trans-cyclooctene conjugate

Abstract

The present disclosure generally relates to trans-cyclooctene conjugates for bioorthogonal delivery of payloads to target locations in subjects. 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 Nos. 63 / 511,439, filed June 30, 2023, 63 / 511,436, filed June 30, 2023, and 63 / 486,233, filed February 21, 2023, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure generally relates to trans-cyclooctene conjugates for bioorthogonal delivery of payloads to target locations in a subject, which have applications, for example, in the treatment of cancer, tumor growth, and immunotherapy. [Background technology]

[0003] Bioorthogonal conjugation, or "click" reactions, are selective and orthogonal (non-interacting) functionalities found in biological systems that have been used in a variety of applications in chemistry, chemical biology, molecular diagnostics, and medicine 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

[0004] Provided herein is a conjugate for use in bioorthogonal reactions, the conjugate comprising a payload covalently linked to one or more optionally substituted trans-cyclooctene moieties via a linker. In some embodiments, the payload is a taxane such as paclitaxel, a taxane, a topoisomerase inhibitor, or an MMAE payload, or a derivative or analog thereof.

[0005] In some embodiments, a method is provided for delivering an effective amount of a payload (i.e., a taxane such as paclitaxel, a topoisomerase inhibitor, or an MMAE payload, 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 at the target location, and administering to the subject a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof. In some embodiments, the conjugate comprises one or more solubilizing groups.

[0006] In some embodiments, a method for treating cancer is provided, comprising administering to a subject in need of treatment a therapeutic support composition described herein to a target location, and administering to the subject a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof.

[0007] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is melanoma, renal cancer, prostate cancer, ovarian cancer, endometrial carcinoma, breast cancer, glioblastoma, lung cancer, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head / neck squamous cell carcinoma, 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, malignant extrinsic or intrinsic airway compression, or cutaneous T-cell lymphoma.

[0008] 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 lymphoma or leukemia. In some embodiments, the cancer is a hematological malignancy. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 shows the percent body weight change after administration of SQT01 in combination with compound 32 to CrTac:NCr-Foxn1nu mice bearing NCI-N87 subcutaneous xenografts. Error bars represent the standard error of the mean (SEM). [Figure 2] 1 shows tumor volume tracings after administration of SQT01 in combination with compound 32 to CrTac:NCr-Foxn1nu mice bearing NCI-N87 subcutaneous xenografts. DETAILED DESCRIPTION OF THE INVENTION

[0010] 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 is instead provided as a description of exemplary embodiments.

[0011] It is understood that certain features that are described for clarity in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features that are described for brevity in the context of a single embodiment can also be provided separately or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically embraced and disclosed herein as if each and every combination were individually and explicitly disclosed, to the extent that such combinations encompass 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 various embodiments and elements thereof (e.g., elements of chemical groups listed in embodiments describing such variables) are also specifically embraced and disclosed herein as if each and every subcombination were individually and explicitly disclosed herein.

[0012] A.Definition Unless otherwise defined, 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 specification, including definitions, will control. 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 the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0013] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," and "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 "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0014] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., 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" can refer to plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, so that, for example, "about 1" can also mean 0.5 to 1.4.

[0015] The conjunction "or" includes any combination of one or more of the listed items linked by the conjunction. For example, the phrase "a device comprising A or B" can refer to a device with A in the absence of B, a device with A in the absence of A, or a device with B in the absence of A, or a device with both A and B present. The phrases "at least one of A, B, ... and N" or "at least one of A, B, ... N, or combinations thereof" are defined in their broadest sense to mean one or more elements selected from the group including A, B, ... and N, i.e., any combination of one or more of the elements A, B, ..., or N (including any one element alone or in combination with one or more of other elements, which may also include additional unlisted elements in combination).

[0016] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999, and Smith and March's Advanced Organic Chemistry, 5 th Edition, John Wiley&Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modern Methods of Organic Synthesis, 3 rdEdition, Cambridge University Press, Cambridge, 1987, the entire contents of each of which are incorporated herein by reference.

[0017] 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.

[0018] The term "alkyl" as used herein means a straight or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The term "lower alkyl" or "C1-C6-alkyl" means a straight or branched hydrocarbon chain containing 1 to 6 carbon atoms. The term "C1-C3-alkyl" means a straight or branched hydrocarbon chain 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.

[0019] As used herein, the term "alkenyl" refers to a hydrocarbon chain containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. An alkenyl group may be substituted or unsubstituted. For example, an alkenyl group may be substituted with an aryl group, such as phenyl.

[0020] 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 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 (-CHC≡CH), as well as cycloalkynyl moieties, such as, but not limited to, substituted or unsubstituted cyclooctyne moieties.

[0021] 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.

[0022] 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, -CH-, -CH(CH)-, -C(CH)-, -CHCH-, -CH(CH)CH-, -C(CH)CH-, -CHCHCH-, -CH(CH)CHCH-, -C(CH)CHCH-, -CHC(CH)CH-, -CHCHCHCHCH-, and -CHCHCHCHCHCH-.

[0023] 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 similarly 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 naturally occurring amino acids, i.e., by way of example only, an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs can retain the same basic chemical structure as natural amino acids but have modified R groups (e.g., norleucine, for example) or 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))-phenylglycine, and 4-(3-methyl-(1,2,4,5-tetrazine))-phenylalanine.

[0024] 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 the parent molecular moiety and fused to a phenyl group. A tricyclic fused ring system is exemplified by a phenyl group attached to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl. 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.

[0025] As used herein, the term "azido" refers to the functional group -N3.

[0026] The term "cycloalkyl," as used herein, refers to a carbocyclic ring system containing 3 to 10 carbon atoms, 0 heteroatoms, and 0 double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes carbocyclic ring systems in which a cycloalkyl group is appended to a parent molecular moiety and fused to an aryl group, as defined herein, a heteroaryl group, as defined herein, or a heterocycle, as defined herein.

[0027] As used herein, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0028] 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).

[0029] The term "fluoroalkyl" as used herein means an alkyl group, as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms have been 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.

[0030] 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.

[0031] 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.

[0032] The term "halogen" or "halo" as used herein means Cl, Br, I, or F.

[0033] The term "haloalkyl," as used herein, means an alkyl group, as defined herein, in which 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by halogen.

[0034] 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.

[0035] The term "heteroalkyl," as used herein, refers to 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 can be oxidized. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, and alkyl sulfides.

[0036] "Heteroalkylene" refers to a divalent heteroalkyl group. A "heteroalkylene" group must have at least one carbon and at least one heteroatom group within the chain. The term "heteroalkylene" includes unbranched or branched saturated chains containing carbon and heteroatoms. For example, one, two, or three carbon atoms can be independently replaced with the same or different heteroatom groups. A heteroatom group can be -NR y -, -O-, -S-, -S(O)-, -S(O)2-, etc., and R yis hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl, each of which may be optionally substituted as defined herein, for example, with oxo. Examples of heteroalkylene groups include, for example, -CH2OCH2-, -CH(CH3)OCH2-, -CH2CH2OCH2-, -OCH2-, -CH(CH3)O-, -CH2CH2O-, -CH2CH2OC(O)-, -CH2CH2OCH2CH2OCH2-, -CH2CH2OCH2CH2O-, -CH2SCH2-, -CH(CH3)SCH2-, -CH2CH2SCH2-, -CH2CH2SCH2CH2SCH2-, -SCH2-, -CH(CH3)S-, -CH2CH2S-, -CH2CH2SCH2CH2S-, -CH2S(O)2CH2-, -CH(CH3)S(O)2CH2-, -CH2CH2S(O)2CH2-, -CH2CH2S(O)2CH2CH2OCH2-, and -CH2NR y CH2-, -CH2CH2NR y -, -CH2CH2C(O)NR y -, -CH(CH3)NR y CH2-, -CH2CH2NR y CH2-, -CH2CH2NR y CH2CH2NR y Including CH2-, R y is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroalkyl, or heteroaryl, each of which may be optionally substituted as defined herein.) As used herein, heteroalkylene contains 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom. As used herein, the term "heteroalkylene" includes groups such as amide or other functional groups having oxo present on one or more carbon atoms.

[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. A tricyclic heteroaryl group is exemplified by a monocyclic heteroaryl ring attached to a parent molecular moiety and fused to two of a monocyclic cycloalkyl group, a monocyclic aryl group, a monocyclic heteroaryl group, or a monocyclic heterocycle, 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 heteroaryls 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 heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic heteroaryls, bicyclic heteroaryls, and tricyclic heteroaryls 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] As used herein, the term "heterocycle" or "heterocyclic" refers to 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, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles are 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, oxazolidinyl, oxetaniyl, and the like. Examples of thiazolinyl include, but are not limited to, thiazolinyl, 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 are 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 can be 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 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are replaced by hydroxyl groups.

[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 The two notations are used interchangeably and have the same meaning.

[0042] The term "substituted" refers to a group that can be further substituted with one or more non-hydrogen substituents, including, but not limited to, halogen, =0, =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 for delivery to a target site in a subject. The payload includes a therapeutic agent.

[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. Representative diagnostic agents include imaging agents such as paramagnetic agents, optical probes, and radionuclides. Paramagnetic agents are imaging agents that exhibit magnetism under an externally applied magnetic 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 of one wavelength and detection with radiation of 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: 3H, 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 Including but not limited to Am.

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

[0049] The term "target organ or tissue" refers to an organ or tissue targeted for delivery of a payload. Exemplary targeted organs and tissues include those 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 can interact with each other, such as in a non-covalent or covalent interaction or binding reaction. However, it should be understood that the resulting complex or reaction product can be produced directly from the interaction or reaction between the added reagents, or from intermediates that can be produced in the contacting mixture that are 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. Conjugation between binding agents in a biological environment can also be referred to as bioconjugation. Binders 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 partner. The selective reaction between bioorthogonal binding partners can minimize side reactions with other binding agents, biological compounds, or other non-complementary 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 for the formation of triazoles via click chemistry, trans-cyclooctene (TCO), and tetrazine (Tz) (e.g., 1,2,4,5-tetrazine). Binders useful in the present disclosure can be highly reactive with their corresponding binding agents, resulting in rapid reactions.

[0053] The term "functionalized" refers to a moiety having a functional group attached to it, e.g., a moiety having a binder 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 terms "pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or prevent or reduce the risk of occurrence or recurrence of a disease or disorder or symptom(s) thereof. With respect to tumorigenic proliferative disorders, a pharmaceutically effective amount or therapeutically effective amount includes, inter alia, an amount sufficient to shrink a tumor or reduce the rate of tumor growth.

[0056] 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 may include mammals, particularly primates, and particularly humans. For veterinary applications, suitable subjects may include, for example, livestock such as cattle, sheep, goats, cows, and pigs, poultry such as chickens, ducks, geese, and turkeys, and domesticated animals, particularly pets, such as dogs and cats. For diagnostic or research applications, suitable subjects may include mammals such as rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs (such as inbred pigs).

[0057] The term "treat" or "treatment," as used herein, means treating or curing 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 disease or condition or symptom(s) thereof in the patient; (b) suppressing the disease or condition or symptom(s) thereof, e.g., by slowing or arresting the onset of 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.

[0058] The term "physiological conditions" is meant to encompass conditions compatible with living cells, eg, primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0059] For compounds described herein, the groups and substituents thereof may be selected in accordance with the allowed valences of the atoms and substituents, such that selection and substitution results in stable compounds that do not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like.

[0060] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed 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.

[0061] For the recitation of numerical ranges herein, each intervening value therebetween, with the same degree of precision, is expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for 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.

[0062] Compounds may exist as stereoisomers where asymmetric or chiral centers exist. Stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. As used herein, the terms "R" and "S" refer to the configurations defined in Section E of the IUPAC 1974 Recommendations for Fundamental Stereochemistry in Pure Appl. Chem., 1976, 45:13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, which 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 compounds can be synthetically prepared from commercially available starting materials containing asymmetric or chiral centers, or they can be prepared by preparing racemic mixtures followed by resolution methods well known to those skilled in the art. These resolution methods are described in detail in (1) Furniss, Hannaford, Smith, and Tatchell, "Vogel's Textbook of Practical Organic Chemistry," 5 th This method is exemplified by (1) combining a mixture of enantiomers with a chiral auxiliary, recrystallizing or chromatographically separating the resulting mixture of diastereomers, and optionally liberating the optically pure product from the auxiliary, as described in the "Chemical Synthesis of Diastereomers and Diastereomeric Enantiomers" series of "Chemical Synthesis of Diastereomers and Diastereomeric Enantiomers" ...

[0063] It is to be understood that the compounds may have tautomeric forms and geometric isomers and these also form an aspect of the present invention.

[0064] The present disclosure also includes isotopically labeled compounds that are identical to those enumerated herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature. Examples of isotopes suitable for inclusion in compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, for example, but not limited to, 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, can offer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. The compounds may incorporate positron-emitting isotopes for medical imaging studies and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that can be incorporated include: 11 C. 13 N, 15 O, and 18 F. Isotopically labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the accompanying examples, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0065] B. Conjugates Provided herein is a conjugate for use in bioorthogonal reactions.In some embodiments, the conjugate comprises a payload attached to a trans-cyclooctene moiety.In some embodiments, the conjugate comprises a payload (i.e., a taxane such as paclitaxel, a topoisomerase inhibitor, or an MMAE payload, or a derivative or analog thereof) attached to a trans-cyclooctene moiety that comprises one or more solubilizing groups.

[0066] In some embodiments, a conjugate of Formula AI or Formula A-II, or a pharmaceutically acceptable salt thereof, is provided: [ka] During the ceremony, m is an integer from 1 to 10; r is 1 or 2, Each D 1 are independently a taxane, topoisomerase inhibitor, or MMAE payload, or a derivative or analog thereof; L 1 and L 2 are each independently a linker, G is an optionally substituted trans-cyclooctene moiety; Each S 1 are independently solubilizing groups.

[0067] In some embodiments, a conjugate of formula AI, or a pharmaceutically acceptable salt thereof [ka] is provided, wherein m is an integer from 1 to 10; D 1 is a taxane, topoisomerase inhibitor, or MMAE payload, or a derivative or analog thereof; L 1 and L 2 are each independently a linker, G is an optionally substituted trans-cyclooctene moiety; Each S 1 are independently solubilizing groups.

[0068] In some embodiments, a conjugate of formula A-II, or a pharmaceutically acceptable salt thereof: [ka] is provided, wherein m is an integer from 1 to 10; r is 1 or 2, Each D 1 are independently a taxane, topoisomerase inhibitor, or MMAE payload, or a derivative or analog thereof; L 2 is the linker, G is an optionally substituted trans-cyclooctene moiety; Each S 1 are independently solubilizing groups.

[0069] In some embodiments, a conjugate of formula AI, or a pharmaceutically acceptable salt thereof [ka] is provided, wherein m is an integer from 1 to 10; D 1 is a taxane payload, or a derivative or analog thereof; L 1 and L 2 are each independently a linker, G is an optionally substituted trans-cyclooctene moiety; Each S 1 are independently solubilizing groups.

[0070] In some embodiments, the conjugate comprises: [ka] [ka] isn't it.

[0071] In some embodiments, the moiety [ka] is the formula A-IIA [ka] wherein: q is 0, 1, or 2; m is an integer from 1 to 10; R 1A In each occurrence, C 1-4 Alkyl, C 1-4 Haloalkyl, and C 1-4 independently selected from the group consisting of alkoxy; L 2 is the linker, Each S 1 are independently solubilizing groups.

[0072] In some embodiments, the moiety [ka] is the formula A-IIA [ka] wherein: m is an integer from 1 to 10; R 1A is C 1-4 Alkyl, C 1-4 Haloalkyl, and C 1-4 alkoxy; L 2 is the linker, Each S 1 are independently solubilizing groups.

[0073] In some embodiments, each S 1is -NHC(NH)NH2, -P(O)(OH)2, -S(O)2OH, -(OCH2CH2) 30-85 -OCH3, -N(CH2CH2C(O)OH)2, or [ka] are independently selected from the group consisting of:

[0074] In some embodiments, one S 1 is -NHC(NH)NH2.

[0075] In some embodiments, one S 1 is -P(O)(OH)2.

[0076] In some embodiments, one S 1 is -S(O)2OH.

[0077] In some embodiments, one S 1 is -(OCH2CH2) 30-85 -OCH3.

[0078] In some embodiments, one S 1 is -N(CH2CH2C(O)OH)2.

[0079] In some embodiments, one S 1 teeth, [ka] is.

[0080] In some embodiments, each S 1 is -NHC(NH)NH2, -P(O)(OH)2, -S(O)2OH, -N(CH2CH2C(O)OH)2, or [ka] are independently selected from

[0081] In some embodiments, L 1 -OC(O)-, -C(O)O-, -NR 1f C(O)- or -C(O)NR 1f - and R 1f is hydrogen, C 1-6 Alkyl, or C 0-4 It is alkylene-CO2H.

[0082] In some embodiments, L 1 is -OC(O)- aa or -NHC(O)- aa and bond aa is D 1 is bonded to.

[0083] In some embodiments, L 2 is a heteroalkylene linker. In some embodiments, L 2 is a linear or branched heteroalkylene linker.

[0084] In some embodiments, m is 1.

[0085] In some embodiments, m is 2.

[0086] In some embodiments, L 2 teeth, [ka] and During the ceremony, 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)-, -C(O)O-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, each of the alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently represents oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R 100 are independently hydrogen, -C(O)OH, C 1-4 Alkyl, C 1-4 haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; 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; n'', m', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; n' is 2.

[0087] In some embodiments, the moiety [ka] [ka] is.

[0088] In some embodiments, the moiety [ka] [ka] is.

[0089] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] teeth, [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] In some embodiments, the moiety [ka] [ka] is.

[0090] In certain embodiments, D 1 is a taxane, a topoisomerase inhibitor, or MMAE, or a derivative or analog thereof. 1 is paclitaxel, or a derivative or analog thereof. In some embodiments, D 1 is paclitaxel or isotaxel, or a derivative or analog thereof. In some embodiments, D 1 is a topoisomerase inhibitor, or a derivative or analog thereof. In some embodiments, D 1 is campothecin, or a derivative or analog thereof. In certain embodiments, D 1 is MMAE, or a derivative or analog thereof.

[0091] In certain embodiments, the term "derivative" or "analog" or "derived from" when used in reference to a payload refers to a payload 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 1The payload is derived from a known payload and linked by a linker L 1 and modified to be covalently attached to at least one optionally substituted trans-cyclooctene via D. 1 After being modified to arrive at the compounds described herein, the payloads retain biological activity equivalent 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.

[0092] In certain embodiments, a hydrogen atom bonded to a heteroatom (e.g., N, O, or S) of the original, unmodified payload is linked to a linker, L 1 In certain embodiments, a halogen atom on the payload is replaced for attachment to the remainder of the compound. In certain embodiments, a hydrogen atom on the payload is replaced for attachment 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.

[0093] In some embodiments, D 1 teeth, [ka] is.

[0094] In some embodiments, D 1 teeth, [ka] is.

[0095] In some embodiments, D1 teeth, [ka] is.

[0096] In some embodiments, D 1 is the formula D-IA [ka] is a compound of the formula Y is a bond, -CH-, or -CH-CH-; Z is a bond, —O—, or —CH—O—; R 1 , R 2 , R 3 , and R 4 are each independently hydrogen, halo, cyano, nitro, -OR 5 , -SR 5 , -NR 5 R 6 , C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-10 Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(=O)R 5 , -C(=O)OR 5 , -OC(=O)R 5 , -C(=O)NR 5 R 6 , -NR 5 C(=O)R 6 , -NR 5 C(=O)OR 6 , -S(=O) 1-2 R 5 , -S(=O) 1-2 NR 5 R 6 , -NR 5 S(=O) 1-2 R 6 , -Si(R 5 )3, or -C=NOR 5 and R 1 , R 2 , R 3 , and R 4Each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may independently, as valence allows, be one or more R 10 optionally replaced by Or, R 1 and R 2 together with the atoms to which they are attached, can contain one or more R 10 C optionally substituted with 3-10 forming a cycloalkyl, heterocyclyl, aryl, or heteroaryl; Or, R 2 and R 3 together with the atoms to which they are attached, can contain one or more R 10 C optionally substituted with 3-10 forming a cycloalkyl, heterocyclyl, aryl, or heteroaryl; Or, R 3 and R 4 together with the atoms to which they are attached, can contain one or more R 10 C optionally substituted with 3-10 forming a cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 5 and R 6 each independently represents hydrogen, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, or C 3-12 cycloalkyl, wherein each of the alkyl, alkenyl, alkynyl, or cycloalkyl is independently optionally substituted with oxo, halo, hydroxyl, or amino, where valence allows, or R 5 and R 6 together with the atom to which they are attached represent heterocyclyl optionally substituted by halo, or C optionally substituted by oxo, halo, hydroxyl, or amino. 1-12 Forming an alkyl R 7 and R 8 each independently represents hydrogen, C 1-12 Alkyl, C2-12 Alkenyl, C 2-12 Alkynyl, or C 3-12 is cycloalkyl, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, or C 3-12 Each cycloalkyl is optionally substituted with an oxo, halo, hydroxyl, or amino group, or R 7 and R 8 together with the atom to which they are attached, represent heterocyclyl optionally substituted by halo, or C optionally substituted by oxo, halo, hydroxyl, or amino. 1-12 Forming an alkyl Each R 10 are independently halo, cyano, nitro, -OR 7 , -SR 7 , -SF5, -NR 7 R 8 , C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Alkynyl, C 3-10 Cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(=O)R 7 , -C(=O)OR 7 , -OC(=O)OR 7 , -OC(=O)R 7 , -C(=O)NR 7 R 8 , -OC(=O)NR 7 R 8 , -NR 7 C(=O)NR 7 R 8 , -S(=O) 1-2 R 7 , -S(=O) 1-2 NR 7 R 8 , -NR 7 S(=O) 1-2 R 8 , -NR 7 S(=O) 1-2 NR 7 R 8 , -NR 7 C(=O)R 8 , -NR7 C(=O)OR 8 , -Si(R 7 )3, or -C=NOR 7 and R 10 Each of the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups independently is a C optionally substituted by one or more halo, or, where valence allows, oxo, halo, hydroxyl, or amino. 1-12 optionally substituted with alkyl; One or more atoms (e.g., hydrogen atoms) of formula D-IA may be L 1 is replaced by a direct covalent bond to

[0097] In certain embodiments, D 1 teeth, [ka] is. In certain embodiments, D 1 teeth, [ka] is.

[0098] In certain embodiments, D 1 is MMAE, or a derivative or analog thereof.

[0099] In certain embodiments, D 1 teeth, [ka] is.

[0100] In certain embodiments, provided herein are conjugates for use in bioorthogonal reactions, the conjugates comprising a payload attached to a trans-cyclooctene moiety. In some embodiments, the conjugate is of formula BI, or a pharmaceutically acceptable salt thereof: [ka] is provided, wherein G is an optionally substituted trans-cyclooctene moiety; L 1 is the linker, m is 1 or 2, Each D 1 is independently [ka] and [ka] a payload selected from the group consisting of however, D 1 but [ka] m is 2 when D 1 but [ka] and m is 1, G is [ka] or L 1 teeth, [ka] or [ka] or containing a disulfide.

[0101] In some embodiments, G is [ka] is selected from the group consisting of:

[0102] In some embodiments, G is [ka] is.

[0103] In some embodiments, G is [ka] is.

[0104] In some embodiments, G is [ka] is.

[0105] In some embodiments, G is [ka] is.

[0106] In some embodiments, each D 1 is independently [ka] is.

[0107] In some embodiments, each D 1 is independently [ka] is.

[0108] In some embodiments, m is 1 and D 1 teeth, [ka] is.

[0109] In some embodiments, D 1 teeth, [ka] is.

[0110] In some embodiments, D 1 teeth, [ka] is.

[0111] In some embodiments, D 1 teeth, [ka] is.

[0112] In some embodiments, D 1 teeth, [ka] is.

[0113] In some embodiments, D 1 teeth, [ka] is.

[0114] In some embodiments, D 1 teeth, [ka] is.

[0115] In some embodiments, D 1 teeth, [ka] is.

[0116] In some embodiments, D 1 teeth, [ka] is.

[0117] In some embodiments, D 1 teeth, [ka] is.

[0118] In some embodiments, D 1 teeth, [ka] is.

[0119] In some embodiments, D 1 teeth, [ka] is.

[0120] In some embodiments, D 1 teeth, [ka] is.

[0121] In some embodiments, D 1 teeth, [ka] is.

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

[0123] In some embodiments, D 1 teeth, [ka] is.

[0124] In some embodiments, m is 1. In some embodiments, L 1 is a linear linker.

[0125] In some embodiments, m is 2. In some embodiments, L 1 is a branched linker.

[0126] In some embodiments, L 1 is —C(O)O— or —O—.

[0127] Also provided is a pharmaceutical composition comprising a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0128] Linker In some embodiments of the conjugates described herein, each linker L 1 or L 2 can independently have 1 to 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 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.

[0129] In some embodiments of the conjugates described herein, the linker L 1 or L 2 can be a bond.

[0130] In some embodiments of the conjugates described herein, the conjugates may comprise a linker (e.g., L1 or L 2 ) can contain one or more (e.g., 1 to 10 or 1 to 5) chain heteroatoms (e.g., O, N, S) and one or more (e.g., 1 to 10 or 1 to 5) alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene moieties, each of which independently is selected from the group consisting of 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.

[0131] In some embodiments of the conjugates described herein, the conjugates may comprise a linker (e.g., L 1 or L 2 ) is the expression: [ka] It can be of During the ceremony, 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, each of the alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently selected from oxo, halo, C 1-4 Alkyl, C 1-4 Alkoxy, and C 1-4 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; n′ and m″ are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0132] In certain embodiments, the linker L 1 is not a bond. 1 is a cleavable linker. In some embodiments, L 1 is a non-cleavable linker.

[0133] In certain embodiments, the linker L 2 is not a bond. 2 is a cleavable linker. In some embodiments, L 2 is a non-cleavable linker.

[0134] In certain embodiments, each R 110 are independently hydrogen, C 1-4 Alkyl, C 1-4haloalkyl, 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.

[0135] In some embodiments, L 1 is a heteroalkylene linker. In some embodiments, L 1 is a linear or branched heteroalkylene linker. In some embodiments, L 1 is a linear heteroalkylene linker. In some embodiments, L 1 is a branched heteroalkylene linker.

[0136] In some embodiments, L 1 -OC(O)-, -C(O)O-, -NR 1f C(O)- or -C(O)NR 1f - and R 1f is hydrogen, C 1-6 Alkyl, or C 0-4 It is alkylene-CO2H.

[0137] In some embodiments, L 1 is -OC(O)- aa or -NHC(O)- aa and bond aa is D 1 is bonded to.

[0138] Also provided is a pharmaceutical composition comprising a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0139] In some embodiments of the conjugates described herein, the linker L 1 can be a bond.

[0140] Representative linkers include, but are not limited to, those shown below. [ka]

[0141] Representative linkers include, but are not limited to, those shown below. [ka]

[0142] In some embodiments of the conjugates described herein, the conjugates may comprise a linker (e.g., L 1 or L 2 )L 1 or L 2 ) can include one or more of polyethylene glycol (e.g., PEG having an average molecular weight of 200 g / mol to 10,000 g / mol), ethylene-1,2-diylbis(methylcarbamate), arylene (i.e., phenylene), ethylene-oxy, amine, ester, amide, carbamate, ketone (i.e., formyl), or carbonate. Linkers (e.g., L) of the formulae disclosed herein, such as those of Formulae AI, A-II, BI, etc. 1 or L 2 )L 1 or L 2 )teeth, [ka] Linkers (e.g., L 1 or L 2 )teeth, [ka] may include:

[0143] In some embodiments of the conjugates described herein, the conjugates may comprise a linker (e.g., L 1 or L 2 ) may contain one or more natural or unnatural amino acids, which may be referred to as a peptide linker. 1 If the drug contains an amino moiety, the linker can be attached to it 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 can also contain a self-immolative spacer that separates the drug from the protein peptide sequence.

