81 (m, 4H), 2.41 - 2.25 (m, 3H), 2.17 -1.98 (m , 3H), 1.93 - 1.83 (m, 2H), 1.25 (s, 3H). Procedure for preparation of Compound Kadded DMAP (269 mg, 2.20 mmol, 8.00 eq.) and compound 8 (89.3 mg, 303 μmol, 1.10 eq.), DIEA (284 mg, 2.20 mmol, 384 μL, 8.00 eq.). The mixture was stirred at 25 °C for 1 hr. LCMS showed compound 7 (Rt= 0.43 min) was remained and desired mass (MS cal.: 1134.6, MS observed: [M+H]+= 1135.8) was detected. The reaction mixture was concentrated under reduced pressure to give a residue-1. Another residue-2 was obtained from 80 mg scale. The two batches were purified by prep-HPLC (AcOH condition) to give Compound K (47.0 mg, 37.7 μmol, 13.6% yield, 95.9% purity, AcOH) as a white solid which was confirmed via LCMS.

[0752] LCMS: Rt= 0.50 min, MS cal.: 1134.6, MS observed: [M+H]+= 1135.7.

[0753] HPLC: Rt = 3.57 min, purity: 95.9%.Procedure for preparation of compound 101.00 eq.) in DCM (20 mL) and MeOH (5 mL) was added PAB (992 mg, 8.06 mmol, 2.00 eq.) and EEDQ (1.99 g, 8.06 mmol, 2.00 eq.). The mixture was stirred at 35 °C for 16 hrs in the dark. TLC (DCM / MeOH = 10:1, product Rf = 0.32) indicated compound 9 was consumed completely and one new spot formed. The reaction mixture was filtered and concentrated under reduced pressure to give compound 10 (2.00 g, 3.02 mmol, 75.1% yield, 91.0% purity) as a yellow solid which was confirmed via LCMS.

[0755] LCMS: Rt = 0.42 min, MS cal.: 601.3, MS observed: [M+H]+= 602.3.

[0756] HPLC: Rt = 2.72 min, purity: 91.0%. Procedure for preparation of compound 11

[0757] To a solution of compound 10 (2.00 g, 3.32 mmol, 1.00 eq.) in DMF (20 mL) was added DIEA (429 mg, 3.32 mmol, 578 μL, 1.00 eq.) and PNP (1.01 g, 3.32 mmol, 1.00 eq.). The mixture was stirred at 20-25 °C for 16 hrs. TLC (DCM / MeOH = 10:1, product Rf = 0.42) indicated compound 10 was consumed completely and one new spot formed. The crude product was triturated with isopropyl ether (200 mL) to give compound 11 (1.80 g, 2.35 mmol, 70.6% yield) as a yellow solid which was confirmed via LCMS. LCMS: Rt= 0.51 min, MS cal.: 766.3, MS observed: [M+H]+= 767.5. HPLC: Rt= 3.65 min, purity: 88.4%.Procedure for preparation of compound 12MMAE (1.69 g, 2.35 mmol, 1.00 eq.) and DIEA (910 mg, 7.04 mmol, 1.23 mL, 3.00 eq.). The mixture was stirred at 20-25 °C for 16 hrs. The reaction was monitored by TLC (DCM / MeOH = 10:1, product Rf= 0.27), TLC indicated the reactant was consumed. The crude product was triturated with isopropyl ether (200 mL) to give compound 12 (3.0 g, crude) as a yellow solid. Procedure for preparation of compound 13

[0759] A solution of compound 12 (3.00 g, 2.23 mmol, 1.00 eq.) in TEA (4.19 g, 41.4 mmol, 6.00 mL, 18.5 eq.) and DMF (24.0 mL) was stirred at 20-25 °C for 16 hrs. The reaction was monitored by TLC,TLC (DCM: MeOH = 10:1, product Rf = 0...

Claims

What is claimed is:

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

2. The tetrazine-based targeting agent of claim 1, wherein at least one targeting moiety is selected from Table 1.

3. The tetrazine-based targeting agent of claim 1 or 2, wherein each tetrazine moiety is independently of Formula I: I wherein:R1, at each occurrence, is independently selected from the group consisting of 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'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''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1; R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present; Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa;Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R21; Ra, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl; R21, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from 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''; R' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; and R''', at each occurrence, is independently selected from alkyl and aryl.

