trans-Cyclooctene conjugates

Abstract

The present disclosure relates generally to trans-cyclooctene conjugates for bioorthogonal delivery of payloads to targeted locations in a subject. The compositions and methods have applications in the treatment of cancer, tumor growth, and immunotherapy.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 273,777, filed October 29, 2021, and is incorporated by reference in its entirety.

[0002] [Reference to electronic sequence listing] The contents of the electronic format sequence listing (2022-10-31_Sequence_Listing_63XT-342804-WO.xml, size: 4881 bytes, and creation date: October 31, 2022) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates generally to trans-cyclooctene conjugates for bioorthogonal delivery of payloads to targeted locations in a subject, with applications, for example, in the treatment of cancer, tumor growth, and immunotherapy. [Background technology]

[0004] Bioorthogonal conjugation or click reactions are selective and orthogonal (non-interacting) functionalities found in biological systems that are used in a variety of applications in chemistry, chemical biology, molecular diagnostics, and medicine, where they can be used to facilitate the selective manipulation of molecules, cells, particles, and surfaces, as well as the tagging and tracking of biomolecules in vitro and in vivo. These reactions include the Staudinger ligation reaction, the azide-cyclooctyne cycloaddition reaction, and the inverse electron demand Diels-Alder reaction. Summary of the Invention

[0005] Provided herein are conjugates comprising a payload covalently attached via a linker to one or more optionally substituted trans-cyclooctene moieties for use in bioorthogonal reactions. In some embodiments, the payload is selected from an inhibitor of poly(ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, and a monoclonal antibody, or a derivative or analog thereof.

[0006] In some embodiments, a method is provided for delivering an effective amount of a payload (i.e., an inhibitor of poly(ADP-ribose) polymerase (PARP inhibitor), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, and a monoclonal antibody, or a derivative or analog thereof) to a target location in a subject, comprising administering to the subject a therapeutic support composition described herein to the target location, and administering to the subject a conjugate or a pharma- ceutically acceptable salt thereof or a composition described herein.

[0007] In some embodiments, a method of treating cancer is provided comprising administering to a subject in need of treatment a therapeutic support composition described herein at a target location and administering to the subject a conjugate or a pharma- ceutically acceptable salt thereof or a composition described herein.

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

[0009] In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a lymphoma or leukemia. In some embodiments, the cancer is a hematopoietic malignancy. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the effect of the trans-cyclooctene conjugate of Example 20 on the proliferation of fresh mouse splenocytes versus unmodified gardiquimod in the absence or presence of a tetrazine activator. In Figure 1, concentrations up to 10 μg / mL are tested. [Diagram 2] Figure 2 shows the effect of the trans-cyclooctene conjugate of Example 20 on the proliferation of fresh mouse splenocytes versus unmodified gardiquimod in the absence or presence of a tetrazine activator. In Figure 2, conjugates up to 50 μg / mL with or without tetrazine are tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The following description sets forth exemplary embodiments of the present technology, however, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but rather is provided as a description of exemplary embodiments.

[0012] 1.Definition It is understood that certain features that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. In contrast, various features that are described for brevity in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically included and disclosed herein as if each and every combination were individually and explicitly disclosed, to the extent that such combinations include subject matter that is, for example, a stable compound (i.e., a compound that can be made, isolated, characterized, and tested for biological activity). Furthermore, all subcombinations of the various embodiments and elements thereof (e.g., elements of chemical groups listed in the embodiments describing such variables) are also specifically included and disclosed herein as if each and every subcombination were individually and explicitly disclosed herein.

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

[0014] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0015] The modifier "about" used in connection with a quantity is inclusive of the specified value and has the meaning dictated by the context (e.g., it includes at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses a range of "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 0.9 to 1.1. Other meanings of "about" (e.g., rounding) may be apparent from the context, so for example, "about 1" may mean 0.5 to 1.4.

[0016] The conjunction term "or" includes any and all combinations of one or more of the listed elements associated by the conjunction term. For example, the phrase "a device comprising A or B" may refer to a device comprising A but not B, a device comprising B but not A, or a device comprising both A and B. The phrase "at least one of A, B, ... and N" or "at least one of A, B, ... N, or combinations thereof" is defined in its broadest sense to mean any combination including one or more elements selected from the group comprising A, B, ... and N, i.e., one or more of the elements A, B, ... or N, including any one element alone or in combination with one or more other elements, which may include additional unlisted elements in combination.

[0017] The definitions of specific functional groups and chemical terms are explained in more detail below.For the purpose of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version) on the inside cover of Handbook of Chemistry and Physics (75th edition), and specific functional groups are generally defined as described therein.Furthermore, the 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, 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987 (the entire contents of each are incorporated herein by reference).

[0018] 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.

[0019] The term "alkyl" as used herein means a linear or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The term "lower alkyl" or "C1-C6-alkyl" means a linear or branched hydrocarbon containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" means a linear or branched hydrocarbon containing 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0020] The term "alkenyl" as used herein means a hydrocarbon chain containing 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 phenyl.

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

[0022] 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.

[0023] 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, e.g., 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-.

[0024] The term "amino acid" refers to both natural and unnatural amino acids, protected natural and unnatural amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids include the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., by way of example only, an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group. While retaining the same basic chemical structure as a natural amino acid, such analogs may have modified R groups (e.g., norleucine, by way of example only) or may retain a modified peptide backbone. Non-limiting examples of amino acid analogs include citrulline, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, homophenylalanine, ornithine, formylglycine, phenylglycine, para-azidophenylglycine, para-azidophenylalanine, para-acetophenylalanine, 4-(3-methyl-(1,2,4,5-tetrazine))-phenylglyine, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.

[0025] The term "aryl" as used herein refers to a phenyl group, or a bicyclic aryl fused ring system or a tricyclic aryl fused ring system. A bicyclic fused ring system is exemplified by a phenyl group attached to a parent molecular moiety and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to a 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. Monocyclic aryls, bicyclic aryls, and tricyclic aryls are connected to the parent molecular moiety through any carbon atom contained within the ring and can be unsubstituted or substituted.

[0026] As used herein, the term "azide" refers to the functional group -N3.

[0027] The term "cycloalkyl" as used herein refers to a carbocyclic ring system containing 3 to 10 carbon atoms, no heteroatoms, and no double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes a carbocyclic ring system in which a cycloalkyl group is attached to a 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.

[0028] The term "cycloalkenyl" as used herein means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having from 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0029] 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).

[0030] 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, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, such as 3,3,3-trifluoropropyl.

[0031] 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.

[0032] The term "fluoroalkoxy" as used herein means at least one fluoroalkyl group, as defined herein, 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.

[0033] The term "halogen" or "halo" as used herein means Cl, Br, I, or F.

[0034] 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 are replaced by halogen.

[0035] The term "haloalkoxy," as used herein, means at least one haloalkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.

[0036] The term "heteroalkyl" as used herein means an alkyl group, as defined herein, in which one or more carbon atoms are 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.

[0037] The term "heteroaryl" as used herein refers to an aromatic monocyclic ring, or an aromatic bicyclic ring system, or an aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. A bicyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring attached to a 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. Tricyclic heteroaryl groups are exemplified by monocyclic heteroaryl rings attached to the parent molecular moiety and fused to two of the monocyclic cycloalkyl groups, as defined herein, monocyclic aryl groups, as defined herein, monocyclic heteroaryl groups, or monocyclic heterocycles, as defined herein. Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-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, benzoxadiazolyl, and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic heteroaryl, bicyclic heteroaryl, and tricyclic heteroaryl are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring and can be unsubstituted or substituted.

[0038] The term "heterocycle" or "heterocyclic" as used herein means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero or one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The seven- and eight-membered rings contain zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, o- ... Examples of such radicals include xazolidinyl, 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-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A 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 heterocyclic group, or a bridged monocyclic heterocyclic 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 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles 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 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane), and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 ]decane). Monocyclic heterocycles, bicyclic heterocycles, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0039] As used herein, the term "hydroxyl" refers to an --OH group.

[0040] 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.

[0041] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix “C x ~C y -" or "C x~y " where x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms in the substituent. Thus, for example, "C1-C3-alkyl" and "C 1~3 "Alkyl" refers to an alkyl substituent containing 1 to 3 carbon atoms. x ~C y -" and "C x~y " notations are used interchangeably and have the same meaning.

[0042] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents, including, but 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.

[0043] The term "tetrazine" refers to a substituted or unsubstituted aromatic cyclic group of two carbon atoms and four 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.

[0044] The term "selectively deliver" refers to the delivery of an agent (e.g., payload) to an organ or tissue (or portion thereof) requiring treatment or diagnosis without significant binding to other non-target organs or tissues (or portions thereof).

[0045] The term "payload" refers to an agent that is delivered to a target site in a subject. Payloads include therapeutic agents.

[0046] 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 the therapeutic agent.

[0047] The term "diagnostic agent" refers to an agent that aids in the diagnosis of a condition or disease. Exemplary diagnostic agents include imaging agents such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are imaging agents that exhibit magnetism 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 with radiation at one wavelength and detection with radiation at a second, different wavelength. Optical probes of the present disclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate), and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. Radionuclides are elements that undergo detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 89 Zr, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu, and 241 Am is one example.

[0048] 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 a particular target. "Stably associated" or "stable association" means that a moiety binds to or associates with another moiety or structure under standard physiological conditions. Bonds can include covalent or 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. A targeting agent can be one member of a specific binding pair such as, but not limited to, one member of a receptor / ligand pair, a ligand-binding portion of a receptor, one member of an antibody / antigen pair, an antigen-binding fragment of an antibody, a hapten, one member of a lectin / carbohydrate pair, one member of an enzyme / substrate pair, biotin / avidin, biotin / streptavidin, digoxin / antidigoxin, one member of a DNA or RNA aptamer binding pair, one member of a peptide aptamer binding pair, etc. Targeting agents include ligands that specifically bind (or substantially specifically bind) to a particular clinically relevant target receptor or cell surface target. Ligands can be antibodies, peptides, nucleic acids, phages, bacteria, viruses, or other molecules that have specific affinity for a target receptor or cell surface target. Examples of receptors and cell surface targets include, but are not limited to, PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, LT4, a human monoclonal antibody against the inhibitory immune checkpoint receptor immunoglobulin-like transcript 4 (ILT4), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), lymphocyte immunoglobulin-like receptor 2 (LIR2), monocyte / macrophage immunoglobulin-like receptor 10 (MIR-10), CD85d, or other cell receptors or cell surface targets. Additional examples are included in the various embodiments disclosed herein.

[0049] The term "targeted organ or tissue" refers to an organ or tissue that is targeted for delivery of a payload. Exemplary targeted organs and tissues include organs and tissues that can be targeted by chemical or biological targeting agents, as well as organs and tissues that cannot be targeted by chemical or biological targeting agents.

[0050] The term "implant" refers to surgical implantation into the body of a subject.

[0051] The term "contacting" or "contacting" refers to the process of bringing at least two different species into contact so that they may interact with each other, for example, in a non-covalent or covalent interaction or binding reaction. However, it should be understood that the resulting complex or reaction product may be produced directly from the interaction or reaction between the added reagents, or may be produced from an intermediate that may be produced in the contacting mixture that is derived from one or more of the added reagents or moieties.

[0052] The term "binding agent" refers to an agent having a functional group capable of forming a covalent bond with a complementary functional group of another binding agent in a biological environment. The binding between binding agents in a biological environment may also be referred to as a bioconjugation reaction. Binding agents include bioorthogonal binding agents, which are binding agents having a bioorthogonal functional group. The bioorthogonal functional group of a bioorthogonal binding agent selectively reacts with the complementary bioorthogonal functional group of another bioorthogonal binding counterpart. The selective reaction between bioorthogonal binding counterparts 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, azides and alkynes that form triazoles via click chemistry, trans-cyclooctene (TCO) and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine), and the like. Binding agents useful in the present disclosure may have high reactivity with the corresponding binding agent, such that the reaction is rapid.

[0053] The term "functionalized" refers to a moiety having a functional group attached to it, e.g., a moiety having a binding agent functional group (e.g., a bioorthogonal functional group) attached thereto.

[0054] The term "administering" refers to any suitable route of administration to a subject, including, but not limited to, oral administration, administration as a suppository, topical contact, parenteral administration, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intranasal or subcutaneous administration, intrathecal administration, or implantation of a sustained release device, e.g., a mini-osmotic pump, into a subject.

[0055] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0056] The term "leaving group" refers to an atom (or group of atoms) with electron-withdrawing capability that can be displaced as a stable chemical species and therewith accept a bonding electron. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.

[0057] The terms "pharmaceutical 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 symptoms thereof and / or prevent or reduce the risk of onset or recurrence of a disease or disorder or symptom(s) thereof. With respect to tumorigenic proliferative disorders, a pharmaceutical effective amount or therapeutically effective amount includes, inter alia, an amount sufficient to shrink a tumor or reduce the rate of tumor growth.

[0058] As used herein, the terms "subject", "patient" or "organism" include humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical subjects to which the agent(s) of the present disclosure may be administered include mammals, particularly primates, especially humans. For veterinary applications, suitable subjects may include, for example, livestock animals such as cattle, sheep, goats, cows, pigs, etc., poultry such as chickens, ducks, geese, turkeys, etc., and domesticated animals, particularly pets, such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs, such as inbred pigs.

[0059] The term "treat" or "treatment" as used herein means treating or the care of a disease or condition or symptom(s) thereof in a patient, such as a mammal (particularly a human), including (a) ameliorating the disease or condition or symptom(s) thereof, e.g., eliminating or causing the regression of the disease or condition or symptom(s) thereof in the patient, (b) inhibiting the disease or condition or symptom(s) thereof, e.g., by slowing the onset of or arresting the disease or condition or symptom(s) thereof in the patient, or (c) alleviating the symptom of the disease or condition or symptom(s) thereof in the patient.

[0060] The term "physiological conditions" is intended to encompass conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0061] For the compounds described herein, the groups and substituents thereof may be selected in accordance with the allowed valences of the atoms and substituents, such that the selection and substitution results in stable compounds, e.g., compounds that do not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, and the like.

[0062] When a range of values ​​is given, it is understood that each intervening value between the upper and lower limit of that range, and any other specified or intervening value in that stated range, to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included within the invention, 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 invention.

[0063] In the description of numerical ranges herein, each intervening value therebetween is expressly contemplated to the same degree of accuracy, for example, in the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0064] Compounds may exist as stereoisomers in which asymmetric or chiral centers exist. Stereoisomers are "R" or "S" depending on the configuration of the substituents around the chiral carbon atom. The terms "R" and "S" as used herein are configurations as defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem., 1976, 45: 13-30. Various stereoisomers and mixtures thereof are contemplated in the present disclosure and are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds may be prepared by synthesis from commercially available starting materials containing asymmetric or chiral centers, or by preparation of racemic mixtures followed by resolution methods well known to those skilled in the art. These resolution methods are described in (1) Furniss, Hannaford, Smith, and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5 thedition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) by the direct separation of the mixture of optical enantiomers on a chiral chromatographic column, or (3) by fractional recrystallization methods.

[0065] It is understood that the compounds may have geometric isomers as well as tautomeric forms and these also form an aspect of the present invention.

[0066] The present disclosure also includes isotopically labeled compounds, which are identical to the compounds enumerated herein except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, including, but not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively. 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Cl. Deuterium (i.e. 2 Substitution with heavier isotopes such as H) may be preferred in some circumstances as it may confer certain therapeutic advantages resulting from greater metabolic stability, for example increased half-life in vivo or reduced dosage requirements. The compounds may also include positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that may be included are: 11 C. 13 N, 15 O, and18 F. Isotopically labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0067] B. Conjugates Provided herein is a conjugate for use in a bioorthogonal reaction. In some embodiments, the conjugate has the formula I: [ka] (In the formula, m is an integer from 1 to 150; G is independently at each occurrence an optionally substituted trans-cyclooctene moiety; D 1 is a payload selected from an inhibitor of poly(ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, a monoclonal antibody, a topoisomerase inhibitor, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, mitomycin C, etoposide, and exatecan, or a derivative or analog thereof; L 1 is, in each instance, independently a linker), or a pharma- ceutically acceptable salt thereof.

[0068] Provided herein is a conjugate for use in a bioorthogonal reaction. In some embodiments, the conjugate has the formula I: [ka] (In the formula, m is an integer from 1 to 150; G is independently at each occurrence an optionally substituted trans-cyclooctene moiety; D 1is a payload selected from an inhibitor of poly(ADP-ribose) polymerase (PARP), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, and a monoclonal antibody, or a derivative or analog thereof; L 1 is, in each instance, independently a linker), or a pharma- ceutically acceptable salt thereof.

[0069] In some embodiments of the conjugates described herein, each trans-cyclooctene moiety is independently selected from the group consisting of: [ka] (In the formula, R 1A is, in each case independently, C 1~4 Alkyl, C 1~4 Haloalkyl and C 1~4 alkoxy; q is 0, 1, or 2; q1 is 0 or 1, R 1B In each case, independently, G 1 , -OH, -NR 1c -C 1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -NR 1c -C 1~6 Alkylene-N(C 1~4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R 1c )-C 1~6 Alkylene-COH, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-COH)-C 2~4 Alkylene) n -N(C 1~4 Alkylene -COH)2, -N(R1c )CHR 1e C(O)OC 1~6 Alkyl, -N(R 1c )-C 1~6 Alkylene-C(O)OC 1~6 Alkyl, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-C(O)OC 1~6 Alkyl)-C 2~4 Alkylene) n -N(C 1~4 Alkylene-C(O)OC 1~6 alkyl)2, -N(R 1c )-C 1~6 Alkylene-SO3H, -N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene -COH) and -N(R 1c )-CH(CH2O-(CH2CH2O) 0~2 -C 1~6 alkylene-COH), R 1c and R 1d is, in each occurrence, independently hydrogen or C 1~4 is alkyl, R 1e is, in each case independently, -C 1~4 Alkylene-COH, -C 1~4 Alkylene-CONH2 or -C 1~4 alkylene-OH, R 1f is, in each occurrence, independently hydrogen, C 1~6 Alkyl or C 1~4 alkylene-COH, n is independently at each occurrence 0, 1, 2, or 3; L 2 is, independently in each occurrence, -C(O)- and C 1~3 alkylene; and G 1is independently at each occurrence an optionally substituted heterocyclyl.

[0070] In some embodiments of the conjugates described herein, each trans-cyclooctene moiety (G) is independently [ka] (In the formula, R 1A is, in each case independently, C 1~4 Alkyl, C 1~4 Haloalkyl and C 1~4 alkoxy; q is 0, 1, or 2; q1 is 0 or 1, R 1B In each case, independently, G 1 , -OH, -NR 1c -C 1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -NR 1c -C 1~6 Alkylene-N(C 1~4 Alkyl)3 + , -N(R 1c )CHR 1e CO2H, -N(R 1c )-C 1~6 Alkylene-COH, -N(R 1f )-C 2~4 Alkylene-(N(C 1~4 Alkylene-COH)-C 2~4 Alkylene) n -N(C 1~4 Alkylene -COH)2, -N(R 1c )CHR 1e C(O)OC 1~6 Alkyl, -N(R 1c )-C 1~6 Alkylene-C(O)OC 1~6 Alkyl, -N(R 1f )-C 2~4 Alkylene-(N(C1~4 Alkylene-C(O)OC 1~6 Alkyl)-C 2~4 Alkylene) n -N(C 1~4 Alkylene-C(O)OC 1~6 alkyl)2, -N(R 1c )-C 1~6 Alkylene-SO3H, -N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene -COH) and -N(R 1c )-CH(CH2O-(CH2CH2O) 0~2 -C 1~6 alkylene-COH), R 1c and R 1d is, in each occurrence, independently hydrogen or C 1~4 is alkyl, R 1e is, in each case independently, -C 1~4 Alkylene-COH, -C 1~4 Alkylene-CONH2 or -C 1~4 alkylene-OH, R 1f is, in each occurrence, independently hydrogen, C 1~6 Alkyl or C 1~4 alkylene-COH, n is independently at each occurrence 0, 1, 2, or 3; L 2 is, independently in each occurrence, -C(O)- and C 1~3 alkylene; and G 1 is independently at each occurrence an optionally substituted heterocyclyl.