[0144] In some embodiments of the conjugates described herein, the conjugates may comprise a linker (e.g., L 1 or L 2 ) is a carboxylic acid acyl unit, "Y" and "Z" are each one or more natural or unnatural amino acids, taken together to form a peptide sequence, and "X" and "W" are the units that bind the peptide and 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 from the bioorthogonal moiety. In certain embodiments, one or more of the amino acids in the peptide linker is N-methylated.

[0145] 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.

[0146] 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.

[0147] In some embodiments, the YZ combinations include valine-citrulline, valine-alanine, and alanine-alanine.

[0148] In certain embodiments, A is —OC(O)—.

[0149] In certain embodiments, X is —OC(O)—.

[0150] In certain embodiments, W is -OC(O)-. In certain embodiments, X is absent and W is -OC(O)-.

[0151] In certain embodiments, -XW is [ka] is.

[0152] In certain embodiments, -XW is [ka] is.

[0153] In certain embodiments, the peptide linker is specifically tailored to be selectively cleaved (e.g., enzymatically cleaved) to release the drug, such as by one or more tumor-associated proteases.

[0154] In certain embodiments, the peptide linker has a chain length of 2 to 4 amino acid residues (i.e., a dipeptide, tripeptide, or tetrapeptide), although 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.

[0155] 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.

[0156] In certain embodiments, linkers (e.g., L 1 or L 2 )teeth, [ka] (for example, [ka] is.

[0157] The above-mentioned linkers may contain a D 1 can be attached to the right side of an amino acid side chain (e.g., [ka]

[0158] In some embodiments, 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-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, 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.

[0159] In some embodiments, R 1Bis -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.

[0160] In some embodiments, R 1A is C 1-4 It is alkyl.

[0161] In some embodiments, R 1A is CH3.

[0162] In some embodiments, R 1c is hydrogen.

[0163] In some embodiments, R 1A is C 1-4 It 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 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 1is a 4-8 membered monocyclic heterocyclyl containing a first nitrogen and optionally one additional heteroatom selected from nitrogen, oxygen, and sulfur; G 1 is attached to the first nitrogen, and C 1-4 Alkyl, C 1-4 Haloalkyl, halo, cyano, OH, -OC 1-4 optionally substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo; n is 0, 1, or 2.

[0164] In some embodiments, R 1A is CH3, R 1e is -CH2CO2H, R 1f is hydrogen or CH2CO2H, G 1 is bonded through the ring nitrogen atom, and C 1-4 Alkyl, C 1-4 Haloalkyl, halo, cyano, -OH, -OC 1-4 and piperazinyl, morpholinyl, piperidinyl, azepanyl, or pyrrolidinyl substituted with 1 to 4 substituents independently selected from the group consisting of alkyl, and oxo.

[0165] 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, and -N(CH2CO2H)-CH2CH2-N(CH2CO2H)2; R 1e is -CH2CO2H.

[0166] In some embodiments, this portion [ka] [ka] is selected from the group consisting of:

[0167] In certain embodiments, a conjugate, or a pharmaceutically acceptable salt thereof, is provided, wherein the conjugate is selected from Table A-1. TIFF2026508188000089.tif129170TIFF2026508188000090.tif249170TIFF20265081880 00091.tif249170TIFF2026508188000092.tif249170TIFF2026508188000093.tif248170 TIFF2026508188000094.tif233170TIFF2026508188000095.tif217170TIFF20265081880 00096.tif249170TIFF2026508188000097.tif217170TIFF2026508188000098.tif249170

[0168] In certain embodiments, a conjugate, or a pharmaceutically acceptable salt thereof, is provided, wherein the conjugate is selected from Table B-1. TIFF2026508188000099.tif234170TIFF2026508188000100.tif249170TIFF2026508188000101.tif23417 0TIFF2026508188000102.tif39170TIFF2026508188000103.tif244170TIFF2026508188000104.tif97170

[0169] In some embodiments, a method is provided for delivering an effective amount of a payload (i.e., a taxane such as paclitaxel, or a camptothecin such as exatecan, or a derivative or analog thereof) to a target location in a subject, the method comprising administering to the subject at the target location a therapeutic support composition described herein, and administering to the subject a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof.

[0170] C. Therapeutic support composition The therapeutic support composition includes a support. The support can be a biocompatible support composition, i.e., one that is 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 can be, inter alia, a hydrogel. The support is implanted into the subject's body and not only supports a binding agent (e.g., a tetrazine-containing group) but can also support a payload after the binding agent is conjugated. Representative supports include, but are not limited to, polymers, viscous or non-viscous liquid materials, gels, hydrogels, polysaccharide hydrogels, cross-linked polymer matrices, metals, ceramics, plastics, bone graft materials, alginate, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Supports also include particles, such as nanoparticles and microparticles.

[0171] The hydrogel can be a polysaccharide hydrogel, alginate, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, etc. Other suitable sugar-based biomaterials include those described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that can be used as supports include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphates, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Representative polyethers include poly(ethylene glycol) (PEG), polypropylene glycol (PPG), triblock pluronics ([PEG]), and the like. n -[PPG] m -[PEG] n), PEG diacrylate (PEGDA), and PEG dimethacrylate (PEGDMA), where n and m are each independently an integer between 1 and 100. The support may also comprise proteins such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(gamma-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), and other poly(amino acids).

[0172] In some embodiments, the support is a hydrogel. In some embodiments, the support is alginate. In some embodiments, the support is chitin. In some embodiments, the support is hyaluronic acid (e.g., non-hydrogel hyaluronic acid substantially free of cross-links). In some embodiments, the support is chitosin. In some embodiments, the support is chitosan.

[0173] In certain embodiments, the support is a particle. The particles of the present disclosure can have a diameter of 2 cm or less, for example, 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particles can be nanoparticles or microparticles. Nanoparticles include particles having an average size on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles having an average size on the micrometer scale (e.g., 1000 μm or less). "Average" 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, microparticles have diameters ranging from 1 μm to 1 mm, e.g., 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, particles as small as 10 to 100 nm in diameter may aggregate to form larger complexes, such as clusters or aggregates, on the order of 1 to 10 μm. Particles of the present disclosure may be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes, such as, but not limited to, ellipsoids, cubes, cylinders, cones, needles, or other irregular shapes, may also be used.

[0174] A "particle" can take the form of any engineered 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 made from inert materials such as alginate or iron oxide. In some examples, 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, such as spherical, rod-like, or asymmetric. Particles, or groups of particles in a composite, can be functionalized with receptors that have 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 unique affinity for a particular substrate. Additionally or alternatively, particles can be functionalized by covalently or otherwise binding or associating with receptors that specifically bind to or otherwise recognize specific clinically relevant substrates. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule 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, alginate 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 do not limit their concentration, their crosslinking with different drugs, their administration methods, their tailored degradation profiles, and other properties known to those skilled in the art.

[0175] The particle, or a group of particles in the complex, can 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 can be directly attached to the particle itself.

[0176] In some embodiments, the biocompatible support is a targeting agent.

[0177] The targeting agent can be an antibody, peptide, nucleic acid, phage, bacterium, virus, or any other molecule that has specific affinity for a 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.

[0178] 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), PSMA. (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 10457), Gene 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 (NCBI Gene ID 10440), and IFN-γ (NCBI Gene ID 10441). and antibodies or antibody fragments that target one or more of IL-1R (NCBI Gene ID 7124), 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).

[0179] 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.

[0180] 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.

[0181] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CEACAM5, such as tusamitamab or CC4, or an antibody fragment derived therefrom.

[0182] 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.

[0183] In certain embodiments, the targeted 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets BCMA, such as belantamab, or an antibody fragment derived therefrom.

[0191] 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.

[0192] 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.

[0193] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD19, such as loncastuximab, XMAB-5574, MOR208, coltuximab, denintuzumab, taplitumomab, or MDX-1342, or an antibody fragment derived therefrom.

[0194] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD79b, such as polatuzumab, or an antibody fragment derived therefrom.

[0195] 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.

[0196] 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.

[0197] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets endoglin, such as carotuximab, or an antibody fragment derived therefrom.

[0198] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets FGFR2, such as bemarituzumab or apultuzumab, or an antibody fragment derived therefrom.

[0199] 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.

[0200] 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.

[0201] In certain embodiments, the targeting agent is an antibody or antibody fragment, or an antibody fragment derived therefrom, that targets MMP9, such as andecaliximab.

[0202] 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.

[0203] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets DLL3, such as rovalpituzumab, or an antibody fragment derived therefrom.

[0204] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD138, such as indatuximab, or an antibody fragment derived therefrom.

[0205] 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.

[0206] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD37, such as BI836826, otlertuzumab, or naratuximab, or an antibody fragment derived therefrom.

[0207] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD74, such as milatuzumab, or an antibody fragment derived therefrom.

[0208] 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.

[0209] 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.

[0210] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets SLAMF7, such as elotuzumab, or an antibody fragment derived therefrom.

[0211] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets BAFF, such as belimumab, or an antibody fragment derived therefrom.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] In certain embodiments, the targeted agent is an antibody or antibody fragment that targets HER3, such as patritumab, seribantumab, lumletuzumab, elgemtumab, AV-203, CDX-3379, or GSK284933, or an antibody fragment derived therefrom.

[0216] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD30, such as brentuximab, or an antibody fragment derived therefrom.

[0217] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD33, such as gemtuzumab, BI835858, vadastuximab, or lintuzumab, or an antibody fragment derived therefrom.

[0218] 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.

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

[0220] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets cMET, such as telisotuzumab, onartuzumab, or SAIT301, or an antibody fragment derived therefrom.

[0221] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD142, such as tisotumab, or an antibody fragment derived therefrom.

[0222] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets NaPi2B, such as rifastuzumab, or an antibody fragment derived therefrom.

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

[0224] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets STEAP1, such as bundurumab, or an antibody fragment derived therefrom.

[0225] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets MUC16, such as sofituzumab, or an antibody fragment derived therefrom.

[0226] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD70, such as borsetuzumab, or an antibody fragment derived therefrom.

[0227] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CD44, such as bivatuzumab, or an antibody fragment derived therefrom.

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

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets ADAMTS5, such as M6495, or an antibody fragment derived therefrom.

[0233] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets CX3CR1, such as BI655088, or an antibody fragment derived therefrom.

[0234] 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.

[0235] In certain embodiments, the targeting agent is an antibody or antibody fragment, or an antibody fragment derived therefrom, that targets TfR1, such as TXB4.

[0236] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets a VEGFR, such as CDP791, or an antibody fragment derived therefrom.

[0237] In certain embodiments, the targeting agent is an antibody or antibody fragment that targets PSMA, such as GY1, or an antibody fragment derived therefrom.

[0238] Other compounds or molecules, such as fluorophores or autofluorescent or luminescent markers, that may aid in the detection of the particle (e.g., in vivo detection) may also be attached to the particle. The ligands and / or detectable labels may be attached directly to the particle or may be attached to the particle via a bioorthogonal functional group as described herein.

[0239] In certain embodiments, the support is a bone graft material, such as a bone graft substitute material. Bone graft substitute materials are materials structurally similar to bone. In some cases, bone graft substitute materials are bioabsorbable, allowing the bone graft substitute material to dissolve or be absorbed by the body over time. Bone graft substitute materials can be osteoinductive, promoting blood vessel and new bone formation within the bone graft substitute material. In some cases, bone graft substitute materials are osteoinductive, promoting new bone formation by actively recruiting mesenchymal stem cells from surrounding tissues. For example, growth factors such as bone morphogenetic proteins can be included in the bone graft substitute material. Bone graft substitute materials include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and the like, and combinations thereof.

[0240] 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 bound 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 linked to the support. For example, the binding agent may be covalently linked to the surface of the support via a covalent bond, such as an amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, or thiourea. In some cases, the binding agent is covalently linked 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. Representative linkers can have 1 to 100 linking atoms and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the linker can have 1 to 50 linking atoms, 5 to 50 linking atoms, or 10 to 50 linking atoms. Representative linkers include, but are not limited to, those shown below. [ka]

[0241] In certain embodiments, the therapeutic support composition comprises a support and a compound of the formula: [ka] and a tetrazine-containing group of the formula: 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 selected from the group consisting of aryl, alkyl, and alkyl; R' and R'' are ... 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.

[0242] In certain embodiments, the therapeutic support composition has the following formula: [ka] wherein 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 selected from the group consisting of aryl, alkyl, and alkyl; R' and R'' are ... 22 is a linker of 1 to 100 linked atoms and can include ethylene-oxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the linker can have 1 to 50 linked atoms, or 5 to 50 linked atoms, or 10 to 50 linked atoms.

[0243] In certain embodiments, the therapeutic support composition has the following formula: [ka] wherein 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 selected from the group consisting of aryl, alkyl, and alkyl; R' and R'' are ... 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.

[0244] In certain embodiments, the therapeutic support composition has the formula: [ka] and / or [ka] or a salt thereof, In the formula, R20 is 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 and NR'C(=S)NR''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'' at each occurrence are independently selected from hydrogen, aryl, and alkyl, and R''' at each occurrence is independently selected from aryl and alkyl.

[0245] In certain embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0246] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0247] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0248] In some embodiments, the therapeutic support composition has the following formula: [ka] and a substituted hyaluronic acid having units of the formula: 2 teeth, [ka] and R 22 is a linker of 1 to 100 connecting atoms, and R 20 is as defined in this disclosure.

[0249] In a further embodiment, G 2 teeth, [ka] is.

[0250] In still further embodiments, G 2 teeth, [ka] and R 20 is hydrogen or C 1-4 It is alkyl.

[0251] The compound of formula (II) has the following formula: [ka] wherein 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 and NR'C(=S)NR"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", where R' and R" at each occurrence are independently selected from hydrogen, aryl, and alkyl, and R'" at each occurrence is independently selected from aryl and alkyl. In a further embodiment according to Formula (II-A), R 20 is hydrogen or C 1-4 It is alkyl.

[0252] In some embodiments, the therapeutic support composition has the formula: [ka] Includes units of.

[0253] Additional therapeutic support compositions are exemplified in WO2017 / 044983, WO / 2015 / 139025A1, and WO / 2014 / 205126A1, the entire contents of each of which applications are incorporated herein by reference.

[0254] Hyaluronic acid derivatives include hyaluronic acid that has a plurality of glucuronic acid units and tetrazine-containing groups that are linked or directly bonded to the glucuronic acid units of hyaluronic acid.Hyaluronic acid can also have a plurality of N-acetylglucosamine units.In certain embodiments, the N-acetylglucosamine units of hyaluronic acid are not linked or bonded to tetrazine-containing groups.

[0255] The tetrazine-containing group can be linked or directly bonded through the carboxylic acid of the glucuronic acid unit. The tetrazine-containing group can be incorporated into hyaluronic acid at about 0.1% to about 80%, as measured by the percentage 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 percentage of carboxylic acid linked or conjugated to the tetrazine-containing group.

[0256] D. Treatment method Aspects of the present disclosure include methods for delivering a payload to a target location in a subject. In certain embodiments, the method includes selectively delivering a payload to a target location in a subject. Selective delivery of a payload includes delivering the payload to a target location (e.g., an organ or tissue, or a portion thereof) without targeting other locations in the subject that do not require administration of the payload (e.g., other organs or tissues, or portions thereof). Selective delivery of a payload can be achieved through the use of the support compositions and functionalized payloads described herein.

[0257] In some cases, the support composition of the present disclosure can be localized to a desired target location in a subject. For example, the method of the present disclosure can include administering a support composition 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 into a subject at a desired target location in the subject. In some embodiments, the support composition can be bound to a targeting agent described herein, and the method can include administering (e.g., systemically administering) the support composition to a subject. In these embodiments, the support composition bound to the targeting agent can be localized to 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 the desired target through specific binding of the targeting agent to its target (e.g., antibody-antigen interaction, etc.).

[0258] As described herein, selective binding can occur between bioorthogonal binding partners (e.g., between the tetrazine binding agent of the support composition and its complementary trans-cyclooctene binding agent of the functionalized payload). For localized administration of the support composition to a desired location in a subject, as described above, selective binding between the binding agent of the support composition and its complementary binding agent of the functionalized payload localizes the payload to the desired target location. Thus, in certain embodiments, the method includes administering a functionalized payload to a 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. Administration of the functionalized payload to a subject can result in contact between the binding agent of the support composition and the complementary binding agent of the functionalized payload, causing the binding agent and its complementary binding agent to bind to each other and 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 greater concentration of the payload at the target location than the concentration of the payload elsewhere in the subject (e.g., non-targeted areas of the subject).

[0259] Provided herein are methods of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof, and a therapeutic support composition.

[0260] 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, head / neck squamous cell carcinoma, 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, malignant extrinsic or intrinsic airway compression, or cutaneous T-cell lymphoma.

[0261] In certain embodiments, the present approach can be used to treat / diagnose hematological malignancies such as myelodysplastic syndromes, acute myeloid leukemia, myelodysplastic 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 others.

[0262] 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.

[0263] In some embodiments, the cancer is a solid tumor.

[0264] In some embodiments, the cancer is lung cancer.

[0265] In some embodiments, the cancer is a soft tissue sarcoma.

[0266] In some embodiments, the soft tissue sarcoma is fibrosarcoma, rhabdomyosarcoma, or Ewing's sarcoma.

[0267] In some embodiments, the methods also include enhancing or eliciting an immune response, hi some embodiments, the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

[0268] 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. Anti-cancer agents, immunomodulatory agents, and trans-cyclooctene prodrugs thereof are known in the art.

[0269] The indications for this approach include both blood cancer and solid cancer. In certain embodiments, this approach can be used for the treatment and / or diagnosis of soft tissue sarcoma (rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma, and all different subtypes of soft tissue sarcoma, and osteosarcoma). These compositions can be used for the treatment and / or diagnosis of pigmented villonodular synovitis.

[0270] The compositions of the present disclosure are used in the treatment and / or diagnosis of a condition or disease in a subject amenable to 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 achieving at least amelioration of symptoms associated with the condition afflicting the subject, where amelioration is used in a broad sense to mean a decrease in at least a parameter associated with the condition being treated, e.g., the magnitude of the symptoms. Thus, treatment also includes situations in which a pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or arrested (e.g., terminated), such that the subject is no longer afflicted by the condition, or at least the symptoms characterizing the condition. Treatment can include inhibiting clinical symptoms, i.e., halting their progression or further progression, e.g., mitigating or completely inhibiting active disease. Treatment can include alleviating 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 overall tumor burden; extending survival; and ameliorating one or more symptoms associated with cancer.

[0271] The subject to be treated can be a subject that needs therapy, and the subject to be treated is a subject that is suitable for treatment with the parent drug.Therefore, various subjects may be suitable for treatment with the compositions disclosed herein.Generally, such subjects are "mammals", and humans are of interest.Other subjects include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0272] In certain embodiments, the functionalized payloads, therapeutic support compositions, additional therapeutic agents, and methods are directed to, among others, 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., glioblastoma multiforme), and lung cancer (e.g., non-small cell lung cancer). The disclosed approach may be used to treat, prevent, and / or diagnose solid tumors, including, but not limited to, soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, gastric cancer, head / neck squamous cell carcinoma, 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, malignant extrinsic or intrinsic airway compression, and cutaneous T-cell lymphoma. The disclosed approach is well suited to adjuvant / neoadjuvant systems. For example, the particles disclosed herein can be placed during a biopsy, and once the test results are returned, the physician can deliver the appropriate cocktail to the desired site in the body. This minimizes tumor size, particularly in the setting of surgically resectable tumors. Then, at the end of the surgery, the surgeon can place more particles around the surgical cavity and treat the patient with additional therapeutic doses (e.g., chemotherapy with the disclosed approach) to minimize the risk of any cancer cells that may be missed at the surgical margins.

[0273] In certain embodiments, the disclosed methods can deploy the particles disclosed herein at the time of biopsy. Once the results are returned, physicians can deliver immunomodulators, such as TLR agonists, STING agonists, chemokines (agents that attract cancerous cells and / or immune cells), and adjuvants to the biopsy site to boost the immune system, similar to the combination of chemotherapy and immunotherapy, but with fewer side effects. This combination approach would be beneficial to patients. While chemotherapy agents treat solid tumors or specific locations, the enhanced response of immunotherapy would be helpful at distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can enhance the effectiveness of one or more immunomodulators, such as ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C, Pfizer), with or in combination with anthracyclines, taxanes, gemcitabine, and other agents.

[0274] 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 suppressors, resisting cell death, achieving immortalization 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.

[0275] Cancers that may be treated by the disclosed methods include astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brainstem cancer, brainstem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, diffuse pontine glioma, ductal carcinoma, endometrial cancer, ependymoma, Ewing's sarcoma, esophageal cancer, eye cancer, fibrosarcoma, gallbladder cancer, gastric adenocarcinoma, 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 cancer, lung cancer, lymphoma, macrophage tumors, and the like. These include, but are not limited to, globulinemia, melanoma, mesothelioma, oral cavity 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.

[0276] 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 a diffuse pontine glioma. In some embodiments, the cancer is a metastatic cancer.

[0277] Without being bound by any particular theory, the 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, and 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 the 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 migrates to the surface of dying cells after the induction of immunogenic apoptosis, where it functions as an "eat me" signal for professional phagocytes. Other important surface-exposed DAMPs are heat shock proteins (HSPs), namely HSP70 and HSP90, which also translocate to the plasma membrane under stress conditions. On the cell surface, they have immunostimulatory effects based on their interaction with numerous 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, subsequently triggering CD8+ T cell responses. Other important DAMPs characteristic of ICD are secreted amphoterin (HMGB1) and ATP. HMGB1 is considered a late apoptosis marker, and its release into the extracellular space is thought to be necessary for optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs), such as Toll-like receptors (TLR) 2 and 4, expressed on APCs. A more recently discovered DAMP released during immunogenic cell death is ATP, which, when secreted, functions as a "find-me" signal for monocytes, inducing their attraction to sites of apoptosis. Kroemer et.al.Curr.Op.Immunol.2008(20),504-511.

[0278] Thus, local delivery of ICD-inducing factors using the compounds and methods of the present invention may be beneficially combined with one or more immunomodulatory agents.

[0279] 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.

[0280] The disclosed approach lends itself well to adjuvant / neoadjuvant systems. For example, the therapeutic support composition disclosed herein can be placed into a biopsy, and once the results from the study are returned, a physician can administer the appropriate cocktail to deliver treatment to the desired site in the body (a compound of Formula AI, A-2, or a subformula disclosed herein, and any additional therapeutic agent(s)). The biopsy results can indicate the amount and type of treatment to deliver to the tumor site. For example, chemokines (agents that attract cancerous and / or immune cells) and adjuvants, which strengthen the immune system similar to chemotherapy but with fewer side effects, can be delivered and combined with immunotherapeutic agents.

[0281] The disclosed compounds and compositions can be administered before surgical resection. The disclosed methods can minimize tumor size before surgical resection, thereby minimizing tumor size, particularly 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. The therapeutic support composition can be placed around the surgical cavity at the end of surgical resection, and the subject can then be treated with additional therapeutic doses to minimize the risk of any cancer cells that may be missed at the resection margin.

[0282] The disclosed method can include multiple systemic doses of a functionalized payload concentrated at one location. The disclosed method can be used to deliver a second payload. The disclosed method can be used to administer a second functionalized payload if the tumor is resistant to the first payload. The second payload can be a TCO-labeled payload of gemcitabine or docetaxel. The TCO-labeled payload of gemcitabine or docetaxel can be administered in combination with doxorubicin. The second functionalized payload can be activated by the therapeutic support composition used for the first prodrug.

[0283] The functionalized payloads disclosed herein can function as adjuvants. This combination approach will be beneficial to patients. While chemotherapeutic agents may treat solid tumors or specific locations and enhance or induce an immune response, enhancing the immunotherapy response of functionalized payloads and / or separate agents may be useful for distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can enhance the efficacy of ipilimumab, nivolumab, pembrolizumab, and avelumab (also known as MSB0010718C, Pfizer) with or in combination with anthracyclines, auristatins, vinca alkaloids, taxanes, gemcitabine, campothecin analogs, and other drugs.

[0284] The disclosed method can be used to treat diffuse pontine glioma (DIPG). Diffuse pontine glioma (DIPG) is a pediatric brainstem tumor that can be highly malignant and difficult to treat. There is no known curative treatment for DIPG, and survival rates have remained dismal over the past 40 years. The median overall survival for DIPG patients is only 11 months, with a two-year survival rate of less than 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 survival rates remaining unchanged over the past 40 years. Diagnosis of DIPG can begin 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, diplopia, abnormal or limited eye movements, asymmetric smiling, impaired balance, and weakness. Alternatively, severe neurologic deterioration may occur more rapidly, with symptoms present within a month before diagnosis. Clinical examination may reveal a triad of multiple cranial nerve disorders, long-pathway signs such as hyperreflexia and clonus, and ataxia. Expansion of the pontine portion of the brainstem may cause obstructive hydrocephalus and increased intracranial pressure.

[0285] Nuclei essential for vital functions such as breathing and heartbeat are located within the pons, and if untreated, DIPGs can impair breathing and heartbeat.

[0286] 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. A biomaterial can be placed during a biopsy. The results of the biopsy can indicate the amount and type of treatment to deliver to the tumor site. The disclosed compounds and compositions can be administered before surgical resection. The disclosed methods can minimize tumor size before surgical resection. The disclosed compounds and compositions can be administered during surgical resection. The disclosed compounds and compositions can be administered after surgical resection. A biomaterial can be placed around the surgical cavity at the end of the surgical resection, after which the subject can 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.

[0287] The disclosed methods can involve multiple systemic doses of a functionalized payload concentrated at one location. The disclosed methods can be used to deliver a second payload. The disclosed methods can be used to administer a second functionalized payload if the tumor is resistant to the first payload. The second payload can be a TCO-labeled payload of paclitaxel, docetaxel, an anthracycline, an auristatin, a vinca alkaloid, a taxane, gemcitabine, a camptothecin analog, or other drug. A TCO-labeled payload of gemcitabine, paclitaxel, or docetaxel can be administered in combination with doxorubicin. The second functionalized payload can be activated by the therapeutic support composition used for the first prodrug.

[0288] Mode of administration Therapeutic methods can include any number of modes of administration of the disclosed conjugates, compounds, or compositions. Modes of administration can 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 dispersions, or dispersible powders. In pharmaceutical compositions, the conjugates, compounds, or compositions disclosed herein can also be dispersed in microparticles, such as nanoparticle compositions.

[0289] For parenteral administration, the conjugates, compounds, or compositions disclosed herein can be dissolved or suspended in physiologically acceptable diluents such as water, buffer, oil, etc., with or without solubilizers, surfactants, dispersants, or emulsifiers. Suitable oils can 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 can be administered in the form of aqueous, lipid, oily, or other types of solutions or suspensions, or even in the form of liposomes or nanosuspensions.

[0290] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0291] 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 the formula disclosed herein, for example, Formula AI or A-II, can be administered by any convenient route, taking into account the condition of the subject and the judgment of a medical professional. Parenteral administration is a suitable means of administering the conjugate of the formula disclosed herein, for example, Formula AI or A-II.