4. The tetrazine-based targeting agent of claim 3, wherein R3and R4are both absent, and the dotted lines are optional double bonds to form a tetrazine.

5. The tetrazine-based targeting agent of claim 3, wherein R3and R4are each independently hydrogen or a group capable of being removed after a triggering event, and the dotted lines are optional double bonds to form a dihydrotetrazine.

6. The tetrazine-based targeting agent of any one of claims 3-5, wherein R2, at each occurrence, is independently cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to three Z1.

7. The tetrazine-based targeting agent of any one of claims 1-6, wherein each tetrazine moiety is independently of Formula II:wherein:R1, at each occurrence, is independently selected from the group consisting of 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'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''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1; R21, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; Ring A, at each occurrence, is independently cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to three Z1; t, at each occurrence, is independently 0, 1, 2, 3, or 4; each Z1is independently selected from 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''; R' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; andR''', at each occurrence, is independently selected from alkyl and aryl.

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

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

10. The tetrazine-based targeting agent of any one of claims 1-9, wherein the tetrazine-based targeting agent comprises more than one tetrazine moiety.

11. The tetrazine-based targeting agent of any one of claims 1-7, wherein the linker is linear.

12. A tetrazine-based targeting agent of Formula III: III wherein:X is a targeting agent selected from Table 1; L is a linker; p is 1-16; R1, at each occurrence, is independently selected from the group consisting of 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'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''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, heterocyclyl, cycloalkyl, aryl, or heteroaryl, is independently optionally substituted with one to three Z1; R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; the dotted lines represent optional double bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present;Y1, at each occurrence, is independently selected from the group consisting of a direct bond, O, S, NRa, or CR31aR31bNRa; Y2, at each occurrence, is independently a direct bond, alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, haloalkylene, heteroalkylene, cycloalkylene, heterocyclylene, arylene, or heteroarylene, is independently optionally substituted with one to four R21; Ra, R31a, and R31b, at each occurrence, are independently selected from the group consisting of hydrogen, alkyl, and haloalkyl; R21, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; each Z1is independently selected from 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''; R' and R", at each occurrence, are independently selected from hydrogen, alkyl, and aryl; and R''', at each occurrence, is independently selected from alkyl and aryl.

13. A tetrazine-based targeting agent of Formula IA, Formula IIA, or Formula VA: VAwherein: ring A is aryl, cycloalkyl, heterocyclyl, or heteroaryl;the dotted lines represent additional bonds to form a tetrazine when R3and R4are both absent, or a dihydrotetrazine when R3and R4are both present; X is a targeting agent selected from Table 1; p is 1-16; L, at each occurrence, is independently a linker; R1, at each occurrence, is independently selected from the group consisting of 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", 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''; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; R2, at each occurrence, is 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; wherein each alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1; R3and R4are both absent; or R3and R4are each independently hydrogen or a group capable of being removed after a triggering event; R20, at each occurrence, is independently selected from the group consisting of 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', 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''; R22, at each occurrence, is independently a linker of 1 to 100 linking atoms optionally comprising one or more ethylene-oxy, amine, ester, amide, carbamate, carbonate, or ketone functional group; R30, at each occurrence, is independently halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, or cycloalkenyl; Ra, R31a, and R31bare each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl; each Z1is independently selected from 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''; R' and R", at each occurrence, are independently selected from hydrogen, aryl, and alkyl; R''', at each occurrence, is independently selected from aryl and alkyl; and t, at each occurrence, is independently 0, 1, 2, 3, or 4.

14. The tetrazine-based targeting agent of claim 12, wherein the moiety: is represented by:.

15. The tetrazine-based targeting agentof claim 3, wherein the moiety:is represented by, , , 16.e e a e-ase age g age o a y oe o ca s - , wee , a eac occurrence, is independently hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl; wherein each alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl is optionally substituted with one to three Z1.

17. The tetrazine-based targeting agent of any one of claims 3-16, wherein R1, at each occurrence, is independently hydrogen or alkyl optionally substituted with one to three Z1.

18. The tetrazine-based targeting agent of any one of claims 3-17, wherein Z1, at each occurrence, is independently selected from halo, hydroxy, alkoxy, and OC(=O)OR'.

19. The tetrazine-based targeting agent of any one of claims 3-18, wherein R21, at each occurrence, is independently halo, alkyl, or haloalkyl.