[0071] In some embodiments, the trans-cyclooctene moiety (G) is [ka] and R2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 may optionally be further substituted with a polyether.

[0072] In some embodiments, the trans-cyclooctene moiety (G) is [ka] It is.

[0073] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0074] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0075] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0076] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0077] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0078] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0079] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0080] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0081] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0082] In some embodiments, the trans-cyclooctene moiety is [ka] It is.

[0083] In some embodiments, GL 1 is, in each case independently, [ka] and R 2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 may optionally be further substituted with a polyether.

[0084] In some embodiments, m is 1 to 20. In some embodiments, m is 1 to 10. In some embodiments, m is 1 to 5. In some embodiments, m is 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, m is 1.

[0085] Also provided is a pharmaceutical composition comprising a conjugate disclosed herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0086] payload The term "payload" as used herein is intended to refer to inhibitors of poly(ADP-ribose) polymerase (PARP inhibitors), duocarmycins, pyrrolobenzodiazepines (PBDs), hemiasterlin, HTI-286, and monoclonal antibodies, or derivatives or analogs thereof.

[0087] In certain embodiments, the term "derivative" or "analog" or "derived from" as used in reference to a payload refers to a compound in which one or more atoms, including hydrogen or non-hydrogen atoms, of the original unmodified payload are substituted with one or more linkers L. 1 D 1 The payload is derived from a known payload and the linker L 1 is modified to be covalently attached to at least one optionally substituted trans-cyclooctene via D 1 After modification to arrive at the compounds described herein, the payloads retain biological activity comparable to that observed in the original unmodified payload. In certain embodiments, D 1 The payloads exhibit binding activity or inhibition that is at least about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% of the binding activity or inhibition observed in the original unmodified payload.

[0088] In certain embodiments, a hydrogen atom bonded to a heteroatom (e.g., N, O, or S) of the original unmodified payload is linked to the linker L 1 In certain embodiments, a halogen atom on the payload is replaced and attached to the remainder of the compound. In certain embodiments, a hydrogen atom on the payload is replaced and attached to the remainder of the compound. In certain embodiments, 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.

[0089] In some embodiments, at least one payload is selected from inhibitors of poly(ADP-ribose) polymerase (PARP), duocarmycins, pyrrolobenzodiazepines (PBDs), hemiasterin, HTI-286, anti-CD3 (αCD3) monoclonal antibodies, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, paclitaxel, gemcitabine, mitomycin C, etoposide, exatecan, and MMAE, or derivatives or analogs thereof.

[0090] In some embodiments, at least one payload is selected from inhibitors of poly(ADP-ribose) polymerase (PARP), duocarmycins, pyrrolobenzodiazepines (PBDs), hemiasterlin, HTI-286, and anti-CD3 (αCD3) monoclonal antibodies, or derivatives or analogs thereof.

[0091] 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.

[0092] The monoclonal antibody used as a payload herein can be a whole monoclonal antibody, or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In some embodiments, the antibody is an immune cell engager and will itself induce or elicit an immune response. In some embodiments, the antibody or fragment thereof is selected from the group consisting 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 1443 ... 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).

[0093] In certain embodiments, the payload is an antibody or antibody fragment that targets CD3, such as OKT3, SP34, UCHT1, teplizumab, otelixizumab, vicilizumab, or foralumab, or an antibody fragment derived therefrom.

[0094] In certain embodiments, the payload is an antibody or antibody fragment that targets CD28, such as celalizumab, TGN1412, or FR104, or an antibody fragment derived therefrom.

[0095] In certain embodiments, the payload is an antibody or antibody fragment that targets CD137 (4-1BB), such as utomirumab, urelumab, LVGN6051, or AGEN2373, or an antibody fragment derived therefrom.

[0096] In certain embodiments, the payload is an antibody or antibody fragment that targets CD16, such as AFM13, or an antibody fragment derived therefrom.

[0097] In certain embodiments, the payload is an antibody or antibody fragment that targets NKG2D, such as NNC0152-0002 or JNJ-64304500, or an antibody fragment derived therefrom.

[0098] In certain embodiments, the payload is an antibody or antibody fragment that targets CD64, such as H22, or an antibody fragment derived therefrom.

[0099] In certain embodiments, the payload is an antibody or antibody fragment that targets GITR / TNFRSF18, such as MK-4166, TRX518, MS-986156, AMG-228, or INCAGN01876, or an antibody fragment derived therefrom.

[0100] In certain embodiments, the payload is an antibody or antibody fragment that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0101] In certain embodiments, the payload is an antibody or antibody fragment that targets CD40, such as iscalimab, ABBV-323, bleselumab (ASKP-1240), BI-655064, FFP-104, BMS986090, dacetuzumab, or lucatumumab, or an antibody fragment derived therefrom.

[0102] In certain embodiments, the payload is an antibody or antibody fragment that targets CD4, such as MAX.16H5, IT1208, zanolimumab (HuMax-CD4), UB-421, or MTRX1011A, or an antibody fragment derived therefrom.

[0103] In certain embodiments, the payload is an antibody or antibody fragment that targets CXCR4, such as F50067, or an antibody fragment derived therefrom.

[0104] In certain embodiments, the payload is an antibody or antibody fragment that targets G-CSFR, such as CSL324, or an antibody fragment derived therefrom.

[0105] In certain embodiments, the payload is an antibody or antibody fragment that targets GM-CSFR, such as mavrilimumab, or an antibody fragment derived therefrom.

[0106] In certain embodiments, the payload is an antibody or antibody fragment that targets CD122, such as Hu-Mik(β)1, or an antibody fragment derived therefrom.

[0107] In certain embodiments, the payload is an antibody or antibody fragment that targets PD-1, such as CC-90006, cemiplimab, camrelizumab, or TSR-042, or an antibody fragment derived therefrom.

[0108] In certain embodiments, the payload is an antibody or antibody fragment that targets CTLA4, such as tremelimumab or ipilimumab, or an antibody fragment derived therefrom.

[0109] In certain embodiments, the payload is an antibody or antibody fragment that targets LAG3, such as leratolimab (BMS-986016), GSK2831781, cemiplimab (REGN3767), favezelimab, yelamirimab, or mavezelimab, or an antibody fragment derived therefrom.

[0110] In certain embodiments, the payload is an antibody or antibody fragment targeting TIGIT, such as BMS-986207, tiragolumab, vibostolimab, etigilimab, domvanalimab, ASP-8374, IBI939, BGB-A1217, COM902, or M6223, or an antibody fragment derived therefrom.

[0111] In certain embodiments, the payload is an antibody or antibody fragment that targets NCR1, such as hNKp46.02, or an antibody fragment derived therefrom.

[0112] In certain embodiments, the payload is an antibody or antibody fragment that targets TIM3, such as covolimab, Sym023, LY3321367, BMS-986258, SHR-1702, dabatolimab, or INCAGN02390, or an antibody fragment derived therefrom.

[0113] In certain embodiments, the payload is an antibody or antibody fragment that targets VISTA, such as SG7, K01401-020, CI-8993, or JNJ-61610588, or an antibody fragment derived therefrom.

[0114] In certain embodiments, the payload is an antibody or antibody fragment that targets CD134, such as KHK4083 or ISB830, or an antibody fragment derived therefrom.

[0115] In certain embodiments, the payload is an antibody or antibody fragment that targets CD27, such as varlilumab, MK-5890, or CDX-527, or an antibody fragment derived therefrom.

[0116] In certain embodiments, the payload is an antibody or antibody fragment that targets CD40L, such as dapirolizumab, or an antibody fragment derived therefrom.

[0117] In certain embodiments, the payload is an antibody or antibody fragment that targets ICOS, such as MEDI-570, KY1044, JTX-2011, or GSK3359609, or an antibody fragment derived therefrom.

[0118] In certain embodiments, the payload is an antibody or antibody fragment that targets BAFFR, such as ianalumab, or an antibody fragment derived therefrom.

[0119] In certain embodiments, the payload is an antibody or antibody fragment that targets LFA-1, such as efalizumab, or an antibody fragment thereof.

[0120] In certain embodiments, the payload is an antibody or antibody fragment that targets a BTLA, such as icatolimab, or an antibody fragment derived therefrom.

[0121] In some embodiments, the 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.

[0122] In some embodiments, D 1 teeth, [ka] It is.

[0123] In some embodiments, the payload is a duocarmycin, or a derivative or analog thereof, hi some embodiments, the duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, carzelesin, bizeresin, or a derivative or analog thereof.

[0124] In some embodiments, D 1 teeth, [ka] The file is TIFF2024543016000021.tif158170.

[0125] In some embodiments, the payload is a pyrrolobenzodiazepine (PBD), or a derivative or analog thereof. In some embodiments, the pyrrolobenzodiazepine (PBD) is a [1,2]diazepino[3,4-e]indole, or a derivative or analog thereof.

[0126] In some embodiments, D 1 teeth, [ka] It is.

[0127] In some embodiments, the payload is an inhibitor of tubulin polymerization, hi some embodiments, the payload is hemiasterlin, HTI-286, or a derivative or analog thereof.

[0128] In some embodiments, D 1 teeth, [ka] is derived from.

[0129] In some embodiments, D 1 is a topoisomerase inhibitor. In some embodiments, D 1 is camptothecin, or a derivative or analog thereof. 1 is topotecan, irinotecan, ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, or rubitecan.

[0130] In some embodiments, D 1 teeth, [ka] It is.

[0131] In some embodiments, D 1 teeth, [ka] It is.

[0132] In some embodiments, D 1 teeth, [ka] It is.

[0133] In some embodiments, D 1 teeth, [ka] It is.

[0134] In some embodiments, D 1 teeth, [ka] It is.

[0135] In some embodiments, D 1 teeth, [ka] It is.

[0136] In some embodiments, D 1 teeth, [ka] It is.

[0137] In some embodiments, the payload comprises a polypeptide. In some embodiments, the polypeptide comprises one or more lysine, serine, threonine, or tyrosine residues. In some embodiments, the linker L 1 is covalently linked 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 L 1 is covalently attached to a lysine residue present on the payload.

[0138] In some embodiments, the payload comprises an N-terminal amino acid, wherein the linker L 1 is covalently attached to the N-terminal amino acid.

[0139] In some embodiments, m is 1-20.

[0140] In some embodiments of the conjugates described herein, the linker L 1 may be a bond.

[0141] In some embodiments of the conjugates described herein, the linker L 1 may have 1 to 100 linking atoms and may include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the linker may have 1 to 50 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms, or 1 to 40 linking atoms, or 1 to 30 linking atoms, or 1 to 20 linking atoms, or 1 to 10 linking atoms, or 1 to 5 linking atoms, or 5 to 30 linking atoms, or 10 to 30 linking atoms, or 5 to 40 linking atoms, or 5 to 50 linking atoms, or 10 to 50 linking atoms.

[0142] In some embodiments of the conjugates described herein, the linker L in formula I 1 may contain 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, where each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moiety is independently selected from oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy and C 1~4 It may be optionally substituted with 1 to 5 substituents independently selected from haloalkyl.

[0143] In some embodiments of the conjugates described herein, the linker L 1is the expression: -Y 10 -(CH2) n’ -Y 20 -(CH2) m’’ -Y 30 - (In the formula, Y 10 , Y 20 , and Y 30 each independently represents a bond, -NR 110 -, -O-, -S(O) 0~2 -, -NR 110 C(O)-, -C(O)NR 110 -, -NR 110 S(O)2-, -S(O)2NR 110 -, -CR 120 =N-NR 110 -, -NR 110 -N=CR 120 -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene is independently oxo, halo, C 1~4 Alkyl, C 1~4 Alkoxy and C 1~4 is optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and n′ and m″ may each independently be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0144] In certain embodiments, the linker L 1 is not a bond. In some embodiments, L 1 is a cleavable linker. In some embodiments, L 1 is a non-cleavable linker.

[0145] In certain embodiments, each R 110 are independently hydrogen, C 1~4 Alkyl, C 1~4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and each R 120 are independently hydrogen, C 1~4 Alkyl, C 1~4 It is haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

[0146] Exemplary linkers include, but are not limited to, those shown below: [ka] Examples include:

[0147] Exemplary linkers include, but are not limited to, those shown below: [ka] Examples include:

[0148] In some embodiments of the conjugates described herein, the linker in formula I can include one or more of polyethylene glycol (e.g., PEG having an average molecular weight of 300 g / mol to 10,000 g / mol), ethylene-1,2-diylbis(methylcarbamate), arylene (i.e., phenylene), ethyleneoxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. [ka] may include.

[0149] In some embodiments of the conjugates described herein, the linker L 1 may contain one or more natural or unnatural amino acids, sometimes referred to as a peptide linker. 1 When the drug comprises an amino moiety, the linker may be attached to the amino moiety using a peptide linker composed of a carboxylic acid acyl unit and one or more amino acids that make up the protein or peptide sequence. The linker may also include a self-immolative spacer that separates the drug from the protein peptide sequence.

[0150] In some embodiments of the conjugates described herein, the linker L 1 is defined as "AYZXW" (wherein "A" is a carboxylic acid acyl unit, "Y" and "Z" are each one or more natural or unnatural amino acids that together form a peptide sequence, and "X" and "W" are the units that bind the peptide and the drug D). 1 or any additional linker having 1-50 linked atoms, or 5-10 linked atoms, or 1-10 linked atoms separating the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.

[0151] In some embodiments, Y can 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 can be at least one amino acid selected from the group consisting of phenylalanine, alanine, and valine.

[0152] In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, acetyl- or formyl-protected lysine, arginine, tosyl- or nitro-protected arginine, histidine, ornithine, acetyl- or formyl-protected ornithine, and citrulline. In some embodiments, Z can be at least one amino acid selected from the group consisting of alanine, lysine, and citrulline.

[0153] In some embodiments, YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0154] In certain embodiments, A is -OC(O)-.

[0155] In certain embodiments, X is -OC(O)-.

[0156] In certain embodiments, W is -OC(O)-. In certain embodiments, X is absent and W is -OC(O)-.

[0157] In certain embodiments, -XW is [ka] It is.

[0158] In certain embodiments, -XW is [ka] It is.

[0159] In certain embodiments, the peptide linker is specifically tailored to be selectively cleaved (eg, enzymatically cleaved) by, for example, one or more tumor-associated proteases to release the drug.

[0160] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide), however, it will be understood that peptide linkers of up to 5, up to 6, up to 7, or up to 8 amino acid residues may also be suitably used.

[0161] In certain embodiments, the peptide linker is selected from the group consisting of Phe-Lys, Val-Lys, Val-Ala, Ala-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Gly-Phe-Leu-Gly [SEQ ID NO: 1], Ala-Leu-Ala-Leu [SEQ ID NO: 2], Phe-N 9 -Tosyl-Arg or Phe-N 9 -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.

[0162] In certain embodiments, the linker L in formula I 1 teeth, [ka] It is.

[0163] The above-mentioned linkers may be D such as lysine or cysteine. 1 may be attached on the right side to an amino acid side chain of [ka] ).

[0164] In some embodiments, L 1 is -OC(O)L 4 -or-OC1~6 Alkylene C(O)L 4 - and L 4 is a bond, -N(R 12 )-C 2~3 Alkylene-N(R 13 )C(O)-, -CH(NHC(O)R 14 ) C 1~4 Alkylene-SSC 1~4 Alkylene -OC(O)-, -NHNHC(O)CH(NHC(O)R 15 )CH2C(O)-, -C 1~6 Alkylene-CH(G x )OC(O)-, [ka] and R 12 , R 13 , R 14 , R 15 , and R 19 are each independently hydrogen or C 1~4 is alkyl, R 16 is hydrogen, C 1~4 Alkyl, -C 1~4 Alkylene -OH, -C 1~4 Alkylene-OC 1~4 Alkyl, -C 1~4 Alkylene-COH or -C 1~4 alkylene-CONH2, and G x is a halogen, C 1~4 Alkyl, C 1~4 Haloalkyl, C 1~4 and phenyl optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

[0165] In some embodiments, GL 1 is, in each case independently, [ka] It is.

[0166] In some embodiments, GL 1 is, in each case independently, [ka] When attached to a lysine residue, the conjugate has the formula: [ka] where PPM is a polypeptide moiety having lysine residues and lysine side chains, and PPM also represents the group: [ka] may have an additional lysine or other amino acid side chain conjugated to

[0167] In some embodiments, R 1B is G 1 , OH, -NR 1c -C 1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -N(R 1c )CHR 1e CO2H, -N(R 1c )CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n -N(CH2CO2H)2; R 1e is -CH2CO2H, -CH2CH2CO2H, -CH2CONH2, -CH2CH2CONH2, -CH2OH, or -CH(CH3)OH, and R 1f is hydrogen or CH2CO2H.

[0168] In some embodiments, R 1B -NR 1c -C 2~4 Alkylene-N(C 1~4 Alkyl)3 + , -N(R1c )-C 1~6 Alkylene-SO3H, -N(R 1c )-(CH2CH2O) 1~3 -CH2CH2N((CH2CH2O) 1~3 -C 1~6 Alkylene -COH) and -N(R 1c )-CH(CH2O-(CH2CH2O) 0~2 -C 1~6 alkylene-COH).

[0169] In some embodiments, R 1B -NR 1c -CH2CH2-N(CH3)3 + , -N(R 1c )-CH2CH2-SO3H, -N(R 1c )-(CH2CH2O)3-CH2CH2N((CH2CH2O)3-CH2CH2-CO2H)2, and -N(R 1c )-CH(CH2O-CH2CH2-CO2H)2.

[0170] In some embodiments, R 1A is C 1~4 It is an alkyl.

[0171] In some embodiments, R 1A is CH3.

[0172] In some embodiments, R 1c is hydrogen.

[0173] In some embodiments, R 1A is C 1~4 is alkyl, R 1B is G 1 , OH, -NR 1c -C 1~4 Alkylene-G 1 , -NR 1c -C 1~4 Alkylene-N(R 1d )2, -N(R 1c )CHR1e CO2H, -N(R 1c )CH2CO2H, and -N(R 1f )-CH2CH2-(N(CH2CO2H)CH2CH2) n -N(CH2CO2H)2; R 1e -C 1~4 alkylene-COH, R 1f is hydrogen or C 1~4 alkylene-COH, G 1 is a 4-8 membered monocyclic heterocyclyl containing a first nitrogen and optionally one additional heteroatom selected from nitrogen, oxygen, and sulfur, where G 1 is attached at the first nitrogen, and optionally C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 Substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo; and n is 0, 1, or 2.

[0174] In some embodiments, R 1A is CH3, R 1e is -CH2CO2H, R 1f is hydrogen or CH2CO2H, and G 1 is attached via a ring nitrogen atom and optionally C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, -OH, -OC 1~4 and piperazinyl, morpholinyl, piperidinyl, azepanyl, or pyrrolidinyl, each of which is substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo.

[0175] In some embodiments, L 2 is -C(O)-.

[0176] In some embodiments, R 1B are OH, N(H)CH2CO2H, -N(H)CHR 1e CO2H, -N(H)-CH2CH2-(N(CH2CO2H)CH2CH2) n -N(CH2CO2H)2, -N(CH2CO2H)-CH2CH2-N(CH2CO2H)2, and R 1e is -CH2CO2H.

[0177] In certain embodiments, the compound of formula IIA: [ka] (In the formula, R 2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 is optionally further substituted with a polyether), or a pharma- ceutically acceptable salt thereof.

[0178] In certain embodiments, the compound of formula IIB: [ka] (In the formula, R 2 is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 is optionally further substituted with a polyether, and R 17 is hydrogen, optionally substituted alkyl, or optionally substituted aryl), or a pharma- ceutically acceptable salt thereof.