[0292] The amount of the composition to be administered to the subject can be determined based on the dosage and / or dosage schedule guideline of the parent drug first.Generally, this composition can achieve the target delivery and / or serum half-life extension of the linked drug, thereby leading to at least one of the following: reduced dosage or reduced administration in the dosage schedule.Therefore, this composition can lead to the reduced dosage and / or reduced administration in the dosage schedule compared with the parent drug before being conjugated in the composition of the present disclosure.

[0293] Pharmaceutical preparations can 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.Unit dosage form can be a packaged preparation, and the package contains individual amounts of preparations, such as tablets, capsules, and powders, packaged in pouches, vials, or ampoules.

[0294] In some embodiments, a kit is provided that includes a conjugate described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, and instructions for use thereof.

[0295] In some embodiments, the kit further comprises a therapeutic support composition.

[0296] The compositions of the present disclosure can be present in any suitable amount, which can depend on a variety of factors, including, but not limited to, the subject's weight and age, 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 1000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, suitable dosages for the 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.

[0297] In some embodiments, multiple doses of the composition are administered. The frequency of administration of the composition can vary depending on various 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).

[0298] The compositions of the present disclosure can be administered at any suitable frequency, interval and period.For example, the compositions of the present disclosure can be administered once an hour, or twice, three times or more per hour, once a day, or twice, three times or more per day, or once every 2, 3, 4, 5, 6 or 7 days, to provide the desired dosage level to the subject.When the compositions of the present disclosure are administered more than once a day, representative intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes, and 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 over the course of 1 hour, 1-6 hours, 1-12 hours, 1-24 hours, 6-12 hours, 12-24 hours, 1 day, 1-7 days, 1 week, 1-4 weeks, 1 month, 1-12 months, 1 year or more, or even indefinitely.

[0299] 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 twice, three times, or more times a day to provide the desired daily dosage level.

[0300] Co-administration can be achieved by co-implantation or co-injection.

[0301] In some embodiments, simultaneous 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. In other embodiments, the composition of the present disclosure and the active agent can be formulated separately and administered simultaneously to a subject.

[0302] The composition of the present disclosure and the active agent can 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 can 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.

[0303] Combination therapy In one aspect, the present invention provides a method of treating cancer or enhancing or eliciting an immune response, comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate disclosed herein (e.g., of formula AI, A-II, etc.), or a pharmaceutically acceptable salt or composition thereof; a therapeutic support composition 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.

[0304] The present invention also provides a pharmaceutical combination comprising a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof, a therapeutic support composition 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, for use in treating or preventing cancer or for use in enhancing or eliciting an immune response.

[0305] The present invention also provides the use of a pharmaceutical combination comprising a conjugate described herein, or a pharmaceutically acceptable salt or composition thereof, 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 eliciting an immune response.

[0306] In the methods and uses described herein, the components of the pharmaceutical combination 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 disease progression according to the present invention may involve administering a first active ingredient in free form or pharmaceutically acceptable salt form and a second active ingredient in free form or pharmaceutically acceptable salt form simultaneously or sequentially in any order in a combined therapeutically effective amount or effective amount, for example, in a daily dosage corresponding to the amounts described herein. The individual active ingredients of the combination may be administered separately at different times during the course of treatment or simultaneously in divided or single dosage forms. Therefore, the present disclosure should be understood to encompass all such regimes of simultaneous or alternating treatment, and the term "administering" should be interpreted accordingly. Thus, the pharmaceutical combination as used herein defines either a fixed combination in a single unit dosage form, or separate dosage forms in the case of combined administration, where the combined administration may be independently simultaneous or at different times. As a further example, the therapeutic support composition and the conjugate may 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.

[0307] The method and use in cancer treatment includes administering / localizing the therapeutic support composition to tumor.In the method and use disclosed herein, the administration of the conjugate, or its pharmaceutically acceptable salt or composition, the therapeutic support composition, and an additional therapeutic agent can inhibit tumor growth.

[0308] The additional therapeutic agent(s) may be administered simultaneously with the disclosed conjugates and compositions or sequentially. 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(s) may be administered in the same composition as the disclosed conjugates. In other embodiments, there may be a time interval between the administration of the additional therapeutic agent and the administration of the disclosed conjugates or compositions. In some embodiments, administering the additional therapeutic agent with the disclosed conjugates or compositions may allow for lower doses and / or less frequent administration 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 containing one or more other active ingredients in addition to the conjugates of the present disclosure.

[0309] anticancer drugs Exemplary anticancer drugs include abiraterone acetate, avitrexate (methotrexate), Abraxane (paclitaxel-albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), adrsil (fluorouracil), afatinib maleate, 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), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase Erwinia chrysanthemi, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexar (tocopherol) Situmomab and I-131 iodine 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 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, Cyphos (ifosfamide), cytarabine, cytarabine liposome, Cytosar-U (cytarabine),Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine) ), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase Erwinia chrysanthemi), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Ebacet (doxorubicin hydrochloride liposomal) rom), everolimus, Evista (raloxifene hydrochloride), exemestane, Fairston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), Forfiri, Forfiri-bevacizumab, Forfiri-cetuximab, Forfirinox, Forfox (leucovorin) , fluorouracil, oxaliplatin), Folotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Giotrif (afatinib maleate), Glivec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab),Recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, Hycamtin (topotecan hydrochloride), HyperCVAD, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), ifosfamide, ifosfamidam (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alfa-2b), iodine-131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib) , irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivance (palifermin), Kyprolis (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Levran (aminolevulinic acid), Lympholidine (chloridin) Lambucil), 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, Megace (megestrol acetate), megestrol acetate, Mekinist (tramethicone) Nib), mercaptopurine, mesna, Mesnex (Mesna), Metazolastone (Temozolomide), methotrexate, methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mastergen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Myrosal (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin),Nanoparticle paclitaxel (paclitaxel-albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), Nelarabine, Neosar (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omacetaxine mepesuxinate, Oncasper (pegaspargase), Ontak (denileukin diftitox), OEPA, OPPA, oxaliplatin tin, paclitaxel, paclitaxel-albumin-stabilized nanoparticle formulation, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, poma Lidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), Provenzi (sipuleucel-T), Purinetol (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Revlimid (lenalidomide), rheumatoid arthritis Trex (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 mepesuxinate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and 1 131-iodine tositumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (dexrazoxane hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), vandetanib, VAMP, Vectibix (panitumumab), VelP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Bepcid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincristine PFS (vincristine sulfate), vincristine sulfate, vincristine liposomal sulfate, vinorelbine tartrate, vismodegib, and Boraxase (glucarpidase) These include, but are not limited to, vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xerox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), and Zytiga (abiraterone acetate).

[0310] 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, filantside, alimta, T2 toxin, MMC, vantaranib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, Iressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, Gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustard, etoposide, or 5-FU.

[0311] In certain embodiments, the anticancer agent is an anthracycline. In certain embodiments, the anticancer agent is a taxane. In certain embodiments, the anticancer agent is gemcitabine. In certain embodiments, the anticancer agent is doxorubicin. In certain embodiments, the anticancer agent is docetaxel. In certain embodiments, the anticancer agent is paclitaxel. In certain embodiments, the anticancer agent is SN38. In certain embodiments, the anticancer agent is monomethyl auristatin E. In certain embodiments, the anticancer agent is an alkylating agent, an antimetabolite (folate antagonist, purine folate antagonist, pyrimidine folate antagonist), antibiotic, taxane, vinca alkaloid, or campothecin analog.

[0312] E. 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 also be used. The synthesis of typical compounds described herein can be achieved 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.

[0313] Where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, it will be understood that other process conditions can also 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.

[0314] Furthermore, 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. For example, Wuts, PGM, Greene, TW, & Greene, TW (2006). Greene's protective groups in organic synthesis. Hoboken, NJ, Wiley-Interscience, and the references cited therein, describe numerous protecting groups.

[0315] 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 within the scope of the present disclosure, unless otherwise specified. 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, etc.

[0316] 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, California, USA), Emka-Chemce, or Sigma (St. Louis, Missouri, USA). Other starting materials can be prepared by procedures described in standard references such as Fieser and Fieser, Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd, Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March, Advanced Organic Chemistry (John Wiley and Sons, 5th Edition, 2001), and Larock, Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof. [Example]

[0317] The following examples are included to demonstrate 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 practice of the present disclosure, and can therefore be considered to constitute specific modes for its practice. However, in light of the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the present disclosure.

[0318] General procedure for the preparation of intermediate 5 [ka] General procedure for the preparation of intermediate i-6 To a solution of intermediate i-5 (150 g, 689 mmol, HCl) in NaOH (1 M, 1.38 L) and NaHCO3 (1 M, 1.38 L) was added (2,5-dioxopyrrolidin-1-yl) 2,2,2-trichloroethyl carbonate (210 g, 723 mmol) in dioxane (1 L). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove dioxane. The residue was extracted with MTBE (5 L), and the aqueous phase was adjusted to pH 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 product 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 analysis showed the reaction was complete, with one major peak having the desired mass detected. The reaction mixture was adjusted to pH 9-10 with saturated aqueous NaHCO3 and then extracted with EtOAc (5 L). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was precipitated with PE (10 Vol) to give intermediate i-6 (190 g, 74.4% yield).

[0319] 1 H NMR: (400MHz, CDCl3): δ3.25(br s,1H)3.85(s,3H)4.64-4.83(m,2H)5.30(dd,J=9.51,1.13Hz,1H)5.92(br d,J=9.38Hz,1H)7.30-7.45(m,5H).

[0320] LCMS (m / z): 391.9 / 393.9 (M+H) + .

[0321] General procedure for the preparation of intermediate i-7 To a solution of intermediate i-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 major 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 HO (500 mL) and then extracted with EtOAc (500 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give intermediate i-7 (285 g, crude), which was carried forward directly.

[0322] General procedure for the preparation of intermediate i-8 To a solution of intermediate i-7 (285 g, crude product) in MeOH (2000 mL) was added KOH (42.5 g, 758 mmol) in HO (1000 mL). The mixture was stirred at 25 °C for 1 h. LC-MS showed that intermediate i-7 was completely consumed, with one major peak having the desired mass being 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 KHSO (1 L) and extracted with EtOAc (5 L). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the crude product. The crude product was precipitated with PE (10 Vol) to give intermediate i-8 (95.0 g, 34.3% yield).

[0323] 1 H NMR(400MHz,MeOD):δ3.82(s,3H)4.41-4.47(m,1H)4.50-4.56(m,1H)4.60(d,J=4 .88Hz,1H)5.47(d,J=4.75Hz,1H)6.46(s,1H)6.86-6.94(m,2H)7.34-7.46(m,7H).

[0324] LCMS(m / z):495.9(M+Na) + .

[0325] General procedure for the preparation of 7-Troc-baccatin III [ka] To a solution of baccatin III (30.0 g, 51.1 mmol) in DCM (300 mL), DMAP (625 mg, 5.11 mmol), pyridine (14.2 g, 179 mmol), and 2,2,2-trichloroethyl carbonochloridate (15.2 g, 71.6 mmol) were added. The mixture was stirred at 25 °C for 0.5 h. LC-MS showed that baccatin III was completely consumed, with one major peak having the desired mass being 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).

[0326] LCMS(m / z):761.5 / 763.5(M+Na) + .

[0327] General procedure for the preparation of intermediate i-9 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 i-8 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was filtered. The crude product was washed with saturated aqueous NH4Cl (100 mL) and water (1000 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give intermediate i-9 (35.0 g, crude).

[0328] LCMS(m / z):1240.0 / 1242.0(M+Na) + .

[0329] General procedure for the preparation of intermediate i-10 To a solution of intermediate i-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 approximately 50% of intermediate i-9 remained, and one major peak with the desired mass was detected. The reaction mixture was filtered and concentrated, and the residue was purified by preparative HPLC (water (0.1% TFA)-ACN). The eluate was concentrated under reduced pressure to remove the solvent, and then extracted with EtOAc (500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give intermediate i-10 (13.0 g, 17.9% yield).

[0330] LCMS(m / z):1120.2(M+Na) + .

[0331] General procedure for the preparation of intermediate i-11 A solution of intermediate i-10 (13.0 g, 11.8 mmol), DMAP (722 mg, 5.90 mmol), EDCI (2.70 g, 14.2 mmol), and benzoic acid (1.70 g, 14.2 mmol) in DCM (260 mL) was stirred at 25 °C for 1 h. LC-MS showed that intermediate i-10 was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was washed with saturated aqueous citric acid (100 mL), saturated aqueous NaHCO (100 mL), and water (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give intermediate i-11 (11.0 g, 77.3% yield).

[0332] LCMS(m / z):1204.1(M+H) + .

[0333] General procedure for the preparation of intermediate i-12 To a solution of intermediate i-11 (20.0 g, 16.6 mmol) in MeOH (200 mL) and AcOH (200 mL) was added Zn dust (21.6 g, 331 mmol). The mixture was stirred at 25 °C for 1 h. LC-MS showed that intermediate i-11 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was filtered, diluted with HO (500 mL), and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with saturated aqueous NaHCO (200 mL) and brine (100 mL), dried over NaSO, 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 i-12 (5.0 g, 21% yield).

[0334] LCMS(m / z):854.3(M+H) + .

[0335] General procedure for the preparation of intermediate 5 A solution of intermediate 12 (5.00 g, 5.90 mmol), DIEA (1.50 g, 11.7 mmol), and intermediate i-3 (3.90 g, 8.80 mmol) in DMF (50 mL) was added. The mixture was stirred at 25 °C for 16 h. LC-MS showed that approximately 50% of intermediate 12 remained, and one major peak with the desired mass was detected. The residue was purified by preparative HPLC (water (0.1% TFA)-ACN) to give intermediate 5 (505 mg, 7.4% yield).

[0336] LCMS(m / z):1161.4(M+H) + .

[0337] General procedure for the preparation of compound 1 [ka] General procedure for the preparation of intermediate 2 [ka] To a solution of intermediate 1 (10.0 g, 41.6 mmol) in MeOH (50 mL) was added NaOMe (5.40 M, 46.2 mL, 249 mmol) in HO (50 mL). The mixture was stirred at 25 °C for 16 h. TLC showed that compound 1 was completely consumed and two new spots were detected. The reaction mixture was diluted with HO (50 mL) and extracted with MTBE (3 × 100 mL). While cooling in an ice-water bath (T < 7 °C), the aqueous layer was acidified with 1 M HCl to pH = 4. The aqueous layer was extracted with MTBE (3 × 100 mL). The combined MTBE layers were dried over NaSO, filtered, and concentrated under reduced pressure to give intermediate 2 (3.50 g, 45.6% yield).

[0338] 1 HNMR:400MHz,CDCl3δ 1.11(s,3H),1.16-1.29(m,1H),1.65(dd,J=15.82,6.19Hz,1H),1.83-2.03(m,4H),2.11-2.37(m,4H),4.49(br s, 1H), 5.64 (dd, J=16.63, 2.25Hz, 1H), 6.02-6.12 (m, 1H).

[0339] General procedure for the preparation of intermediate 3 [ka] To a solution of intermediate 2 (3.50 g, 19.0 mmol) in MeCN (60 mL) was added DIEA (17.2 g, 133 mmol) and DSC (20.9 g, 81.7 mmol). The resulting mixture was stirred at 25 °C for 16 h. HPLC showed that compound 2 was completely consumed and one major peak containing the desired product was detected. The reaction mixture was purified by recrystallization from HO (600 mL × 4) at 25 °C to give intermediate 3 (6.90 g, 89.5% yield).

[0340] 1HNMR:400MHz, CDCl3δ1.28(s,3H),1.97-2.16(m,4H),2.26-2.47(m,4H),2.84(s,8H),5.29(br s, 1H), 5.62 (dd, J=16.70, 2.31Hz, 1H), 6.02-6.15 (m, 1H).

[0341] General procedure for the preparation of compound 5 [ka] To a solution of intermediate 4 (1.50 g, 1.76 mmol) in DCM (30 mL) were added DMAP (472 mg, 3.86 mmol) and intermediate 3 (816 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 major peak with the desired mass was detected. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO, DCM:MeOH = 50 / 1 to 10 / 1) to give intermediate 5 (1.10 g, 51.7% yield).

[0342] LCMS:ESI-LCMS:MH + Calculated value 1161.4, measured value 1161.7

[0343] General procedure for the preparation of compound 1 [ka] To a solution of intermediate 6 (314 mg, 1.38 mmol) in DMF (1.0 mL), DMAP (168 mg, 1.38 mmol), intermediate 5 (200 mg, 172 μmol), and DIEA (178 mg, 1.38 mmol) were added. The mixture was stirred at 25° C. for 16 hours. LC-MS showed that one major peak with the desired mass was detected. The mixture was filtered and purified by preparative HPLC (TFA conditions) to give compound 1 (20 mg, 8.31% yield).

[0344] LCMS:ESI-LCMS:MH + Calculated value 1176.5, measured value 1176.7

[0345] General procedure for the preparation of compound 2 [ka] To a solution of intermediate 7 (145 mg, 689 μmol) in DMF (0.5 mL), DMAP (84.2 mg, 689 μmol), intermediate 5 (100 mg, 86.1 μmol), and DIEA (89.0 mg, 689 μmol) were added. The mixture was stirred at 35° C. for 40 hours. LC-MS showed that intermediate 5 was completely consumed, with one major peak having the desired mass being detected. The mixture was filtered and purified by preparative HPLC (TFA conditions) to give compound 2 (30 mg, 28.1% yield).

[0346] LCMS:ESI-LCMS:MH + Calculated value 1220.5, measured value 1220.8

[0347] General procedure for the preparation of compound 3 [ka] To a solution of intermediate 8 (192 mg, 1.38 mmol) in DMF (1.0 mL), DMAP (168 mg, 1.38 mmol), intermediate 5 (200 mg, 172 μmol), and DIEA (178 mg, 1.38 mmol) were added. The mixture was stirred at 35° C. for 60 hours. LC-MS showed that one major peak with the desired mass was detected. The mixture was filtered and purified by preparative HPLC (TFA conditions) to give compound 3 (19 mg, 7.71% yield).

[0348] LCMS:ESI-LCMS:MH + Calculated value: 1185.5, measured value: 1185.6.

[0349] General procedure for the preparation of compound 4 [ka] General procedure for the preparation of intermediate 9-3 [ka] To a solution of intermediate 9-1 (1.0 g, 4.01 mmol) in DCM (10 mL) was added intermediate 9-2 (568 mg, 4.01 mmol). The mixture was stirred at 0 °C for 1 h. TLC showed that intermediate 9-1 was completely consumed and one new spot was formed. The mixture was used in the next step without any workup.

[0350] General procedure for the preparation of intermediate 9-5 [ka] To a solution of intermediate 9-4 (500 mg, 3.81 mmol) in DCM (5.0 mL) were added EtN (771 mg, 7.62 mmol) and intermediate 9-3 (1.49 g, 3.81 mmol). The mixture was stirred at 0 °C for 2 h. TLC showed that intermediate 9-3 was completely consumed and one new spot was formed. The reaction mixture was partitioned between 20 mL of DCM and 20 mL of HO. The organic layer was separated, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, DCM:MeOH = 0 / 1 to 100 / 1) to give intermediate 9-5 (1.0 g, 51.6% yield).

[0351] 1 H NMR:400MHz,DMSO-d6δ11.28(s,1H),8.12(t,J=6.07Hz,1H),6.67-6.81(m,1H),4.15(dd,J= 5.44,3.81Hz,2H),3.70(d,J=6.00Hz,2H),3.58-3.63(m,2H),3.48-3.54(m,4H),3.37(t,J= 6.07Hz, 2H), 3.06 (q, J=5.88Hz, 2H), 1.41 (s, 9H), 1.37 (s, 9H).

[0352] LCMS:ESI-LCMS:MH + Calculated value 486.2, measured value 486.2

[0353] General procedure for the preparation of intermediate 9 [ka] To a solution of intermediate 9-5 (700 mg, 1.38 mmol) in dioxane (7.0 mL), HCl / dioxane (4 M, 7.0 mL) was added. The mixture was stirred at 0-25 °C for 48 h. LC-MS analysis showed that intermediate 9-5 was completely consumed, with one major peak having the desired mass detected. The reaction solution was precipitated with 200 mL of isopropyl ether (100 mL × 2), filtered, and concentrated under reduced pressure to give intermediate 9 (500 mg, crude product).

[0354] 1 H NMR:400MHz,DMSO-d6δ11.25(s,1H),8.02-8.16(m,3H),4.16(br dd,J=5.32,3.69Hz,2H),3.72(br d,J=5.88Hz,2H),3.62(br t,J=4.88Hz,4H),3.56(s,4H),2.86-3.01(m,2H).

[0355] LCMS:ESI-LCMS:MH + Calculated value 330.1, measured value 330.1

[0356] General procedure for the preparation of compound 4 [ka] To a solution of intermediate 9 (252 mg, 689 μmol) in DMF (0.5 mL), DMAP (84.2 mg, 689 μmol) and intermediate 5 (100 mg, 86.1 μmol), and DIEA (89.0 mg, 689 μmol) were added. The mixture was stirred at 25° C. for 16 hours. LC-MS showed that compound 5 was completely consumed, and one major peak with the desired mass was detected. The mixture was filtered and purified by preparative HPLC (TFA conditions) to give compound 4 (20.5 mg, 16.5% yield).

[0357] LCMS: ESI-LCMS: MH+ calculated 1375.5, found 1375.7

[0358] General procedure for the preparation of compound 5 [ka] General procedure for the preparation of intermediate 10-2 [ka] To stirred THF (50 mL) under a N2 atmosphere at 0 °C, NaH (153 mg, 3.83 mmol, 60% purity) was added portionwise, and MeNO2 (35.6 g, 583 mmol) was added dropwise to the stirred suspension. After 1 h, intermediate 10-1 (5.0 g, 16.6 mmol) was added in one portion, and the mixture was stirred at 25 °C for 16 h. LC-MS showed that intermediate 10-1 was completely consumed, with one major peak having the desired mass being detected. The reaction mixture was diluted with saturated aqueous NH4Cl (100 mL) and extracted with DCM (200 mL × 2). The combined organic phase was washed with brine (400 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, DCM:MeOH = 100 / 1 to 50 / 1) to give intermediate 10-2 (3.0 g, 48.2% yield).

[0359] 1 HNMR:400MHz, CDCl3-dδ4.70(t,J=6.82Hz,2H),4.14-4.26(m,8H),2.42-2.65(m,3H),1.35(t,J=7.07Hz,12H).

[0360] LCMS:ESI-LCMS:MH + Calculated value 362.1, measured value 362.1

[0361] General procedure for the preparation of intermediate 10-3 [ka] A 100 mL round-bottom flask was purged with Ar three times, and Pd / C (300 mg, 10% purity) was carefully added. Then, THF (30 mL) was added to completely infiltrate the Pd / C, followed by slowly dissolving a THF solution of intermediate 10-2 (3.0 g, 8.30 mmol) under an Ar atmosphere. The resulting mixture was degassed and purged with H2 three times. The mixture was then stirred at 70 °C under an H2 atmosphere for 16 h. LC-MS analysis showed that intermediate 10-2 was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was carefully filtered and concentrated under reduced pressure to obtain the crude product. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50 / 1 to 5 / 1) to obtain intermediate 10-3 (1.1 g, 39.5% yield).

[0362] 1 HNMR:400MHz, CDCl3-dδ4.09-4.29(m,8H),2.85-3.03(m,2H),2.45-2.65(m,1H),2.02-2.11(m,2H),1.34(br t,J=5.69Hz,12H).

[0363] LCMS:ESI-LCMS:MH + Calculated value 332.1, measured value 332.1

[0364] General procedure for the preparation of intermediate 10 [ka] A solution of intermediate 10-3 (1.1 g, 3.32 mmol) in HCl (6 M, 22 mL) was stirred at 100 °C for 40 h. LC-MS analysis showed that intermediate 10-3 was completely consumed, with one major peak having the desired mass detected. H2O was added to the reaction mixture, which was then washed three times with DCM (5 mL x 3). The aqueous layer was then lyophilized to give intermediate 10 (0.65 g, 78.5% yield).

[0365] 1 HNMR: 400MHz, D2O δ3.16 (br t, J=7.19Hz, 2H), 2.05-2.26 (m, 3H).

[0366] LCMS:ESI-LCMS:MH + Calculated value 220.0, measured value 220.0

[0367] General procedure for the preparation of compound 5 [ka] To a solution of intermediate 10 (302 mg, 1.38 mmol) in ACN (1.0 mL) were added DIEA (178 mg, 1.38 mmol) and intermediate 5 (200 mg, 172 μmol). The mixture was stirred at 50° C. for 60 hours. LC-MS showed that one major peak with the desired mass was detected. The mixture was filtered and purified by preparative HPLC (TFA conditions) to give compound 5 (22 mg, 10.1% yield).

[0368] LCMS: ESI-LCMS: MH+ calculated 1265.4, found 1265.8.

[0369] General procedure for the preparation of compound 27 [ka]

[0370] Procedure for the preparation of compound 3 [ka] To a solution of compound 1 (200 mg, 490 μmol, 1.0 equiv.) in DMF (2.0 mL) was added compound 2 (248 mg, 589 μmol, 1.2 equiv.) and DIEA (253 mg, 1.96 mmol, 4.0 equiv.). The mixture was stirred at 25° C. for 16 hours. LC-MS confirmed the desired mass (MS calculated: 714.2, MS found: [M+H] + One peak (R = 715.3) t =0.38 min). The reaction mixture was added to 40 mL of isopropyl ether and filtered to give compound 3 (320 mg, yield 87.9%, purity 96.4%) as a brown solid. LCMS:Rt =0.38 min, MS calculated value: 714.2, MS observed value: [M+H] + =715.3. HPLC:R t =2.26 minutes, purity: 96.4%

[0371] Procedure for the preparation of compound 27 [ka] To a solution of compound 3 (630 mg, 881 μmol, 1.0 equiv.) in DMF (6.0 mL) was added compound 4 (1.58 g, 2.21 mmol, 2.50 equiv.), DMAP (215 mg, 1.76 mmol, 2.0 equiv.), and DIEA (683 mg, 5.29 mmol, 6.0 equiv.). The mixture was stirred at 25° C. for 16 hours. LC-MS showed that target 152 had formed in 34.0% and compound 3 remained in 62.6%. Compound 4 (1.58 g, 2.21 mmol, 2.5 equiv.) and DIEA (228 mg, 1.76 mmol, 2.0 equiv.) were then added to the solution. The mixture was stirred at 30° C. for 48 hours. LC-MS showed that compound 27 had formed in 64.4% and compound 3 remained in 30.0%. The reaction mixture was added to 150 mL of isopropyl ether and filtered to obtain the crude product. The residue was purified by preparative HPLC (0.1% TFA condition) to obtain Compound 27 (200 mg, yield 17.9%, purity 94.7%).

[0372] LCMS:R t =0.349 min, MS calculated value: 1199.6, MS observed value: [M+2H] 2+ =601.1, [M+H] + =1200.5.

[0373] HPLC:R t =1.837 minutes, purity: 94.7%.

[0374] Example 1: Synthesis of Compound 104 [ka] [ka]

[0375] General procedure for the preparation of compound 2 [ka] To a solution of compound 1 (5.0 g, 29.7 mmol, 1 equiv.) in DMF (50 mL) was added imidazole (4.65 g, 68.4 mmol, 2.3 equiv.) and TBSCl (9.86 g, 65.4 mmol, 8.05 mL, 2.2 equiv.). The mixture was stirred at 0-25 °C for 2 h. LCMS confirmed the desired mass (MS calculated: 396.2, MS found: [M-H]). + One peak (R = 395.1) t = 0.703 min) was detected. The reaction mixture was partitioned between 100 mL of MTBE and 100 mL × 3 of H2O. The organic phase was separated, washed with brine (100 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 = 100 / 0 to 95 / 5). (petroleum ether / ethyl acetate = 10:1, R f =0.84 min) LCMS showed compound 2 (9.2 g, 23.1 mmol, 78.0% yield, 100% purity) as a colorless oil.