20. The tetrazine-based targeting agent of any one of claims 3-18, wherein t, at each occurrence, is 0.

21. The tetrazine-based targeting agent of any one of claims 3-20, wherein R2or Ring A is pyridyl or phenyl.

22. The tetrazine-based targeting agent of any one of claims 3-20, wherein R2or Ring A is other than pyridyl or phenyl.

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

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

25. The tetrazine-based targeting agent of any of claims 1-24, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises one or more amino acids.

26. The tetrazine-based targeting agent of any of claims 1-25, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises a polypeptide.

27. The tetrazine-based targeting agent of any of claims 1-26, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises one or more of a hydrazone, a hydrazide, a disulfide, a N-succinimidyl-4-(2- pyridyldithio)pentanoate (SPP), a N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), a 4-(4’- acetylphenoxy)butanoic acid (AcBut), one or more linear or branched, natural or unnatural amino acid, a valine-citrulline (Val-Cit) moiety, or a phenylalanine-lysine (Phe-Lys) moiety.

28. The tetrazine-based targeting agent of any one of claims 1-27, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises 1 to 100 linking atoms, from 1 to 50 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms, or from 1 to 40 linking atoms, or from 1 to 30 linking atoms, or from 1 to 20 linking atoms, or from 1 to 10 linking atoms, or from 1 to 5 linking atoms, or from 5 to 30 linking atoms, or from 10 to 30 linking atoms, or from 5 to 40 linking atoms, or from 5 to 50 linking atoms, or from 10 to 50 linking atoms.

29. The tetrazine-based targeting agent of any one of claims 1-28, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl.

30. The tetrazine-based targeting agent of any one of claims 1-29, wherein L, -Y2-Y1-L, -Y2-L, or the linker comprises one or more chain heteroatoms and one or more alkylene, alkenylene, alkynylene,arylene, or heteroarylene, moieties; wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene moiety, may be independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4 alkyl, C1-4 alkoxy, and C1-4 haloalkyl.

31. The tetrazine-based targeting agent of any one of claims 1-30, wherein L, -Y2-Y1-L, -Y2-L, or the linker is an alkylene linker optionally comprising one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, thio-succinimide, or ketone functional groups.

32. The tetrazine-based targeting agent of any one of claims 1-31, wherein L, -Y2-Y1-L, -Y2-L, or the linker is of the formula: -Y10-(CHR130)n’-Y20-(CHR140)n''-Y30-(CHR150)m''-Y40- wherein: each of Y10, Y20, Y30, and Y40are independently a bond, -NR110-, -O-, -S(O)0-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-, -OC(O)O-, -(CH2CH2O)1-5-, -C(O)O-, alkylene, alkenylene, alkynylene, arylene, or heteroarylene; wherein each alkylene, alkenylene, alkynylene, arylene, or heteroarylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4alkyl, C1-4alkoxy, and C1-4haloalkyl; each R110is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R120is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; each R130is independently hydrogen, C1-4alkyl, C1-4haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; each R140is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; each R150is independently hydrogen, C1-4alkyl, C1-4haloalkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, or an amino acid side chain; and n', n'', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

33. The tetrazine-based targeting agent of any one of claims 1-31, wherein L, -Y2-Y1-L, -Y2-L, or the linker is of the formula: -Y10-(CH2)n’-Y20-(CH2)m''-Y30- wherein: each of Y10, Y20, and Y30are independently a bond, -NR110-, -O-, -S(O)0-2-, -NR110C(O)-, -C(O)NR110-, -NR110S(O)2-, -S(O)2NR110-, -CR120=N-NR110-, -NR110-N=CR120-, -C(O)-, -OC(O)-,-OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene; wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently optionally substituted with one to five substituents independently selected from oxo, halo, C1-4alkyl, C1-4alkoxy, and C1-4haloalkyl; each R110is independently hydrogen, C1-4 alkyl, C1-4 haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; each R120is independently hydrogen, C1-4alkyl, C1-4haloalkyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

34. The tetrazine-based targeting agent of any one of claims 1-33, wherein L, -Y2-Y1-L, -Y2-L, or the linker is or comprises: , , ,35. The tetrazine-based targeting agent of any one of claims 1-33, wherein L, -Y2-Y1-L, -Y2-L, or the linker is: ,, orID wherein X i37. A tetrazine-based targeting agent of Formula IIH: IH wherein X is.