[0179] In certain embodiments, the compound of formula IIC: [ka] (In the formula, R 2is -OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R 2 is optionally further substituted with a polyether, and R 17 is hydrogen, optionally substituted alkyl, or optionally substituted aryl), or a pharma- ceutically acceptable salt thereof.

[0180] In some embodiments, R 17 is hydrogen, alkyl, or aryl, where alkyl or aryl is C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 In some embodiments, R is optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, aryl, aryloxy ... 17 is hydrogen, alkyl, or aryl, where alkyl or aryl is C 1~4 Alkyl, C 1~4 Haloalkyl, halo, cyano, OH, -OC 1~4 It is optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo.

[0181] In certain embodiments, a conjugate selected from Table 1, or a pharma- ceutically acceptable salt thereof, is provided.

[0182] [Table 1] TIFF2024543016000047.tif228170TIFF2024543016000048.tif208170TIFF2024543016000049.tif249170TIFF2024543016000050.tif227170 TIFF2024543016000051.tif242170TIFF2024543016000052.tif239170TIFF2024543016000053.tif244170TIFF2024543016000054.tif102170

[0183] In some embodiments, a method is provided for delivering an effective amount of a payload (i.e., an inhibitor of poly(ADP-ribose) polymerase (PARP inhibitor), a duocarmycin, a pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, and a monoclonal antibody, or a derivative or analog thereof) to a target location in a subject, the method comprising administering to the subject a therapeutic support composition described herein to the target location, and administering to the subject a conjugate or a pharma- ceutically acceptable salt thereof or a composition described herein.

[0184] C. Therapeutic support composition The therapeutic support composition includes a support. The support may be a biocompatible support composition, i.e., compatible with the subject's body. In some cases, the support is non-toxic to the subject and does not substantially react with tissues or biological compounds in the subject. For example, the support may be, inter alia, a hydrogel. The support may be implanted into the subject's body and may support not only the binding agent (e.g., a tetrazine-containing group) but also the payload after the binding agent is conjugated. Exemplary supports include, but are not limited to, polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, crosslinked polymer matrices, metals, ceramics, plastics, bone graft materials, alginates, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports may also include particles, such as nanoparticles, microparticles, and the like.

[0185] The hydrogels may be polysaccharide hydrogels, alginates, 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 supports include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphates, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Exemplary polyethers include, but are not limited to, poly(ethylene glycol) (PEG), polypropylene glycol (PPG), triblock pluronics ([PEG] n -[PPG] m -[PEG] n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA). Supports can also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(γ-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), and the like.

[0186] In some embodiments, the support is a hydrogel. In some embodiments, the support is an alginate. In some embodiments, the support is chitin. In some embodiments, the support is hyaluronic acid (e.g., non-hydrogel hyaluronic acid having substantially no crosslinks). In some embodiments, the support is chitosin.

[0187] In certain embodiments, the support is a particle. The particle of the present disclosure may have a diameter of 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 particle may be a nanoparticle or a microparticle. Nanoparticles include particles with an average dimension on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles with an average dimension on the micrometer scale (e.g., 1000 μm or less). "Average" refers to the arithmetic mean. In some embodiments, the nanoparticles have a diameter in the range of 1 nm to 1 μm, e.g., 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have diameters ranging from 1 μm to 1 mm, for example, 10 μm to 1 mm, or 25 μm to 1 mm, or 50 μm to 1 mm, or 75 μm to 1 mm, or 100 μm to 1 mm, or 150 μm to 1 mm, or 200 μm to 1 mm, or 250 μm to 1 mm, or 300 μm to 1 mm, or 350 μm to 1 mm, or 400 μm to 1 mm, or 450 μm to 1 mm, or 500 μm to 1 mm. In further embodiments, small particles with diameters on the order of 10 nm to 100 nm may aggregate to form larger complexes, such as clusters or aggregates on the order of 1 μm to 10 μm. The particles of the present disclosure may be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes may be used, such as, but not limited to, ellipsoids, cubes, cylinders, cones, needles, or other irregular shapes.

[0188] A "particle" can take the form of any manufactured material, molecule, cryptophane, virus, phage, etc. Particles can be composed of materials such as, but not limited to, metals, ceramics, plastics, glass, composites, polymers, hydrogels, etc. For example, particles can be composed of inert materials such as alginate or iron oxide. In some instances, particles can be magnetic and formed from paramagnetic, superparamagnetic, or ferromagnetic materials, or other materials that respond to magnetic fields. Furthermore, particles can be of any shape, e.g., spheres, rods, asymmetric shapes, etc. Particles, or groups of particles in a composite, can be functionalized with receptors that have a specific affinity to bind to or interact with clinically relevant substrates. The receptors can be inherent to the particles themselves. For example, the particles themselves can be viruses or phages that have an inherent affinity for certain substrates. Additionally or alternatively, particles can be functionalized by covalently or otherwise attaching or associating with receptors that specifically bind to or otherwise recognize certain clinically relevant substrates. The functionalized receptors can be antibodies, peptides, nucleic acids, phages, bacteria, viruses, or any other molecules with a defined affinity for the target substrate. Examples of materials that can be used for the "particles" and / or "carriers" include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, poloxamers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. These examples are not intended to limit their concentrations, their crosslinking with different agents, their methods of administration, their tailored degradation profiles, and other properties known to those skilled in the art.

[0189] The particle, or a group of some particles in the complex, may be functionalized with a targeting agent (e.g., a ligand or antibody) that specifically binds (or substantially specifically binds) to a target (e.g., a target receptor or cell surface target, such as a clinically relevant receptor or cell surface target (e.g., an antigen)). The targeting agent may be directly attached to the particle itself. The targeting agent may be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule that has specific affinity for the target receptor or cell surface target. In some cases, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, 4-1BB, GITR, or other cell receptor or cell surface target.

[0190] In some embodiments, the targeting agent is a monoclonal antibody. The monoclonal antibody can be a whole monoclonal antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab)). In certain embodiments, the targeting agent is selected from the group consisting of CD25 (NCBI gene ID 3559), CEA (NCBI gene ID 634), CEACAM5 (NCBI gene ID 1048), ASPH (NCBI gene ID 444), EGFR (NCBI gene ID 1956), EPCAM (NCBI gene ID 4072), VEGFR (NCBI gene ID 3791), PDGFR (NCBI gene ID 5159), TROP2 (NCBI gene ID 4070), Nectin 4 (NCBI gene ID 81607), P SMA (NCBI Gene ID 2346), BCMA (NCBI Gene ID 608), CD22 (NCBI Gene ID 933), CD20 (NCBI Gene ID 920), CD19 (NCBI Gene ID 930), CD79b (NCBI Gene ID 974), CD38 (NCBI Gene ID 952), CD45 (NCBI Gene ID 5788), endoglin (NCBI Gene ID 2022), FGFR2 (NCBI Gene ID 14183), C4.4A (NCBI Gene ID 27076), and claudin-18.2 (NCBI gene ID 51208), MMP9 (NCBI gene ID 4318), folate receptor (NCBI gene ID 2348), DLL3 (NCBI gene ID 10683), CD138 (NCBI gene ID 6382), CD56 (NCBI gene ID 4684), CD37 (NCBI gene ID 951), CD74 (NCBI gene ID 972), mesothelin (NCBI gene ID 10232), IL-6R (NCBI gene ID 3570), SLAMF7 ( NCBI Gene ID 57823), BAFF (NCBI Gene ID 10673), MUC1 (NCBI Gene ID 4582), GPC3 (NCBI Gene ID 2719), HER2 (NCBI Gene ID 2064), HER3 (NCBI Gene ID 2065), CD30 (NCBI Gene ID 943), CD33 (NCBI Gene ID 945), CD123 (NCBI Gene ID 3563), GPNMB (NCBI Gene ID 10457), cMET (NCBI Gene ID 10461), and HER2 (NCBI Gene ID 2064). ID 4233), CD142 (NCBI gene ID 2152), NaPi2B (NCBI gene ID 10568), GCC (NCBI gene ID 2984), STEAP1 (NCBI gene ID 26872), MUC16 (NCBI gene ID 94025), CD70 (NCBI gene ID 970), CD44 (NCBI gene ID 960), (NCBI gene ID), antibody fragment (NCBI gene ID), vWF (NCBI gene ID 7450), TNF (N and antibodies or antibody fragments that target one or more of: IL-6R (NCBI Gene ID 3570), BCMA (NCBI Gene ID 608), ADAMTS5 (NCBI Gene ID 11096), CX3CR1 (NCBI Gene ID 1524), CXCR4 (NCBI Gene ID 7852), TfR1 (NCBI Gene ID 7037), VEGFR (NCBI Gene ID 3791), or PSMA (NCBI Gene ID 2346).

[0191] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD25, such as daclizumab, RG6292, basiliximab, or HuMax-TAC, or an antibody fragment derived therefrom.

[0192] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CEA, such as labetuzumab, 15-1-32, PR1A3, or cT84.66, or an antibody fragment derived therefrom.

[0193] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CEACAM5, such as Tusamitiamab or CC4, or an antibody fragment derived therefrom.

[0194] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets ASPH, such as PAN-622, or an antibody fragment derived therefrom.

[0195] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets EGFR, such as cetuximab, necitumumab, nimotuzumab, matuzumab, AMG595, depatuxizumab, dapatuxizumab, durigotuzumab, futuximab, GC1118, imgatuzumab, panitumumab, altumumab, tomzotuximab, or laprituximab, or an antibody fragment derived therefrom.

[0196] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets EPCAM, such as oportuzumab, sitatuzumab, tucotuzumab, catumaxomab, edrecolomab, or adecatumumab, or an antibody fragment derived therefrom.

[0197] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets VEGFR, such as ramucizumab, ramucirumab, or blinacimab, or an antibody fragment derived therefrom.

[0198] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets PDGFR, such as olaratumab or ramucirumab, or an antibody fragment derived therefrom.

[0199] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets TROP2, such as sacituzumab or Pr1E11, or an antibody fragment derived therefrom.

[0200] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets Nectin-4, such as Enfortumab, or an antibody fragment derived therefrom.

[0201] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets PSMA, such as J591 or MLN591, or an antibody fragment derived therefrom.

[0202] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets BCMA, such as belantamab, or an antibody fragment derived therefrom.

[0203] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD22, such as moxetumomab, inotuzumab, epratuzumab, or pinatuzumab, or an antibody fragment derived therefrom.

[0204] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD20, such as ublituximab, ofatumumab, rituximab, obinutuzumab, tositumomab, or ibritumomab, or an antibody fragment derived therefrom.

[0205] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD19, such as loncastuximab, XMAB-5574, MOR208, cortuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment derived therefrom.

[0206] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD79b, such as polatuzumab, or an antibody fragment derived therefrom.

[0207] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD38, such as isatuximab, daratumumab, MOR202, or TAK-079, or an antibody fragment derived therefrom.

[0208] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD45, such as I-131-BC8 or Iomab-B, or an antibody fragment derived therefrom.

[0209] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets endoglin, such as carotuximab, or an antibody fragment derived therefrom.

[0210] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets FGFR2, such as bemarituzumab or apurutumab, or an antibody fragment derived therefrom.

[0211] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets C4.4A, such as rupartumab, or an antibody fragment derived therefrom.

[0212] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets claudin-18.2, such as zolbetuximab or claudiximab, or an antibody fragment derived therefrom.

[0213] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets MMP9, such as andecaliximab, or an antibody fragment derived therefrom.

[0214] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets the folate receptor, such as mirvetuximab, farletuzumab, MORAb-202, MORAb-003, or SP8166, or an antibody fragment derived therefrom.

[0215] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets DLL3, such as rovalpituzumab, or an antibody fragment derived therefrom.

[0216] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD138, such as indatuximab, or an antibody fragment derived therefrom.

[0217] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD56, such as lorvotuzumab, promiximab, or an antibody fragment derived therefrom.

[0218] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD37, such as BI 836826, otlertuzumab, or naratuximab, or an antibody fragment derived therefrom.

[0219] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD74, such as milatuzumab, or an antibody fragment derived therefrom.

[0220] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets mesothelin, such as anetumab, amatuximab, or MMOT-0530A, or an antibody fragment derived therefrom.

[0221] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets IL-6R, such as tocilizumab or sarilumab, or an antibody fragment derived therefrom.

[0222] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets SLAMF7, such as elotuzumab, or an antibody fragment derived therefrom.

[0223] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets BAFF, such as belimumab, or an antibody fragment derived therefrom.

[0224] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets MUC1, such as KL-6, MY.1E12, hMUC1-1H7, TAB004, huC242, clivatuzumab, 8HuDS6, gatipotuzumab, AR20.5, or cantuzumab, or an antibody fragment derived therefrom.

[0225] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets GPC3, such as codrituzumab, ECT204, or MDX-1414, or an antibody fragment derived therefrom.

[0226] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets HER2, such as pertuzumab, trastuzumab, or margetuximab, or an antibody fragment derived therefrom.

[0227] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets HER3, such as patritumab, seribantumab, lumuletuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom.

[0228] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD30, such as brentuximab, or an antibody fragment derived therefrom.

[0229] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD33, such as gemtuzumab, BI 835858, vadastuximab, or lintuzumab, or an antibody fragment derived therefrom.

[0230] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD123, such as KHK2823, taclotuzumab, or G4723A, or an antibody fragment derived therefrom.

[0231] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets GPNMB, such as glembatumumab, or an antibody fragment derived therefrom.

[0232] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets cMET, such as terisotuzumab, onartuzumab, or SAIT301, or an antibody fragment derived therefrom.

[0233] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD142, such as tisotumab, or an antibody fragment derived therefrom.

[0234] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets NaPi2B, such as rifastuzumab, or an antibody fragment derived therefrom.

[0235] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets GCC, such as indusatumab, or an antibody fragment derived therefrom.

[0236] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets STEAP1, such as buntuzumab, or an antibody fragment derived therefrom.

[0237] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets MUC16, such as sofituzumab, or an antibody fragment derived therefrom.

[0238] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD70, such as borsetuzumab, or an antibody fragment derived therefrom.

[0239] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD44, such as bivatuzumab, or an antibody fragment derived therefrom.

[0240] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets vWF, such as caplacizumab, or an antibody fragment derived therefrom.

[0241] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets TNF, such as ozoralizumab, V565, or PF-05230905, or an antibody fragment derived therefrom.

[0242] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets IL-6R, such as bovalilizumab, or an antibody fragment derived therefrom.

[0243] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets BCMA, such as LCAR-B38M, or an antibody fragment derived therefrom.

[0244] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets ADAMTS5, such as M6495, or an antibody fragment derived therefrom.

[0245] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CX3CR1, such as BI 655088, or an antibody fragment derived therefrom.

[0246] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CXCR4, such as AD-214 or ALX-0651, or an antibody fragment derived therefrom.

[0247] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets TfR1, such as TXB4, or an antibody fragment derived therefrom.

[0248] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets VEGFR, such as CDP791, or an antibody fragment derived therefrom.

[0249] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets PSMA, such as GY1, or an antibody fragment derived therefrom.

[0250] Other compounds or molecules may be attached to the particles, such as fluorophores or autofluorescent or luminescent markers, that may aid in the detection of the particle (e.g., in vivo detection). The ligands and / or detectable labels may be attached directly to the particles or may be attached to the particles via bioorthogonal functional groups as described herein.

[0251] In certain embodiments, the support is a bone graft material, such as a bone graft replacement material. A bone graft replacement material is a material that is structurally similar to bone. In some cases, the bone graft replacement material is bioabsorbable, thus the bone graft replacement material can dissolve or be absorbed in the body over time. The bone graft replacement material can be osteoconductive, so that blood vessels and new bone formation are promoted into the bone graft replacement material. In some cases, the bone graft replacement material is osteoinductive, so that new bone formation is promoted through active recruitment of mesenchymal stem cells from the surrounding tissue. For example, growth factors such as bone morphogenetic proteins can be included in the bone graft replacement material. Bone graft replacement 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.

[0252] The therapeutic support composition of the present disclosure includes a support and a first binding agent covalently linked to the support. The binding agent may be attached to the support on a surface of the support, such as a solvent accessible surface of the support (e.g., a surface of the support in contact with the surrounding solvent). In some cases, the binding agent is directly attached to the support. For example, the binding agent may be covalently attached to the surface of the support via a covalent bond, such as, for example, an amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, thiourea, etc. In some cases, the binding agent is covalently attached to the support via an amide bond. In other cases, the binding agent may be linked to the support via a linker. Any suitable linker can be used to link the binding agent to the support. Exemplary linkers can have 1-100 linking atoms and can include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, a linker can have 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms. Exemplary linkers include, but are not limited to, those shown below: [ka] Examples include:

[0253] In certain embodiments, the therapeutic support composition comprises a support and a compound of the formula: [ka] (In the formula, R 20is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, 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' and R'' are selected from the group consisting of hydrogen, aryl, and alkyl, and R''' is independently selected from aryl and alkyl, and R' and R'' are independently selected from hydrogen, aryl, ... 30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; R a , R 31a , and R 31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl, and t is 0, 1, 2, 3, or 4).

[0254] In certain embodiments, the therapeutic support composition has the formula: [ka] (In the formula, R 20is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, 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' and R'' are selected from the group consisting of hydrogen, aryl, and alkyl, R''' is independently selected at each occurrence from aryl and alkyl, and R' and R'' are independently selected at each occurrence from aryl and alkyl, and R' and R'' are independently selected at each occurrence from aryl and alkyl, and R 22 is a linker of 1 to 100 linked atoms, and may include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functionalities. For example, the linker may have 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0255] In certain embodiments, the therapeutic support composition has the formula: [ka] (In the formula, R 20is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, 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'', N R'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'' are independently selected from hydrogen, aryl, and alkyl, and R''' is independently selected from aryl and alkyl, and R 30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; R a , R 31a , and R 31b are each independently hydrogen, C1-C6-alkyl, or C1-C6-haloalkyl, and t is 0, 1, 2, 3, or 4.

[0256] In certain embodiments, the therapeutic support composition has the formula: [ka] (In the formula, R 20is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, 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'', wherein R and R are independently selected from hydrogen, aryl, and alkyl, and R''' is independently selected from aryl and alkyl, or a salt thereof.

[0257] In certain embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0258] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0259] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0260] In some embodiments, the therapeutic support composition has formula (II): [ka] (In the formula, G 2 teeth, [ka] and R 22 is a linker of 1 to 100 linking atoms, and R 20 is as defined herein).

[0261] In a further embodiment, G 2 teeth, [ka] It is.

[0262] In yet a further embodiment, G 2 teeth, [ka] and R 20 is hydrogen or C 1~4 It is an alkyl.

[0263] Compounds of formula (II) include those of formula (II-A): [ka] (In the formula, R 20is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, 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'', N and R' and R'' are independently selected from hydrogen, aryl, and alkyl, and R''' is independently selected from each occurrence of aryl and alkyl. In a further embodiment according to Formula (II-A), R 20 is hydrogen or C 1~4 It is an alkyl.

[0264] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0265] Additional therapeutic support compositions are exemplified in WO 2017 / 044983, WO 2015 / 139025, and WO 2014 / 205126, the entire contents of each of which are incorporated herein by reference in their entirety.

[0266] Hyaluronic acid derivatives include hyaluronic acid having multiple glucuronic acid units and tetrazine-containing groups linked or directly bonded to the glucuronic acid units of hyaluronic acid.Hyaluronic acid may have multiple N-acetylglucosamine units.In certain embodiments, the N-acetylglucosamine units of hyaluronic acid are not linked or conjugated to tetrazine-containing groups.

[0267] The tetrazine-containing group can be linked through the carboxylic acid of the glucuronic acid unit or can be directly attached. The tetrazine-containing group can be incorporated into hyaluronic acid at about 0.1% to about 80% as measured by the % of carboxylic acid linked or conjugated to the tetrazine-containing group, for example, at 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 acid linked or conjugated to the tetrazine-containing group.