[0376] LCMS:R t = 0.703 min, (MS calculated: 396.2, MS observed: [MH] + =395.1.

[0377] HPLC:R t =4.769 minutes, purity: 100%

[0378] 1 H NMR(400MHz,DMSO-d6):δ ppm8.08(s,1H),6.97(s,2H),4.69(s,4H),2.20(s,3H),0.90(s,18H),0.07(s,12H)

[0379] General procedure for the preparation of compound 3 [ka] To a solution of compound 2 (8.0 g, 20.2 mmol, 1.0 equiv.) in THF (80 mL) was added TEA (6.12 g, 60.5 mmol, 8.42 mL, 3.0 equiv.) and PNP-Cl (6.10 g, 30.2 mmol, 1.5 equiv.). The mixture was stirred at 0-25 °C for 2 h. LCMS confirmed the desired mass (MS calculated: 561.2, MS found: [M+18]). + One peak (R = 579.1) t = 0.576 min) was detected. The residue was obtained by filtration and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 100 / 1) (petroleum ether / ethyl acetate: 20:1, R f =0.65) LCMS showed compound 3 (10.1 g, 17.9 mmol, 89.1% yield, 100% purity) as a white solid.

[0380] LCMS:R t = 0.578 min, MS calculated value: 561.2, MS observed value: [M+Na] + =584.3.

[0381] HPLC:R t =4.363 minutes, purity: 100%

[0382] 1 H NMR(400MHz,CDCl3):δ ppm8.27-8.36(m,2H),7.43-7.54(m,2H),7.23(s,2H),4.73(s,4H),2.39(s,3H),0.93(s,17H),0.10(s,12H)

[0383] General procedure for the preparation of compound 5 [ka] To a solution of compound 3 (5.0 g, 8.90 mmol, 1.0 equiv.) in DMF (50 mL) was added compound 4 (2.51 g, 13.3 mmol, 1.5 equiv.). The mixture was stirred at 25° C. for 1 h. LCMS showed the desired mass (MS calculated: 610.3, MS found: [M+Na] + One peak (R = 633.3) t =0.577 min). The reaction mixture was partitioned between 100 mL of MTBE and 100 mL of H2O. The organic phase was separated, washed with brine (100 mL × 2), 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 = 100 / 0 to 20 / 1) (petroleum ether:ethyl acetate: 5:1, R f =0.56) as shown by LCMS to give compound 5 (3.4 g, 5.23 mmol, 58.7% yield, 94.0% purity) as a colorless oil.

[0384] LCMS:R t = 0.577 min, (MS calculated: 610.3, MS observed: [M+Na] + =633.3)

[0385] HPLC:R t =4.389 minutes, purity: 94.0%

[0386] HNMR(400MHz,DMSO-d6):δppm7.14(s,2H),4.55(br s,4H),3.52-3.60(m,1H),3.36-3.50(m,3H),3.07(br s,1H),2.74-2.97(m,5H),2.30(s,3H),1.31-1.42(m,9H),0.89(d,J=2.1Hz,18H),0.06(d,J=3.4Hz,12H)

[0387] General procedure for the preparation of compound 6 [ka] To a solution of compound 5 (3.4 g, 5.56 mmol, 1.0 equiv.) in MeOH (75 mL) was added Amberlyst 15 (75 mg, 5.56 mmol, 1.0 equiv.). The mixture was stirred at 25° C. for 12 h. LCMS showed the desired mass (MS calculated: 382.2, MS found: [M+Na] + One peak (R = 405.0) t =0.355 min). Filtration and concentration under reduced pressure gave a residue. The crude product was used in the next step without further purification. LCMS showed compound 6 (2.1 g, 5.38 mmol, 96.6% yield, 97.9% purity) as a colorless gum. LCMS:R t =0.355 min, MS calculated: 382.2, MS observed: [M+Na] + = 405. HPLC:R t =2.143 minutes, purity: 97.9%. 1 H NMR(400MHz,DMSO-d6):δ ppm7.15(s,2H),5.02-5.11(m,2H),4.35(br s,4H),3.54(br t,J=5.9Hz,1H),3.40(br d,J=5.0Hz,3H),3.05(br d,J=7.1Hz,1H),2.91(br s,2H),2.77-2.88(m,3H),2.30(s,3H),1.34-1.44(m,9H)

[0388] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (1.0 g, 2.61 mmol, 1.0 equiv.) in THF (10 mL), DIEA (2.70 g, 20.9 mmol, 3.64 mL, 8.0 equiv.) and pyridine (103 mg, 1.31 mmol, 105 μL, 0.5 equiv.) were added, followed by PNP-Cl (3.16 g, 15.6 mmol, 6.0 equiv.) in an ice bath. The mixture was stirred at 0-25 °C for 2 h. LCMS revealed the desired mass (MS calculated: 712.2, MS found: [M+Na]). + One peak (R = 735.2) t=0.556 min). The residue was diluted with DCM (20.0 mL) and extracted with H2O (20.0 mL × 2). The combined organic layers were washed with brine (20.0 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 = 100 / 1 to 1 / 1, petroleum ether:ethyl acetate = 2:1, R f =0.29). LCMS showed compound 7 (1.1 g, 1.45 mmol, 55.4% yield, 94.0% purity) was a white solid.

[0389] LCMS:R t =0.556 min, MS calculated: 712.2, MS observed: [M+Na] + =735.2.

[0390] HPLC:R t =4.387 minutes, purity: 94.0%

[0391] 1 H NMR:(400MHz,DMSO-d6):δ ppm8.32(d,J=8.8Hz,4H),7.54(br d,J=8.9Hz,4H),7.40(s,2H),5.23(br d,J=9.8Hz,4H),3.59(br s,1H),3.35-3.50(m,3H),3.09(s,1H),2.69-2.97(m,5H),2.36(s,3H),1.28-1.39(m,9H)

[0392] General procedure for the preparation of compound 8 [ka] To a solution of compound 7 (500 mg, 701 μmol, 1.0 equiv.), MMAE (1.06 g, 1.47 mmol, 2.1 equiv.) in DMF (10 mL) was added HOBt (227 mg, 1.68 mmol, 2.4 equiv.) and DIEA (362 mg, 2.81 mmol, 488 μL, 4.0 equiv.). The mixture was stirred at 25° C. for 16 h. LCMS showed the desired mass (MS calculated: 1869.1, MS found: [M+H]).+ One peak (R = 1871.5) t = 0.470 min). The reaction mixture was quenched by adding HCl (4 M, 1.0 mL) at 0-5 °C until the pH reached 5-6. The residue was purified by preparative HPLC (TFA conditions). LCMS showed compound 8 (900 mg, 457 μmol, 65.1% yield, 95.0% purity) as a white solid.

[0393] LCMS:R t =0.470 min, MS calculated: 1869.1, MS observed: [M+H] + =1871.5).

[0394] HPLC:R t =3.664 minutes, purity: 95.0%

[0395] General procedure for the preparation of compound 9 [ka] To a solution of compound 8 (700 mg, 374 μmol, 1.0 equiv.) in DCM (8.0 mL) was added HCl / EtOAc (4 M, 2.0 mL). The mixture was stirred at 0-5°C for 1 h. LCMS showed the desired mass (MS calculated: 1769.1, MS found: [M+Na] + One peak (R = 1771.3) t =0.486 min). The mixture was concentrated under reduced pressure to give a residue. LCMS showed compound 9 (600 mg, 305 μmol, 81.5% yield, 90.0% purity) as a white solid.

[0396] LCMS:R t =0.486 min, MS calculated value: 1869.1, MS observed value: [M+H] + =1871.5).

[0397] HPLC:R t =3.610 minutes, purity: 90.0%

[0398] General procedure for the preparation of compound 11 [ka] To a solution of compound 9 (600 mg, 339 μmol, 1.0 equiv.) in DMF (6.0 mL) was added DIEA (87 mg, 678 μmol, 118 μL, 2.0 equiv.) and DMAP (41.4 mg, 339 μmol, 1.0 equiv.). Then, TCO-NHS ester (186 mg, 440 μmol, 1.3 equiv.) was added. The mixture was stirred at 25° C. for 3.0 hours. LCMS confirmed the desired mass (MS calculated: 2076.2, MS found: [M+H / 2]). + One peak (R = 1039.7) t =0.590 min). The mixture was concentrated under reduced pressure to give a residue. LCMS showed compound 11 (610 mg, 251 μmol, 74.3% yield, 85.8% purity) as a white solid.

[0399] LCMS:R t =0.486 min, MS calculated value: 2076.2, MS observed value: [M+2H] 2+ =1039.7.

[0400] HPLC:R t =4.484 min, purity: 85.8%

[0401] General procedure for the preparation of compound 104 [ka] To a solution of glycine (144 mg, 1.93 mmol, 8.0 equiv.) in DMF (5.0 mL) and HO (1.0 mL) was added DIEA (186 mg, 1.44 mmol, 251 μL, 6.0 equiv.) and DMAP (58.8 mg, 481 μmol, 2.0 equiv.). Compound 11 (500 mg, 240 μmol, 1.0 equiv.) was then added. The mixture was stirred at 25° C. for 16 hours. LCMS confirmed the desired mass (MS calculated: 2036.2, MS found: [M+2H]). 2+ One peak (R = 1019.5) t=0.561 min). The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (TFA conditions). LCMS showed that compound 104 (210 mg, 103 μmol, yield 42.8%, purity 100%) was a white solid.

[0402] LCMS:R t =0.561 min, MS calculated value: 2036.2, MS observed value: [M+2H] 2+ =1019.5.

[0403] HRMS:R t =2.374 minutes, MS calculated value: [M+H] + =2037.2425, MS measured value: [M+H] + =2037.2447

[0404] HPLC:R t =3.107 minutes, purity: 100%

[0405] Example 2: Synthesis of Compound 112 [ka] [ka] 3-(Methoxycarbonyl)-4-(2-methylphenyl)but-3-enoic acid (3) [ka] 2-Methylbenzaldehyde (1) (50 g, 0.42 mol) and dimethyl succinate (2) (103 g, 0.71 mol) in methanol (130 ml) were treated with NaOMe (29.5 g, 0.55 mol) at 80 °C for 1 h. The mixture was cooled to 20 °C, neutralized with 3 M hydrochloric acid, and diluted with water. Unreacted 2 was removed by extraction with CHCl. ​​The mixture was acidified with 3 M hydrochloric acid, and the product was extracted with CHCl. ​​The extract was dried over MgSO, filtered, and concentrated to give a 9:1 mixture of (E) isomer 3a and (Z) isomer 3b (93 g, 95% yield). ESI m / z: 235.1 (M+1).+ .

[0406] Methyl 4-hydroxy-8-methylnaphthalene-2-carboxylate (4) [ka] The crude product 3 (93 g, 0.40 mol) was dissolved in THF (350 mL), and the solution was treated with TFAA (55 mL, 0.40 mol) at reflux temperature until complete conversion to 4. The reaction mixture was neutralized with aqueous KCO. The product was extracted with EtOAc. The organic layer was dried over MgSO, filtered, concentrated, and the residue was crystallized by cooling to 5 °C. The whitish-yellow crystals were filtered, washed with acetonitrile, and dried to give alcohol 4 (23.4 g, 27%). ESI m / z: 217.1 (M+1). + .

[0407] 1 H NMR (400MHz, DMSO-d6) δ10.53(s,1H),8.15-8.11(m,1H),8.05(d,J=8.2Hz,1H),7.51-7.42(m,2H),7.40(d,J=1.4Hz,1H),3.90(s,3H),2.67(s,3H).

[0408] Methyl 4-(benzyloxy)-8-methylnaphthalene-2-carboxylate (5) [ka] Alcohol 4 (23.4 g, 0.11 mol) was treated with benzyl chloride (13.4 mL, 117 mmol) in DMF (95 mL) in the presence of KCO (21.5 g, 155 mmol) at 80 °C. Upon completion of the reaction, the mixture was cooled and diluted with CHCl and water. The layers were separated and the organic layer was concentrated to give crude product 5 (33.1 g, 100% yield). ESI m / z: 307.1 (M+1). + .

[0409] 4-(benzyloxy)-8-methylnaphthalene-2-carboxylic acid (6) [ka] The residue containing methyl 4-(benzyloxy)-8-methyl-2-naphthoate (5) (33.1 g, 0.11 mol) was dissolved in toluene (150 mL) and methanol (200 mL), and the resulting solution was treated with 4 M aqueous NaOH (200 mL) under reflux for 2 h. Water (800 mL) was added in portions, and the methanol and toluene were removed by distillation. The crude product 6 was precipitated by adding 4 M hydrochloric acid (100 mL), the suspension was cooled to 10 °C, and the precipitate was filtered off, washed with cold water, collected, and dried. The crude product 6 was crystallized from toluene to give off-white crystals. The crystals were filtered, washed with toluene, and dried to give 6 (30.2 g, 95%). ESI m / z: 293.1 (M+1). + .

[0410] 1 H NMR (400MHz, DMSO-d6) δ13.14(s,1H),8.30(s,1H),8.14(d,J=8.3Hz,1H),7.62-7.34(m,8H),5.37(s,2H),2.70(s,3H).

[0411] tert-Butyl N-[4-(benzyloxy)-8-methylnaphthalen-2-yl]carbamate (7) [ka] Acid 6 (28 g, 96 mmol) was reacted with diphenylphosphoryl azide (30.6 g, 111 mmol), tert-butyl alcohol (18.5 g, 249 mmol), and EtN (14.7 mL, 106 mmol) in toluene (140 mL) at 85 °C for 3 h. After the mixture was cooled to 30 °C, EtOAc and water were added and the layers were separated. The organic layer was washed with aqueous NaCO and saturated aqueous NaCl, dried over MgSO, filtered, and concentrated. The residue was triturated with isopropyl alcohol. The solid was filtered off, washed with isopropyl alcohol, and dried to give 7 (28.8 g, 83%). ESI m / z: 364.1 (M+1).+ .

[0412] 1 H NMR(400MHz,DMSO-d6)δ9.52(s,1H),7.95(d,J=8.3Hz,1H),7.77(s,1H),7.56(d,J=7.2Hz,2H),7.44(t,J=7. 4Hz, 2H), 7.36 (t, J=7.2Hz, 1H), 7.33-7.27 (m, 2H), 7.25-7.19 (m, 1H), 5.22 (s, 2H), 2.53 (s, 3H), 1.51 (s, 9H).

[0413] tert-Butyl N-[4-(benzyloxy)-1-bromo-8-methylnaphthalen-2-yl]carbamate (8) [ka] Carbamate 7 (34.1 g, 94 mmol) was treated with NBS (17.6 g, 98.6 mmol) in THF (550 mL) at 10 °C. After completion of the reaction, the reaction was quenched by adding aqueous NaSO followed by 1 M NaOH solution. EtOAc was added and the layers were separated. The organic layer was washed with saturated aqueous NaCl, dried over MgSO, and concentrated to give crude product 8 (41.4 g, 100% yield) as a yellow solid. ESI m / z: 442.0 (M+1). + .

[0414] tert-Butyl N-[4-(benzyloxy)-1-bromo-8-methylnaphthalen-2-yl]-N-[(2R)-oxiran-2-ylmethyl]carbamate (10) [ka] The crude product 8 (41.4 g, 94 mmol) was dissolved in THF (100 mL), deprotonated with K₄t-Bu (13.7 g, 122 mmol) at 10° C., and alkylated with (S)-glycidyl nosylate (29.2 g, 113 mmol) at 25° C. for 3 h. The reaction was quenched by adding aqueous NH₄Cl, and the mixture was extracted with EtOAc. The organic layer was washed with water and saturated aqueous NaCl. After evaporation of the solvent, the crude product 10 was obtained. Crystallization from heptane gave 10 (37.3 g, 80%). ESI m / z: 498.0 (M+1). + .

[0415] 1 H NMR(400MHz,CDCl3)δ8.38-8.27(m,1H),7.51(t,J=6.8Hz,2H),7.45-7.34(m,5H),6.92(s,1H),6.80(s,1H),5.31-5.21(m,2H),4.23-4.09(m,1) H),3.47-3.35(m,1H),3.12(d,J=4.6Hz,4H),2.75(t,J=4.5Hz,1H),2.6 8-2.63(m,1H),2.45-2.40(m,1H),1.60-1.56(m,3H),1.34-1.31(m,6H).

[0416] tert-Butyl (1S)-5-(benzyloxy)-1-(hydroxymethyl)-9-methyl-1H,2H,3H-benzo[e]indole-3-carboxylate (11) [ka] Epoxide 10 (37.3 g, 83 mmol) was dissolved in THF (400 mL). The solution was cooled to -25 °C under a nitrogen atmosphere. n-Butyllithium (40 mL, 2.5 M in hexanes) was then added slowly while maintaining the temperature at -25 to -20 °C. After stirring the mixture for an additional 10 min, the reaction was quenched with saturated aqueous NH4Cl. The mixture was extracted twice with EtOAc (2 × 200 mL). Aqueous p-toluenesulfonic acid (4.8 g of monohydrate in 20 mL of water) was added to the combined organic layers, and the reaction mixture was stirred for 1 h. The reaction was quenched by adding 1 M aqueous Na2CO3. The layers were separated, and the organic layer was washed with saturated aqueous NaCl, dried over MgSO4, and concentrated. The crude product 11 was dissolved in CHCl2 and filtered through silica gel (0.063–0.1 mm, 60 Å). Elution was with CHCl (1 L), followed by CHCl / EtOAc (1 L, 9:1, v / v) and CHCl / EtOAc (1 L, 4:1, v / v). Fractions containing 11 were combined, the resulting solution was concentrated to dryness, and the residue was crystallized from CHCl / pentane. The crystals were collected and dried to give alcohol 11 (9.75 g, 31%). ESI m / z: 420.1 (M+1). + .

[0417] 1 H NMR(400MHz,CDCl3)δ8.22(d,J=8.0Hz,1H),8.07-7.88(m,1H),7.58-7.50(m,2H),7.46-7.29(m,4H),7.23-7.17(m,1H),5.26(s ,2H),4.25-4.21(m,1H),4.08-3.96(m,2H),3.81-3.73(m,1H),3.49-3.42(m,1H),2.81(s,3H),2.70-2.54(m,1H),1.60(s,9H).

[0418] tert-Butyl (1S)-5-(benzyloxy)-1-[(methanesulfonyloxy)methyl]-9-methyl-1H,2H,3H-benzo[e]indole-3-carboxylate (12) [ka] Alcohol 11 (5.0 g, 11.9 mmol) was treated with methanesulfonyl chloride (1.2 mL, 15.5 mmol) and EtN (4.3 mL, 30.9 mmol) in CHCl (40 mL) at 0–5° C. for 90 min. The reaction mixture was washed with hydrochloric acid, water, and saturated aqueous NaCl, dried over MgSO, and concentrated to give 12 (5.93 g, 100%). ESI m / z: 442.0 (M-tBu). + .

[0419] tert-Butyl (1S)-5-(benzyloxy)-1-(chloromethyl)-9-methyl-1H,2H,3H-benzo[e]indole-3-carboxylate (13) [ka] The residue 12 was dissolved in DMF (35 mL), and the solution was treated with LiCl (2.53 g, 59.7 mmol) at 80 °C for 90 min. After evaporation of DMF, the residue was partitioned between CHCl and water. The layers were separated, and the organic layer was washed with saturated aqueous NaCl, dried over MgSO, and concentrated. The residue was dissolved in hot heptane (100 mL), and the mixture was treated with activated charcoal and filtered. The activated charcoal was further washed with heptane. The combined filtrate was cooled to approximately 50 °C, seeded, and maintained at this temperature for 1 h. The suspension was cooled to 7 °C over 2 h and stirred at this temperature for an additional 1 h. The crystals were filtered, washed with heptane, collected, and dried. The dried crystals were recrystallized from heptane using the procedure described above to give chloride 13 (3.88 g, 74%). ESI m / z = 438.1 [M+H] + .

[0420] 1H NMR(400MHz,DMSO-d6)δ8.08(d,J=8.0Hz,1H),7.56(d,J=7.4Hz,2H),7.45(t,J=7.4Hz,2H),7.41-7.32(m,2H),7.29-7.20(m,1H),5.31 -5.24(m,2H),4.28-4.20(m,1H),4.14-4.07(m,1H),4.06-3.97(m,1H),3.75-3.69(m,1H),3.45-3.37(m,1H),2.75(s,3H),1.56(s,9H).

[0421] (1S)-5-(benzyloxy)-1-(chloromethyl)-9-methyl-1H,2H,3H-benzo[e]indole (14) [ka] Compound 13 (3.88 g, 8.8 mmol) was dissolved in 4 M HCl in dioxane (30 mL) and the solution was stirred for 4 h. A suspension formed, which was concentrated to dryness to yield amine 14 (2.99 g, 100%) as a yellow solid. ESI m / z: 302.1 (M-Cl) + .

[0422] Ethyl 6-[4-(methoxymethoxy)benzamido]imidazo[1,2-a]pyridine-2-carboxylate (17) [ka] To a solution of amine 15 (5 g, 24.4 mmol) in DMA (75 mL) was added 4-(methoxymethoxy)benzoic acid 16 (4.45 g, 24.4 mmol) and EDC·HCl (5.62 g, 29.3 mmol). The resulting mixture was stirred at room temperature for 18 h. The reaction mixture was then concentrated. The residue was dissolved in water (100 mL) and CHCl (100 mL), and the layers were separated. The organic layer was washed with water, dried over MgSO, and concentrated. The residue was transferred to a filter and rinsed with EtOAc. The residue was dried under vacuum to give 17 (7.74 g, 86%). ESI m / z: 370.2 (M+H). + .

[0423] 6-[4-(methoxymethoxy)benzamido]imidazo[1,2-a]pyridine-2-carboxylic acid (18) [ka] Compound 17 (7.74 g, 21.0 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), and 2 M aqueous NaOH (40 mL) was added. The mixture was stirred at 70° C. for 30 min. The mixture was then cooled to room temperature, water was added, and the mixture was acidified with 4 M hydrochloric acid solution. The resulting suspension was filtered, and the residue was dried to give acid 18 (6.37 g, 89%). ESI m / z: 342.1 (M+H). + .

[0424] 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),9.47(s,1H),8.66(s,1H),7.99(d,J=8.9Hz,2H),7.67(s,2H),7.18(d,J=8.9Hz,2H),5.30(s,2H),3.40(s,3H).

[0425] N-{2-[(1S)-5-(benzyloxy)-1-(chloromethyl)-9-methyl-1H,2H,3H-benzo[e]indole-3-carbonyl]imidazo[1,2-a]pyridin-6-yl}-4-(methoxymethoxy)benzamide (19) [ka] Compound 14 (2.99 g, 8.8 mmol) was then dissolved in DMA (80 mL). The solution was cooled to 0 °C, and compound 18 (3.30 g, 9.7 mmol) and EDC·HCl (5.05 g, 26.3 mmol) were added. The mixture was stirred for 18 h, and the temperature was slowly raised to 20 °C. Subsequently, the reaction mixture was concentrated, and the crude product was dissolved in CHCl / water (1 L, 1:1, v / v), and the layers were separated. The organic layer was dried over MgSO, filtered, and concentrated. The crude product was purified by column chromatography (CHCl / methanol, 1:0 to 39:1, v / v). Compound 19 was obtained (5.27 g, 90%). ESI m / z: 661.3 (M+H). + .

[0426] 1 H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.50(s,1H),8.70(s,1H),8.31(s,1H),8.14(d ,J=8.2Hz,1H),8.00(d,J=8.7Hz,2H),7.75(d,J=9.7Hz,1H),7.63-7.54(m,3H),7.50- 7.27(m,6H),7.19(d,J=8.7Hz,2H),5.31(s,4H),5.19-5.11(m,1H),4.67-4.57(m,1H) ),4.41-4.34(m,1H),3.82-3.75(m,1H),3.52-3.45(m,1H),3.41(s,3H),2.82(s,3H).

[0427] N-{2-[(1S)-1-(chloromethyl)-5-hydroxy-9-methyl-1H,2H,3H-benzo[e]indole-3-carbonyl]imidazo[1,2-a]pyridin-6-yl}-4-(methoxymethoxy)benzamide (20) [ka] A suspension of Pd / C (10 wt%, 0.527 g, 0.476 mmol) and ammonium formate (5.03 g, 79.8 mmol) in methanol (20 mL) was heated at 95 °C for 5 min. The mixture was then cooled to room temperature. Subsequently, additional ammonium formate (5.03 g, 79.8 mmol) was added, followed by a suspension of compound 19 (5.27 g, 7.98 mmol) in THF (100 mL). The resulting mixture was stirred at room temperature for 3 h. Upon completion of the reaction, the mixture was filtered through Hyflo Celite. The Hyflo Celite pad was rinsed with THF, and the combined filtrate was concentrated. The crude product was purified by column chromatography (CHCl / methanol, 39:1 to 9:1, v / v) to yield compound 20 (3.34 g, 73%). ESI m / z: 571.0 (M+H). + .

[0428] (1S)-1-(Chloromethyl)-3-{6-[4-(methoxymethoxy)benzamido]imidazo[1,2-a]pyridine-2-carbonyl}-9-methyl-1H,2H,3H-benzo[e]indol-5-yl 4-nitrophenyl carbonate (21) [ka] A solution of compound 20 (2.69 g, 4.72 mmol) in anhydrous THF (250 mL) was cooled to 0 °C under a nitrogen atmosphere, and then 4-nitrophenyl chloroformate (1.24 g, 6.18 mmol) and EtN (3.23 mL, 23.7 mmol) were added. The mixture was stirred at 0 °C for 1.5 h. Subsequently, the reaction mixture was concentrated, and the crude product was dissolved in CHCl / water (1 L, 1:1, v / v), and the layers were separated. The organic layer was dried over MgSO, filtered, and concentrated. The crude product was purified by column chromatography (CHCl / methanol, 1:0 to 39:1, v / v). Compound 21 was obtained (2.24 g, 64%). ESI m / z: 736.3 (M+H). + .

[0429] 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.49(s,1H),8.77-8.65(m,2H),8.44-8.35(m,2H ),8.06-7.94(m,3H),7.85-7.78(m,2H),7.75(d,J=9.7Hz,1H),7.62-7.55(m,1H),7.53- 7.41(m,2H),7.18(d,J=8.8Hz,2H),5.31(s,2H),5.21(d,J=11.9Hz,1H),4.76-4.66(m, 1H),4.56-4.45(m,1H),3.90-3.83(m,1H),3.65-3.56(m,1H),3.41(s,3H),2.87(s,3H).

[0430] tert-Butyl N-{2-[({[(1S)-1-(chloromethyl)-3-{6-[4-(methoxymethoxy)benzamido]imidazo[1,2-a]pyridine-2-carbonyl}-9-methyl-1H,2H,3H-benzo[e]indol-5-yl]oxy}carbonyl)[2-(2-hydroxyethoxy)ethyl]amino]ethyl}-N-methylcarbamate (23) [ka] Compound 21 (2.24 g, 3.05 mmol) was dissolved in THF (100 mL), followed by the addition of compound 22 (1.20 g, 4.57 mmol) and DIPEA (0.79 g, 6.10 mmol), and the mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated, and the crude product was purified by column chromatography (CHCl / methanol, 1:0 to 97:3, v / v) to yield compound 23 (2.12 g, 81%). ESI m / z: 859.2 (M+H). + .