38. A tetrazine-based targeting agent, or a pharmaceutically acceptable salt thereof, as shown in Table 2.

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

40. A method of treating cancer or enhancing or eliciting an immune response, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the tetrazine- based targeting agent of any of claims 1-38, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 39, and a conjugate comprising a payload linked to one or more trans-cyclooctene moieties, or a pharmaceutically acceptable salt thereof.

41. The method of claim 40, wherein the conjugate is of Formula X, or a pharmaceutically acceptable salt thereof:X wherein:G, at each occurrence, is independentl ; L1, at each occurrence, is independentlm is an integer from 1-150; D is a payload; R1A, at each occurrence, is independently selected from the group consisting of C1-4alkyl, C1-4haloalkyl, and C1-4alkoxy; q is 0, 1, or 2; q1 is 0 or 1; R1B, at each occurrence, is independently selected from the group consisting of G1, OH, –NR1c–C1-4alkylene–G1, –NR1c–C1-4alkylene–N(R1d)2, –NR1c–C1-6alkylene–N(C1-4alkyl)3+, –N(R1c)CHR1eCO H –N(R1c)–C alk lene–CO H –N(R1f)–C alk lene–(N(C alk lene–CO H)– ne–C1-6alkylene–CO2H)2, and –N(R1c)–CH(CH2O–(CH2CH2O)0-2–C1-6alkylene–CO2H)2; R1cand R1d, at each occurrence, are independently hydrogen or C1-4alkyl; R1e, at each occurrence, is independently –C1-4alkylene–CO2H, –C1-4alkylene–CONH2, or –C1-4alkylene–OH; R1f, at each occurrence, is independently hydrogen, C1-6alkyl, or C1-4alkylene–CO2H; n, at each occurrence, is independently 0, 1, 2, or 3; L2, at each occurrence, is independently selected from the group consisting of –C(O)– and C1-3alkylene; and G1, at each occurrence, is independently an optionally substituted heterocyclyl.

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

43. The method of any one of claims 40-42, wherein the payload is a therapeutic monoclonalantibody, cytokine, chemokine, chemokine antagonist, and immune checkpoint inhibitor payload; or a pharmaceutically acceptable salt thereof.

44. The method of any one of claims 40-43, wherein the payload is selected from a therapeutic agent for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, Carmustine, Ixabepilone, Patupilone (epothelone class), platinum drugs, exatecan, auristatin (dolastatin 10, MMAE, MMAD, MMAF) mitomycin C, bleomycin, calicheamicin, staurosporine, hemiasterlin, and the like), an immunosuppressant (e.g., cyclosporin A, rapamycin, and the like), an anti-fungal agent (e.g., Amphotericin, and the like), an antibiotic (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin Β, flucytosine, emtricitabine, gentamicin, colistin, and the like), lurbinectedin, gardiquimod, a matrix metalloproteinase (ΜΜΡ) inhibitor, L-dopa, oseltamivir, cefalexin, 5- aminolevulinic acid, cysteine, celecoxib, nimodipine, vancomycin, daptomycin, and cyclic-adenosine monophosphatidyl (c-AMP).

45. The method of claim 40, wherein the conjugate is selected from: ,,47. The method of claim 46, wherein the cancer is a melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer , glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric carcinoma, squamous cell carcinoma of head / neck, anal / vulvar carcinoma, esophageal carcinoma, pancreatic adenocarcinoma, cervical carcinoma, hepatocellular carcinoma, Kaposi’s sarcoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma Wilm’s tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, Prostatic adenocarcinoma, Nasopharyngeal carcinoma, or Cutaneous T-cell lymphoma.

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

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

50. The method of claim 49, wherein the cancer is a hematological malignancy such as myelodysplastic syndrome, acute myeloid leukemia, myelodisplastic syndromes, chronic myelogenous leukemia, chronic myelomonocytic leukemia, primary myelofibrosis, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, monoclonal gammopathy, plasma cell myeloma, follicular lymphoma, marginal zone lymphoma, classical Hodgkin’s 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’s lymphoma.

51. The method of any of claims 40-50, wherein the method is a method of enhancing or eliciting an immune response.

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

53. The method of any of claims 40-52, further comprising administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anticancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

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

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

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