[0268] D. Treatment method Aspects of the present disclosure include methods of delivering a payload to a target location in a subject. In certain embodiments, the methods include selectively delivering a payload to a target location in a subject. Selective delivery of a payload includes delivering a payload to a target location (e.g., an organ or tissue, or a portion thereof) without targeting other locations (e.g., other organs or tissues, or portions thereof) in the subject that do not require administration of the payload. Selective delivery of a payload can be achieved through the use of the support compositions and functionalized payloads described herein.

[0269] In some cases, the support composition of the present disclosure can be localized at a desired target location in a subject. For example, the method of the present disclosure can include administering a support composition as described herein to a subject. The support composition can be administered to a subject at a desired target location in the subject. In some cases, the support composition can be implanted in a subject at a desired target location in the subject. In some embodiments, the support composition can be attached to a targeting agent as described herein, and the method can include administering (e.g., systemically administering) the support composition to a subject. In these embodiments, the support composition attached to a targeting agent can be localized at a desired target location in a subject through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, etc.), or can be localized on the surface (e.g., cell surface) of a desired target through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, etc.).

[0270] As described herein, selective binding between bioorthogonal binding counterparts (e.g., between the tetrazine binding agent of the support composition and its complementary trans-cyclooctene binding agent of the functionalized payload) can occur. Due to the localization of administration of the support composition to a desired location in the subject as described above, selective binding between the binding agent of the support composition and its complementary binding agent of the functionalized payload will result in the payload being localized to the desired target location. Thus, in certain embodiments, the method includes administering a functionalized payload to the subject, such that the functionalized payload binds to the support composition to form a support complex. For example, the functionalized payload can be administered systemically to the subject. When the functionalized payload is administered to the subject, contact between the binding agent of the support composition and the complementary binding agent of the functionalized payload can occur, such that the binding agent and its complementary binding agent bind to each other to form a support complex, thereby selectively delivering the payload to the target location in the subject. In some embodiments, selective delivery of the functionalized payload results in a concentration of the payload at the targeted location that is greater than the concentration of the payload elsewhere in the subject (e.g., non-targeted areas of the subject).

[0271] Provided herein is a method of treating cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of a conjugate described herein or a pharma- ceutically acceptable salt thereof and a therapeutic support composition.

[0272] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

[0273] In certain embodiments, this approach can be used to treat and / or diagnose hematopoietic malignancies, such as myelodysplastic syndromes, acute myeloid leukemia, myeldysplastic 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 lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, nodular lymphocyte-predominant Hodgkin lymphoma, and the like.

[0274] In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, breast cancer, glioma, lung cancer, soft tissue cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer.

[0275] In some embodiments, the cancer is a solid tumor.

[0276] In some embodiments, the cancer is a soft tissue sarcoma.

[0277] In some embodiments, the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0278] In some embodiments, the method also includes enhancing or eliciting an immune response, hi some embodiments, the immune response is an increase in one or more of white blood cells, lymphocytes, monocytes, and eosinophils.

[0279] In some embodiments, the method further comprises administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, wherein the anti-cancer agents, immunomodulatory agents, and trans-cyclooctene prodrugs thereof are known in the art.

[0280] Indications for this approach include both hematopoietic and solid cancers. In certain embodiments, this approach can be used to treat and / or diagnose soft tissue sarcomas, i.e. rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all the various subtypes of soft tissue sarcomas, as well as osteosarcoma. The composition can be for treating and / or diagnosing pigmented villonodular synovitis.

[0281] The compositions of the present disclosure are used in the treatment and / or diagnosis of a condition or disease in a subject suitable for treatment or diagnosis by administration of a payload (e.g., a parent drug (i.e., the drug prior to conjugation to the composition)). "Treatment" means that at least an amelioration of symptoms associated with the condition suffered by the subject is achieved, where amelioration is used in a broad sense to refer to at least a reduction in a parameter associated with the condition being treated, e.g., the magnitude of the symptoms. Thus, treatment also includes a situation in which a pathological condition, or at least symptoms associated therewith, are completely arrested, e.g., prevented from developing or arrested, e.g., terminated, such that the subject is no longer afflicted by the condition, or at least symptoms characterizing the condition. Treatment may include arresting clinical symptoms, i.e., arresting their progression or further progression, e.g., alleviating or completely arresting active disease. Treatment may include buffering clinical symptoms, i.e., causing their regression. For example, in the context of cancer, the term "treating" includes any or all of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing total tumor burden, extending survival, and ameliorating one or more symptoms associated with cancer.

[0282] The subject to be treated may be a subject in need of therapy, where the subject to be treated is a suitable subject for treatment with the parent drug. Thus, a variety of subjects may be suitable for treatment with the compositions disclosed herein. In general, such subjects are "mammals", where humans are of interest. Other subjects may include household pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., animal models of disease, etc.), as well as non-human primates (e.g., chimpanzees and monkeys, etc.).

[0283] In certain embodiments, the functionalized payloads, therapeutic support compositions, additional therapeutic agents and methods can be used to treat, but are 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 cancer, breast cancer (e.g., triple-negative breast cancer), glioblastoma (e.g., multicenter, open cell, ovarian cancer ... Solid tumors, including glioblastoma, gliomas of the ovary, and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcomas, fibrosarcomas, osteosarcomas, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumors, prostate adenocarcinoma, nasopharyngeal carcinoma, and cutaneous T-cell lymphoma, may be treated, prevented, and / or diagnosed. The disclosed approach is well suited as an adjuvant / neoadjuvant system. For example, the particles disclosed herein may be placed during a biopsy, and once the results from the study are back, the physician can deliver the appropriate cocktail to the desired site in the body. This will minimize the size of the tumor, especially in the setting of tumors that are surgically resectable. Then, at the end of the operation, the surgeon can place more particles around the surgical cavity and treat the patient with additional therapeutic doses (e.g., chemotherapy with the disclosed approaches) to minimize the risk that any cancer cells may be missed at the surgical margins.

[0284] In certain embodiments, the disclosed methods provide the ability to place the particles disclosed herein at the time of biopsy. Once the results are returned, the physician can deliver and deliver immunomodulatory agents, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous and / or immune cells), and adjuvants to the biopsy site to boost the immune system with fewer side effects, similar to the combination of chemotherapy and immunotherapy agents. This combination approach can be beneficial to the patient. Chemotherapeutic agents treat solid tumors or specific sites, while enhanced immunotherapy responses can be helpful at distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used or utilized with anthracyclines, taxanes, gemcitabine, and other agents that enhance the efficacy of one or more immunomodulatory agents, such as ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).

[0285] cancer The disclosed method can be used to treat or prevent cancer, including metastatic cancer. Cancer is a group of related diseases that may involve maintaining proliferation signaling, evading growth inhibition, resisting cell death, achieving immortality through replication, inducing angiogenesis, and activating invasion and metastasis. The disclosed method can enhance or induce an immune response against cancer in a subject. The immune response can cause an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0286] Cancers that may be treated by the disclosed methods include, but are 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 carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumors, glioma, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver pancreatic cancer, lung cancer, lymphoma, macroglobulinemia, melanoma, mesothelioma, oral cancer, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thyroid cancer, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0287] 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 cancer, soft tissue sarcoma, osteosarcoma, or pancreatic cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a soft tissue cancer. In some embodiments, the cancer is a fibrosarcoma. In some embodiments, the cancer is diffuse intrinsic pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0288] Without being bound to a particular theory, local release of certain anticancer drugs using the compounds and methods of the present invention may cause or contribute to immunogenic cell death (ICD). For example, certain anticancer drugs (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 effective antitumor immune responses through activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. ICD is characterized by the secretion of damage-associated molecular patterns (DAMPs). Three important DAMPs are exposed on the cell surface during ICD. One of the DAMP molecules, calreticulin (CRT), is normally located in the lumen of the endoplasmic reticulum (ER), and after induction of immunogenic apoptosis, it migrates to the surface of dying cells, where it functions as an "eat me" signal for professional phagocytes. Other important surface-exposed DAMPs are the heat shock proteins (HSPs), namely HSP70 and HSP90, which also translocate to the plasma membrane under stress conditions. At the cell surface, they have immune stimulatory effects based on their interaction with a number of antigen-presenting cell (APC) surface receptors, such as CD91 and CD40, and also promote cross-presentation of tumor cell-derived antigens on MHC class I molecules, which then translocate to CD8. + It triggers T cell responses. Other important DAMPs characteristic of ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered a late apoptotic marker, and its release into the extracellular space is thought to be necessary for optimal release and presentation of tumor antigens to dendritic cells. HMGB1 binds to several pattern recognition receptors (PRRs), such as Toll-like receptor (TLR) 2 and TLR4, expressed on APCs. A very recently discovered DAMP released during immunogenic cell death is ATP, which, when secreted, acts as a "find me" signal for monocytes, inducing their attraction to the site of apoptosis. Kroemer et. al. Curr. Op. Immunol. 2008 (20), 504-511.

[0289] Thus, localized delivery of ICD-inducing factors using the compounds and methods of the present invention may be advantageously combined with one or more immunomodulatory agents.

[0290] In certain embodiments, the functionalized payloads, therapeutic support compositions, and methods can be used to treat, prevent, and / or diagnose 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), 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.

[0291] The disclosed approach is well suited as an adjuvant / neoadjuvant system. For example, the therapeutic support composition disclosed herein can be placed in a biopsy, and once the results from the study are back, the physician can administer the appropriate cocktail to deliver the treatment to the desired site in the body (the compound of formula I and any additional therapeutic agent(s)). The results of the biopsy can indicate the amount and type of treatment delivered to the tumor site. For example, chemokines (agents that attract cancerous cells and / or immune cells) and adjuvants can be delivered and combined with immunotherapeutic agents to enhance the immune system with fewer side effects, similar to chemotherapy agents.

[0292] The disclosed compounds and compositions can be administered prior to surgical resection. The disclosed methods can minimize tumor size prior to surgical resection. This minimizes tumor size, especially in the context of surgically resectable tumors. The disclosed conjugates, compounds, and compositions can be administered during surgical resection. The disclosed conjugates, compounds, and compositions can be administered after surgical resection. At the end of surgical resection, the therapeutic support composition can be placed around the surgical cavity, and the subject can then be treated with additional therapeutic doses to minimize the risk that any cancer cells may be missed at the surgical margin.

[0293] The disclosed method may include multiple systemic doses of functionalized payloads that are concentrated at one location. The disclosed method may be used to deliver a second payload. If the tumor is resistant to the first payload, the disclosed method may be used to administer a second functionalized payload. The second payload may be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by the therapeutic support composition used for the first prodrug.

[0294] The functionalized payloads disclosed herein can act as adjuvants. This combination approach will benefit patients. Chemotherapeutic agents will treat solid tumors or specific locations and may enhance or induce immune responses, while functionalized payloads and / or enhanced immunotherapy responses of separate agents may be useful at distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used or utilized with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, campothecin analogues, and other agents that enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer).

[0295] The disclosed methods can be used to treat diffuse intrinsic pontine glioma. Diffuse intrinsic pontine glioma (DIPG) is a pediatric brainstem tumor that can be aggressive and difficult to treat. There is no known curative treatment for DIPG, and chances of survival have remained dismal over the past 40 years. DIPG patients have an overall median survival of only 11 months, with a 2-year survival rate below 10%. DIPG accounts for 75%-80% of pediatric brainstem tumors, affecting an estimated 200-300 children in the United States each year. Research has been hampered by the rarity of this devastating disease and the lack of experimental model systems to date, with chances of survival remaining stable over the past 40 years. Diagnosis of DIPG begins with clinical symptoms and can be confirmed by MRI. The disease may begin with several months of systemic symptoms, including behavioral changes and school difficulties, double vision, abnormal or restricted eye movements, asymmetry in smiling, loss of balance, and weakness. In other cases, severe neurological deterioration occurs earlier, with symptoms present for less than a month before diagnosis. Laboratory examination may reveal a triad of multiple cranial nerve disorders, tract signs such as hyperreflexia and clonus, and ataxia. Enlargement of the pontine portion of the brainstem may cause obstructive hydrocephalus and elevated intracranial pressure.

[0296] Nuclei important for vital functions such as breathing and heart rate are located within the pons, and if left untreated, DIPGs can impair breathing and heart rate.

[0297] The disclosed methods can be used to deliver molecular payloads to the site of DIPG. The disclosed methods can include systemic delivery of drugs that are activated only at the tumor site. The disclosed methods can be used as neoadjuvant or adjuvant therapy. The biomaterial can be placed during a biopsy. The results of the biopsy can indicate the amount and type of treatment delivered to the tumor site. The disclosed compounds and compositions can be administered prior to surgical resection. The disclosed methods can minimize the size of the tumor prior to surgical resection. The disclosed compounds and compositions can be administered during surgical resection. The disclosed compounds and compositions can be administered after surgical resection. At the end of the surgical resection, the biomaterial can be placed around the surgical cavity, and the subject can then be treated with additional therapeutic doses. The disclosed biodegradable gel can be implanted at the time of biopsy or surgery. The disclosed methods may not require additional invasive procedures to deliver additional doses of the disclosed compounds and compositions.

[0298] The disclosed method may include multiple systemic doses of functionalized payloads that are concentrated at one location. The disclosed method may be used to deliver a second payload. If the tumor is resistant to the first payload, the disclosed method may be used to administer a second functionalized payload. The second payload may be a TCO-labeled payload of paclitaxel, docetaxel, anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, campothecin analogs, or other agents. A TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel may be administered in combination with doxorubicin. The second functionalized payload may be activated by the therapeutic support composition used for the first prodrug.

[0299] Administration Method The method of treatment may include any number of administration modes of the disclosed conjugates, compounds, or compositions. Administration modes may include tablets, pills, dragees, hard and soft gel capsules, granules, pellets, skin patches, skin creams, skin gels, aqueous, lipid, oily, or other liquids, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixirs, solid emulsions, solid solutions, or dispersible powders. In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein may be dispersed in microparticles, e.g., nanoparticulate compositions.

[0300] For parenteral administration, the conjugates, compounds, or compositions disclosed herein may be dissolved or suspended in a physiologically acceptable diluent, such as water, a buffer, an oil, or the like, with or without a solubilizing agent, surfactant, dispersing agent, or emulsifying agent. 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 type of solution or suspension, or may also be administered in the form of a liposome or nanosuspension.

[0301] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0302] The therapeutic support composition is preferably administered locally to the tumor site, such as by injection or implantation. The functionalized payload, such as the conjugate of formula I or formula (III), can be administered by any suitable route, taking into account the condition of the subject and the judgment of the medical professional. Parenteral administration is a suitable means of administering the conjugate of formula I.

[0303] The amount of the composition administered to a subject can be determined based on the dosage and / or dosing schedule guidelines of the parent drug.In general, the composition can provide targeted delivery and / or serum half-life extension of the conjugated drug, resulting in at least one of reduced dosage or reduced administration in the dosing schedule.Therefore, the composition can provide reduced dosage and / or reduced administration in the dosing schedule compared to the parent drug before conjugation in the composition of the present disclosure.

[0304] Pharmaceutical preparations may be provided in unit dosage form.In this form, pharmaceutical preparations can be divided into unit doses that contain appropriate amounts of the compositions of the present disclosure.The unit dosage form can be a packaged preparation, and the package contains individual amounts of preparations, such as tablets, capsules, and powders, which are divided into pouches, vials, or ampoules.

[0305] In some embodiments, a kit is provided that includes a conjugate described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising same, and instructions for its use.

[0306] In some embodiments, the kit further comprises a therapeutic support composition.

[0307] The compositions of the present disclosure can be present in any suitable amount, which may depend on a variety of factors including, but not limited to, the weight and age of the subject, disease state, etc. Suitable dosage ranges for the compositions of the present disclosure include 0.1 mg to 10,000 mg, or 1 mg to 1,000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, suitable dosages for compositions 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.

[0308] In some embodiments, multiple doses of the composition are administered. The number of times the composition is administered may vary depending on any of a variety of factors, such as the severity of symptoms, the condition of the subject, etc. For example, in some embodiments, the composition is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), every day (qd), twice a day (qid), or three times a day (tid).

[0309] The composition of the present disclosure can be administered at any suitable number of times, intervals, and duration.For example, the composition of the present disclosure can be administered once per hour, or 2, 3 or more times per hour, once per day, or 2, 3 or more times per day, or once per 2, 3, 4, 5, 6, or 7 days to provide the subject with the desired dosage level.When the composition of the present disclosure is administered more than once per day, representative intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes, as well as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, and 24 hours. The compositions of the present disclosure can be administered once, twice, or three or more times for 1 hour, 1 hour to 6 hours, 1 hour to 12 hours, 1 hour to 24 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day, 1 day to 7 days, 1 week, 1 week to 4 weeks, 1 month, 1 month to 12 months, a year or more, or even indefinitely.

[0310] The composition of the present disclosure can be co-administered with another active agent. Co-administration includes administering the composition of the present disclosure and the active agent within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours of each other. Co-administration also includes administering the composition of the present disclosure and the active agent simultaneously or nearly simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or about 30 minutes of each other), or sequentially in any order. Furthermore, 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 to provide the desired dosage level per day.

[0311] Co-administration can be achieved by co-implantation or co-injection.

[0312] In some embodiments, co-administration can be achieved by co-formulation, e.g., preparing a single pharmaceutical formulation that includes both the composition of the present disclosure and the active agent, hi other embodiments, the composition of the present disclosure and the active agent can be formulated separately and co-administered to a subject.

[0313] The composition of the present disclosure and the active agent may be present in the formulation in any suitable weight ratio, such as 1:100-100:1 (w / w), or 1:50-50:1, or 1:25-25:1, or 1:10-10:1, or 1:5-5:1 (w / w). The composition of the present disclosure and the other active agent may be present in any suitable weight ratio, such as 1:100 (w / w), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1, or 100:1 (w / w). Other dosage amounts and dosage ratios of the composition of the present disclosure and the active agent are suitable for the formulations and methods described herein.

[0314] Combination therapy In one aspect, the present invention provides a method of treating cancer or enhancing or inducing an immune response, comprising administering to a subject in need of treatment or enhancing or inducing a therapeutically effective amount of a conjugate of the present invention (e.g., Formula I) or a pharma- ceutically acceptable salt or composition thereof, a therapeutic support composition as described herein, and a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0315] The present invention also provides a pharmaceutical combination comprising a conjugate or a pharma- ceutical acceptable salt thereof or composition as described herein for use in the treatment or prevention of cancer or for use in enhancing or inducing an immune response, a therapeutic support composition as described herein, and an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0316] The present invention also provides the use of a pharmaceutical combination comprising a conjugate or a pharma- ceutical acceptable salt or composition thereof as described herein, a therapeutic support composition, and a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof, for the treatment or prevention of cancer or for use in enhancing or inducing an immune response.

[0317] In the methods and uses described herein, the components of the combination medicament may be administered / used simultaneously, separately or sequentially in any order, and these components may be administered separately or as a fixed combination. For example, delaying or treating the progression of a disease according to the present invention may include administering a first active ingredient in free form or in pharma- ceutical acceptable salt form and administering a second active ingredient in free form or in pharma-ceutical acceptable salt form simultaneously or sequentially in any order, in a therapeutically effective amount or effective amount taken together, for example, in a daily dosage amount corresponding to the amount described herein. The individual active ingredients of the combination medicament may be administered separately at different times during the course of treatment, or simultaneously in a divided or single dosage form. Thus, the present disclosure should be understood as embracing all such regimes of simultaneous or alternating treatment, and the term "administer" should be interpreted accordingly. Thus, the combination medicament used herein defines either a fixed combination in one unit dosage form, or separate dosage forms in the case of combination administration, where the combined administration may be independently simultaneous or at different times. As a further example, the therapeutic support composition and the conjugate can be administered / used simultaneously (e.g., by co-injection or co-implantation), separately, or sequentially, followed by administration of an additional therapeutic agent selected from the group consisting of an anti-cancer agent, an immunomodulatory agent, or a trans-cyclooctene prodrug thereof.