[0431] 1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),9.48(s,1H),8.71(s,1H),8.34(s,1H),8.00(d,J=8.8Hz,2H),7.75( d,J=9.8Hz,1H),7.61-7.56(m,1H),7.45-7.41(m,1H),7.39-7.34(m,1H),7.18(d,J=8.8Hz,2H),5.31(s,2 H),5.22-5.15(m,1H),4.71-4.63(m,2H),4.49-4.44(m,1H),3.84(d,J=11.0Hz,1H),3.78-3.73(m,2H),3. 66-3.61(m,1H),3.57-3.48(m,6H),3.41(s,3H),2.88-2.82(m,4H),2.03-1.95(m,1H),1.45-1.23(m,11H).

[0432] (1S)-1-(Chloromethyl)-3-[6-(4-hydroxybenzamido)imidazo[1,2-a]pyridine-2-carbonyl]-9-methyl-1H,2H,3H-benzo[e]indol-5-yl N-[2-(2-hydroxyethoxy)ethyl]-N-[2-(methylamino)ethyl]carbamate (24) [ka] A solution of 23 (2.12 g, 2.47 mmol) in CHCl (50 mL) was cooled to 0 °C, and then TFA (25 mL) was added. The mixture was stirred at 0 °C for 1 h, diluted with CHCl (200 mL), and concentrated. The residue was dissolved in CHCl / toluene, and the mixture was concentrated to give compound 24 (1.53 g, 87%). ESI m / z: 715.2 (M+H). + .

[0433] (1R,4E,6R)-6-Hydroxy-1-methylcyclooct-4-ene-1-carboxylic acid (26) [ka] A three-neck flask (100 mL) was charged with methyl (1R,E)-6-acetoxy-1-methylcyclooct-4-ene-1-carboxylate (2 g, 1 equivalent, 8.33 mmol). To this material was added water (8 mL). A heterogeneous (oil / water) mixture was obtained. To the reaction mixture was added a solution of potassium hydroxide (0.8 g, 1.5 equivalents, 12.5 mmol) in water (8 mL). The flask containing the reaction mixture was covered with aluminum foil to avoid light exposure and stirred at room temperature for 25 hours. 1 The progress of the reaction was monitored by H-NMR analysis. The reaction mixture was extracted with TBME (3 x 100 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the undesired ester (0.87 g, 53% yield) as a colorless / slightly pale yellow oil. While cooling in an ice / water bath (T < 7 °C), the aqueous layer was acidified with 1 M HCl to pH = 2. The aqueous layer was extracted with TBME (3 x 100 mL). The combined TBME layers were washed with water (100 mL) and brine (100 mL). The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the desired acid 26 (1.02 g, 66% yield). The product was stored in the freezer. ESI m / z: 207.3 (M+H) + .

[0434] 1 H NMR(400MHz,DMSO-d6)δ5.93-5.81(m,1H),5.62-5.53(m,1H),4.66(d,J=3.1Hz,1H),4.24(s, 1H), 2.21-2.09 (m, 2H), 2.02-1.95 (m, 1H), 1.85-1.66 (m, 4H), 1.46-1.37 (m, 1H), 0.97 (s, 3H).

[0435] 2,5-Dioxopyrrolidin-1-yl(1R,4E,6R)-6-({[(2,5-dioxopyrrolidin-1-yl)oxy]carbonyl}oxy)-1-methylcyclooct-4-ene-1-carboxylate (27) [ka] A mixture of compound 26 (1.02 g, 5.54 mmol), N,N'-disuccinimidyl carbonate (DSC) (6.1 g, 23.84 mmol), and DIPEA (5.29 g, 41.00 mmol) in anhydrous acetonitrile (20 mL) was stirred at 25 °C for 24 h. LCMS showed the reaction was complete and 26 was consumed. The reaction mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase column chromatography (C18 column (40 g), eluting with 0 to 65% acetonitrile in water with 0.01% TFA for 15 min) to give compound 27 (1.90 g, 81% yield). ESI m / z: 445.1 (M+Na). + .

[0436] 1 H NMR(400MHz,DMSO-d6)δ5.94-5.78(m,2H),5.30-5.24(m,1H),2.81(d,J=4.6Hz,8H),2.39-1.85(m,8H),1.30-1.12(m,4H).

[0437] 2,5-Dioxopyrrolidin-1-yl(1R,4E,6R)-6-[({2-[({[(1S)-1-(chloromethyl)-3-[6-(4-hydroxybenzamido)imidazo[1,2-a]pyridine-2-carbonyl]-9-methyl-1H,2H,3H-benzo[e]indol-5-yl]oxy}carbonyl)[2-(2-hydroxyethoxy)ethyl]amino]ethyl}(methyl)carbamoyl)oxy]-1-methylcyclooct-4-ene-1-carboxylate (28) [ka] To a solution of compound 24 (479 mg, 0.67 mmol, 1.0 equiv.) in DMF (10 mL) was added compound 27 (340 mg, 0.81 mmol, 1.2 equiv.), followed by DIPEA (259 mg, 2.01 mmol, 3.0 equiv.). The mixture was stirred at room temperature for 1.5 hours. LCMS showed the reaction was complete and 24 was consumed. The reaction mixture was used directly in the next step without further purification. ESI m / z: 1022.4 (M+H). + .

[0438] 2-{[(1R,4E,6R)-6-[({2-[({[(1S)-1-(chloromethyl)-3-[6-(4-hydroxybenzamido)imidazo[1,2-a]pyridine-2-carbonyl]-9-methyl-1H,2H,3H-benzo[e]indol-5-yl]oxy}carbonyl)[2-(2-hydroxyethoxy)ethyl]amino]ethyl}(methyl)carbamoyl)oxy]-1-methylcyclooct-4-en-1-yl]formamido}acetic acid (Compound 112) [ka] To the reaction mixture was added a solution of glycine (502 mg, 6.70 mmol, 10.0 equiv) and DIPEA (1.73 g, 13.40 mmol, 20.0 equiv) in HO (10 mL). The resulting mixture was stirred at room temperature for 1 h. LCMS showed the reaction was complete and no intermediate 28 remained. The reaction mixture was purified by reverse-phase column chromatography (C18 column (120 g), eluting with 0-65% acetonitrile (NH4HCO3) in water for 15 min) to give compound 112 (210 mg, 32% yield). ESI m / z: 982.3 (M+H). + .

[0439] 1 H NMR(400MHz,DMSO-d6)δ10.28-10.19(m,1H),9.46(s,1H),8.70(s,1H),8.32(s,1H),7.90(d,J=8.5Hz,2H) ,7.80-7.68(m,2H),7.59(d,J=9.7Hz,1H),7.47-7.40(m,1H),7.38-7.33(m,1H),6.90(d,J=8.7Hz,2H),5.8 7-5.54(m,3H),5.22-5.07(m,2H),4.71-4.63(m,1H),4.50-4.42(m,1H),3.89-3.50(m,16H),3.08-2.94(m, 3H),2.84(s,3H),2.17-2.05(m,2H),1.88-1.70(m,3H),1.65-1.42(m,3H),1.23(s,2H),0.98-0.87(m,3H).

[0440] Example 3: Synthesis of Compound 27 [ka] General procedure for the preparation of compound 3 [ka] To a solution of compound 1 (200 mg, 490 μmol, 1.00 equiv.) in DMF (2.00 mL) was added compound 2 (248 mg, 589 μmol, 1.20 equiv.) and DIEA (253 mg, 1.96 mmol, 4.00 equiv.). The mixture was stirred at 25° C. for 16 hours. LC-MS confirmed the desired mass (MS calculated: 714.2, MS found: [M+H] + One peak (R = 715.3) t =0.38 min) was detected. The reaction mixture was added to 40 mL of isopropyl ether and filtered to give compound 3 (320 mg, yield 87.9%, purity 96.4%) as a brown solid.

[0441] LCMS:R t =0.38 min, MS calculated value: 714.2, MS observed value: [M+H] + =715.3.

[0442] HPLC:R t =2.26 minutes, purity: 96.4%

[0443] General procedure for the preparation of compound 27 [ka] To a solution of compound 3 (630 mg, 881 μmol, 1.00 equiv.) in DMF (6.00 mL), compound 4 (1.58 g, 2.21 mmol, 2.50 equiv.), DMAP (215 mg, 1.76 mmol, 2.00 equiv.), and DIEA (683 mg, 5.29 mmol, 6.00 equiv.) were added. The mixture was stirred at 25° C. for 16 hours. LC-MS (EC16907-6-P1A3) showed that 34.0% of compound 27 was formed and 62.6% of compound 3 remained. Compound 4 (1.58 g, 2.21 mmol, 2.50 equiv.) and DIEA (228 mg, 1.76 mmol, 2.00 equiv.) were then added to the solution. The mixture was stirred at 30° C. for 48 hours. LC-MS showed that 64.4% of compound 27 was formed, and 30.0% of compound 3 remained. The reaction mixture was added to 150 mL of isopropyl ether and filtered to obtain the crude product. The residue was purified by preparative HPLC (0.1% TFA condition) to obtain compound 27 (200 mg, 17.9% yield, 94.7% purity) as a pale yellow solid. LCMS R t =0.331 min, MS calculated value: 1199.6, MS observed value: [M+H] + =1200.5. HPLC:R t =1.837 minutes, purity: 94.7%.

[0444] Example 4: Synthesis of Compound 120 [ka] General procedure for the preparation of compound 3 [ka] To a solution of compound 1 (14.1 g, 54.0 mmol, 1.0 equiv.) and compound 2 (13.0 g, 54.0 mmol, 1.0 equiv.) in toluene (300 mL) and EtOH (300 mL) was added Pd(PPh3)4 (3.12 g, 2.70 mmol, 0.05 equiv.) and K2CO3 (29.8 g, 216 mmol, 4.0 equiv.). The mixture was stirred at 90 °C for 16.0 h. TLC (petroleum ether / EtOAc = 10:1, Rf = 0.50 and 0.57 for the product) showed complete consumption of compound 1 and the formation of two new spots. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with water (500 mL) and extracted with EtOAc (500 mL × 3). The combined organic layers were washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give compounds 3 and 3a (9.50 g, yield 79.5%) as yellow oils.

[0445] Note: 1.8 g of compound 3, 2.8 g of compound 3a, and 4.9 g of a mixture of compounds 3 and 3a were obtained.

[0446] Compound 3 1 H NMR(400MHz,CDCl3)δ:8.41(d,J=2.25Hz,1H),8.12(dd,J=8.57,2.31Hz,1H),8.00(d,J=8.63Hz,1H),7.43(dd,J=17 .51,11.01Hz,1H),5.82(d,J=17.13Hz,1H),5.53(d,J=11.26Hz,1H),4.42(q,J=7.13Hz,2H),1.42(t,J=7.13Hz,3H).

[0447] Compound 3a 1H NMR(400MHz,CDCl3)δ:8.40(d,J=2.13Hz,1H),8.11(dd,J=8.63,2.25Hz,1H),8.00(d,J=8.51Hz,1H ),7.42(dd,J=17.39,11.01Hz,1H),5.82(d,J=17.51Hz,1H),5.52(d,J=11.01Hz,1H),3.95(s,3H).

[0448] General procedure for the preparation of compound 4 [ka] A solution of Pd / C (220 mg, 206 μmol, 10% purity, 0.02 equiv.) in MeOH (2.00 mL) was purged with N three times. Compound 3 (1.8 g, 8.13 mmol, 1.0 equiv.) in MeOH (25 mL) was then slowly added and purged with H three times. The reaction mixture was stirred at 25 °C for 4.0 h under an H atmosphere (15 psi). TLC (petroleum ether / EtOAc = 10:1, R of the product) showed no significant difference. f =0.15), which indicated that compound 3 was completely consumed and one new spot was formed. The reaction was clean by TLC. The reaction mixture was filtered and the solvent was removed by rotary evaporation to give compound 4 (1.50 g, crude) as a yellow solid.

[0449] 1 H NMR(400MHz,CDCl3)δ:7.80(d,J=8.13Hz,1H),6.45-6.50(m,2H),4.29(q,J=7.13Hz,2H), 3.60-3.95(m,2H),2.94(q,J=7.42Hz,2H),1.36(t,J=7.13Hz,3H),1.21(t,J=7.44Hz,3H).

[0450] General procedure for the preparation of compound 5 [ka] To a solution of ethyl compound 4 (1.50 g, 7.76 mmol, 1.0 equiv.) in THF (30.0 mL) was added LiAlH (2.5 M, 9.31 mL, 3.0 equiv.) under a N atmosphere at 0 °C. The mixture was then stirred at 25 °C for 4.0 h. TLC (petroleum ether / EtOAc = 1:1, product Rf = 0.20) showed that compound 4 was completely consumed and one new spot was formed. The reaction mixture was cooled to 0 °C and worked up by adding water (900 μL), 15% NaOH solution (900 μL), and water (2.70 mL) again under a N atmosphere. The resulting slurry was stirred at 25 °C for 15 min, dried over NaSO, filtered, and washed with THF (50.0 mL). The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 0 / 1) to give compound 5 (0.60 g, 3.97 mmol, 48.8% yield in two steps, 90.0% purity) as a yellow oil.

[0451] 1 H NMR(400MHz, CDCl3)δ:7.06-7.13(m,1H),6.55-6.59(m,1H),6.51(br d,J=7.75Hz,1H),4.59(s,2H),2.67(q,J=7.67Hz,2H),1.22(t,J=7.57Hz,3H).

[0452] General procedure for the preparation of compound 6-3 [ka] To a solution of compound 6-1 (4.00 g, 21.7 mmol, 1.0 equiv.) and compound 6-2 (5.45 g, 43.4 mmol, HCl salt, 2.0 equiv.) in DCM (40.0 mL) was added DIEA (11.2 g, 86.8 mmol, 15.1 mL, 4.0 equiv.), HOBt (7.33 g, 54.2 mmol, 2.5 equiv.), and EDCI (10.4 g, 54.2 mmol, 2.5 equiv.). The mixture was stirred at 25 °C for 2.0 h. TLC (petroleum ether / EtOAc = 1:1, R of the product) showed no significant difference. f=0.20), indicating the complete consumption of compound 6-1 and the formation of one new spot. The reaction mixture was partitioned between HCl solution (1.0 M, 100 mL) and DCM (100 mL). The organic phase was separated, washed with saturated NaHCO3 (100 mL) and brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 6-3 (3.20 g, crude product) as a yellow oil.

[0453] 1 H NMR(400MHz,CDCl3)δ:6.08-6.15(m,1H),6.00-6.08(m,1H),5.63(dd,J=16.57,2.31Hz,1H),4.45(br s,1H),3.96(d,J=5.25Hz,2H),3.73(s,3H),2.20-2.33(m,2H),2.05-2.12(m,2H), 1.87-1.99(m,2H),1.78-1.86(m,2H),1.57(dd,J=15.51,6.25Hz,1H),1.11(s,3H).

[0454] General procedure for the preparation of compound 6 [ka] To a solution of compound 6-3 (3.2 g, 12.5 mmol, 1.0 equiv.) in DMF (30.0 mL) were added bis(4-nitrophenyl)carbonate (7.63 g, 25.0 mmol, 2.0 equiv.) and DIEA (4.86 g, 37.6 mmol, 6.55 mL, 3.0 equiv.). The mixture was stirred at 25 °C for 2.0 h. TLC (petroleum ether / EtOAc = 1:2, product Rf = 0.20) showed that compound 6-3 was completely consumed and one new spot was formed. The reaction mixture was quenched with saturated NH4Cl (50 mL) and extracted with EtOAc (50.0 mL × 3). The combined organic layers were washed with brine (50 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 = 100 / 1 to 0 / 1) to give compound 6 (3.60 g, 8.56 mmol, 39.4% yield in two steps, 98.6% purity) as a yellow oil.

[0455] LCMS:R t =0.41 min, MS calculated value: 420.1, MS observed value: [M+Na] + =443.1.

[0456] HPLC:R t =2.55 minutes, purity: 98.6%.

[0457] 1 H NMR(400MHz,CDCl3)δ:7.26-7.30(m,2H),7.19-7.24(m,1H),6.99(br s,1H),6.10(br t,J=4.69Hz,1H),5.89-5.99(m,1H),5.64(dd,J=16.63,2.38Hz,1H),5.26(br s,1H),4.66(s,2H),3.98(dd,J=5.00,2.25Hz,2H),3.75(s,3H),2.69(q,J=7.50Hz,2H),2. 22-2.32(m,2H),2.06-2.15(m,2H),1.82-1.99(m,4H),1.22(t,J=7.57Hz,3H),1.16(s,3H).

[0458] General procedure for the preparation of compound 7 [ka] To a solution of compound 5 (1.17 g, 7.73 mmol, 1.3 equiv.) and compound 6 (2.50 g, 5.95 mmol, 1.0 equiv.) in DMF (25.0 mL) was added HOAt (1.21 g, 8.92 mmol, 1.25 mL, 1.5 equiv.) and DIEA (1.54 g, 11.8 mmol, 2.07 mL, 2.0 equiv.). The mixture was stirred at 50° C. for 2.0 hours. LC-MS showed that compound 6 was completely consumed and the desired mass (MS calculated: 432.2, MS found: [M+Na]) was obtained. + One main peak (R = 455.1) t =0.37 min). The reaction mixture was quenched with saturated NH4Cl (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 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 = 100 / 1 to 0 / 1) to give compound 7 (2.20 g, 4.86 mmol, 81.7% yield, 95.6% purity) as a yellow oil.

[0459] LCMS:R t =0.38 min, MS calculated value: 432.2, MS observed value: [M+Na] + =455.2.

[0460] HPLC:R t =2.06 minutes, purity: 95.6%.

[0461] General procedure for the preparation of compound 8 [ka] To a solution of compound 7 (2.20 g, 5.09 mmol, 1 equiv.) in DMF (20.0 mL) was added bis(4-nitrophenyl)carbonate (4.64 g, 15.2 mmol, 3 equiv.) and DIEA (1.97 g, 15.26 mmol, 2.66 mL, 3 equiv.). The mixture was stirred at 25° C. for 2.0 h. LC-MS showed that compound 7 was completely consumed and the desired mass (MS calculated: 597.2, MS found: [M+Na] + One main peak (R = 620.3) t =0.49 min). The reaction mixture was extracted with water and EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 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 = 100 / 1 to 0 / 1) to give compound 8 (2.60 g, 2.82 mmol, yield 55.4%, purity 64.8%) as a yellow oil.

[0462] LCMS:R t =0.49 min, MS calculated value: 597.2, MS observed value: [M+Na] + =620.2.

[0463] HPLC:R t =3.30 minutes, purity: 64.8%.

[0464] General procedure for the preparation of compound 9 [ka] To a solution of compound 8 (720 mg, 1.21 mmol, 1.0 equiv) and MMAE (517 mg, 722 μmol, 0.6 equiv) in DMF (7.00 mL) was added HOAt (245 mg, 1.81 mmol, 252 μL, 1.5 equiv) and DIEA (311 mg, 2.40 mmol, 419 μL, 2.0 equiv). The mixture was stirred at 25° C. for 4.0 h. LC-MS showed that compound 8 was completely consumed and had the desired mass (MS calculated: 1175.7, MS found: [M+H]). + One peak (R = 1176.8)t =0.52 min) was detected. The reaction mixture was purified by preparative HPLC (TFA conditions) to give compound 9 (300 mg, 255 μmol, yield 19.7%, purity 99.2%) as a white solid.

[0465] LCMS:R t =0.52 min, MS calculated value: 1175.7, MS observed value: [M+H] + =1176.8.

[0466] HPLC:R t =2.38 minutes, purity: 99.2%.

[0467] General procedure for the preparation of compound 120 [ka] To a solution of compound 9 (400 mg, 339 μmol, 1.0 equiv.) in THF (4.00 mL) was added LiOH·HO (1.0 M, 1.36 mL, 4.0 equiv.). The mixture was stirred at 0 °C for 2.0 h. LC-MS showed that compound 9 was completely consumed and the desired mass (MS calculated: 1161.6, MS found: [M+Na] + One peak (R = 1184.7) t =0.51 min). The reaction mixture was acidified with 10% aqueous TFA while stirring at 0 °C and then concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TFA conditions) to give compound 120 (323 mg, 278 μmol, yield 82.0%, purity 99.8%, TFA salt) as a white solid.

[0468] LCMS:R t =0.36 min, MS calculated value: 1161.6, MS observed value: [M+Na] + =1184.8.

[0469] HPLC:R t =2.76 minutes, purity: 99.8%.

[0470] Example 5: Synthesis of Compound 33 [ka] General procedure for the preparation of compound 2 [ka] To a solution of triphosgene (25.8 g, 87.2 mmol, 1.45 equiv.) in THF (150 mL) was added pyridine (6.47 g, 81.7 mmol, 6.60 mL, 1.36 equiv.) under a N2 atmosphere at 0°C. After the addition, compound 1 (10.0 g, 60.1 mmol, 8.54 mL, 1.00 equiv.) in THF (150 mL) was added dropwise at 0°C. The mixture was stirred at 0°C for 2 hours. TLC (petroleum ether:ethyl acetate=3:1) confirmed the identity of compound 1 (R f =0.35) was completely consumed, and many new spots (R f =0.01, R f =0.3, R f =0.59, R f =0.75, R f =0.85). The reaction mixture was diluted with 1.0 M HCl (150 mL) at 0 °C and extracted with DCM (150 mL × 3). The combined organic layers were washed with brine (150 mL × 2) and dried over Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1, petroleum ether:ethyl acetate = 3:1, R f =0.75, by plate 2) to give compound 2 (6.10 g, crude) as a yellow oil which was used without further purification.

[0471] General Procedure for the Preparation of Compound 1-1 [ka] To a solution of compound 1A (7.10 g, 53.3 mmol, 1.00 equiv.) and BocO (17.5 g, 80.0 mmol, 18.4 mL, 1.50 equiv.) in EtOH (112 mL) was added Raney nickel (710 mg, 8.29 mmol) under a N atmosphere. The suspension was degassed and purged with H three times. The mixture was stirred under H (50 Psi) at 25 °C for 16 h. LCMS showed that compound 1A was completely consumed and the desired mass was detected. (R t =0.23 min). The reaction mixture was filtered and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, petroleum ether:ethyl acetate = 1:1, R f =0.30). HNMR and HPLC showed that compound 1-1 (8.20 g, 32.0 mmol, yield 60.0%, 92.6%) was a white solid.

[0472] LCMS:R t =0.22 min, MS calculated value: 237.2, MS observed value: [M+H] + =238.0.

[0473] HPLC:R t =0.78 min, purity: 92.6%. 1 H NMR(400MHz,CDCl3)δ:ppm8.10(dd,J=5.1,1.8Hz,1H),7.18(dd,J=7.1,1.3Hz,1H),6.47(dd,J=7.0,5.3Hz,1H),5.75(br s,1H),4.79(br s,1H),4.17(d,J=6.5Hz,2H),3.00(d,J=4.8Hz,3H),1.46(s,9H).

[0474] General procedure for the preparation of compounds 1-2 [ka] To a solution of compound 1-1 (1.30 g, 5.07 mmol, 1.00 equiv.) in THF (40 mL) was added CsCO (1.98 g, 6.09 mmol, 1.20 equiv.) and compound 2 (5.80 g, 25.3 mmol, 5.00 equiv.). The mixture was stirred at 50 °C for 3 h. LCMS showed that compound 1-1 remained (R t =0.29 min). LCMS showed several new peaks and the desired mass was detected (R t =0.50 min, MS calculated: 429.1, MS observed: [M+H] + =452.1). The reaction mixture was diluted with ice water (60.0 mL) and extracted with DCM (80.0 mL × 3). The combined organic layers were washed with brine (150 mL × 2) and dried over Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1) (petroleum ether:ethyl acetate = 2:1, R f Compound 1-2 (1.39 g, 3.19 mmol, 62.9% yield, 98.5% purity) was obtained as a yellow oil and purified by HNMR, LCMS (R t =0.48 min), and HPLC (R t = 3.46 min).

[0475] LCMS:R t =0.48 min, MS calculated value: 429.1, MS observed value: [M+H] + =452.2.

[0476] HPLC:R t =3.46 minutes, purity: 98.5%.

[0477] 1 H NMR(400MHz,DMSO-d6)δ:ppm8.38-8.36(m,1H),7.72(d,J=6Hz,1H),7.40-7.34 (m,7H),5.16(s,2H),4.80-5.54(m,2H),4.12(s,2H),3.18(s,3H),1.38(s,9H).

[0478] General procedure for the preparation of compounds 1-3 [ka] A 50 mL round-bottom flask was purged with Ar three times, and Pd / C (120 mg, 113 μmol, 10% purity) was carefully added. THF (4.00 mL) was then added to completely infiltrate the Pd / C, followed by the slow addition of compound 1-2 (1.20 g, 2.79 mmol, 1.00 equiv.) in THF (8.00 mL) under an Ar atmosphere. The resulting mixture was degassed and purged with H three times, after which the mixture was stirred at 25 °C for 2 h under an H atmosphere (15 psi). LCMS showed that compound 1-2 was completely consumed (MS calculated: 339.1, MS found: [M+Na]). + =362.0). (R t =0.348 min). The reaction mixture was dissolved in THF (10.0 mL), filtered through diatomaceous earth, the cake was washed with THF (10.0 mL × 2), and the filtrate was concentrated under reduced pressure. The crude product was used in the next step without further purification. Compound 1-3 (1.00 g, crude product) was obtained as a colorless oil by LCMS (R t =0.34 min), HNMR, and HPLC (R t = 1.43 min).

[0479] LCMS:R t =0.34 min, MS calculated value: 339.1, MS observed value: [M+Na] + =362.0.

[0480] HPLC:R t =1.43 minutes, purity: 96.2%.

[0481] 1 H NMR(400MHz,DMSO-d6)δ:ppm8.37(br d,J=3.4Hz,1H),7.72(br s,1H),7.23-7.50(m,2H),4.33-4.67(m,2H),3.98-4.21(m,2H),3.14-3.24(m,3H),1.30-1.45(m,9H).

[0482] General procedure for the preparation of compounds 1-4 [ka] To a solution of compound 1-3 (766 mg, 2.26 mmol, 1.20 equiv.) in DMF (10.0 mL) was added HOBt (305 mg, 2.26 mmol, 1.20 equiv.), DIEA (486 mg, 3.76 mmol, 655 μL, 2.00 equiv.), exatecan (1.00 g, 1.88 mmol, 1.00 equiv.), and EDCI (541 mg, 2.82 mmol, 1.50 equiv.). The mixture was stirred at 25° C. for 2 hours. LCMS showed that compound 1-3 was completely consumed. LCMS showed several new peaks, and the desired mass was detected (R t =0.47 min, MS calculated value: 756.2, MS observed value: [M+Na] + =757.3). The reaction mixture was added to isopropyl ether (100 mL x 2) to precipitate the crude product, which was then centrifuged to obtain the crude product as a residue. The liquid supernatant was discarded. The crude product was used in the next step without further purification. Compound 1-4 (2.0 g, crude product) was obtained as a brown solid, which was analyzed by LCMS (R t =0.47 min) and HPLC (R t = 2.84 min).

[0483] LCMS:R t =0.47 min, MS calculated value: 756.2, MS observed value: [M+Na] + =757.3

[0484] HPLC:R t =2.84 min, purity: 84.3%

[0485] General Procedure for the Preparation of Compounds 1-5 [ka] To a solution of compound 1-4 (2.00 g, 2.64 mmol, 1.00 equiv.) in DCM (10.0 mL) was added TFA (15.3 g, 135 mmol, 10 mL, 50.9 equiv.). The mixture was stirred at 0-25°C for 1 h. LCMS showed that compound 1-4 was completely consumed. LCMS showed several new peaks, and the desired mass was detected (R t =0.34 min, MS calculated: 656.2, MS observed: [M+H] + =657.3). The reaction mixture was added to isopropyl ether (200 mL x 2) to precipitate the crude product, which was then centrifuged to obtain the crude product as a residue. The liquid supernatant was discarded. The crude product was used in the next step without further purification. Compound 1-5 (2.00 g, crude product, TFA salt) was obtained as a gray solid, and was analyzed by LCMS (R t =0.35 min) and HPLC (R t = 1.64 min).

[0486] LCMS:R t =0.35 min, 656.2, MS observed value: [M+Na] + =657.3

[0487] HPLC:R t =2.84 minutes, purity: 92.7%.

[0488] General procedure for the preparation of compounds 1-7 [ka] To a solution of compound 1-5 (1.50 g, 1.95 mmol, 1.00 equiv, TFA) in DMF (15.0 mL) was added N-methylmorpholine (394 mg, 3.89 mmol, 428 μL, 2.00 equiv) and compound 1-6 (411 mg, 973 μmol, 0.50 equiv). The mixture was stirred at 0-5°C for 4 h. LCMS showed that compound 1-5 was completely consumed. LCMS showed several new peaks, and the desired mass was detected (R t =0.49 min, MS calculated: 963.3, MS observed: [M+Na] +=964.5). The reaction mixture was added to isopropyl ether (150 mL x 2) to precipitate the crude product, which was then centrifuged to obtain the crude product as a residue. The liquid supernatant was discarded. The residue was purified by preparative HPLC (TFA conditions). Compound 1-7 (330 mg, 338 μmol, yield 17.40%, purity 98.9%) was obtained as a yellow solid. LCMS (R t =0.47 min) and HPLC (R t =2.88 min) confirmed the desired product.

[0489] LCMS:R t =0.47 min, MS calculated value: 963.3, MS observed value: [M+H] + =964.5.

[0490] HPLC:R t =2.88 minutes, purity: 98.9%.

[0491] General procedure for the preparation of compound 33 [ka] To a solution of compound 1-7 (350 mg, 363 μmol, 1.00 equiv.) and NH2-BiPEG3-OH in DMSO (3.50 mL) was added DIEA (187 mg, 1.45 mmol, 253 μL, 4.00 equiv.) and DMAP (4.44 mg, 36 μmol, 0.10 equiv.). The mixture was stirred at 25 °C for 16 h. LCMS showed that compound 1-7 was completely consumed. LCMS showed several new peaks and the desired mass was detected (R t =0.39 min, MS calculated value: 1448.6, MS observed value: [M+Na] + =1450.0). The reaction filtrate was used directly without workup. The residue was purified by preparative HPLC (0.25% AcOH condition). Target 245 (310 mg, 205.35 μmol, 56.5% yield, 100% purity, AcOH salt) was obtained as a white solid, which was analyzed by LCMS (R t =0.39 min) and HPLC (R t = 2.26 min).

[0492] LCMS:R t =0.39 min, MS calculated value: 1448.6, MS observed value: [M+H] + =1449.9.

[0493] HPLC:R t =2.26 minutes, purity: 100%.

[0494] Example 6: Synthesis of Compound 34 [ka] General procedure for the preparation of compound 2-2 [ka] To a solution of exatecan (5.00 g, 9.41 mmol, 1.00 equiv.) and compound 2-1 (1.62 g, 12.2 mmol, 1.30 equiv.) in DMF (50.0 mL) was added DMAP (574 mg, 4.70 mmol, 0.50 equiv.), TEA (1.43 g, 14.1 mmol, 1.96 mL, 1.50 equiv.), and EDCI (3.61 g, 18.8 mmol, 2.00 equiv.). The mixture was stirred at 25 °C for 2 hours. LCMS showed complete consumption of exatecan. LCMS showed several new peaks, and the desired mass was detected (R t =0.43 min, MS calculated value: 549.2, MS observed value: [M+H] + =550.2). The reaction mixture was added with H2O (150 mL) and extracted with DCM (50.0 mL x 3). The combined organic layers were washed with brine (80.0 mL), dried over Na2SO4, filtered, and concentrated to give a residue. The reaction mixture was poured into isopropyl ether (210 mL) and triturated with petroleum ether (210 mL) at 25 °C for 3 hours. Compound 2 (5.10 g, 8.89 mmol, 94.5% yield, 95.8% purity) was obtained as a gray solid by LCMS (R t =0.44 min), HPLC(R t = 2.79 min).

[0495] LCMS:R t=0.44 min, MS calculated value: 549.2, MS observed value: [M+H] + =550.2.

[0496] HPLC:R t =2.79 minutes, purity: 95.8%

[0497] General procedure for the preparation of compounds 2-3 [ka] A solution of compound 2-2 (5.00 g, 8.72 mmol, 1.00 equiv.) in DCM (25 mL) was added with TFA (38.3 g, 336 mmol, 25.0 mL, 38.6 equiv.) and stirred at 0° C. for 1 h. The mixture was stirred at 25° C. for 5 h. LCMS showed that compound 2-2 was completely consumed. LCMS showed several new peaks and the desired mass was detected (R t =0.38 min, MS calculated value: 493.1, MS observed value: [M+H] + =494.2). The reaction mixture was poured into isopropyl ether (500 mL) and triturated with petroleum ether (150 mL) at 25° C. for 2 hours. Compound 3 (5.00 g, 8.07 mmol, 92.5% yield, 98.0% purity) was obtained as a yellow solid by LCMS (R t =0.35 min) and HPLC (R t = 1.92 min).

[0498] LCMS:R t =0.35 min, MS calculated: 549.2, MS observed: [M+H] + =494.1.

[0499] HPLC:R t =1.92 minutes, purity: 98.0%

[0500] General procedure for the preparation of compounds 2-5 [ka] To a solution of compound 2-3 (500 mg, 806.5 μmol, 1.00 equiv., TFA) in DMF (5.00 mL) was added compound 4 (395 mg, 1.45 mmol, 1.80 equiv.), HOBt (218 mg, 1.61 mmol, 2.00 equiv.), and DMAP (98.5 mg, 806 μmol, 1.00 equiv.) and DIC (203 mg, 1.61 mmol, 249 μL, 2.00 equiv.). The mixture was stirred at 25° C. for 12 hours. LCMS showed that compound 2-3 was completely consumed. LCMS showed several new peaks and the desired mass was detected (R t =0.43 min, MS calculated value: 747.2, MS observed value: [M+H] + =748.3). The reaction mixture was poured into isopropyl ether (100 mL x 2) and crystallized. The crude product, compound 2-5 (2.00 g, crude product), was obtained as a gray solid and used in the next step.

[0501] LCMS:R t =0.43 min, MS calculated value: 747.2, MS observed value: [M+H] + =748.3

[0502] HPLC:R t =2.73 minutes, purity: 83.0%

[0503] General procedure for the preparation of compounds 2-6 [ka] To a solution of compound 2-5 (2.00 g, 2.67 mmol, 1.00 equiv) in DCM (4.00 mL) was added formic acid (19.5 g, 424 mmol, 16.0 mL). The mixture was stirred at 25° C. for 8 hours. LCMS showed that compound 2-5 remained (R t =0.42 min). LCMS showed several new peaks and the desired mass was detected (R t =0.32 min, MS calculated value: 647.2, MS observed value: [M+H] +=648.2). The reaction mixture was poured into isopropyl ether (200 mL) and crystallized. The residue was purified by preparative HPLC (TFA conditions). Compound 2-6 (556 mg, 849 μmol, yield 31.7%, purity 98.9%) was obtained as a yellow solid, and LCMS (R t =0.32 min) and HPLC (R t = 1.60 min).

[0504] LCMS:R t =0.32 min, MS calculated value: 647.2, MS observed value: [M+H] + =648.2.

[0505] HPLC:R t =1.60 minutes, purity: 98.9%

[0506] General procedure for the preparation of compounds 2-7 [ka] To a solution of compound 2-6 (556 mg, 849 μmol, 1.00 equiv.) in DMF (6.00 mL) was added N-methylmorpholine (42.9 mg, 424 μmol, 46.6 μL, 0.50 equiv.) and compound 7 (437 mg, 1.02 mmol, 1.20 equiv.). The mixture was stirred at 0° C. for 36 hours. LCMS showed that compound 2-6 remained (R t =0.33 min). LCMS showed several new peaks and the desired mass was detected (R t =0.45 min, MS calculated: 954.3, MS observed: [M+H] + =955.4). The reaction mixture was poured into isopropyl ether (60.0 mL × 1) and crystallized. The residue was purified by preparative HPLC (TFA conditions). Compound 2-7 (445 mg, 454.8 μmol, yield 53.5%, purity 97.6%) was obtained as a yellow solid, and LCMS (R t =0.47 min) and HPLC (R t = 2.79 min).

[0507] LCMS:R t=0.47 min, MS calculated value: 954.3, MS observed value: [M+H] + =955.3.

[0508] HPLC:R t =2.79 minutes, purity: 97.6%.

[0509] General procedure for the preparation of compound 34 [ka] To a solution of NH-BiPEG-OH (386 mg, 613 μmol, 2.00 equiv.) in DMSO (3.00 mL), DIEA (79.2 mg, 613 μmol, 107 μL, 2.00 equiv.) and DMAP (3.75 mg, 30.6 μmol, 0.10 equiv.) was added compound 2-7 (300 mg, 306 μmol, 1.0 equiv.). The mixture was stirred at 25° C. for 12 hours. LCMS showed that compound 8 remained (R t =0.46 min). LCMS showed several new peaks and the desired mass was detected (R t =0.39 min, MS calculated value: 1439.6, MS observed value: [M+H] + =1440.7). The residue was purified by preparative HPLC (0.25% AcOH condition). Compound 34 (151 mg, 104 μmol, yield 34.0%, purity 99.6%) was obtained as a white solid. t =0.40 min) and HPLC (R t = 2.23 min).

[0510] LCMS:R t =0.40 min, MS calculated: 1439.6, MS observed: [M+H] + =1440.6.

[0511] HPLC:R t =2.23 minutes, purity: 99.6%.

[0512] Example 7: Synthesis of Compound 121 [ka] General procedure for the preparation of compound 3 [ka] To a solution of compound 1 (500 mg, 1.06 mmol) in DCM (5.0 mL) was added compound 2 (374 mg, 3.17 mmol), DIEA (272 mg, 2.11 mmol, 367 μL), EDCI (303 mg, 1.58 mmol), and DMAP (193 mg, 1.58 mmol). The mixture was stirred at 20-25 °C for 2 h. LCMS showed that compound 1 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was partitioned between DCM (10.0 mL) and HCl (1 M) (10.0 mL). The organic layer was separated, washed with NaHCO (10.0 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give compound 3 (550 mg, 90.2% yield) as a yellow oil.

[0513] LCMS:R t =0.474 min, MS calculated value: 573.9, MS observed value: [M+H] + =574.4.

[0514] HPLC:R t= 3.389 minutes, purity: 99.4%.

[0515] General procedure for the preparation of compound 6 [ka] To a solution of compound 4 (2.00 g, 10.8 mmol) in DCM (20.0 mL), compound 5 (2.73 g, 21.7 mmol, HCl), DIEA (5.61 g, 43.4 mmol, 7.56 mL), HOBt (3.67 g, 27.1 mmol), and EDCI (5.20 g, 27.1 mmol) were added. The mixture was stirred at 20-25 °C for 2 h. TLC (petroleum ether / EtOAc = 1:1, R of the product) revealed a 5% methyl ... f=0.32), indicating the complete consumption of compound 4 and the formation of one new spot. The reaction was clean by TLC. The reaction mixture was partitioned between HCl (1 M) (50.0 mL) and DCM (50.0 mL). The organic layer was separated, washed with NaHCO3 (50.0 mL) and brine (50.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 6 (2.00 g, 71.1% yield, crude product) as a white solid.

[0516] 1 H NMR(400MHz,CDCl3)δ:5.98-6.15(m,2H),5.65(br d,J=16.5Hz,1H),4.48(br s,1H),3.98(d,J=5.1Hz,2H),3.75(s,3H),2.23-2.38(m,2H),1.82-2.15(m,6H),1.59(br dd,J=15.5,6.1Hz,1H),1.13(s,3H).

[0517] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (2.00 g, 7.83 mmol) in DMF (20 mL), bis(4-nitrophenyl)carbonate (4.77 g, 15.6 mmol) and DIEA (3.04 g, 23.5 mmol, 4.09 mL) were added. The mixture was stirred at 20-25 °C for 16 h. TLC (petroleum ether / EtOAc = 1:1, R of the product) revealed a 50% methyl ... f =0.63), indicating that compound 6 was completely consumed and one new spot was formed. The reaction mixture was partitioned between HCl (1 M) (50.0 mL) and EtOAc (50.0 mL). The organic phase was separated, washed with brine (50.0 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / EtOAc = 10 / 1 to 1 / 1) to give compound 7 (3.00 g, 90.0% yield) as a yellow oil. LCMS: R t=0.422 min, MS calculated value: 420.4, MS observed value: [M+H] + =443.0. HPLC:R t =2.826 minutes, purity: 98.9%. 1 H NMR(400MHz,CDCl3)δ:8.29(br d,J=9.1Hz,2H),7.41(br d,J=9.1Hz,2H),5.99-6.15(m,2H),5.66(dd,J=16.6,1.8Hz,1H),4.00(dd,J=4.9,1.6 Hz,2H),3.77(s,3H),2.28-2.43(m,2H),2.16-2.25(m,2H),1.89-2.04(m,3H),1.76(br dd,J=14.9,6.3Hz,1H),1.19(s,3H).

[0518] General procedure for the preparation of compound 8 [ka] To a solution of compound 3 (280 mg, 407 μmol) in DMF (3.00 mL) was added DIEA (210 mg, 1.63 mmol, 283 μL), compound 7 (342 mg, 814 μmol), and HOAt (110 mg, 814 μmol). The mixture was stirred at 40 °C for 16 h. LCMS showed 28.7% compound 3 remaining. LC-MS showed several new peaks, with 22.7% of the desired compound detected. Three parallel reactions were combined for workup. The mixture was purified by preparative HPLC (0.1% HCOOH) to give compound 8 (450 mg, 41.5% yield) as a white solid.

[0519] LCMS:R t =0.626 min, MS calculated value: 855.2, MS observed value: [M+H] + =855.6.

[0520] HPLC:R t =5.081 minutes, purity: 96.5%.

[0521] General procedure for the preparation of compound 121 [ka] To a solution of compound 8 (450 mg, 526 μmol) in THF (3.0 mL) was added LiOH·HO (110 mg, 2.63 mmol) in HO (3.0 mL). The mixture was stirred at 20-25 °C for 2 h. TBAF (1 M, 2.10 mL) was added to the mixture, and the mixture was stirred for 16 h. LCMS showed that compound 8 was completely consumed, and one major peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (0.1% AcOH) to give compound 121 (200 mg, 44.9% yield) as a colorless oil. LCMS: R t =0.487 min, MS calculated value: 740.9, MS observed value: [M+H] + =741.4. HPLC:R t =3.282 minutes, purity: 99.5%.

[0522] Example 8: Synthesis of Compound 116 [ka] General procedure for the preparation of compound 12 [ka] To a solution of 116a (80 mg, 191 μmol, 1.0 equiv.) in DMF (1.0 mL) was added compound 11 (118 mg, 248 μmol, 1.3 equiv.) and DIEA (55.8 mg, 382 μmol, 2.0 equiv.). The mixture was stirred at 25° C. for 6.0 h. LCMS showed complete consumption of 116a and the desired mass (MS calculated: 723.3, MS found: [M+H] + One main peak (R = 724.4) t =0.41 min) was detected. The reaction mixture was triturated with isopropyl ether (10 mL) to give compound 12 (117 mg, crude product) as a white solid, which was confirmed by LCMS.

[0523] LCMS:R t =0.41 min, MS calculated value: 723.3, MS observed value: [M+H]+ =724.4.

[0524] General procedure for the preparation of compound 116 [ka] To a solution of compound 12 (117 mg, 207 μmol, 1.0 equiv.) in DMF (1.0 mL) and HO (0.2 mL) was added 12A (77.8 mg, 1.04 mmol, 5.0 equiv.), DMAP (75.9 mg, 622 μmol, 3.0 equiv.), and DIEA (1334 mg, 1.04 mmol, 5.0 equiv.). The mixture was stirred at 25° C. for 2.0 h. LCMS showed that compound 12 was completely consumed, with one major peak (Rt = 0.37 min) having the desired mass (MS calculated: 683.3, MS found: [M+H] = 684.4). The reaction mixture was filtered and purified by preparative HPLC (TFA conditions), followed by exchange to the AcOH salt using an ion exchange resin to give compound 116 (42 mg, 58.9 μmol, 28.4% yield, 95.9% purity) as a white solid, which was confirmed by LCMS and HPLC.

[0525] LCMS:R t =0.37 min, MS calculated value: 683.3, MS observed value: [M+H] + =684.4.

[0526] HPLC:R t =1.99 minutes, purity: 95.9%.

[0527] Example 9: Synthesis of Compound 115 [ka] General procedure for the preparation of compounds 1-3 [ka] To a solution of compound 1-2 (116 mg, 235 μmol, 0.50 equiv., HCl salt) in DMF (3.00 mL) was added DIEA (121 mg, 940 μmol, 163 μL, 2.0 equiv.), powdered molecular sieves, and compound 1-1 (200 mg, 470 μmol, 1.0 equiv.). The mixture was stirred at 25° C. for 12 hours. LCMS showed that compound 1-2 was consumed (R t =0.46 min), the desired mass was detected (R t =0.47 min). The reaction mixture was filtered to remove the powdered molecular sieves. The filtrate was triturated with isopropyl ether (60 mL) to give compound 1-3 (400 mg, crude product) as a yellow oil.

[0528] LCMS:R t =0.473 min, MS calculated value: 739.3, MS observed value: [M+H] + =740.4.

[0529] General procedure for the preparation of compound 115 [ka] To a solution of compound 1-3 (400 mg, 540 μmol, 1.0 equiv.) in THF (4.0 mL) was added LiOH·HO (45.3 mg, 1.08 mmol, 2.0 equiv.) in HO (1.0 mL) at 0° C. The mixture was stirred at 0° C. for 2.0 h. LCMS showed that compound 1-3 was completely consumed and the desired mass (R t =0.45 min). The reaction mixture was adjusted to pH 6 with AcOH and then purified by preparative HPLC (AcOH conditions) to give compound 115 (40.0 mg, 48.5 μmol, 9.43% yield, 95.3% purity, AcOH salt) as a pale yellow solid, which was analyzed by LCMS (R t =0.446 min) and HPLC (R t = 3.21 min).

[0530] LCMS:R t =0.45 min, MS calculated: 725.3, MS observed: [M+H] + =726.4.

[0531] HPLC:R t =3.21 minutes, purity: 95.3%.

[0532] Example 10: Synthesis of Compound 118 [ka] General procedure for the preparation of compounds 2-3 [ka] To a solution of compound 11 (209 mg, 488 μmol, 1.0 equiv.) and compound 2-2 (170 mg, 488 μmol, 1.0 equiv., HCl) in DMF (2.00 mL) was added DIEA (126 mg, 977 μmol, 170 μL, 2.0 equiv.). The mixture was stirred at 25° C. for 2.0 hours. By LCMS, compound 2-2 was consumed and the desired mass was detected (R t =0.39 min). The reaction mixture was triturated with isopropyl ether (20.0 mL) and concentrated under reduced pressure to give compound 2-3 as a yellow oil, which was identified by LCMS (R t =0.39 min).

[0533] LCMS:R t = 0.391 min, MS calculated: 618.3, MS observed: [M+H]+ = 619.4.

[0534] General procedure for the preparation of compound 118 [ka] To a solution of compound 2-3 (300 mg, 480 μmol, 1.0 equiv.) in DMF (2.40 mL) and HO (0.60 mL) was added compound 12A (216 mg, 2.89 mmol, 6.0 equiv.), DIEA (62.1 mg, 480 μmol, 83 μL, 1.0 equiv.), and DMAP (58.7 mg, 480 μmol, 1.0 equiv.). The mixture was stirred at 25° C. for 1.0 h. LCMS revealed that compound 2-3 remained (R t =0.41 min), the desired mass was detected (Rt =0.37 min). The reaction solution was purified by reverse-phase HPLC (AcOH condition) to give target 187 (120 mg, 181 μmol, yield 37.6%, purity 96.5%, AcOH salt) as a white solid, which was analyzed by LCMS, HPLC, and 1 Confirmed by 1 H NMR.

[0535] LCMS:R t = 0.368 min, MS calculated: 578.3, MS observed: [M+H]+ = 579.4.

[0536] HPLC:R t =1.82 minutes, purity: 96.5%.

[0537] 1 H NMR(400MHz,DMSO-d6):δ:7.99(d,J=8.38Hz,1H),7.60(d,J=8.25Hz,1H),7. 37-7.50(m,2H),7.11-7.27(m,2H),6.48-6.59(m,2H),5.56-5.79(m,2H),4.9 4-5.07(m,1H),4.44-4.55(m,2H),3.57(d,J=5.00Hz,2H),3.00-3.07(m,2H), 2.88-2.94 (m, 2H), 2.09-2.18 (m, 2H), 1.33-1.86 (m, 14H), 0.90-1.01 (m, 6H).

[0538] Example 11: Synthesis of Compound 114 [ka] General procedure for the preparation of compound 29 [ka] To a solution of compound 11 (100 mg, 236 μmol, 1.1 equiv.) and target 185 (123 mg, 214 μmol, 1.0 equiv., TFA salt) in DMF (2 mL) was added DIEA (55.3 mg, 428 μmol, 74.5 μL, 2.0 equiv.). The mixture was stirred at 25° C. for 1.0 h. LC-MS analysis indicated complete consumption of target 185 and the desired mass (R t =0.37 min) was detected. The mixture was used in the next step without workup or purification. Compound 29 was confirmed by LCMS (R t =0.37 min) and HPLC (R t = 1.97 min).

[0539] LCMS:R t =0.37 min, MS calculated value: 767.3, MS observed value: [M+H] + =768.5.

[0540] HPLC:R t =1.97 minutes, purity: 89.8%.

[0541] General procedure for the preparation of compound 114 [ka] To the above solution of compound 29 (164 mg (theoretical), 213 μmol, 1.0 equiv.) in DMF (2.0 mL) was added HO (0.5 mL), compound 12A (79.7 mg, 1.06 mmol, 5.0 equiv.), DMAP (78.1 mg, 639 μmol, 3.0 equiv.), and DIEA (82.6 mg, 639 μmol, 111 μL, 3.0 equiv.). The mixture was stirred at 25° C. for 1.0 h. LC-MS analysis showed that compound 29 was completely consumed and the desired mass (R t =0.34 min). The mixture was purified by preparative HPLC (AcOH condition) to give compound 114 (67 mg, 92.0 μmol, 43.2% yield, 96.6% purity, AcOH salt) as a white solid, which was analyzed by LCMS (R t =0.34 min) and HPLC (R t= 2.65 min).

[0542] LCMS:R t =0.34 min, MS calculated value: 727.37, MS observed value: [M+H] + =728.5.

[0543] HPLC:R t =2.65 minutes, purity: 96.6%.

[0544] Example 12: Synthesis of Compound 113 [ka] General procedure for the preparation of compound 11A [ka] To a solution of target 183 (120 mg, 256 μmol, 1.0 equiv., AcOH salt) and 11 (119 mg, 282 μmol, 1.1 equiv.) in DMF (1.0 mL), DMAP (31.3 mg, 256 μmol, 1.0 equiv.) and DIEA (66.2 mg, 512 μmol, 89.2 μL, 2.0 equiv.) were added, and the mixture was stirred at 25 °C for 1.0 h. LC-MS showed that target 183 was completely consumed, and several new peaks were observed in LC-MS, with approximately 97.3% of the desired compound detected. The resulting reaction mixture was triturated twice with isopropyl ether (10 mL × 2). The precipitated solid was filtered and dried in vacuo to give compound 11A (115 mg, crude product) as a brown solid, as determined by LC-MS (R t =0.41 min) and HPLC (R t = 3.09 min).

[0545] LCMS:R t =0.41 min, MS calculated value: 675.3, MS observed value: [M+H] + =676.4.

[0546] HPLC:R t =3.09 minutes, purity: 94.7%.

[0547] General procedure for the preparation of compound 113 [ka] To a solution of compound 11A (115 mg, 170 μmol, 1.0 equiv.) and compound 12A (63.9 mg, 851 μmol, 5.0 equiv.) in DMF (1.0 mL) and HO (250 μL) was added DIEA (66.0 mg, 511 μmol, 88.9 μL, 3.0 equiv.) and DMAP (62.4 mg, 510 μmol, 3.0 equiv.), and the mixture was stirred at 25° C. for 1.0 h. LC-MS indicated approximately 0% of compound 11A remained. LC-MS showed several new peaks, and the desired compound was detected in approximately 96.4%. The residue was purified by preparative HPLC (AcOH conditions) to give compound 113 (45 mg, 64.7 μmol, 38.0% yield, AcOH salt) as a white solid, which was analyzed by LC-MS (R t =0.38 min) and HPLC (R t = 2.19 min).

[0548] LCMS:R t =0.38 min, MS calculated value: 635.3, MS observed value: [M+H] + =636.2.

[0549] HPLC:R t =2.19 minutes, purity: 96.3%.

[0550] Example 13: Synthesis of Compound 117 [ka] General procedure for the preparation of compound 45-2A [ka] To a solution of compound 45-1A (20.0 g, 114 mmol, 1.0 equiv.) in ACN (120 mL) were added DCC (25.9 g, 125 mmol, 25.4 mL, 1.1 equiv.), pyridine (27.1 g, 342 mmol, 27.6 mL, 3.0 equiv.), and compound 45-1B (14.8 g, 125 mmol, 18.0 mL, 1.1 equiv.). The mixture was stirred at 25° C. for 3.0 hours. TLC (petroleum ether / ethyl acetate=3:1) confirmed that compound 45-1A (R f =0.20) is completely consumed and one new spot (R f =0.57) was formed. The reaction mixture was diluted with HO (400 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with saturated NaHCO solution (200 mL), brine (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure at 35 °C to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20:1 to 1:3) to give compound 45-2A (20.0 g, 72.6 mmol, yield 60.0%, purity 94.1%) as a yellow oil, which was identified by LCMS (R t =0.48 min), HPLC(R t = 3.80 min).

[0551] LCMS:R t =0.48 min, MS calculated value: 275.42, MS observed value: [M+Na] + =298.0.

[0552] HPLC:R t =3.80 minutes, purity: 94.1%.

[0553] General procedure for the preparation of compound 45-3 [ka] Compound 45-2A (5.0 g, 18.1 mmol, 1.0 equiv.) was dissolved in HCl / dioxane (2.0 M, 10 mL). The mixture was stirred at 25° C. for 3.0 h. TLC (petroleum ether / ethyl acetate=3:1) confirmed that compound 45-2A (R f=0.57) is completely consumed and one new spot (R f =0.21) was formed. The reaction mixture was concentrated in vacuo to give compound 45-3 (4.0 g, crude product) as a yellow oil, which was identified by LCMS (R t =0.22 min).

[0554] LCMS:R t =0.22 min, MS calculated value: 175.10, MS observed value: [M+Na] + =198.3.