[0318] The methods and uses for treating cancer include administering / localizing the therapeutic support composition to the tumor. In the methods and uses disclosed herein, administration of the conjugate or a pharma- ceutically acceptable salt or composition thereof, the therapeutic support composition, and an additional therapeutic agent may inhibit tumor growth.

[0319] The 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. The additional therapeutic agent may be administered before the disclosed conjugates and compositions. The additional therapeutic agent may be administered after the disclosed conjugates and compositions. The additional therapeutic agent may be administered simultaneously with the disclosed conjugates and compositions. In some embodiments, the additional therapeutic agent may be administered in the same composition as the disclosed conjugates. In other embodiments, there may be a time interval between administration of the additional therapeutic agent and administration of the disclosed conjugates or compositions. In some embodiments, administration of the disclosed conjugates or compositions with an additional therapeutic agent may allow for administration of a lower dose and / or less frequent intervals of the other therapeutic agent. When used in combination with one or more other active ingredients, the conjugates or compositions of the present invention and the other active ingredients may be used in lower doses than when each is used alone. Thus, pharmaceutical compositions of the present invention include those that contain one or more other active ingredients in addition to the conjugates of the present disclosure.

[0320] Anticancer drugs Exemplary anticancer agents include, but are not limited to, abiraterone acetate, avitrexate (methotrexate), Abraxane (albumin-stabilized nanoparticle formulation of paclitaxel), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), adrsil (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), Alanone (nelarabine), arsenic trioxide, Arzera (ofatumumab), Erwinia chrysanthemi asparaginase, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexar (tositumomab and I 131Iodine tositumomab), bicalutamide, bleomycin, bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib S-malate, CAF, Camptus (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV V bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, Cometriq (cabozantinib S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, liposomal cytarabine, Cytosar-U (cytarabine), Cytoxan (cytarabine) Clophosphamide), 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, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), Efdec (fluorouracil), Erytech (rasburicase), Elence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (Erwinia chrysanthemi asparaginase), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Ebaset (doxorubicin hydrochloride liposome), everolimus,Evista (raloxifene hydrochloride), exemestane, Fairston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Forex (methotrexate), ForexPFS (methotrexate), Forfili, Forfili-bevacizumab, Forfili-cetuximab, Forfirinox, Forfox (leucovorin, fluorouracil, oxaliplatin), Forotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, Gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Jilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, Hycamtin (topotecan hydrochloride), Hy Per-CVAD, Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), Ifosfamide, Ifosfamidum (ifosfamide), Imatinib mesylate, Imbruvica (ibrutinib), Imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), Iodine, 131Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivans (palifermin), Kyprolis (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leucovorin acetate Prolide, 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 months) (leuprolide acetate), Lupron Depot (4 months) (leuprolide acetate), Marchibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megase (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), Metazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), mitomycin C, Mitozytrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (brinjal) Sulfane), Myrosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (vinorelbine tartrate), Nelarabine, Neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omacetaxine mepeccate, Oncaspar (pegaspargase),Ontac (denileukin diftitox), OEPA, OPPA, oxaliplatin, paclitaxel, albumin-stabilized nanoparticle formulation of paclitaxel, palifermin, palonosetron hydrochloride, disodium pamidronate, panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pem Trexed 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), Provenzi (sipuleucel-T), Purinetol (mercaptopurine), radium dichloride, 223, 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), Silatron (peginterferon alfa-2b), Sinovir (thalidomide), Synribo (omacetaxine mepeccate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomi (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and I 131Iodine 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), Versal (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate) ), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine liposomal sulfate, vinorelbine tartrate, vismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xerox, Xgeva (denosumab), Xofigo (radium dichloride) 223 ), 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).

[0321] The anticancer drug can be a PBD dimer, calicheamicin, speromycin, tubulysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothilone B, taxol, cryptophycin, SN38, velcade, bruceanthin, DAVLBH, DM1, filanthoside, alimta, T2 toxin, MMC, vantalanib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, iressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, gleevec, topotecan, bleomycin, doxorubicin, cisplatin, nitrogen mustard, etoposide, or 5-FU.

[0322] In certain embodiments, the anti-cancer agent is an anthracycline. In certain embodiments, the anti-cancer agent is a taxane. In certain embodiments, the anti-cancer agent is gemcitabine. In certain embodiments, the anti-cancer agent is doxorubicin. In certain embodiments, the anti-cancer agent is docetaxel. In certain embodiments, the anti-cancer agent is SN38. In certain embodiments, the anti-cancer agent is monomethylauristatin E. In certain embodiments, the anti-cancer agent is an alkylating agent, an antimetabolite (folate antagonist, purine antagonist, pyrimidine antagonist), antibiotic, taxane, vinca alkaloid, or campothecin analog.

[0323] 7. Compound Synthesis Conjugates can be prepared using the methods disclosed herein and routine modifications thereof, which will be apparent in light of the disclosure herein and methods well known in the art. In addition to the teachings herein, conventional, well known synthetic methods can be used. The synthesis of typical compounds described herein can be accomplished as described in the following examples. Where available, reagents and starting materials can be purchased commercially, for example from Sigma Aldrich or other chemical suppliers.

[0324] It will also be understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, other process conditions can be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0325] In addition, conventional protecting groups may be required 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 specific functional groups, are well known in the art. Numerous protecting groups are described, for example, in Wuts, PGM, Greene, TW, & Greene, TW (2006). Greene's protective groups in organic synthesis. Hoboken, NJ, Wiley-Interscience, and references cited therein.

[0326] Furthermore, the conjugates of the present disclosure may contain one or more chiral centers. Thus, if desired, such conjugates can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present disclosure, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) can be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such conjugates can be separated, for example, using chiral column chromatography, chiral resolving agents, and the like.

[0327] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, Calif., USA), Emka-Chemce, or Sigma (St. Louis, Missouri, USA). Others can be prepared by procedures described in standard reference texts such as Fieser and Fieser, Reagents for Organic Synthesis, vols. 1-15 (John Wiley, and Sons, 1991), Rodd, Chemistry of Carbon Compounds, vols. 1-5 and supplements (Elsevier Science Publishers, 1989), Organic Reactions, vols. 1-40 (John Wiley, and Sons, 1991), March, Advanced Organic Chemistry (John Wiley, and Sons, 5th ed., 2001), and Larock, Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof. EXAMPLES

[0328] The following examples are included to illustrate specific embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that work well in the implementation of the present disclosure and therefore can be considered as constituting specific aspects for its implementation. However, those skilled in the art should understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0329] Example 1: Synthesis of TCO(Gly)-PABC-MMAE (Compound 1) [ka]

[0330] Methyl ((1R,6R,E)-6-hydroxy-1-methylcyclooct-4-ene-1-carbonyl)glycinate (1.2). Step-1: To a mixture of methyl glycinate (5.1 g, 32.6 mmol, 2.0 equiv.) in DMF (100.0 mL) and DIPEA (14.9 mL, 85.5 mmol, 5.3 equiv.), (1R,6R,E)-6-hydroxy-1-methylcyclooct-4-ene-1-carboxylic acid (1.1) (3.0 g, 16.3 mmol, 1.0 equiv.) and HATU (12.4 g, 32.6 mmol, 2.0 equiv.) were added sequentially. The mixture was stirred at room temperature overnight and diluted with EtOAc (400 mL) and water (400 mL). The aqueous layer was extracted once with EtOAc (400 mL). The combined organic layers were dried over Na2SO4 and filtered. The filtrate was concentrated and purified by flash chromatography (220 g ISCO column) eluting with a gradient of EtOAc in hexanes (0% to 100%) and an isocratic gradient of 100% EtOAc in hexanes to give 3.25 g (78% yield) of methyl ((1R,6R,E)-6-hydroxy-1-methylcyclooct-4-ene-1-carbonyl)glycinate.

[0331] Methyl ((1R,6R,E)-1-methyl-6-(((4-nitrophenoxy)carbonyl)oxy)cyclooct-4-ene-1-carbonyl)glycinate (1.3). Step-2: To a solution of compound 1.2 (1.0 g, 4.0 mmol, 1.0 equiv) in anhydrous DCM (30 mL) was added pyridine (0.9 g, 12 mmol, 3.0 equiv). The mixture was cooled in an ice bath. To this mixture was added a solution of p-nitrophenyl chloroformate (1.0 g, 5 mmol, 1.3 equiv) in DCM (5 mL) over 2 min. The mixture was stirred at room temperature for 1 h and partitioned with EtOAc and water. The organic phase was washed with aqueous sodium bicarbonate, water, then dried over sodium sulfate, filtered and concentrated. The resulting residue was dissolved in a minimum amount of DCM and purified by flash chromatography on a silica gel column (40 g, ISCO) using a stepwise gradient of EtOAc in DCM (0% to 20%) as eluent to give 1.6 g (95%) of methyl ((1R,6R,E)-1-methyl-6-(((4-nitrophenoxy)carbonyl)oxy)cyclooct-4-ene-1-carbonyl)glycinate. (+ESI)[M+H] + = 421.1.

[0332] Methyl ((1R,6R,E)-6-(((4-(hydroxymethyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycinate (1.4). step-3: To a solution of compound-1.3 (138 mg, 0.33 mmol, 1.0 equiv), (4-aminophenyl)methanol (40.4 mg, 0.33 mmol, 1.0 equiv), and HOBt (111 mg, 0.66, 2.0 equiv) in DMF (2.0 mL) was added DIEA (85 mg, 0.66 mmol, 2.0 equiv) sequentially. The mixture was stirred at room temperature overnight while being monitored by LCMS. Upon completion of the reaction, the mixture was loaded directly on a C18 flash chromatography (25 g, Agela) using a stepwise gradient of acetonitrile in water (0% to 60% in 16 min; product eluted at approximately 40% acetonitrile in water). Fractions were collected and diluted with EtOAc (approximately 200 mL). The aqueous layer was extracted two more times with EtOAc (100 mL each). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give 130 mg (98%) of methyl ((1R,6R,E)-6-(((4-(hydroxymethyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycinate. (+ESI)[M+Na] + = 427.3.

[0333] Methyl ((1R,6R,E)-1-methyl-6-(((4-(((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)cyclooct-4-ene-1-carbonyl)glycinate (1.5). step-4: To a solution of compound 1.4 (130 mg, 0.32 mmol, 1.0 equiv) and bis(4-nitrophenyl)carbonate (108 mg, 0.35 mmol, 1.1 equiv) in dry DCM (8 mL) was added DIEA (83 mg, 0.64 mmol, 2.0 equiv). The mixture was stirred at room temperature overnight, diluted with DCM (30 mL), washed twice with NaHCO3 (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography using a gradient of EtOAc and hexanes as eluent (0% to 80%; product elutes around 70%) to give 160 mg (87%) of methyl ((1R,6R,E)-1-methyl-6-(((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)carbamoyl)oxy)cyclooct-4-ene-1-carbonyl)glycinate. (+esi)[M+H] + =570.2 (trace ions). 1 H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.32 (d, J = 9.1 Hz, 2H), 7.79 (t, J = 5.8 Hz, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), 5.91 (ddd, J = 14.3, 9.9, 3.9 Hz, 1H), 5.79 - 5.67 (m, 1H), 5.24 (s, 2H), 5.15 (s, 1H), 3.80 - 3.65 (m, 2H), 3.60 (s, 3H), 2.30-2.03 (m, 3H), 2.02-1.93 (m, 1H), 1.92-1.78 (m, 2H), 1.70 (d, J = 13.4 Hz, 1H), 1.58 (dd, J = 14.4, 6.2 Hz, 1H), 1.05 (s, 3H).

[0334] Methyl ((1R,6R,E)-6-(((4-(((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycinate (1.6). Step 5: Synthesis of compound 1.5 (40 mg, 0.07 mmol, 1.0 equiv.) and (2S)-N-((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy To a solution of si-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide (50 mg, 0.07 mmol, 1.0 equiv.) and HOBt (27 mg, 0.14 mmol, 2.0 equiv.; 80% purity) was added DIPEA (14 mg, 0.14 mmol, 2.0 equiv.). The mixture was stirred at room temperature overnight and monitored by LCMS. Once compound 1.5 was consumed, the mixture was directly loaded onto a C18 cartridge (25 g, Agela) and purified with a step gradient (0%→100%; compound elutes around 65%) using acetonitrile and water. Fractions were collected and partially concentrated to remove most of the acetonitrile and carried directly to the next step without complete drying. (+esi)[M+H] + =1248.6.

[0335] ((1R,6R,E)-6-(((4-(((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycine (TCO(Gly)-PABC-MMAE). Step 6: Compound 1.6 in water from above step was diluted with MeOH (3.0 mL). To the mixture was added LiOH (7 mg, 0.28 mmol, 4 equiv). The mixture was stirred at room temperature for 1 h while being monitored by LCMS. Upon completion of the reaction, the mixture was partially concentrated to remove most of the MeOH and acidified to pH 3 with HCl (1N), where a sticky solid was observed. The aqueous solution was extracted 4 times with EtOAc (10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The product was transferred to a 40 mL vial, redissolved in a mixture of acetonitrile and water (1:1; 8 mL), and lyophilized to give 45 mg (57% over two steps) of ((1R,6R,E)-6-(((4-(((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy- The compound was obtained as follows: 2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycine. (+esi)[M+H] - = 1134.8. 1H NMR (400 MHz, メタノール-d4) δ 7.98-7.71 (m, 2H), 7.57-7.44 (m, 2H), 7.44 - 7.27 (m, 4H), 7.26-7.16 (m, 1H), 6.10-5.97 (m, 1 H), 5.74 (d, J = 16.7 Hz, 1H), 5.26-5.12 (m, 3 H), 5.06 (d, J = 13.2 Hz, 1H), 4.80 - 4.50 (m, 5H), 4.45 - 4.15 (m, 4H), 4.13-4.02 (m, 1H), 3.91 - 3.65 (m, 5H), 3.61-3.39 (m, 5H), 3.36 (s, 3H), 3.25-3.08 (m, 2H), 3.02 - 2.85 (m, 2H), 2.60 - 2.38 (m, 2H), 2.36-2.13 (m, 4H), 2.13 - 1.77 (m, 6H), 1.77 - 1.53 (m, 3H), 1.51-1.26 (m, 5H), 1.26 - 1.09 (m, 8H), 1.09 - 0.69 (m, 15H).

[0336] Example 2: Synthesis of TCO(gly)-ニラパリブ(Compound 2)

change

[0337] (1R,6R,E)-6-((2-Methoxy-2-oxoethyl)carbamoyl)-6-methylcyclooct-2-en-1-yl (S)-3-(4-(7-carbamoyl-2H-indazol-2-yl)phenyl)piperidine-1-carboxylate glycinate (2.1). Step-1: To a solution of compound-1.3 (39 mg, 0.09 mmol, 1.0 eq), niraparib (30 mg, 0.09 mmol, 1.0 eq), and HOBt (111 mg, 0.66, 2.0 eq) in DMF (1.0 mL), DIEA (12 mg, 0.19 mmol, 2.0 eq) was added sequentially. The mixture was stirred at room temperature overnight while being monitored by LCMS. Upon completion of the reaction, the mixture was directly loaded onto a C18 flash chromatograph (12 g, ISCO) using a step gradient of acetonitrile in water (0% to 100% in 12 min; product elutes at approximately 70% acetonitrile in water). Fractions were collected and partially concentrated to remove most of the acetonitrile and carried directly into the next step without complete drying. (+ESI)[M+H] + =602.6.

[0338] ((1R,6R,E)-6-(((S)-3-(4-(7-carbamoyl-2H-indazol-2-yl)phenyl)piperidine-1-carbonyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycine (TCO(Gly)-Niraparib). Step 2: Compound 2.1 in water from above step was diluted with MeOH (3.0 mL). To the mixture was added LiOH (9 mg, 0.37 mmol, 4 equiv). The mixture was stirred at room temperature for 1 h while being monitored by LCMS. Upon completion of the reaction, the mixture was partially concentrated to remove most of the MeOH and acidified to pH 3 with HCl (1N). The precipitate was collected and dried to give 40 mg (73% over two steps) of ((1R,6R,E)-6-(((4-(((((2S)-1-(((2S)-1-(((4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine- 1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)oxy)-1-methylcyclooct-4-ene-1-carbonyl)glycine was obtained as a white powder. (+esi)[M+H] - = 588.7. 1 H NMR (400 MHz, methanol-d4) δ 9.03 (s, 1H), 8.19 (d, J = 7.0 Hz, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.75-7.62 (m, 1H), 7.57 (s, 2H), 7.29 (t, J = 7.7 Hz, 1H), 5.92 (s, 1H), 5.75 (d, J = 16.7 Hz, 1H), 5.22 (s, 1H), 4.43 - 4.08 (m, 3H), 3.81 (d, J = 15.3 Hz, 2H), 3.23 - 2.79 (m, 4H), 2.33 (s, 2H), 2.25 - 1.80 (m, 6H), 1.80-1.53 ​​(m, 2H), 1.18 (s, 3H).

[0339] Example 3: Gly-TCO-HTI-286 conjugate (compound 3) [ka]

[0340] Gly(OMe)-TCO (2). A solution of trans-cyclooctene 1, glycine methyl ester, DIPEA, and HOBt in DCM is placed in a round-bottom flask equipped with a magnetic stir bar. EDC is added to the solution, and the resulting mixture is stirred at ambient temperature for 1 h. Consumption of the starting material is observed by HPLC. The reaction mixture is partitioned between ethyl acetate and citrate buffer (pH 4.5). The organic layer is then washed with citrate buffer (2x), followed by sodium bicarbonate (2x) and brine. The organic layer is dried over sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography to give intermediate 2.

[0341] Gly(OMe)-TCO-para-nitrophenyl carbonate (3). A solution of intermediate 2 and pyridine in DCM is placed in a round-bottom flask equipped with a magnetic stir bar. To this solution is added para-nitrophenyl chloroformate, and the resulting mixture is stirred at ambient temperature. Consumption of the starting material is monitored by HPLC. The reaction mixture is partitioned between ethyl acetate and citrate buffer (pH 4.5). The organic layer is then washed with citrate buffer (2x), followed by sodium bicarbonate (3x) and brine. The organic layer is dried over sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography to give carbonate 3.

[0342] Gly-TCO-HTI-286 (4). Carbonate 3 is added to a solution of HTI-286 and pyridine in DMF in a round bottom flask equipped with a magnetic stir bar. The reaction mixture is stirred at ambient temperature until the starting material is consumed as monitored by HPLC. Lithium hydroxide (aq) and THF are added to the reaction mixture. The mixture is stirred at ambient temperature for an additional period of time. The reaction mixture is then concentrated under reduced pressure and the resulting residue is purified by reverse phase chromatography (10%→100% MeCN / water with 0.1% formic acid) to give the desired product, compound 3.

[0343] Example 4: Gly-TCO-spacelink-DuoTM conjugate (compound 4) [ka]

[0344] Seco-duocarmycin TM-PNP carbonate (5). To a solution of seco-duocarmycin TM in DCM, pyridine and para-nitrophenyl chloroformate are added. The reaction mixture is stirred at ambient temperature until the starting material is consumed as monitored by HPLC. The reaction mixture is then partitioned between ethyl acetate and citrate buffer. The organic layer is washed with citrate buffer (2x), followed by sodium bicarbonate (3x) and brine. The organic layer is then dried over sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure to give carbonate 5, which is used without further purification.

[0345] Gly(OFm)-TCO-para-nitrophenyl carbonate (7). A solution of alcohol 6 and pyridine in DCM is placed in a round-bottom flask equipped with a magnetic stir bar. To this solution is added para-nitrophenyl chloroformate, and the resulting mixture is stirred at ambient temperature. Consumption of the starting material is monitored by HPLC. The reaction mixture is then partitioned between ethyl acetate and buffer (pH 4.5). The organic layer is washed with citrate buffer (2x), followed by sodium bicarbonate (3x) and brine. The organic layer is dried over sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography to give carbonate 7.