[0555] General procedure for the preparation of compound 45-5 [ka] To a solution of compound 45-4 (1.7 g, 9.23 mmol, 1.0 equiv.) in DMF (4.0 mL), DIEA (3.58 g, 27.7 mmol, 4.82 mL, 3.0 equiv.) and HATU (4.21 g, 11.1 mmol, 1.2 equiv.) were added, and finally compound 45-3 (2.93 g, 13.8 mmol, 1.50 equiv., HCl salt) was added. The mixture was stirred at 25° C. for 2.0 h. LCMS showed the desired mass (R t =0.42 min). The residue was purified by preparative HPLC (TFA condition) to give compound 45-5 (2.76 g, 7.73 mmol, 83.82% yield, 95.7% purity) as a white solid, which was identified by LCMS (R t =0.42 min) and HPLC (R t = 3.06 min). t =0.42 min, MS calculated value: 341.20, MS observed value: [M+Na] + =364.1.

[0556] HPLC:R t =3.06 minutes, purity: 99.5%.

[0557] General procedure for the preparation of compound 45-6 [ka] To a solution of compound 45-5A (670 mg, 2.26 mmol, 1.1 equiv.) in THF (7.0 mL), DCC (507 mg, 2.46 mmol, 497 μL, 1.20 equiv.) and DMAP (25.0 mg, 205 μmol, 0.1 equiv.), compound 45-5 (700 mg, 2.05 mmol, 1.0 equiv.) were added. The mixture was stirred at 25° C. for 1.0 h. LCMS showed the desired mass (R t =0.55 min). The reaction mixture was filtered and dried under vacuum to give a residue, which was purified by preparative HPLC (TFA conditions) to give compound 45-6 (790 mg, 1.21 mmol, 59.1% yield, 95.2% purity) as a white solid, which was identified by LCMS (R t =0.55 min), HPLC(R t = 4.58 min).

[0558] LCMS:R t =0.55 min, MS calculated: 620, MS observed: [M+Na] + =643.3.

[0559] HPLC:R t =4.58 minutes, purity: 91.3%.

[0560] General procedure for the preparation of compound 45 [ka] To a solution of compound 45-6 (600 mg, 966 μmol, 1.0 equiv.) in DMF (6.0 mL) was added TEA (97.8 mg, 966 μmol, 134 μL, 1.0 equiv.). The mixture was stirred at 25° C. for 1.0 h. LCMS confirmed that compound 45-6 was completely consumed and the desired mass (R t =0.36 min). The reaction mixture was purified by preparative HPLC (FA conditions) to give compound 45 (500 mg, 827 μmol, 85.7% yield, 66.1% purity) as a yellow oil, which was identified by LCMS (R t =0.36 min), HPLC(R t= 1.94 min).

[0561] LCMS:R t =0.36 min, MS calculated value: 398.22, MS observed value: [M+Na] + =421.2.

[0562] HPLC:R t =1.94 minutes, purity: 66.1%.

[0563] General procedure for the preparation of compound 46 [ka] To a solution of compound 44 (210 mg, 589 μmol, 1.0 equiv.) and compound 45 (392 mg, 766 μmol, 1.3 equiv., TFA salt) in DMF (3.0 mL) was added DIC (112 mg, 883 μmol, 137 μL, 1.5 equiv.), HOBt (120 mg, 883 μmol, 1.5 equiv.), and DIEA (190 mg, 1.47 mmol, 256 μL, 2.5 equiv.). The mixture was stirred at 25° C. for 4.0 h. LCMS showed that compound 44 was completely consumed and the desired mass (R t =0.43 min). The reaction mixture was triturated with isopropyl ether (30 mL × 2), and the precipitated solid was centrifuged and purified by preparative HPLC (TFA conditions) to give compound 46 (150 mg, 176 μmol, 29.9% yield, 93.7% purity) as a white solid, which was analyzed by LCMS (R t =0.42 min) and HPLC (R t = 2.77 min).

[0564] LCMS:R t =0.42 min, MS calculated value: 736.37, MS observed value: [M+H] + =737.4.

[0565] HPLC:R t =2.77 minutes, purity: 93.7%.

[0566] General procedure for the preparation of compound 117 [ka] To a solution of compound 46 (150 mg, 203 μmol, 1.0 equiv.) in THF (1.0 mL) was added TBAF (1 M, 1.02 mL, 5 equiv.). The mixture was stirred at 25° C. for 4.0 h. LC-MS showed that compound 46 was completely consumed and the desired mass (R t =0.35 min), one major peak was detected. The reaction mixture was concentrated under reduced pressure to remove THF. The residue was purified by preparative HPLC (AcOH condition) to give compound 117 (42 mg, 60.3 μmol, yield 29.7%, purity 99.3%, AcOH salt) as a white solid.

[0567] LCMS:R t =0.35 min, MS calculated: 636.30, MS observed: [M+H] + =637.4.

[0568] HPLC:R t =2.70 minutes, purity: 99.3%.

[0569] Example 14: Synthesis of Compound 101 [ka] General procedure for the preparation of compound 3 [ka] To a solution of compound 1 (1.22 g, 11.4 mmol, 1.00 equiv) in EtOH (12.0 mL) was added AcOH (614 mg, 10.2 mmol, 585 μL, 0.89 equiv) and compound 2 (3.77 g, 17.1 mmol, 1.49 equiv). The mixture was stirred at 25° C. for 16 h. LCMS showed that compound 1 was completely consumed and the desired mass (MS calculated: 215.0, MS found: [M+H] + = 216.0) was detected. The reaction mixture was concentrated under reduced pressure to give compound 3 (5.67 g, crude product) as a yellow oil.

[0570] LCMS-1:R t =0.36 min, MS calculated value: 215.0, MS observed value: [M+H] + =216.0

[0571] General procedure for the preparation of compound 4 [ka] To a solution of compound 3 (5.67 g, 26.3 mmol, 1.00 equiv.) in DMF (25.0 mL) was added DIEA (10.2 g, 78.9 mmol, 13.7 mL, 3.00 equiv.) and PNP (16.0 g, 52.6 mmol, 2.00 equiv.). The mixture was stirred at 25° C. for 7 h. LCMS showed that compound 3 was completely consumed and the desired mass (MS calculated: 380.05, MS found: [M+H] + The reaction mixture was diluted with HO (100 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (0.1% TFA condition) to give compound 4 (1.40 g, 3.66 mmol, 31.8% yield in two steps, 99.4% purity) as a yellow solid, which was confirmed by LCMS, HPLC, and HNMR.

[0572] LCMS:R t =0.48 min, MS calculated value: 380.0, MS observed value: [M+H] + =381.1.

[0573] HPLC:R t =3.53 minutes, purity: 99.4%.

[0574] 1 H NMR (400MHz, CDCl3)δ:8.47-8.45(m,1H),8.30-8.26(m,2H),7.76-7.74(m,1H),7.65-7.60(m,2H),7.39-7.35(m,2H),7.11-7.07(m,1H).

[0575] General procedure for the preparation of compound 5 [ka] To a solution of compound 4 (1.40 g, 3.66 mmol, 1.00 equiv.) in DMF (14.0 mL) was added MMAE (2.63 g, 3.66 mmol, 1.00 equiv.), HOBt (741 mg, 5.49 mmol, 1.50 equiv.), and DIEA (945 mg, 7.32 mmol, 1.27 mL, 2.00 equiv.). The mixture was stirred at 25° C. for 12 hours. LCMS showed that compound 4 was completely consumed and the desired mass (MS calculated: 958.53, MS found: [M+H] + =959.5) was detected. The residue was purified by preparative HPLC (0.1% TFA condition) to obtain compound 5 (2.50 g, 2.54 mmol, yield 69.3%, purity 97.4%) as a white solid, which was confirmed by LCMS and HPLC.

[0576] LCMS:R t =0.55 min, MS calculated: 958.5, MS observed: [M+H] + =959.7.

[0577] HPLC:R t =3.93 minutes, purity: 97.4%.

[0578] General procedure for the preparation of compound 7 [ka] To a solution of compound 6 (2.40 g, 15.2 mmol, 10.0 equiv, HCl) in EtOH (15.0 mL) was added AcOH (81.4 mg, 1.36 mmol, 77.6 μL, 0.89 equiv) and compound 5 (1.50 g, 1.52 mmol, 1.00 equiv). The mixture was stirred at 80° C. for 3 h. LCMS showed that compound 5 was completely consumed and the desired mass (MS calculated: 968.5, MS found: [M+H] +=969.5) was detected. The residue was purified by preparative HPLC (0.1% TFA condition) to obtain compound 7 (780 mg, 767 μmol, yield 50.4%, purity 99.0%, HCl) as a white solid, which was confirmed by LCMS and HPLC.

[0579] LCMS:R t =0.41 min, MS calculated value: 968.5, MS observed value: [M+H] + =969.6.

[0580] HPLC:R t =2.62 minutes, purity: 99.0%.

[0581] General procedure for the preparation of compound 8 [ka] To a solution of compound 8a (2.00 g, 10.8 mmol, 1.00 equiv.) in ACN (10.0 mL) was added DIEA (4.21 g, 32.5 mmol, 5.67 mL, 3.00 equiv.) and DSC (2.78 g, 10.8 mmol, 1.00 equiv.) in ACN (10.0 mL). The mixture was stirred at 25 °C for 1 h. Analysis by LCMS indicated the desired mass (MS calculated: 281.1, MS found: [M+Na] + The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether: EtOAc = 1:1) to give compound 8 (1.82 g, 6.18 mmol, 56.9% yield, 95.5% purity) as a pale yellow solid, which was confirmed by LCMS, HPLC, and HNMR.

[0582] LCMS:R t =0.44 min, MS calculated value: 281.1, MS observed value: [M+H] + =304.0.

[0583] HPLC:R t =1.77 minutes, purity: 95.5%.

[0584] 1 H NMR(400MHz,CDCl3)δ:6.13-6.06(m,1H),5.68-5.64(m,2H),4.51(s,1H),2.82-2 .81(m,4H),2.41-2.25(m,3H),2.17-1.98(m,3H),1.93-1.83(m,2H),1.25(s,3H).

[0585] General procedure for the preparation of compound 101 [ka] To a solution of compound 7 (280 mg, 275 μmol, 1.00 equiv., HCl) in DMF (1.00 mL) were added DMAP (269 mg, 2.20 mmol, 8.00 equiv.), compound 8 (89.3 mg, 303 μmol, 1.10 equiv.), and DIEA (284 mg, 2.20 mmol, 384 μL, 8.00 equiv.). The mixture was stirred at 25° C. for 1 hour. LCMS showed that compound 7 (R t =0.43 min) remained, and the desired mass (MS calculated: 1134.6, MS observed: [M+H] + The reaction mixture was concentrated under reduced pressure to give residue-1. Another residue-2 was obtained from an 80 mg scale. Two batches were purified by preparative HPLC (AcOH condition) to give compound 101 (47.0 mg, 37.7 μmol, 13.6% yield, 95.9% purity, AcOH) as a white solid, which was confirmed by LCMS and HPLC.

[0586] LCMS:R t =0.50 min, MS calculated: 1134.6, MS observed: [M+H] + =1135.7.

[0587] HPLC:R t =3.57 minutes, purity: 95.9%.

[0588] Example 15: Synthesis of Compound 14 [ka] [ka] General procedure for the preparation of compound 10 [ka] To a solution of compound 9 (2.00 g, 4.03 mmol, 1.00 equiv.) in DCM (20 mL) and MeOH (5 mL) was added 4-aminobenzyl alcohol (992 mg, 8.06 mmol, 2.00 equiv.) and EEDQ (1.99 g, 8.06 mmol, 2.00 equiv.). The reaction mixture was stirred in the dark at 35 °C for 16 h. TLC (DCM / MeOH = 10:1, R of the product) showed no significant difference. f =0.32), indicating the complete consumption of compound 9 and the formation of one new spot. The reaction mixture was filtered and concentrated under reduced pressure to give compound 10 (2.00 g, 3.02 mmol, 75.1% yield, 91.0% purity) as a yellow solid, which was confirmed by LCMS and HPLC.

[0589] LCMS:R t =0.42 min, MS calculated value: 601.3, MS observed value: [M+H] + =602.3.

[0590] HPLC:R t =2.72 minutes, purity: 91.0%.

[0591] General procedure for the preparation of compound 11 [ka] To a solution of compound 10 (2.00 g, 3.32 mmol, 1.00 equiv.) in DMF (20 mL), DIEA (429 mg, 3.32 mmol, 578 μL, 1.00 equiv.) and PNP (1.01 g, 3.32 mmol, 1.00 equiv.) were added. The mixture was stirred at 20-25 °C for 16 h. TLC (DCM / MeOH = 10:1, R of the product) revealed a 50% methyl ... f=0.42), indicating the complete consumption of compound 10 and the formation of one new spot. The crude product was triturated with isopropyl ether (200 mL) to give compound 11 (1.80 g, 2.35 mmol, 70.6% yield) as a yellow solid, which was confirmed by LCMS and HPLC.

[0592] LCMS:R t =0.51 min, MS calculated value: 766.3, MS observed value: [M+H] + =767.5.

[0593] HPLC:R t =3.65 minutes, purity: 88.4%.

[0594] General procedure for the preparation of compound 12 [ka] To a solution of compound 11 (1.80 g, 2.35 mmol, 1.00 equiv.) in DMF (18.0 mL), MMAE (1.69 g, 2.35 mmol, 1.00 equiv.) and DIEA (910 mg, 7.04 mmol, 1.23 mL, 3.00 equiv.) were added. The mixture was stirred at 20-25 °C for 16 h. The reaction was monitored by TLC (DCM / MeOH = 10:1, R of the product). f =0.27) and TLC showed that the reactants were consumed. The crude product was triturated with isopropyl ether (200 mL) to give compound 12 (3.0 g, crude) as a yellow solid.

[0595] General procedure for the preparation of compound 13 [ka] A solution of compound 12 (3.00 g, 2.23 mmol, 1.00 equiv.) in TEA (4.19 g, 41.4 mmol, 6.00 mL, 18.5 equiv.) and DMF (24.0 mL) was stirred at 20-25 °C for 16 h. The reaction was monitored by TLC (DCM:MeOH = 10:1, R of the product). f=0.19), indicating that the reactants had been consumed. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was triturated with isopropyl ether (200 mL). The residue was purified by preparative HPLC (0.1% TFA condition) to give compound 13 (620 mg, 551 μmol, 24.7% yield over two steps) as a white solid, which was confirmed by LCMS and HPLC.

[0596] LCMS:R t =0.40 min, MS calculated: 1122.7, MS observed: [M+H] + =1123.7.

[0597] HPLC:R t =2.58 minutes, purity: 96.7%.

[0598] General procedure for the preparation of compound 16 [ka]

[0599] Peptide synthesis Peptides were synthesized using standard Fmoc chemistry.

[0600] Resin preparation: To a vessel containing 2-CTC resin (1.50 mmol, Sub: 0.81 mmol / g, 1.85 g) and Fmoc-7-Ahp-OH (1.50 mmol, 0.55 g, 1.00 equiv.) in DCM (30.0 mL), DIEA (4.00 equiv.) was added with N bubbling for 2 h at 15 °C. MeOH (15.0 mL) was added and mixed for 30 min. Next, 20% piperidine in DMF (50.0 mL) was added, and the mixture was bubbled with N for 30 min at 15 °C. The mixture was then filtered to obtain the resin. The resin was washed with DMF (50.0 mL) × 5 before proceeding to the next step.

[0601] Coupling: A solution of Fmoc-D-Glu(OAll)-OH (4.50 mmol, 1.84 g, 3.00 equiv.) and HBTU (4.27 mmol, 1.62 g, 2.85 equiv.) in DMF (30.0 mL) was added to the resin while bubbling with N2. Next, DIEA (9.00 mmol, 6.00 equiv.) was added dropwise to the mixture, and N2 was bubbled through at 20 °C for 30 min. The coupling reaction was monitored by the ninhydrin test, and completion was indicated by the absence of color. The resin was then washed with DMF (50.0 mL) × 5.

[0602] Deprotection: A solution of PhSiH3 (10.0 equiv.) and Pd(PPh3)4 (0.10 equiv.) in DCM (20 mL) was added to the resin, and the mixture was bubbled with N2 for 30 min at 15 °C. The resin was then washed with DCM (50.0 mL) × 5 and DMF (50.0 mL) × 5.

[0603] Coupling: A solution of TFP (15.0 mmol, 2.49 g, 10.0 equiv.) in DMF (30.0 mL) was added to the resin while bubbling with N2. DIC (10.0 equiv.) was then added dropwise to the mixture, and N2 was bubbled through at 20 °C for 16 h. The resin was then washed with DMF (50.0 mL) x 5. A solution of bisPEG2-OtBu (3.00 mmol, 2.14 g, 2.00 equiv.) in DMF (30.0 mL) was added to the resin while bubbling with N2. DIEA (4.00 equiv.) was then added dropwise to the mixture, and N2 was bubbled through at 20 °C for 2 h.

[0604] The resin was then washed with DMF (50.0 mL) x 5, MeOH (50.0 mL) x 5, and then dried under vacuum.

[0605] Peptide cleavage Cleavage buffer (HFIP / DCM, 2 / 8, v / v, 100 ml) is added to the flask containing the side-chain protected peptide at room temperature and stirred twice for 0.5 hours.

[0606] Collect the filtrate by filtration and concentrate the mixture under reduced pressure.

[0607] The residue was dissolved in ACN (100 ml) and H2O (150 ml) and then lyophilized to give compound 16 (520 mg, purity 93.2%, yield 27.3%), which was confirmed by LCMS and HPLC.

[0608] LCMS: Rt=1.71 min, MS calculated: 1190.7, MS observed: [M+H] + =1191.4

[0609] HPLC: Rt=15.3 min, purity: 95.6%

[0610] General procedure for the preparation of compound 17 [ka] To a solution of compound 16 (370 mg, 310 μmol, 1.00 equiv.) in DCM (4.00 mL) was added TFP (103 mg, 621 μmol, 2.00 equiv.) and DIC (117 mg, 931 μmol, 144 μL, 3.00 equiv.). The mixture was stirred at 20-25 °C for 16 h. LC-MS analysis showed that compound 16 was completely consumed and had the desired m / z (MS calculated: 1338.7, MS found: [M+H]). + One main peak (R = 1339.6) t =0.51 min). The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (0.1% TFA condition) to give compound 17 (350 mg, 257 μmol, yield 82.9%, purity 98.6%) as a colorless oil, which was confirmed by LCMS and HPLC.

[0611] LCMS:R t =0.51 min, MS calculated value: 1338.7, MS observed value: [M+H] + =1339.6.

[0612] HPLC:R t =3.87 minutes, purity: 98.6%

[0613] General procedure for the preparation of compound 18 [ka] A solution of compound 17 (350 mg, 261 μmol, 1.00 equiv.) in HCOOH (10 mL) was stirred at 20-25 °C for 16 h. LC-MS analysis confirmed that compound 17 was completely consumed and had the desired m / z (MS calculated: 1226.6, MS found: [M+H]). + One main peak (R = 1227.6) t =0.45 min) was detected. The reaction mixture was concentrated under reduced pressure to give compound 18 (300 mg, 150 μmol, 57.5% yield, 61.5% purity) as a colorless oil, which was confirmed by LCMS and HPLC.

[0614] LCMS:R t =0.45 min, MS calculated: 1226.6, MS observed: [M+H] + =1227.6.

[0615] HPLC:R t =3.09 minutes, purity: 61.5%.

[0616] General procedure for the preparation of compound 14 [ka] To a solution of compound 13 (300 mg, 242 μmol, 1.00 equiv., TFA) in DMF (3.00 mL) was added compound 18 (297 mg, 242 μmol, 1.00 equiv.) and DIEA (94.0 mg, 727 μmol, 126 μL, 3.00 equiv.). The mixture was stirred at 20-25 °C for 16 h. LCMS showed complete consumption of compound 13 and the desired m / z (MS calculated: 2183.3, MS found: (M+2H)). 2+ One main peak (R = 1093.2) t =0.46 min). The crude product was triturated with isopropyl ether (30 mL) to give compound 14 (610 mg, crude) as a colorless oil, which was confirmed by LCMS.

[0617] LCMS:R t =0.46 min, MS calculated: 2183.3, MS observed: (M+2H) 2+ =1093.1.

[0618] General procedure for the preparation of compound 15 [ka] A solution of compound 14 (610 mg, 279 μmol, 1.00 equiv.) in DMF (4 mL) and TEA (727 mg, 7.18 mmol, 1.00 mL, 25.700 equiv.) was stirred at 20-25 °C for 16 h. LC-MS analysis confirmed that compound 14 was completely consumed and that one major peak (R) with the desired m / z (MS calculated: 1961.2, MS found: (M+2H) = 982.0) was observed. t =0.39 min). The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (0.1% TFA condition) to give compound 15 (200 mg, 94.9 μmol, 34.0% yield in two steps, 98.6% purity, TFA) as a white solid, which was confirmed by LCMS and HPLC.

[0619] LCMS:R t =0.40 min, MS calculated: 1961.2, MS observed: (M+2H) 2+ =982.0.

[0620] HPLC:R t =2.46 minutes, purity: 98.6%.

[0621] General procedure for the preparation of target compound 14 [ka] To a solution of compound 15 (200 mg, 96.3 μmol, 1.00 equiv., TFA) in THF (0.3 mL) and HO (0.3 mL) was added compound 8 (81.2 mg, 288 μmol, 3.00 equiv.), DIEA (37.3 mg, 288 μmol, 50.3 μL, 3.00 equiv.), and DMAP (35.3 mg, 288 μmol, 3.00 equiv.). The mixture was stirred at 20-25 °C for 2 h. LC-MS-1 indicated approximately 14.1% compound 15 remaining and one major peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to give the residue (portion 1). Another residue (portion 2) was obtained from a 120 mg scale. The combined residues were purified by preparative HPLC (0.1% TFA) and then salt-exchanged via preparative HPLC (0.1% CH3COOH) to give compound 14 (41.0 mg, 19.0 μmol, 12.4% yield, AcOH salt) as a white solid.

[0622] LCMS:R t =0.45 min, MS calculated: 2127.3, MS observed: (M+2H) 2+ =1065.1.

[0623] HPLC:R t =2.90 minutes, purity: >99.0%.

[0624] Example 16: Synthesis of Compound 109 [ka] To a solution of compound 1 (500 mg, 487 μmol, 1.00 equiv.) and compound 2 (1.07 g, 7.31 mmol, 15 equiv.) in DMSO (5.0 mL) was added DIEA (0.501 g, 3.90 mmol, 0.68 mL, 8.00 equiv.) and DMAP (476 mg, 3.90 mmol, 8.00 equiv.). The mixture was stirred at 25° C. for 32 h. LCMS showed that compound 1 was completely consumed and the desired mass (R t=1.54 min). The mixed layer was filtered, and the organic filtrate was separated. The crude product was purified by reverse-phase HPLC (FA conditions). Compound 109 (200 mg, 188 μmol, 38.6% yield, 98.4% purity; 1H NMR showed that compound 109 contained no HCOOH residues) was obtained as a white solid by LCMS (R t = 1.54 min) and HPLC (Rt = 3.02 min).

[0625] LCMS:R t = 1.54 min, MS calculated value: 1056.6, MS observed value: [M+Na] + =1079.7

[0626] HPLC:R t =3.02 minutes, purity: 98.4%

[0627] Example 17: Synthesis of Compound 30 [ka] General procedure for the preparation of compound 3-2 [ka] D To a solution of compound 3 (500 mg, 555 μmol, 1.00 equiv.) in MF (10.0 mL) was added DIEA (143 mg, 1.11 mmol, 193 μL, 2.00 equiv.) and compound 3-1 (375 mg, 888 μmol, 1.60 equiv.). The mixture was stirred at 25° C. for 12 hours. LC-MS revealed that compound 3-1 remained (R t =0.46 min), compound 3 was completely consumed and the desired mass was detected (R t =0.53 min). The reaction mixture was used directly for purification. The residue was purified by preparative HPLC (FA conditions). Compound 3-2 (252 mg, 213 μmol, yield 38.4%, purity 98.3%) was obtained as a brown solid and was confirmed by LC-MS and HPLC.

[0628] LCMS:R t=0.52 min, MS calculated value: 1160.44, MS observed value: [M+H] + =1161.5.

[0629] HPLC:R t =4.30 minutes, purity: 98.3%.

[0630] General procedure for the preparation of compound 4-2 [ka] To a solution of compound 4-1 (20.0 g, 85.3 mmol, 1.00 equiv.) in DCM (200 mL) at 0 °C, TEA (12.9 g, 128 mmol, 17.8 mL, 1.50 equiv.) and 4-methylbenzenesulfonyl chloride (17.9 g, 93.9 mmol, 1.10 equiv.) were added under N2. The mixture was stirred at 25 °C for 2 h. LCMS showed that compound 4-1 was completely consumed and the desired mass (R t =0.50 min). The reaction mixture was diluted with HO (300 mL) and extracted with DCM (120 mL × 2). The combined organic layers were washed with brine (150 mL × 2), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. Compound 4-2 (36.0 g, 82.3 mmol, 96.5% yield, 88.9% purity) was obtained as a white oil and was confirmed by LCMS (R t =0.47 minutes).

[0631] LCMS:R t =0.47 min, MS calculated value: 388.1, MS observed value: [M+Na] + =411.1.

[0632] General procedure for the preparation of compound 4-4 [ka] To a solution of compound 4-2 (35.3 g, 80.9 mmol, 3.00 equiv.) and compound 4-3 (6.70 g, 26.9 mmol, 1.00 equiv.) in ACN (350 mL) was added CsCO (2.64 g, 8.09 mmol, 0.30 equiv.) and KCO (11.1 g, 80.9 mmol, 3.00 equiv.), and NaI (404 mg, 2.70 mmol, 0.10 equiv.). The mixture was stirred at 85 °C for 12 h. LCMS showed that compound 4-3 was completely consumed and the desired mass (R t1 =0.42 min). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1). Compound 4-4 (12.2 g, 17.9 mmol, yield 65.9%, purity 99.3%) was obtained as a yellow oil, which was analyzed by LCMS (R t =0.42 min).

[0633] LCMS:R t =0.42 min, MS calculated value: 680.4, MS observed value: [M+H] + =681.4.

[0634] General procedure for the preparation of compound 4 [ka] Compound 4-4 (6.00 g, 8.75 mmol, 1.00 equiv) in HCl / dioxane (60.0 mL) was stirred at 25° C. for 2 h. LC-MS showed that compound 4-4 was completely consumed and the desired mass (R t1 =0.07 min). The reaction mixture was concentrated under reduced pressure to give a residue. Compound 4 (4.50 g, crude product, HCl) was obtained as a yellow oil and was confirmed by HNMR.

[0635] LCMS:R t =0.07 min, MS calculated value: 468.2, MS observed value: [M+H] + =469.3.

[0636] 1 H NMR (400MHz, CDCl3)δ:8.19-8.10(m,2H),4.56-3.88(m,10H),3.77-3.71(m,12H),3.66-3.62(m,12H),3.31-3.23(m,2H),2.60-2.57(m,2H).

[0637] General procedure for the preparation of compound 30 [ka] To a solution of compound 3-2 (310 mg, 266 μmol, 1.00 equiv.) in DMSO (0.50 mL) were added DIEA (69.0 mg, 533 μmol, 93.0 μL, 2.00 equiv.), DMAP (65.2 mg, 533 μmol, 2.00 equiv.), and compound 4 (250 mg, 533 μmol, 2.0 equiv.). The mixture was stirred at 25° C. for 12 hours. LC-MS revealed that compound 3-2 (R t =0.53 min) remained at 19.9% ​​and the desired mass was detected (R t =0.43 min). The reaction mixture was used directly for purification. The residue was purified by preparative HPLC (AcOH conditions). Compound 30 (248 mg, 163 μmol, 47.2% yield, 95.5% purity) was obtained as a white solid and was characterized by LC-MS and HPLC.