[0346] Gly(OFm)-TCO-N-Boc-spacelink carbamate (8). A solution of carbonate 7 and pyridine in DCM is placed in a round-bottom flask equipped with a magnetic stir bar. To this solution is added N-Boc-N,N'-dimethyl-1,2-diaminoethane and the resulting mixture is stirred at ambient temperature. Consumption of starting material is observed by HPLC. The reaction mixture is then partitioned between ethyl acetate and citrate buffer (pH 4.5). The organic layer is washed with citrate buffer (2x), followed by sodium bicarbonate (3x) and brine. The organic layer is dried over sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The resulting residue is purified by flash chromatography to give carbamate 8.

[0347] Gly(OFm)-TCO-spacelink-DuoTM (9). A solution of carbamate 8 in dioxane is placed in a round-bottom flask equipped with a magnetic stir bar. HCl (4M in dioxane) is added to the solution and the resulting mixture is stirred at ambient temperature. Consumption of the starting material is monitored by HPLC. The reaction mixture is then concentrated under reduced pressure and the resulting residue is used without further purification.

[0348] The residue is resuspended in DCM and carbonate 5 is added to the solution. 2,6-lutidine is added to this mixture and the resulting reaction mixture is stirred at ambient temperature until the carbonate is consumed. The reaction mixture is then concentrated under reduced pressure and the resulting residue is purified by flash chromatography to give carbamate 9.

[0349] Gly-TCO-spacelink-DuoTM (10). A solution of carbamate 9 in DMF is placed in a round-bottom flask equipped with a magnetic stir bar. Piperidine is added to the solution and the resulting mixture is stirred at ambient temperature. Consumption of the starting material is monitored by HPLC. The reaction mixture is then concentrated under reduced pressure and the resulting residue is purified by reverse phase chromatography (10%→100% MeCN / water with 0.1% formic acid) to give the desired product, compound 4.

[0350] Example 5: TCO-PBD conjugate (Compound 5) [ka]

[0351] TCO-PBD (12). To a solution of SG3199 in DMF, bis-NHS-TCO 11 and HOBt are added. The reaction mixture is stirred at ambient temperature protected from light until the starting material is consumed. Lithium hydroxide (aq) and methanol are added to the reaction mixture, and the resulting solution is stirred at ambient temperature for an additional hour. The reaction mixture is then concentrated under reduced pressure, and the resulting residue is purified by reverse phase chromatography (10%→100% MeCN / water with 0.1% ammonium formate) to give the desired product, compound 5.

[0352] Example 6: TCO(ammonium)-exatecan conjugate (compound 6) [ka]

[0353] To a mixture of triphosgene (177 mg, 0.6 mmol) in THF (10 mL) was added (1R,6R,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. This 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 hours. The mixture was concentrated and purified by preparative HPLC (CHCN / H0(FA) 0%→70%) to give intermediate A (110 mg, 14% yield). LCMS: (m / z, C 35 H 36 FN3O8) = 646.3 [M+H] +

[0354] [ka]

[0355] To a solution of intermediate A (65 mg, 100 μmol) and HOSU (18 mg, 150 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-trimethylethane-1-aminium (21 mg, 110 μmol) was added. The mixture was stirred at 25° C. for another 24 h. The mixture was concentrated and purified by preparative HPLC (CH3CN / H2O with 0.01% formic acid) 0%→70%) to give compound 6 (21 mg, 29%).

[0356] Example 7: TCO(PEG)-Exatecan Conjugate (Compound 7) [ka]

[0357] A solution of 13-(2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-yl)-4,7,10,16,19,22-hexaoxa-13-azapentacosane diacid (42 mg, 60 μmol) in TEA:DCM (1:5) (5 mL) was stirred at 0° C. for 1 h. The mixture was concentrated to give crude intermediate B. Then, to a solution of intermediate A (65 mg, 100 μmol) and HOSU (18 mg, 150 μmol) in DMF (5 mL), DIEA (38 mg, 300 μmol) was added. The mixture was stirred at 25° C. for 30 min. Then, the above crude intermediate B (21 mg, 110 μmol) was added. The mixture was stirred at 25° C. for 24 h. The mixture was concentrated and purified by preparative HPLC (CH3CN / H2O with 0.05% TFA) 0%→70%) to give compound 7 (20 mg, 27.2%).

[0358] Example 8: TCO(gly)-Exatecan Conjugate (Compound 8) [ka]

[0359] To a solution of intermediate A (65 mg, 100 μmol) and HOSU (18 mg, 150 μmol) in 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) were added. The mixture was stirred at 25° C. for 5 h. The mixture was concentrated and purified by preparative HPLC (CH3CN / H2O with 0.01% formic acid) 0%→70%) to give compound 8 (32 mg, 45.2%).

[0360] Example 9: TCO-PABC-spacelink-etoposide (Compound 9) [ka]

[0361] To a solution of 1 (1.70 g, 9.23 mmol) in DCM (20.0 mL) was added 2-trimethylsilylethanol (1.64 g, 13.8 mmol), DMAP (1.69 g, 13.8 mmol), EDCI (2.65 g, 13.8 mmol), and DIEA (1.79 g, 13.8 mmol) at 0° C. The mixture was stirred at 25° C. for 16 h. TLC (petroleum ether:ethyl acetate=1:1, R f =0.4) indicated that compound 1 was completely consumed. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1). Intermediate 2 (1.80 g, 68.6% yield) was obtained.

[0362] 1 H NMR: (400 MHz, CDCl3) δ 6.15 - 6.00 (m, 1H), 5.63 (dd, J = 2.0, 16.4 Hz, 1H), 4.48 (s, 1H), 4.13 - 4.08 (m, 2H), 2.31 - 2.15 (m, 3H), 1.94 - 1.81 (m, 4H), 1.58 - 1.56 (m, 2H), 1.09 (s, 3H), 0.98 - 0.94 (m, 2H), 0.03 (s, 9H).

[0363] [ka]

[0364] To a solution of intermediate 2 (1.80 g, 6.33 mmol) in DCM (40.0 mL) was added (4-nitrophenyl) carbonochloridate (5.10 g, 25.3 mmol) and pyridine (2.50 g, 31.6 mmol). The mixture was stirred at 25° C. for 12 h. TLC (petroleum ether:ethyl acetate=3:1, R f=0.4) indicated that intermediate 2 was completely consumed and one major new spot was formed. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1). Intermediate 3 (1.80 g, 63.3% yield) was obtained.

[0365] 1 H NMR: (400 MHz, CDCl3) δ 8.29 (dd, J = 9.2, 3.2 Hz, 2H), 7.40 (dd, J = 9.2, 2.8 Hz, 2H), 6.11- 6.03 (m, 1H), 5.64 (dd, J = 2.4, 16.4 Hz, 1H), 5.29 (s, 1H), 4.15 - 4.10 (m, 2H), 2.40 - 2.17 (m, 4H), 1.98 - 1.92 (m, 3H), 1.71 - 1.65 (m, 1H), 1.14 (s, 3H), 1.03 - 0.95 (m, 2H), 0.09 (s, 9H).

[0366] [ka]

[0367] To a solution of (4-aminophenyl)methanol (2.47 g, 20.0 mmol) in DMF (15.0 mL) was added HOBt (811 mg, 6.01 mmol), DIEA (2.59 g, 20.0 mmol), and Intermediate 3 (1.80 g, 4.00 mmol). The mixture was stirred at 20° C. for 12 h. TLC (petroleum ether:ethyl acetate=3:1, R f=0.2) indicated that intermediate 3 was completely consumed and one major new spot was formed. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with H2O (100 mL) and extracted with EtOAc (30.0 mL x 4), and the combined organic phase was washed with brine (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 -> 3 / 1). Intermediate 4 (1.60 g, 92.2% yield) was obtained.

[0368] [ka]

[0369] To a solution of intermediate 4 (1.40 g, 3.23 mmol) in DCM (20.0 mL), DIEA (1.25 g, 9.69 mmol) and 4A (1.96 g, 6.46 mmol) were added. The mixture was stirred at 25 °C for 2 h. LCMS (ES20203-16-P1C, product: RT = 1.214 min) showed that one main peak with the desired mass was detected. The mixture was diluted with DCM (100 mL), washed with 1 M HCl (30.0 mL x 2) and brine (20.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 → 5 / 1). Intermediate 5 (1.40 g, 67.2% yield) was obtained.

[0370] 1H NMR: (400 MHz, CDCl3) δ 8.30- 8.26 (m, 2H), 7.48 - 7.36 (m, 6H), 6.79 (s, 1H), 6.01 - 5.93 (m, 1H), 5.66 (dd, J = 2.8, 16.8Hz, 1H), 5.30 (s, 1H), 5.26 (s, 2H), 4.15 - 4.11 (m, 2H), 2.32 - 2.21 (m, 2H), 2.21 - 2.13 (m, 2H), 1.93 - 1.81 (m, 3H), 1.70 - 1.61 (m, 1H), 1.14 (s, 3H), 1.03 - 0.96 (m, 2H), 0.05 (s, 9H).

[0371] [ka]

[0372] To a solution of intermediate 6 (2.00 g, 3.40 mmol) in THF (70.0 mL), TEA (5.16 g, 51.0 mmol) and (4-nitrophenyl) carbonochloridate (1.03 g, 5.10 mmol), THF (30.0 mL) were added. The mixture was stirred at 25° C. for 2 h. LCMS (ES19514-19-p2a2, product: RT=0.866 min) showed that one main peak with the desired mass was detected. After the mixture was treated with 200 mL of isopropyl ether, the mixture was filtered and the filter cake was collected to obtain the product. The crude product was used in the next step without further purification. Intermediate 7 (2.00 g, crude) was obtained.

[0373] LCMS: [M+H] + 754.0

[0374] [ka]

[0375] To a solution of intermediate 7 (1.00 g, 930 μmol) and 7A (432 mg, 1.39 mmol) in DMF (10.0 mL) was added TEA (188 mg, 1.86 mmol). The mixture was stirred at 25° C. for 0.5 h. LCMS (ES20203-20-P1B, product: RT=1.005 min) showed that one main peak with the desired mass was detected. After pouring the mixture into water (100 mL) and separating the solid, the mixture was filtered, the solid was washed with water (20.0 mL×2), dissolved in EtOAc (60.0 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 (SiO2, petroleum ether / ethyl acetate=20 / 1→5 / 1). Intermediate 8 (600 mg, 69.8% yield) was obtained.

[0376] LCMS: [M+H] + 925.3

[0377] [ka]

[0378] To a mixture of intermediate 5 (170 mg, 280 μmol), intermediate 8 (300 mg, 260 μmol) in DMF (5.0 mL) was added TEA (363 mg, 3.59 mmol) at 20° C. The mixture was stirred at 20° C. for 12 h. LCMS (ES20203-21-P1A, product: RT=1.093 min) showed that one main peak with the desired mass was detected. The mixture was filtered and the filtrate was purified by preparative HPLC (Welch's XB-C18 7 μm 110 Å 250 mm×50 mm; mobile phase: [water (NH3HCO3 at 0.01 mol / L in H2O)-ACN]; B%: 55%→75%, retention time: 37 min, flow rate 60 mL / min). Intermediate 9 (180 mg, 59.7% yield) was obtained.

[0379] Note: The Fmoc group of intermediate 8 was removed in situ and the resulting amine was reacted with nitrophenyl carbonate in one pot.

[0380] [ka]

[0381] To a mixture of intermediate 9 (300 mg, 258 μmol) in DMF (10.0 mL) was added TBAF (1 M, 1.29 mmol) at 20° C. The mixture was stirred at 20° C. for 3 h. LCMS showed that one main peak with the desired mass was detected. The mixture was filtered and purified by preparative HPLC (Welch's XB-C18 7 μm 110 Å 250 mm×50 mm; mobile phase: [water (0.1% TFA in H2O)-ACN]; B%: 38%→58%, retention time: 20 min, 20 mL / min). Compound 9 (120 mg, 41.8% yield) was obtained.

[0382] LCMS: [M+Na] + 1084.3

[0383] 1H NMR: (400 MHz, DMSO-d6) δ 9.80 - 9.61 (m, 1H), 7.52 - 7.38 (m, 2H), 7.34 - 7.19 (m, 3H), 6.74 (s, 1H), 6.55 (s, 2H), 6.00 (d, J = 5.6 Hz, 2H), 5.95 - 5.84 (m, 1H), 5.75 - 5.65 (m, 1H), 5.15 (s, 1H), 5.00 (s, 2H), 4.75 - 4.66 (m, 2H), 4.43 (d, J = 3.2 Hz, 1H), 4.38 (d, J = 7.2 Hz, 1H), 4.29 (t, J = 8.8 Hz, 1H), 4.14 (dd, J = 3.2, 9.6 Hz, 1H), 3.94 (d, J = 2.4 Hz, 1H), 3.77 - 3.73 (m, 1H), 3.68 (d, J = 4.4 Hz, 5H), 3.33 (s, 2H), 3.25 (s, 6H), 2.80 (s, 6H), 2.49 - 2.40 (m, 2H), 2.26 - 2.16 (m, 2H), 2.07 - 1.93 (m, 2H), 1.90 - 1.78 (m, 3H), 1.63 - 1.54 (m, 2H), 1.34 - 1.28 (m, 1H), 1.22 (d, J = 5.2 Hz, 3H), 1.02 (s, 3H), 0.94 (t, J = 7.2 Hz, 2H).

[0384] Example 10: TCO-PABC-ゲムシタビン (Compound 10)

change

[0385] To a solution of intermediate 10 (500 mg, 1.90 mmol) in pyridine (30.0 mL) was added compound 11 (629 mg, 1.99 mmol) at 0° C. The mixture was stirred at 25° C. for 12 h. LCMS showed that compound 10 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO2, DCM / MeOH=20 / 1). Intermediate 12 (800 mg, 83.3% yield) was obtained.

[0386] LCMS: [M+H] + 506.1

[0387] [ka]

[0388] To a mixture of intermediate 12 (304 mg, 600 μmol) and compound 5 (300 mg, 501 μmol) in THF (20.0 mL) was added LiHMDS (1M, 1.50 mmol) at −30° C. The mixture was stirred at −30° C. for 0.5 h. And then a solution of compound 5 (300 mg, 501 μmol) in THF (20.0 mL) was added dropwise to the reaction and stirred at −30° C. for 1 h. LCMS showed that one main peak with the desired mass was detected. The mixture was quenched by the addition of saturated ammonium chloride solution (100 mL), diluted with H2O (100 mL), the mixture was extracted with EtOAc (40.0 mL×3), and the combined organic phase was washed with brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO2, petroleum ether / ethyl acetate=20 / 1→5 / 1) to obtain intermediate 13 (260 mg, 53.8% yield).

[0389] LCMS: [M+Na] + 987.3

[0390] [ka]

[0391] To a mixture of intermediate 13 (260 mg, 270 μmol) in DMF (10.0 mL) was added TBAF (1M, 2.15 mmol) at 20° C. The mixture was stirred at 20° C. for 2 h. LCMS showed that one main peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch's Xtimate C18 100 mm×40 mm×3 μm; mobile phase: [water (TFA)-ACN]; B%: 26%→66%, 8 min). Compound 10 (105 mg, 62.5% yield) was obtained.

[0392] LCMS: [M+H] + 623.2

[0393] HNMR: (400 MHz, DMSO-d6) δ 12.63 - 11.69 (m, 1H), 11.19 - 10.72 (m, 1H), 9.91 - 9.66 (m, 1H), 8.23 ​​(d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 7.6 Hz, 1H), 6.44 - 6.24 (m, 1H), 6.20 - 6.10 (m, 1H), 5.99 - 5.82 (m, 1H), 5.76 - 5.65 (m, 1H), 5.16 (s, 1H), 5.12 (s, 2H), 4.29 - 4.10 (m, 1H), 3.92 - 3.86 (m, 1H), 3.81 (d, J = 13.2 Hz, 1H), 3.66 (dd, J = 3.6, 12.4 Hz, 1H), 2.22 (d, J = 6.0 Hz, 2H), 2.09 - 1.78 (m, 6H), 1.65 - 1.57 (m, 1H), 1.03 (s, 3H).

[0394] Example 11: TCO-Mitomycin C (Compound 11) [ka]

[0395] To a mixture of intermediate 3A (350 mg, 1.05 mmol), DIEA (345 mg, 2.67 mmol), and DMAP (109 mg, 890 μmol) in DMF (5.0 mL) was added a solution of intermediate 3 (400 mg, 890 μmol) in DMF (5.0 mL) at 0 °C. The mixture was stirred at 20 °C for 12 h. Then, TBAF (1 M, 4.45 mmol) was added to the reaction mixture at 0 °C and stirred at 20 °C for 2 h. LCMS showed that one main peak with the desired mass was detected. The reaction mixture was diluted with ice water (100 mL) and then extracted with DCM (100 mL x 4), and the organic layer was dried, filtered, and concentrated under reduced pressure to give a residue. The aqueous phase was quenched by the addition of 1 M HCl (100 mL) at 0 °C and NaClO solution (100 mL) at 0 °C. The residue was purified by preparative HPLC (Welch XB-C18 7 μm 110 Å 250 mm × 50 mm; mobile phase: [water (0.01 mol / L NH3HCO3 in H2O)-ACN]; B%: 10% to 30% in 40 min, retention time: 20 min, 20 mL / min). Compound 11 (102 mg, 20.8% yield) was obtained.

[0396] LCMS: [M+H] + 545.2

[0397] 1HNMR: (400 MHz, DMSO-d6) δ 7.25 - 6.97 (m, 2H), 6.67 - 6.31 (m, 2H), 5.99 - 5.79 (m, 1H), 5.65 - 5.53 (m, 1H), 5.10 - 5.04 (m, 1H), 5.02 - 4.95 (m, 1H), 4.35 - 4.28 (m, 1H), 4.04 - 3.95 (m, 1H), 3.68 (d, J = 4.8 Hz, 1H), 3.53 (d, J = 6.4 Hz, 2H), 3.14 (s, 3H), 2.58 - 2.56 (m, 1H), 2.24 - 2.01 (m, 4H), 1.88 - 1.72 (m, 3H), 1.68 (s, 3H), 1.60 - 1.52 (m, 1H), 0.97 (s, 3H).

[0398] Example 12: TCO-(taurine)-gemcitabine (compound 12) [ka]

[0399] To a mixture of intermediate 15 (200 mg, 422 μmol) and intermediate 15A (106 mg, 845 μmol) in DMF (2.0 mL), EDCI (162 mg, 845 μmol), HOBt (114 mg, 845 μmol), and DIEA (164 mg, 1.27 mmol) were added at 20° C. The mixture was stirred at 20° C. for 12 h. LCMS showed that one main peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch's Xtimate C18 100 mm×40 mm×3 μm; mobile phase: [water (TFA)-ACN]; B%: 10%→40%, 8 min). Compound 12 (102 mg, 39.8% yield) was obtained.

[0400] 1HNMR: (400 MHz, DMSO-d6) δ 8.23 ​​(d, J = 7.6 Hz, 1H), 7.51 (s, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.17 (t, J = 7.6 Hz, 1H), 5.99 - 5.86 (m, 1H), 5.68 (d, J = 16.4 Hz, 1H), 5.16 (s, 1H), 4.27 - 4.12 (m, 1H), 3.88 (d, J = 8.4 Hz, 1H), 3.82 (s, 1H), 3.66 (s, 3H), 3.30 - 3.26 (m, 2H), 2.55 (s, 1H), 2.26 - 2.14 (m, 2H), 2.11 - 1.79 (m, 5H), 1.66 (d, J = 12.8 Hz, 1H), 1.51 - 1.38 (m, 1H), 1.31 - 1.12 (m, 1H), 1.00 (s, 3H).

[0401] Example 13: TCO-(taurine)-Ptx (Compound 13) [ka]

[0402] DIEA (491 mg, 3.80 mmol) was added to a stirred suspension of compound 1 (100 mg, 540 μmol) and DSC (598 mg, 2.33 mmol) in MeCN (2.0 mL), and the mixture was stirred at 20° C. for 12 h. TLC (petroleum ether:ethyl acetate=1:1, R f =0.3) showed the formation of one major new spot. The mixture was poured into water (50.0 mL) and extracted with EA (30.0 mL x 3), and the combined organic phase was washed with brine (30.0 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1 -> 1 / 1). Intermediate 16 (130 mg, 56.7% yield) was obtained.

[0403] 1HNMR: (400 MHz, CDCl3) δ 6.14 - 6.01 (m, 1H), 5.63 (dd, J = 2.4, 16.8 Hz, 1H), 5.29 (s, 1H), 2.87 - 2.81 (m, 8H), 2.51 - 2.26 (m, 4H), 2.20 - 1.94 (m, 4H), 1.28 (s, 3H).

[0404] [ka]

[0405] To a mixture of intermediate 16A (106 mg, 124 μmol) and DMAP (34.0 mg, 275 μmol) in DCM (2.0 mL) was added intermediate 16 (58.0 mg, 137 μmol). The mixture was stirred at 20° C. for 12 h. LCMS showed that one main peak with the desired mass was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH=10 / 1, R f =0.4) to give intermediate 17 (130 mg, 79.5% yield). LCMS: [M-CO2-OSu] + 1020.9.

[0406] [ka]

[0407] A mixture of intermediate 17 (50.0 mg, 43.1 μmol), intermediate 15A (16.2 mg, 129 μmol), DMAP (10.5 mg, 86.1 μmol), and DIEA (33.4 mg, 258 μmol) in DMF (5.0 mL) was stirred at 20° C. for 12 h. LCMS showed that one major peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Boston Green ODS 150 mm x 30 mm x 5 μm; mobile phase: [water (TFA)-ACN]; B%: 40% to 70%, 10 min) and further purified by preparative HPLC (column: YMC-Actus Triart C18, 250 mm x 30 mm, 5 μm, 120 Å; mobile phase: [water-ACN]; B%: 20% to 70% in 40 min, retention time: 22 min, 20 ml / min). Compound 13 (6.80 mg, 12.0% yield) was obtained. LCMS: [M+H] + 1171.2

[0408] Example 14: TCO-Ammonium-Ptx (Compound 14) [ka]

[0409] A mixture of intermediate 17 (50.0 mg, 43.1 μmol), intermediate 20 (18.0 mg, 129 μmol, HCl), DIEA (33.4 mg, 258 μmol), and DMAP (10.5 mg, 86.1 mmol) in DMF (1.0 mL) was stirred at 20° C. for 12 h. LCMS showed that one main peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch's Xtimate C18 150 mm×25 mm×5 μm; mobile phase: [water (TFA)-ACN]; B%: 33%→63%, retention time: 11 min). Compound 14 (24.0 mg, 45.3% yield) was obtained. LCMS: [M] + 1148.4

[0410] Example 15: TCO-bisacid-Ptx (Compound 15) [ka]

[0411] A mixture of intermediate 17 (40.0 mg, 34.5 μmol), intermediate 17B (37.4 mg, 138 μmol, HCl), DMAP (8.42 mg, 68.9 μmol), and DIEA (26.7 mg, 207 μmol) in DMF (1.0 mL) was stirred at 20 °C for 12 h. LCMS showed that one main peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch's Xtimate C18 100 mm × 40 mm × 3 μm; mobile phase: [water (TFA)-ACN]; B%: 32% → 62%, 8 min), and then the mixture was adjusted to pH = 8-9 with NH3·H2O before being lyophilized. Compound 15 (14 mg, 30.6% yield). LCMS: [M+H] + 1282.4

[0412] Example 16: TCO-bisPEG-Ptx (Compound 16) [ka]

[0413] A mixture of intermediate 17 (40.0 mg, 34.5 μmol), intermediate 17C (41.4 mg, 68.9 μmol), DMAP (8.42 mg, 68.9 μmol), and DIEA (4.45 mg, 34.5 μmol) in DMF (5.0 mL) was stirred at 20° C. for 12 h. LCMS showed that one major peak with the desired mass was detected. The mixture was filtered and concentrated under reduced pressure. The mixture was purified by preparative HPLC (column: YMC-Actus Triart C18 150 mm x 30 mm x 5 μm; mobile phase: [water (TFA)-ACN]; B%: 43% → 63%, 10.5 min) and further purified by preparative HPLC (column: YMC-Actus Triart C18, 250 mm x 10 mm, 5 μm, 120 Å; mobile phase: [water-ACN]; B%: 30% → 60% in 50 min, retention time: 30 min, 2 mL / min). Compound 16 (10.8 mg, 14.3% yield) was obtained.

[0414] LCMS: [M+H] + 1282.4

[0415] Example 17: TCO-Cipro (Compound 17) [ka]

[0416] To a solution of intermediate 2 (ciprofloxacin, 180 mg, 489 μmol) in DMF (0.3 mL), DIEA (172 mg, 232 μL) and intermediate 1 (200 mg, 445 μmol) in DMF (0.6 mL) were added, and the mixture was stirred at 25° C. for 12 h, then HOBt (120 mg, 890 μmol) in DMF (0.3 mL) was added to the solution and stirred at 25° C. for 2 h, then TBAF (1M, 3.5 mL) was added to the solution and the mixture was stirred at 25° C. for 10 h. LCMS showed that the reactants were consumed and one main peak was the desired product. The residue was purified by preparative HPLC (TFA condition) to give compound 17 (106 mg, 44.0% yield).

[0417] 1HNMR: (400 MHz, DMSO-d6) δ ppm 15.20 (br s, 1 H), 12.02 (br s, 1 H), 8.68 (s, 1 H), 7.95 (d, J = 13.05 Hz, 1 H), 7.61 (d, J = 7.53 Hz, 1 H), 5.78-5.90 (m, 1 H), 5.66-5.74 (m, 1 H), 5.16 (br s, 1 H), 3.73-3.87 (m, 2 H), 3.60 (br s, 3 H), 3.36-3.40 (m, 4 H), 2.14-2.25 (m, 2 H), 2.03-2.12 (m, 1 H), 1.90-1.97 (m, 1 H), 1.72-1.88 (m, 3 H), 1.57-1.64 (m, 1 H), 1.31-1.36 (m, 2 H), 1.19 (br s, 2 H), 1.02 (s, 3 H). LCMS: [M+H] + 542.1

[0418] Example 18: TCO-TLR7 / 8a (Compound 18) [ka]

[0419] To a solution of gardiquimod (100 mg, 319 μmol) in DMF (1 mL) was added DIEA (82.5 mg, 638 μmol) and intermediate 3 (202 mg, 479 μmol). The mixture was stirred at 25° C. for 4 h. LC-MS showed that gardiquimod was completely consumed and one main peak with the desired mass was detected. Intermediate 4 (200 mg, crude) was used in the subsequent step without further purification.

[0420] [ka]

[0421] To a solution of intermediate 4 (200 mg, 322 μmol) in DMF (4 mL) was added intermediate 5 (242 mg, 3.22 mmol), DIEA (250 mg, 1.93 mmol) and DMAP (236 mg, 1.93 mmol). The mixture was stirred at 25° C. for 16 h. LC-MS showed that intermediate 4 was completely consumed and one main peak with the desired mass was detected. The product was purified by preparative HPLC (0.1% TFA condition) to give compound 18 (200 mg, 53.4% ​​yield).

[0422] 1 HNMR: (400 MHz, MeOD) δ 1.02 - 2.31 (m, 20 H), 3.52 - 3.90 (m, 4 H), 4.90 - 5.27 (m, 5 H), 5.40 - 6.04 (m, 2 H), 7.29 - 7.40 (m, 1 H), 7.52 - 7.64 (m, 1 H), 7.67 - 7.82 (m, 2 H), 8.51 (br d, J = 8.38 Hz, 1 H). HRMS: [M+H] + 583.3102.

[0423] Example 19: TCO-STINGa (Compound 19) [ka]

[0424] To a solution of intermediate 2 (2.0 g, 10.8 mmol) in DCM (10 mL) was added DIEA (4.21 g, 32.5 mmol) and EDCI (4.16 g, 21.7 mmol) and DMAP (2.65 g, 21.7 mmol) and intermediate 6 (1.54 g, 13.0 mmol). The mixture was stirred at 25 °C for 16 h. TLC showed that intermediate 2 was completely consumed and one new spot was formed. The reaction mixture was partitioned between DCM (10 mL) and H2O (10 mL). The organic phase was separated, washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 → 10 / 1) to give intermediate 7 (800 mg, 25.9% yield).

[0425] Note: After storing compound 7 at 0° C. for 12 h, TLC showed the formation of one new spot that corresponds to pure compound 7.

[0426] 1 HNMR: (400MHz, CDCl3) δ 0.03 - 0.07 (m, 9 H), 0.94 - 1.00 (m, 2 H), 1.10 (s, 3 H), 1.56 (br dd, J = 15.57, 6.07 Hz, 2 H), 1.77 - 2.00 (m, 5 H), 2.14 - 2.35 (m, 3 H), 4.05 - 4.17 (m, 2 H), 4.48 (br s, 1 H), 5.64 (dd, J = 16.63, 2.38 Hz, 1 H), 5.98 - 6.14 (m, 1 H).

[0427] [ka]

[0428] To a solution of MSA-2 (250 mg, 849 μmol) in DCM (10 mL) was added intermediate 7 (725 mg, 2.55 mmol), DIEA (329 mg, 2.55 mmol), DMAP (156 mg, 1.27 mmol), and HATU (646 mg, 1.70 mmol). The mixture was stirred at 25° C. for 2 h. LC-MS showed that MSA-2 was completely consumed and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (0.1% TFA condition) to give intermediate 8 (210 mg, 44.1% yield).

[0429] [ka]

[0430] To a solution of intermediate 8 (200 mg, 357 μmol) in DMF (2 mL) was added TBAF (1 M, 1.43 mL, 1.43 mmol). The mixture was stirred at 25° C. for 2 h. LC-MS showed that intermediate 8 was completely consumed and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (0.1% TFA condition) to give intermediate 9 (88 mg, 53.6% yield).

[0431] LCMS: [M+Na] + 483.0

[0432] [ka]

[0433] To a solution of intermediate 9 (88 mg, 191 μmol) in DMF (0.2 mL) was added DSC (97.9 mg, 382 μmol) and DIEA (49.4 mg, 382 μmol). The mixture was stirred at 25° C. for 16 h. LC-MS showed that intermediate 9 was completely consumed and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (0.1% TFA) to give intermediate 10 (60 mg, 56.3% yield).

[0434] LCMS: [M+H] + 558.0

[0435] [ka]

[0436] To a solution of intermediate 10 (60 mg, 107.60 μmol) in DMF (1 mL) was added intermediate 5 (80.7 mg, 1.08 mmol), DIEA (139 mg, 1.08 mmol) and DMAP (131 mg, 1.08 mmol). The mixture was stirred at 25° C. for 24 h. LC-MS showed that about 20% of intermediate 10 remained, then intermediate 5 (80.7 mg, 1.08 mmol) was added and the mixture was stirred at 25° C. for 24 h. LC-MS showed that intermediate 10 was completely consumed and one main peak with the desired mass was detected. The crude material was purified by preparative HPLC (0.1% TFA condition) to give compound 19 (30 mg, 80% purity) (containing DIEA residues), and then repurified by preparative HPLC (0.1% TFA condition) to give compound 19 (10 mg, 17.9% yield).

[0437] 1HNMR: (400MHz, CDCl3) δ 1.14 (s, 3 H), 1.16 - 1.31 (m, 1 H), 1.58 - 1.65 (m, 1 H), 1.62 - 1.62 (m, 1 H), 1.81 - 1.89 (m, 3 H), 1.96 - 2.12 (m, 3 H), 2.19 - 2.25 (m, 1 H), 2.20 - 2.33 (m, 1 H), 2.88 (br t, J = 6.38 Hz, 2 H), 3.35 (br t, J = 6.32 Hz, 2 H), 3.46 - 3.69 (m, 11 H), 3.96 (s, 3 H), 3.98 (s, 3 H), 5.27 (br s, 1 H), 5.60 (dd, J = 16.45, 2.06 Hz, 1 H), 5.82 - 5.95 (m, 1 H), 6.16 (br s, 1 H), 7.93 (s, 1 H).

[0438] LCMS: [M+Na] + 540.1

[0439] Example 20: Synthesis of Compound 20

change

[0440] General recipe for intermediate 6

change

[0441] To a solution of intermediate 5 (150 g, 689 mmol, HCl) in NaOH (1M, 1.38 L) and NaHCO3 (1M, 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 h. The reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was extracted with MTBE (5 L), and then the aqueous phase was adjusted to a pH of about 4 with saturated aqueous KHSO4 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 the above crude in MeOH (2 L) was added SOCl2 (90.2 g, 758 mmol) and the mixture was stirred at 25° C. for 2 h. LC-MS showed the reaction was complete with one main peak detected with the desired mass. The reaction mixture was adjusted to pH 9-10 with saturated aqueous NaHCO3, then extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude. The crude was precipitated with PE (10 vol) to give intermediate 6 (190 g, 74.4% yield).

[0442] 1 H 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, 5H).

[0443] LCMS (m / z): 391.9 / 393.9 (M+H) + .

[0444] General procedure for the preparation of intermediate 7 [ka]

[0445] To a solution of intermediate 6 (185 g, 499 mmol) in toluene (1.9 L) was added 4-methylbenzenesulfonic 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 h. LC-MS showed that one main peak with the desired mass was detected. The reaction mixture was then cooled to 25° C. The reaction mixture was concentrated under reduced pressure to remove toluene. The residue was diluted with H2O (500 mL) and then extracted with EtOAc (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give intermediate 7 (285 g, crude), which was carried forward as is.

[0446] General procedure for the preparation of intermediate 8 [ka]

[0447] To a solution of intermediate 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 h. LC-MS showed that intermediate 7 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was extracted with MTBE (5 L). The aqueous layer was diluted with saturated aqueous KHSO4 (1 L) and extracted with EtOAc (5 L), and the combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude. The crude was precipitated with PE (10 vol) to give intermediate 8 (95.0 g, 34.3% yield).

[0448] 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).

[0449] LCMS (m / z): 495.9 (M+Na) + .

[0450] General procedure for the preparation of 7-Troc-baccatin III [ka]

[0451] To a solution of baccatin III (30.0 g, 51.1 mmol) in DCM (300 mL) was added DMAP (625 mg, 5.11 mmol), 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 h. LC-MS showed that baccatin III was completely consumed and one main peak with the desired mass was detected. The residue was diluted with water (300 mL), extracted with DCM (300 mL), washed with water (200 mL) and brine (200 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 7-Troc-baccatin III (45.0 g, 34.3% yield).

[0452] LCMS (m / z): 761.5 / 763.5 (M+Na) + .

[0453] General procedure for the preparation of intermediate 9 [ka]

[0454] To a solution of 7-Troc-baccatin III (26.0 g, 34.1 mmol) and intermediate 8 (32.4 g, 68.2 mmol) 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 h. LC-MS showed that intermediate 8 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was filtered. The crude was washed with saturated aqueous NH4Cl (100 mL) and water (1000 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give intermediate 9 (35.0 g, crude).

[0455] LCMS (m / z): 1240.0 / 1242.0 (M+Na) + .

[0456] General procedure for the preparation of intermediate 10 [ka]

[0457] To a solution of intermediate 9 (80.0 g, 65.6 mmol) in MeOH (350 mL) was added 4-methylbenzenesulfonic acid hydrate (24.9 g, 131 mmol). The mixture was stirred at 25° C. for 16 h. LC-MS showed that about 50% of intermediate 9 remained and one main peak with the desired mass was detected. The reaction mixture was filtered, concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluent was concentrated under reduced pressure to remove the solvent, followed by extraction with EtOAc (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give intermediate 10 (13.0 g, 17.9% yield).

[0458] LCMS (m / z): 1120.2 (M+Na) + .

[0459] General procedure for the preparation of intermediate 11 [ka]

[0460] Intermediate 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) were dissolved in DCM (260 mL). The mixture was stirred at 25° C. for 1 h. LC-MS showed that intermediate 10 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was washed with saturated aqueous citric acid (100 mL), saturated aqueous NaHCO3 (100 mL), and water (200 mL), dried over NaSO4, filtered, and concentrated under reduced pressure to give intermediate 11 (11.0 g, 77.3% yield).

[0461] LCMS (m / z): 1204.1 (M+H) + .

[0462] General procedure for the preparation of intermediate 12 [ka]

[0463] To a solution of intermediate 11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL) was added zinc dust (21.6 g, 331 mmol). The mixture was stirred at 25 °C for 1 h. LC-MS showed that intermediate 11 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was filtered and diluted with H2O (500 mL), then extracted with EtOAc (100 mL x 3). The combined organic layers were washed with saturated aqueous NaHCO3 (200 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give intermediate 12 (5.0 g, 21% yield).

[0464] LCMS (m / z): 854.3 (M+H) + .

[0465] General procedure for the preparation of intermediate 13 [ka]

[0466] Intermediate 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and 3 (3.90 g, 8.80 mmol) were dissolved in DMF (50 mL). The mixture was stirred at 25° C. for 16 h. LC-MS showed that about 50% of intermediate 12 remained and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give intermediate 13 (505 mg, 7.4% yield).

[0467] LCMS (m / z): 1161.4 (M+H) + .

[0468] General procedure for the preparation of compound 20 [ka]

[0469] To a solution of compound intermediate 13 (150 mg, 0.13 mmol) in DMF (1.50 mL), DMAP (94.7 mg, 0.78 mmol) and compound intermediate 13-1 (66.7 mg, 0.65 mmol) and DIEA (100 mg, 0.78 mmol) were added. The mixture was stirred at 25° C. for 16 h. LC-MS showed that intermediate 13 was completely consumed and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 20 (75.0 mg, 50.5% yield).

[0470] LCMS (m / z): 1148.5 (M) + .

[0471] Example 21: General procedure for the preparation of compound 21 [ka]

[0472] Intermediate 13 (350 mg, 0.30 μmol) and DMAP (221 mg, 1.81 mmol) and intermediate 14 (249 mg, 0.39 mmol, HCl) were dissolved in DMF (0.3 mL). The mixture was stirred at 25° C. for 16 h. LC-MS showed that intermediate 13 was completely consumed and one main peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give compound 21 (205 mg, 41.3% yield).

[0473] LCMS (m / z): 1646.5 (M+H) + .

[0474] Example 22: TCO-lurbinectedin conjugate (compound 22) [ka]

[0475] TCO-lurbinectedin (compound 22). To a solution of lurbinectedin in DMF, bis-NHS-TCO 11 and HOBt are added. The reaction mixture is stirred at ambient temperature, protected from light, until the starting material is consumed. Glycine and optionally a base are added to the reaction mixture, and the resulting solution is stirred at ambient temperature for an additional hour. The reaction mixture is then concentrated under reduced pressure, and the resulting residue is purified by reverse phase chromatography (10%→100% MeCN / water with 0.1% ammonium formate) to obtain the desired product, compound 22.

[0476] Example 23: Anti-CD3-Fab-TCO therapeutic conjugates [ka]

[0477] Fabs are prepared from OKT3 using a commercially available kit (Pierce™ Fab Preparation Kit, no. 44985) according to the manufacturer's protocol and purified by Protein G resin (no. 6511-25 from BioVision). 10 mM TCO-PEG3-NHS prepared in DMSO is added to the purified Fab. The two components are reacted at a 3:1 drug to protein molar ratio at 25° C. for 2 hours, after which it is dialyzed against PBS (pH 7.4) to remove excess TCO-PEG3-NHS compound from the protein component. The resulting solution of therapeutic targeting moieties is analyzed by SDS-Page and LCMS to confirm the formation of therapeutic targeting moieties. On average, approximately 1-2 TCOs are expected to be covalently attached to each Fab, as confirmed by LCMS.

[0478] Example 24: Preparation of anti-CD3 Fab-PEG3-TCO conjugate The Fab of anti-CD3 antibody 2C11 was synthesized by plasmid construction, HEK293 cell expression, and purification. The Fab-TCO conjugate was prepared by reacting TCO-PEG3-NHS (structure shown below, purchased from SiChem, catalog number SC-8406) with the primary amine on the Fab to form a stable amide bond.

[0479] [ka]

[0480] Synthesis of 2C11 Fab Vector Construction: Coding sequences (listed below) were synthesized and subcloned into expression vectors. Constructed plasmids were transformed and propagated in E. coli. Large scale plasmid production was performed using the NucleoBond Xtra Maxi Plus EF kit. Purified plasmids were verified by agarose gel and confirmed by sequencing.

[0481] HC sequence of 2C11-Fab: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI (SEQ ID NO: 1).

[0482] LC sequence of 2C11-Fab: DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 2).

[0483] Protein expression: Constructs containing the heavy and light chains of Fab were co-transfected into HEK293 cells using PEI. Culture medium was harvested 7 days after transfection.

[0484] Protein purification: The conditioned medium expressing the target Fab was collected by centrifugation and filtration, and then loaded onto a CaptureSelect LC-kappa (mouse) affinity column (Mabselect Prism). The loading buffer was PBS (pH 8.0), followed by washing with PBS (pH 8.0), followed by washing with PBS (pH 8.0) containing 0.2% Triton X-100 / 114, followed by washing with PBS (pH 8.0). The protein was eluted with 50 mM sodium citrate buffer (pH 3.0) containing 150 mM NaCl. The collected solution was neutralized with 1 M Tris, 1 M arginine buffer (pH 9.0). The affinity purified protein was further purified by gel filtration using Superdex S-200 column chromatography. The purified Fab was analyzed by SDS-PAGE, SEC-HPLC, and endotoxin measurement.

[0485] Preparation of the conjugate: The Fab protein was dialyzed overnight against PBS (pH 7.4) with one buffer change at approximately 4 hours from the start. 10 mM TCOt-PEG3-NHS was prepared in DMSO. The two components were reacted at a 3:1 drug to protein molar ratio at 25°C for 2 hours, after which it was dialyzed against PBS (pH 7.4) to remove excess TCO-PEG3-NHS compound from the protein component. LCMS analysis demonstrated an average loading of 1.9 TCO-PEG3 per Fab.

[0486] Biochemical Examples Biochemical Example 1: TLR7 / 8a (Gardiquimod); Effects on proliferation of fresh mouse splenocytes [ka]

[0487] Lymphocytes were isolated from the spleens of C57BL / 6 mice. Spleens were ground and cells were filtered through a 70 μm cell strainer using DBPS. Red blood cells were lysed and cells were washed with DPBS. Isolated lymphocytes were suspended in culture medium. Cells were seeded in 96-well plates at 50000 cells per well in 90 μL per well and then incubated overnight at 37° C., 5% CO2, 95% air, and 100% relative humidity. The following day, compounds were prepared and 10 μL of compound medium was added to wells of a 96-well plate in triplicate. The following conditions were tested: unmodified gardiquimod, TCO-gly-gardiquimod, TCO-gly-gardiquimod+tetrazine. The following concentrations of gardiquimod and TCO-gly-gardiquimod were used: 10 μg / mL, 2.5 μg / mL, 0.83 μg / mL, 0.28 μg / mL, 0.093 μg / mL, and 0.031 μg / mL. DMSO medium was added to blank and control wells to a final concentration of 0.1% DMSO. The plates were incubated for 48 hours. The plates were then analyzed by CellTiter-Glo luminescence assay according to the manufacturer's instructions (Promega-G7573) to assess cell viability. The inhibition rate (IR) of the tested compounds was determined by the following formula: IR(%)=(1-(RLU compound-RLU blank) / (RLU control-RLU blank))×100%. The inhibition of various doses of the tested compounds was calculated in Excel and then used to plot the inhibition curves and obtain the minimum (%), maximum (%), and IC 50 Related parameters such as serotonin level, serotonin concentration, and serotonin level were evaluated. Data were interpreted using GraphPad Prism.

[0488] As shown in Figures 1 and 2, treatment with unmodified gardiquimod resulted in a concentration-dependent change in proliferation / cell viability. Figure 1 shows the results from an experiment where the highest concentration tested was 10 μg / mL. Figure 2 shows a repeat experiment with TCO-gly-gardiquimod in the absence or presence of tetrazine at concentrations up to 50 μg / mL. TCO-gly-gardiquimod had no to minimal effect on cell viability across a range of concentrations. Even at the highest dose (50 μg / mL), its activity was minimal, suggesting an effective attenuation of activity. On the other hand, treatment with TCO-gly-gardiquimod in the presence of tetrazine led to a concentration-dependent increase in cell viability / proliferation. At concentrations above 5 μg / mL, TCO-gly-gardiquimod showed greater activity against proliferation compared to unmodified gardiquimod in the presence of tetrazine, suggesting potentially superior activity compared to the unmodified drug.

[0489] The therapeutic support compositions described herein can be prepared as described in WO 2018 / 187740. Methods for testing and using the conjugates in combination with the support compositions can also be found in WO 2018 / 187740.

[0490] 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 to which this invention belongs.

[0491] The invention illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like are to be read expansively and without limitation. Furthermore, the terms and expressions employed herein are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the claims.

[0492] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety, to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0493] Although the present disclosure has been described in conjunction with the above embodiments, the above description and examples should be understood as illustrative rather than limiting the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

Claims

1. Formula I: 【Chemistry 1】 (In the formula, m is an integer from 1 to 150, G, at each occurrence, is independently an optionally substituted trans-cyclooctene moiety; D １ is a payload selected from an inhibitor of poly(ADP-ribose) polymerase (PARP), duocarmycin, pyrrolobenzodiazepine (PBD), hemiasterin, HTI-286, a monoclonal antibody, a topoisomerase inhibitor, lurbinectedin, MSA-2, gardiquimod, ciprofloxacin, mitomycin C, etoposide, and exatecan, or a derivative or analog thereof; L １ is, in each occurrence, independently a linker), or a pharmaceutically acceptable salt thereof.

2. Each trans-cyclooctene moiety independently comprises: 【Chemistry 2】 (In the formula, R １Ａ is, in each case independently, C １～４ Alkyl, C １～４ Haloalkyl, and C １～４ alkoxy; q is 0, 1, or 2; q1 is 0 or 1; R １Ｂ is, in each case independently, G １ , —OH, —NR １ｃ -C １～４ Alkylene-G １ , -NR １ｃ -C １～４ Alkylene-N(R １ｄ ) ２ , -NR １ｃ -C １～６ Alkylene-N(C １～４ alkyl) ３ ＋ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ )-C １～６ Alkylene-CO ２ H, -N(R １ｆ )-C ２～４ Alkylene-(N(C １～４ Alkylene-CO ２ H)-C ２～４ alkylene) ｎ -N(C １～４ Alkylene-CO ２ H) ２ , -N(R １ｃ ) CHR １ｅ C(O)OC １～６ Alkyl, —N(R １ｃ )-C １～６ Alkylene-C(O)OC １～６ Alkyl, —N(R １ｆ )-C ２～４ Alkylene-(N(C １～４ Alkylene-C(O)OC １～６ alkyl)-C ２～４ alkylene) ｎ -N(C １～４ Alkylene-C(O)OC １～６ alkyl) ２ , -N(R １ｃ )-C １～６ Alkylene-SO ３ H, -N(R １ｃ )-(CH ２ CH ２ O) １～３ -CH ２ CH ２ N ((CH ２ CH ２ O) １～３ -C １～６ Alkylene-CO ２ H) ２ , and −N(R １ｃ )-CH(CH ２ O-(CH ２ CH ２ O) ０～２ -C １～６ Alkylene-CO ２ H) ２ is selected from the group consisting of R １ｃ and R １ｄ is, in each occurrence, independently hydrogen or C １～４ alkyl, and R １ｅ is, independently in each occurrence, -C １～４ Alkylene-CO ２ H, -C １～４ Alkylene -CONH ２ , or -C １～４ alkylene-OH, R １ｆ is, in each occurrence, independently hydrogen, C １～６ Alkyl, or C １～４ Alkylene-CO ２ H, n is independently 0, 1, 2, or 3 at each occurrence; L ２ is, in each occurrence, independently —C(O)— and C １～３ alkylene; and G １ and R is independently at each occurrence an optionally substituted heterocyclyl, or a pharmaceutically acceptable salt thereof.

3. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the payload is an inhibitor of poly(ADP-ribose) polymerase (PARP), or a derivative or analog thereof.

4. 4. The conjugate of claim 3, or a pharmaceutically acceptable salt thereof, wherein the inhibitor of poly(ADP-ribose) polymerase (PARP) is niraparib, talazoparib, olaparib, pamiparib, rucaparib, veliparib, iniparib, 3-aminobenzamide, CEP-9722, E7016, or a derivative or analog thereof.

5. D １ teeth, 【Transformation 3】 4. The conjugate of claim 3, wherein:

6. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the payload is a duocarmycin, or a derivative or analog thereof.

7. 7. The conjugate of claim 6, wherein the duocarmycin is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, carzelesin, bizeresin, or a derivative or analog thereof, or a pharmaceutically acceptable salt thereof.

8. D １ teeth, 【Chemistry 4】 【change】 7. The conjugate of claim 6, wherein:

9. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the payload is a pyrrolobenzodiazepine (PBD) or a derivative or analog thereof.

10. 10. The conjugate of claim 9, or a pharmaceutically acceptable salt thereof, wherein the pyrrolobenzodiazepine (PBD) is [1,2]diazepino[3,4-e]indole, or a derivative or analog thereof.

11. D １ teeth, 【Transformation 5】 10. The conjugate of claim 9, wherein:

12. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the payload is hemiasterlin, HTI-286, or a derivative or analog thereof.

13. D １ teeth, 【Transformation 6】 13. The conjugate of claim 12, wherein:

14. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the payload is an anti-CD3 (αCD3) monoclonal antibody, or a derivative or analog thereof.

15. 15. The conjugate of claim 14, or a pharmaceutically acceptable salt thereof, wherein the anti-CD3 (αCD3) monoclonal antibody is OKT3, SP34, UCHT1, teplizumab, otelixizumab, visilizumab, or foralarumab, or a derivative or analog thereof.

16. L １ comprises from 1 to 100 linking atoms, or 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.

17. L １ 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 independently is selected from the group consisting of oxo, halo, C １～４ Alkyl, C １～４ Alkoxy, and C １～４ 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, optionally substituted with 1 to 5 substituents independently selected from haloalkyl.

18. L １ is an alkylene linker optionally containing one or more -O-, -S-, amine, ester, amide, carbamate, carbonate, or ketone functional groups, or a pharmaceutically acceptable salt thereof.

19. L １ is the formula: -Y １０ —(CH ２ ( ｎ’ -Y ２０ —(CH ２ ( ｍ’’ -Y ３０ — (In the formula, Y １０ , Y ２０ , and Y ３０ each independently represents a bond, —NR １１０ -, -O-, -S(O) ０～２ -, -NR １１０ C(O)-, -C(O)NR １１０ -, -NR １１０ S (O) ２ -, -S(O) ２ NR １１０ -, -CR １２０ = N-NR １１０ -, -NR １１０ -N=CR １２０ -, -C(O)-, -OC(O)-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, wherein each alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently is oxo, halo, C １～４ Alkyl, C １～４ Alkoxy, and C １～４ and optionally substituted with 1 to 5 substituents independently selected from haloalkyl, and each R １１０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; Each R １２０ are independently hydrogen, C １～４ Alkyl, C １～４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl, and n' and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8, or a pharmaceutically acceptable salt thereof.

20. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker comprises one or more amino acids.

21. L １ is -OC(O)L ４ -or-OC １～６ Alkylene C(O)L ４ - and L ４ is a bond, -N(R １２ )-C ２～３ Alkylene-N(R １３ )C(O)-,-CH(NHC(O)R １４ ) C １～４ Alkylene -S-S-C １～４ Alkylene -OC(O)-, -NHNHC(O)CH(NHC(O)R １５ ) CH ２ C(O)-, -C １～６ Alkylene-CH(G ｘ )OC(O)-, 【Transformation 7】 and R １２ , R １３ , R １４ , R １５ , and R １９ are each independently hydrogen or C １～４ is alkyl, R １６ is hydrogen, C １～４ Alkyl, -C １～４ Alkylene -OH, -C １～４ Alkylene-OC １～４ Alkyl, —C １～４ Alkylene-CO ２ H or -C １～４ Alkylene -CONH ２ and G ｘ is a halogen, C １～４ Alkyl, C １～４ Haloalkyl, C １～４ 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein phenyl is optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkoxy, cyano, and nitro.

22. G-L １ are, in each case independently, 【Transformation 8】 2. The conjugate of claim 1, wherein:

23. G-L １ are, in each case independently, 【Chemistry 9】 2. The conjugate of claim 1, wherein:

24. R １Ｂ is G １ , OH, -NR １ｃ -C １～４ Alkylene-G １ , -NR １ｃ -C １～４ Alkylene-N(R １ｄ ) ２ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ ) CH ２ CO ２ H, or -N(R １ｆ )-CH ２ CH ２ -(N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ and R １ｅ is -CH ２ CO ２ H, —CH ２ CH ２ CO ２ H, —CH ２ CONH ２ , -CH ２ CH ２ CONH ２ , -CH ２ OH, or -CH(CH ３ ) OH, and R １ｆ is hydrogen or -CH ２ CO ２ 5. The conjugate of claim 4, wherein R is H, or a pharmaceutically acceptable salt thereof.

25. R １Ａ is C １～４ is alkyl, R １Ｂ is G １ , —OH, —NR １ｃ -C １～４ Alkylene-G １ , -NR １ｃ -C １～４ Alkylene-N(R １ｄ ) ２ , -N(R １ｃ ) CHR １ｅ CO ２ H, -N(R １ｃ ) CH ２ CO ２ H, or -N(R １ｆ )-CH ２ CH ２ -(N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ and R １ｅ is -C １～４ Alkylene-CO ２ H, R １ｆ is hydrogen or -C １～４ Alkylene-CO ２ H, G １ is a 4- to 8-membered monocyclic heterocyclyl containing a first nitrogen and optionally one additional heteroatom selected from nitrogen, oxygen, and sulfur, wherein G １ is attached at the first nitrogen, and optionally C １～４ Alkyl, C １～４ Haloalkyl, halo, cyano, —OH, —OC １～４ substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo; 5. The conjugate of claim 4, or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2.

26. R １Ａ is CH ３ and R １ｅ is -CH ２ CO ２ H, R １ｆ is hydrogen or -CH ２ CO ２ H, and G １ is attached via a ring nitrogen atom, and optionally C １～４ Alkyl, C １～４ Haloalkyl, halo, cyano, —OH, —OC １～４ 5. The conjugate of claim 4, or a pharmaceutically acceptable salt thereof, which is piperazinyl, morpholinyl, piperidinyl, azepanyl, or pyrrolidinyl substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo.

27. L ２ The conjugate of claim 4, or a pharmaceutically acceptable salt thereof, wherein is —C(O)—.

28. R １Ｂ is -OH, N(H)CH ２ CO ２ H, -N(H)CHR １ｅ CO ２ H, -N(H)-CH ２ CH ２ -(N(CH ２ CO ２ H) CH ２ CH ２ ) ｎ -N(CH ２ CO ２ H) ２ , or -N(CH ２ CO ２ H) —CH ２ CH ２ -N(CH ２ CO ２ H) ２ and R １ｅ is -CH ２ CO ２ H, or a pharmaceutically acceptable salt thereof.

29. G, independently in each occurrence, 【Chemistry 10】 and R ２ is —OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R ２ The conjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein

30. G-L １ are, in each case independently, 【Chemistry 11】 and R ２ is —OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R ２ 10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein:

31. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 1 to 20, or 1 to 10, or 1 to 5.

32. 2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein m is 1.

33. G is, 【Chemistry 12】 2. The conjugate of claim 1, wherein:

34. Formula IIA: 【Chemistry 13】 (In the formula, R ２ is —OH, 2-aminoethanesulfonic acid, an N-linked natural or unnatural amino acid, or an optionally substituted ethylenediamine, where R ２ or a pharmaceutically acceptable salt thereof.

35. structure: 【Chemistry 14】 【change】 【change】 【change】 【change】 【change】 【change】 or a pharmaceutically acceptable salt thereof.

36. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 35, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

37. A pharmaceutical composition for treating cancer or enhancing or inducing an immune response, comprising the conjugate of any one of claims 1 to 35 or a pharmaceutically acceptable salt thereof, said pharmaceutical composition comprising a biocompatible support and a compound of formula: 【Chemistry 15】 (In the formula, R ２０ is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF ３ , C.F. ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ is selected from the group consisting of NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R", and NR'C(=S)NR''R'', 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; R ３０ is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; R ａ , R ３１ａ , and R ３１ｂ are each independently hydrogen, C １ ~C ６ - alkyl, or C １ ~C ６ -haloalkyl, and wherein t is 0, 1, 2, 3, or 4).

38. 38. The pharmaceutical composition of claim 37, wherein the tetrazine-containing group is linked or directly bonded to a hyaluronic acid biocompatible support.

39. The therapeutic support composition has the formula (II): 【Chemistry 16】 (In the formula, G ２ teeth, 【Chemistry 17】 and R ２２ 38. The pharmaceutical composition of claim 37, comprising a substituted hyaluronic acid unit of the formula:

40. G ２ teeth, [Chemistry 18] 40. The pharmaceutical composition of claim 39, wherein:

41. G ２ teeth, 【Chemistry 19】 and R ２０ is hydrogen or C １～４ 41. The pharmaceutical composition of claim 40, wherein the alkyl is alkyl.

42. The pharmaceutical composition of claim 37 for treating cancer.

43. 43. The pharmaceutical composition of claim 42, wherein the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, squamous cell carcinoma of the head and neck, anal / vulvar cancer, esophageal cancer, pancreatic adenocarcinoma, cervical cancer, hepatocellular carcinoma, Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Wilms' tumor / neuroblastoma, bladder cancer, thyroid adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, nasopharyngeal carcinoma, or cutaneous T-cell lymphoma.

44. 43. The pharmaceutical composition of claim 42, wherein the cancer is a solid tumor.

45. 43. The pharmaceutical composition of claim 42, wherein the cancer is a soft tissue sarcoma.

46. 43. The pharmaceutical composition of claim 42, wherein the cancer is a hematopoietic malignancy such as myelodysplastic syndrome, acute myeloid leukemia, myelodysplastic syndrome, 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 lymphoma, monoclonal B-cell lymphocytosis, lymphoproliferative disorder NOS, T-cell lymphoma, precursor B-lymphoblastic leukemia, mantle cell lymphoma, plasmacytoma, Burkitt lymphoma, T-cell leukemia, hairy cell leukemia, precursor T-lymphoblastic leukemia, or nodular lymphocyte-predominant Hodgkin lymphoma.

47. The pharmaceutical composition of claim 37 for enhancing or inducing an immune response.

48. 48. The pharmaceutical composition of claim 47, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

49. The pharmaceutical composition of claim 37, further administered in combination with an additional therapeutic agent selected from the group consisting of anticancer agents or trans-cyclooctene prodrugs thereof.

50. A kit comprising the conjugate according to any one of claims 1 to 35 or a pharmaceutically acceptable salt thereof, and instructions for its use.

51. A method for producing a polymerizable composition comprising: 【Chemistry 20】 (In the formula, R 20 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF 3 , CF 2 —R′, NO 2 , OR′, SR′, C(═O)R′, C(═S)R′, OC(═O)R′′, SC(═O)R′′, OC(═S)R′′, SC(═S)R′′, S(═O)R′, S(═O) 2 R′′, S(═O) 2 is selected from the group consisting of NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R'", SC(=S)R'R", NR'C(=O)NR''R", and NR'C(=S)NR''R'', 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; R 30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; R a , R 31a and R 31b are each independently hydrogen, C 1 -C 6 -alkyl or C 1 -C 6 -haloalkyl; and 51. The kit of claim 50, further comprising a therapeutic support composition comprising a tetrazine-containing group of formula (I), wherein t is 0, 1, 2, 3, or 4.

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