[0638] LCMS:R t =0.43 min, MS calculated value: 1513.68, MS observed value: [M+H] + =1514.9.

[0639] HPLC:R t =3.47 minutes, purity: 95.5%.

[0640] Example 18: Synthesis of Compound 31 [ka] General procedure for the preparation of compound 6 [ka] To a solution of compound 5 (2.00 g, 4.66 mmol, 1.00 equiv.) in DMF (20.0 mL) was added DIEA (1.20 g, 9.32 mmol, 1.62 mL, 2.00 equiv.) and exatecan (1.86 g, 3.49 mmol, 0.75 equiv.). The mixture was stirred at 25° C. for 2 hours. LCMS showed that compound exatecan was completely consumed and the desired mass (R t =0.46 min). The crude product was purified by reverse-phase HPLC (FA conditions). Compound 6 (2.1 g, 2.81 mmol, 60.4% yield, 99.5% purity) was confirmed as a yellow solid by LCMS.

[0641] LCMS:R t =0.46 min, MS calculated value: 742.3, MS observed value: [M+H] + =743.4

[0642] General procedure for the preparation of compound 31 [ka] To a solution of compound 4 (prepared in the above example) (1.37 g, 2.68 mmol, 2.00 equiv.) and compound 6 (1.00 g, 1.34 mmol, 1.00 equiv.) in DMSO (10.0 mL) was added DIEA (346.3 mg, 2.68 mmol, 466.7 μL, 2.00 equiv.) and DMAP (327 mg, 2.68 mmol, 2.00 equiv.). The mixture was stirred at 25° C. for 10 h. LCMS showed that compound 6 was completely consumed and the desired mass (R t =0.39 min). The mixed layer was filtered, and the organic filtrate was separated. The crude product was purified by reverse-phase HPLC (AcOH conditions). The crude product was purified by reverse-phase HPLC (FA conditions). Compound 31 (300 mg, 264 μmol, 19.7% yield, 96.5% purity) was confirmed as a yellow solid by LCMS and HPLC.

[0643] LCMS:R t =0.39 min, MS calculated value: 1095.5, MS observed value: [M+H] + =1096.7

[0644] HPLC:R t =2.50 minutes, purity: 96.5%

[0645] Example 19: Synthesis of Compound 28 [ka] [ka] General procedure for the preparation of compound 7-3 [ka] To a solution of compound 7-1 (11.0 g, 36.2 mmol, 3.00 equiv.) and compound 7-2 (3.00 g, 12.0 mmol, 1.00 equiv.) in ACN (120 mL) was added CsCO (1.18 g, 3.62 mmol, 0.30 equiv.) and KCO (5.01 g, 36.2 mmol, 3.00 equiv.), and NaI (181 mg, 1.21 mmol, 0.10 equiv.). The mixture was stirred at 85 °C for 12 h. LCMS showed that compound 7-2 was completely consumed and the desired mass (R t The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 1:0 to 0:1, dichloromethane:methanol = 10:1, R f =0.20) to give compound 7-3 (6.60 g, 10.0 mmol, 82.8% yield, 77.7% purity) as a yellow oil, which was confirmed by LCMS.

[0646] LCMS:R t =0.27 min, MS calculated value: 512.3, MS observed value: [M+H] + =513.3

[0647] General procedure for the preparation of compound 7-5 [ka] To a solution of compound 7-3 (6.60 g, 10.0 mmol, 1.00 equiv) in DCM (70.0 mL) was added NaOH (120 mg, 3.00 mmol, 0.30 equiv) and compound 7-4 (12.8 g, 100 mmol, 14.5 mL, 10.0 equiv). The mixture was stirred at 40° C. for 12 h. LCMS showed that compound 7-3 was completely consumed and the desired mass (R t The reaction mixture was diluted with NH4Cl (20.0 mL) and extracted with DCM (30.0 mL x 2). The combined organic layers were washed with brine (30.0 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 1:0 to 0:1, dichloromethane:methanol = 10:1, R f =0.60) to give compound 7-5 (4.37 g, 5.58 mmol, 55.7% yield, 98.2% purity) as a yellow oil, which was confirmed by LCMS.

[0648] LCMS:R t =0.45 min, MS calculated value: 768.5, MS observed value: [M+H] + =769.2.

[0649] General procedure for the preparation of compound 7 [ka] Compound 7-5 (2.04 g, 2.60 mmol, 1.00 equiv) in HCl / dioxane (20.0 mL) was stirred at 25° C. for 2 h. LC-MS showed that compound 7-5 was completely consumed and the desired mass (R t1 =0.13 min). The reaction mixture was concentrated under reduced pressure to give a residue. Compound 7 (1.90 g, crude product, HCl) was obtained as a yellow oil and was confirmed by HNMR.

[0650] LCMS:R t =0.13 min, MS calculated value: 556.3, MS observed value: [M+H]+ =557.3.

[0651] 1 H NMR (400MHz, CDCl3) δ: 7.96 (s, 2H), 4.03-3.88 (m, 8H), 3.76-3.50 (m, 32H), 3.26-3.25 (m, 2H), 2.65-2.59 (m, 4H). [ka]

[0652] To a solution of compound 6 (500 mg, 669 μmol, 1.00 equiv.) and compound 7 (932 mg, 1.67 mmol, 2.50 equiv.) in DMSO (5.00 mL) was added DIEA (173 mg, 1.34 mmol, 233.3 μL, 2.00 equiv.) and DMAP (163 mg, 1.34 mmol, 2.00 equiv.). The mixture was stirred at 25° C. for 10 h. LCMS showed that compound 6 was completely consumed and the desired mass (R t =0.394 min). The mixed layer was filtered, and the organic filtrate was separated. The crude product was purified by reverse-phase HPLC (AcOH conditions). Compound 28 (200 mg, 184 μmol, 15.5% yield, 97.3% purity) was confirmed as a yellow solid by LCMS and HPLC.

[0653] LCMS:R t =0.39 min, MS calculated value: 1083.6, MS observed value: [M+H] + =1084.8

[0654] HPLC:R t =2.55 minutes, purity: 97.3%,

[0655] Example 20: Synthesis of Compound 29 [ka] To a solution of compound 3-2 (398 mg, 342 μmol, 1.00 equiv.) in DMSO (0.50 mL), DIEA (88.5 mg, 685 μmol, 119 μL, 2.00 equiv.), DMAP (83.7 mg, 685 μmol, 2.00 equiv.), and compound 7 (381 mg, 685 μmol, 2.00 equiv.) were added. The mixture was stirred at 25° C. for 12 hours. LC-MS showed that compound 3-2 was completely consumed and the desired mass (R t =0.43 min). The reaction mixture was used directly for purification. The residue was purified by preparative HPLC (AcOH conditions). Compound 29 (200 mg, 124 μmol, 35.7% yield, 95.3% purity) was obtained as a white solid and was characterized by LC-MS and HPLC.

[0656] LCMS:R t =0.43 min, MS calculated: 1601.73, MS observed: [M+H] + =1602.8.

[0657] HPLC:R t =3.50 minutes, purity: 95.3%.

[0658] Example 21: Synthesis of Compound 105 [ka] [ka] General procedure for the preparation of compound 3 [ka] A mixture of compound 1 (22.0 g, 68.9 mmol, 1.0 equiv.), compound 2 (15.0 g, 75.8 mmol, 1.1 equiv.), and K2CO3 (28.5 g, 206 mmol, 3.0 equiv.) in dioxane (220 mL) and HO (15 mL) was degassed and purged three times with N2. Pd(dppf)Cl2·CHCl2 (2.81 g, 3.45 mmol, 0.05 equiv.) was then added under N2 atmosphere and purged three times with N2. The mixture was stirred under N2 atmosphere at 80 °C for 8.0 h. LCMS showed that compound 1 was completely consumed, and the desired mass (MS calculated: 311.1, MS found: [M-tBu]) was obtained. + One main peak (R = 256.1) t =0.48 min). The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 30 / 1 to 10 / 1) to give compound 3 (19.6 g, 57.4 mmol, 83.2% yield, 90.7% purity) as a white solid, which was confirmed by HNMR, LCMS, and HPLC.

[0659] LCMS:R t =0.48 min, MS calculated value: 311.1, MS observed value: [M-tBu] + =256.0.

[0660] HPLC:R t =3.21 minutes, purity: 90.7%.

[0661] 1 H NMR(400MHz,DMSO-d6)δ:9.96(s,1H),9.28(s,1H),7.83(d,J=8.25Hz,2H),7.37-7.44(m,3H),7.26(br d,J=8.25Hz,1H),7.17(t,J=7.82Hz,1H),6.84(d,J=7.50Hz,1H),3.98(s,2H),1.45(s,9H).

[0662] General procedure for the preparation of compound 5 [ka] A mixture of compound 4 (45.0 g, 99.4 mmol, 1.0 equiv.) and HBF (654 g, 2.98 mol, 464 mL, 40% in HO, 30 equiv.) was stirred at 25 °C for 16 h. LCMS showed that compound 4 was completely consumed and the desired mass (MS calculated: 412.1, MS found: [M+H] + =413.0) t =0.31 min) was detected. The reaction mixture was filtered, and the filter was dried under reduced pressure to give compound 5 (39.0 g, crude product) as a white solid, which was confirmed by LCMS and HPLC.

[0663] LCMS:R t =0.31 min, MS calculated value: 412.1, MS observed value: [M+H] + =413.0.

[0664] HPLC:R t =1.40 minutes, purity: 97.4%.

[0665] General procedure for the preparation of compound 6 [ka] A solution of compound 6 (5.00 g, 12.1 mmol, 1.0 equiv) and MgSO (7.30 g, 60.6 mmol, 5.0 equiv) in ACN (100 mL) was stirred at 25 °C for 1.0 h. A solution of compound 3 (4.15 g, 13.3 mmol, 1.1 equiv) in ACN (100 mL) was then added, followed by the dropwise addition of TfOH (9.10 g, 60.6 mmol, 5.36 mL, 5.0 equiv) to the mixture, maintaining the temperature below 25 °C. The mixture was stirred at 25 °C for 3 h. LCMS showed that compound 5 was completely consumed, with the desired mass (MS calculated: 605.2, MS found: [M+H]). + =606.3) t=0.37 min). The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TFA conditions) to give compound 6 (3.74 g, 6.18 mmol, 50.9% yield, 91.6% purity) as a white solid, which was confirmed by LCMS and HPLC.

[0666] LCMS:R t =0.36 min, MS calculated: 605.2, MS observed: [M+H] + =606.3.

[0667] HPLC:R t =2.31 minutes, purity: 91.9%.

[0668] General procedure for the preparation of compound 7 [ka] A solution of compound 6 (2.40 g, 4.13 mmol, 1.0 equiv) in THF (12.5 mL) was cooled to −40° C., and then POCl (11.3 g, 45.1 mmol, 6.25 mL, 10.9 equiv) was added dropwise. The mixture was stirred at −40° C. for 2.0 h. LCMS showed that compound 6 was completely consumed, with the desired mass (MS calculated: 685.2, MS found: [M+H]). + One main peak (R = 686.4) t =0.49 min). The mixture was quenched with saturated NaHCO3 at 0 °C and then lyophilized to give the crude product. The crude product was purified by preparative HPLC (NH4HCO3 condition) to give compound 7 (750 mg, 1.09 mmol, 26.5% yield, 99.1% purity) as a white solid, which was confirmed by LCMS and HPLC.

[0669] LCMS:R t =0.50 min, MS calculated value: 685.2, MS observed value: [MH] + =684.3.

[0670] HPLC:R t =2.46 minutes, purity: 99.1%.

[0671] General procedure for the preparation of compound 45-2A [ka] To a solution of compound 45-1A (20.0 g, 114 mmol, 1.0 equiv.) in ACN (120 mL) were added DCC (25.9 g, 125 mmol, 25.4 mL, 1.1 equiv.), pyridine (27.1 g, 342 mmol, 27.6 mL, 3.0 equiv.), and compound 45-1B (14.8 g, 125 mmol, 18.0 mL, 1.1 equiv.). The mixture was stirred at 25° C. for 3.0 hours. TLC (petroleum ether / ethyl acetate=3:1) confirmed that compound 45-1A (R f =0.20) is completely consumed and one new spot (R f =0.57) was formed. The reaction mixture was diluted with HO (400 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with saturated NaHCO solution (200 mL) and brine (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure at 35 °C to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20:1 to 1:3) to give compound 45-2A (20.0 g, 72.6 mmol, yield 60.0%, purity 94.1%) as a yellow oil, which was confirmed by LCMS and HPLC.

[0672] LCMS:R t =0.48 min, MS calculated value: 275.42, MS observed value: [M+Na] + =298.0.

[0673] HPLC:R t =3.80 minutes, purity: 94.1%.

[0674] General procedure for the preparation of compound 45-3 [ka] Compound 45-2A (5.0 g, 18.1 mmol, 1.0 equiv.) was dissolved in HCl / dioxane (2.0 M, 10 mL). The mixture was stirred at 25° C. for 3.0 h. TLC (petroleum ether / ethyl acetate=3:1) confirmed that compound 45-2A (R f =0.57) is completely consumed and one new spot (R f =0.21) was formed. The reaction mixture was concentrated in vacuo to give compound 45-3 (4.0 g, crude product) as a yellow oil, which was confirmed by LCMS.

[0675] LCMS:R t =0.22 min, MS calculated value: 175.10, MS observed value: [M+Na] + =198.3.

[0676] General procedure for the preparation of compound 45-5 [ka] To a solution of compound 45-4 (1.7 g, 9.23 mmol, 1.0 equiv.) in DMF (4.0 mL), DIEA (3.58 g, 27.7 mmol, 4.82 mL, 3.0 equiv.) and HATU (4.21 g, 11.1 mmol, 1.2 equiv.) were added, and finally compound 45-3 (2.93 g, 13.8 mmol, 1.5 equiv., HCl salt) was added. The mixture was stirred at 25° C. for 2.0 h. LCMS showed the desired mass (R t =0.42 min), one main peak was detected. The residue was purified by preparative HPLC (TFA condition) to give compound 45-5 (2.76 g, 7.73 mmol, yield 83.8%, purity 95.7%) as a white solid, which was confirmed by LCMS and HPLC.

[0677] LCMS:R t =0.42 min, MS calculated value: 341.20, MS observed value: [M+Na] + =364.1.

[0678] HPLC:R t =3.06 minutes, purity: 99.5%.

[0679] General procedure for the preparation of compound 8 [ka] To a solution of compound 45-5 (1.30 g, 4.39 mmol, 1.0 equiv.) in DCM (4.0 mL), pyridine (1.04 g, 13.1 mmol, 1.06 mL, 3.0 equiv.) was added and cooled to 0 °C. Compound 45-4A (1.33 g, 6.59 mmol, 1.5 equiv.) in DCM (4.0 mL) was then added. The mixture was stirred at 25 °C for 4.0 h. LCMS showed that one peak (Rt = 0.53 min) with the desired mass (MS calculated: 506.2, MS found: [M + Na] + = 529.0) was detected. The solvent was removed under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 30 / 1 to 1 / 1) to give compound 8 (1.7 g, 2.40 mmol, yield 54.5%, purity 94.4%) as a pale yellow oil, which was confirmed by LCMS, HPLC, and HNMR.

[0680] LCMS:R t =0.53 min, MS calculated value: 506.2, MS observed value: [M+Na] + =529.2.

[0681] HPLC:R t =4.27 minutes, purity: 94.4%.

[0682] 1 H NMR(400MHz,chloroform-d)δ:8.25-8.32(m,2H),7.36-7.44(m,2H),6.00-6.14(m,2H),5.65(dd,J=16.70,2.06Hz,1H), 4.21-4.29(m,2H),3.96(dd,J=4.94,1.06Hz,2H),2.28-2.42(m,2H),2.14-2.27(m,2H),1.94-2.03(m,2H),1.89(br s, 1H), 1.75 (dd, J=15.13, 6.25Hz, 1H), 1.18 (s, 3H), 0.98-1.05 (m, 2H), 0.04 (s, 9H).

[0683] General procedure for the preparation of compound 9 [ka] To a solution of compound 8 (1.62 g, 3.21 mmol, 2.0 equiv.) and compound 7 (1.14 g, 1.60 mmol, 1.0 equiv.) in DMF (15 mL) was added HOAt (655 mg, 4.81 mmol, 673 μL, 3.0 equiv.) and DIEA (829 mg, 6.42 mmol, 1.12 mL, 4.0 equiv.). The mixture was stirred at 25° C. for 16 h. LCMS showed that compound 8 was completely consumed and the desired mass (MS calculated: 1052.4, MS found: [M−H]) was obtained. + One main peak (R = 1051.3) t =0.63 min) was detected. The mixture was purified by preparative HPLC (NHHCO conditions) to give compound 9 (829 mg, 787 μmol, 49.0% yield, 99.3% purity) as a white solid, which was confirmed by LCMS and HPLC.

[0684] LCMS:R t =0.63 min, MS calculated value: 1052.4, MS observed value: [MH] + =1051.2.

[0685] HPLC:R t =3.72 minutes, purity: 99.3%.

[0686] General procedure for the preparation of compound 105 [ka] To a solution of compound 9 (410 mg, 389 μmol, 1.0 equiv) in THF (3.0 mL) was added TBAF (1.0 M, 1.95 mL, 5.0 equiv) at 0° C. The mixture was stirred at 25° C. for 16 h. LCMS showed that compound 9 was completely consumed and the desired mass (MS calculated: 952.3, MS found: [M−H] + One main peak (R = 951.5) t=0.48 min). A second batch of compound 9 (455 mg) was run in parallel and combined for workup and purification. The reaction mixture was diluted with DMF (5.0 mL) and purified by preparative HPLC (AcOH conditions) to give compound 105 (210 mg, 220 μmol, 26.8% yield, 99.3% purity) as a white solid, which was confirmed by LCMS, HPLC, and FNMR. TIFF2026508188000283.tif108170

[0687] LCMS:R t =0.48 min, MS calculated value: 952.3, MS observed value: [MH] + =951.5.

[0688] HPLC:R t =2.34 minutes, purity: 99.3%.

[0689] 19 F NMR(376MHz,DMSO-d6)δ:-164.95(brs,1F),-186.36(s,1F).

[0690] Example 22: Synthesis of Compound 122 [ka] General procedure for the preparation of compound 2 [ka] To a solution of compound 1 (289 mg, 1.53 mmol, 1.10 equiv) in DMF (10.0 mL) was added HATU (794 mg, 2.09 mmol, 1.50 equiv). The mixture was stirred at 25° C. for 0.08 h. DIEA (540 mg, 4.18 mmol, 727 μL, 3.00 equiv) and MMAE (1.00 g, 1.39 mmol, 1.00 equiv) were added. The mixture was stirred at 25° C. for 1.5 h. LCMS showed that MMAE was completely consumed and the desired mass (R t=0.50 min). The reaction mixture was purified by preparative HPLC (TFA conditions). Compound 2 (880 mg, 960 μmol, yield 68.9%, purity 97.0%) was obtained as a white solid and was confirmed by LCMS and HPLC.

[0691] LMCS:R t =0.47 min, MS calculated: 888.59, MS observed: [M+H] + =889.5.

[0692] HPLC:R t =3.39 minutes, purity: 97.0%.

[0693] General procedure for the preparation of compound 3 [ka] To a solution of compound 2 (0.88 g, 960 μmol, 1.00 equiv.) in DCM (7.04 mL) was added TFA (2.70 g, 23.6 mmol, 1.76 mL, 24.6 equiv.). The mixture was stirred at 0° C. for 4 h. LCMS showed that compound 2 was completely consumed and the desired mass (R t =0.35 min). The reaction mixture was precipitated with 140 mL x 3 of isopropyl ether. Compound 3 (0.80 g, 701 μmol, yield 73.0%, purity 79.2%, TFA) was obtained as a white solid and was confirmed by LCMS and HPLC.

[0694] LCMS:R t =0.37 min, MS calculated value: 788.54, MS observed value: [M+H] + =789.7.

[0695] HPLC:R t =1.53 minutes, purity: 79.2%.

[0696] General procedure for the preparation of compound 4 [ka] To a solution of compound 4a (2.00 g, 10.8 mmol, 1.00 equiv.) in ACN (10.0 mL) was added DIEA (4.21 g, 32.5 mmol, 5.67 mL, 3.00 equiv.) and DSC (2.78 g, 10.8 mmol, 1.00 equiv.) in ACN (10.0 mL). The mixture was stirred at 25° C. for 1 hour. TLC (dichloromethane:methanol=10:1) confirmed that compound 4a (R f =0.35) is completely consumed and two new spots (R f LCMS showed that the desired mass (R = 0.00, 0.50) was formed. t =0.32 min). The reaction mixture was concentrated under reduced pressure to give a residue at 45 °C. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 1:1, petroleum ether:ethyl acetate = 1:1, R f =0.35) Compound 4 (1.82 g, 6.18 mmol, 56.9% yield, 95.5% purity) was obtained as a pale yellow solid and was characterized by LCMS, HPLC, and HNMR.

[0697] LCMS:R t =0.44 min, MS calculated value: 281.13, MS observed value: [M+H] + =304.0.

[0698] HPLC:R t =1.77 minutes, purity: 95.5%.

[0699] 1 H NMR(400MHz,CDCl3)δ:6.13-6.06(m,1H),5.68-5.64(m,2H),4.51(s,1H),2.82-2 .81(m,4H),2.41-2.25(m,3H),2.17-1.98(m,3H),1.93-1.83(m,2H),1.25(s,3H)

[0700] General procedure for the preparation of compound 122 [ka] To a solution of compound 3 (0.80 g, 701 μmol, 1.00 equiv, TFA) in DMF (8.00 mL) was added compound 4 (206 mg, 701 μmol, 1.00 equiv) and DIEA (181 mg, 1.40 mmol, 244 μL, 2.00 equiv). The mixture was stirred at 25° C. for 5 h. LCMS showed that compound 3 was completely consumed and the desired mass (R t =0.45 min). The reaction mixture was purified by preparative HPLC (AcOH conditions). Compound 122 (Peak 1, 210 mg, 202 μmol, 28.9% yield, 98.1% purity, AcOH) was obtained as a white solid and was characterized by LCMS, HPLC, and HRMS.

[0701] NOTE: Two peaks of compound 122 were obtained after preparative HPLC. TIFF2026508188000289.tif27170

[0702] The spectrum of compound 122_peak 1 is shown below.

[0703] LCMS:R t =0.46 min, MS calculated value: 954.64, MS observed value: [M+H] + =955.8. HRMS:R t =1.80 min, MS calculated value: [M+H] + 955.6478, MS observed value: [M+H] + =955.6491. HPLC:R t =351 minutes, purity: 98.1%.

[0704] The compounds in Table A-1 and Table B-1 can be or were prepared according to the procedures described herein using the appropriate starting materials.

[0705] The therapeutic support compositions described herein can be prepared according to the procedures described in WO2018 / 187740. Methods for testing and using the conjugates in combination with the support compositions can also be found in WO2018 / 187740.

[0706] Example 23: Efficacy evaluation of compound 32 as a single agent and in combination with SQT01 in the NCI-N87 human gastric tumor xenograft model Study Objective: The objective of this study was to evaluate the antitumor activity of compound 32 as a single agent and in combination with SQT01 in the NCI-N87 human gastric tumor xenograft model. The significant endpoint was tumor growth inhibition.

[0707] Experimental design TIFF2026508188000290.tif60170

[0708] Materials: Animals and housing conditions TIFF2026508188000291.tif46170 Test item TIFF2026508188000292.tif55170TIFF2026508188000293.tif61170TIFF2026508188000294.t if31170TIFF2026508188000295.tif51170TIFF2026508188000296.tif109170TIFF2026508188 000297.tif43170TIFF2026508188000298.tif44170TIFF2026508188000299.tif31170TIFF202 6508188000300.tif55170TIFF2026508188000301.tif37170TIFF2026508188000302.tif145170

[0709] Results: Body weight change and tumor growth curves: Body weight change and tumor growth curves are shown in Figures 1 and 2. No obvious weight loss was observed among all groups during this experiment.

[0710] Summary of results: Mouse body weight and weight changes are summarized in Figures 1 and 2. All mice were in good condition, and all treatments were well tolerated by tumor-bearing CrTac:NCr-Foxn1nu mice. No obvious weight loss was observed in any group during the treatment period, indicating that the drugs were well tolerated by animals.

[0711] The tumor volumes in the different groups are shown in Figure 2. Group 2 (SQT01 + Compound 32) shows antitumor effects compared to the vehicle group.

[0712] Example 24: In vitro cell viability assessment Test objectives The purpose of this study was to evaluate the antiproliferative effects of test compounds (compounds 32 and 33) in five cancer cell lines (Calu-3, HCC827, MDA-MB-468, NCI-N87, and T47D) with or without tetrazine reagent. Deruxtecan, exatecan, and staurosporine were included as positive controls. The study was performed based on an internal standard.

[0713] Test Design Cell plating and compound treatment is shown in the plate map below. TIFF2026508188000303.tif62170TIFF2026508188000304.tif57170TIFF2026508188000305.tif62170TIFF2026508188000306.tif57170

[0714] Cancer cells were maintained under culture conditions at 37°C in an atmosphere containing 0% or 5% CO2 in air. Tumor cells were subcultured periodically. Cells growing in the logarithmic growth phase were counted using a hemocytometer using trypan blue staining. After counting, the cell concentration was adjusted to an appropriate cell density. TIFF2026508188000307.tif38170

[0715] 90 μL of cell suspension was plated onto assay plates according to the plate map, and 90 μL of assay medium was plated into blank wells. Plates were incubated overnight at 37°C, 0% or 5% CO2, 95% air, and 100% relative humidity.

[0716] Preparation of compound stock plates For each test compound, an equal volume of 20 mM compound solution in DMSO was mixed with DMSO or 20 mM tetrazine in DMSO. The solution was allowed to age for 15-20 minutes at ambient temperature protected from light. This solution was designated the 10 mM compound stock. Preparation of compound stock plate (1000x stock plate): Stock solutions were serially diluted in DMSO from highest to lowest concentration according to the plate map (Table 4). These were prepared fresh for use. TIFF2026508188000308.tif55170

[0717] To prepare the 10x concentrated compound plate, 198 µL of assay medium was added to each well of a V-bottom plate, followed by the addition of 2 µL of stock compound solution of each concentration from the 500x stock plate. Following this, 2 µL of DMSO was added to blank and control wells.

[0718] For compound treatment, 10 μL of compound-medium from each well of the 10x concentrated compound plate was added to the cells in the 96-well assay plate according to the plate map. 10 μL of DMSO medium was added to blank and control wells. The final DMSO concentration was 0.1%. The assay plate was placed in an incubator and incubated for 3 days.

[0719] From this point on, the procedure was carried out according to the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit manual (Promega-G7573).

[0720] The test article results are summarized in the table below. TIFF2026508188000309.tif65170

[0721] All compounds were stored at −20° C. A summary of the cell viability assay results is shown in Table 5.6. TIFF2026508188000310.tif86170

[0722] Overall, the data indicated that compounds 32 and 33 pretreated with tetrazine reagent inhibited cell proliferation more effectively than compounds without tetrazine reagent pretreatment. The IC50 values ​​for compound 32 pretreated with tetrazine reagent ranged from 0.005 to 0.115 μM, and for compound 32 without tetrazine reagent pretreatment ranged from 0.023 to 0.429 μM in all five cell lines. The IC50 values ​​for compound 33 pretreated with tetrazine reagent ranged from 0.004 to 0.072 μM in all five cell lines. The IC50 values ​​for compound 33 without tetrazine reagent pretreatment were 9.154, 4.388, and 1.545 μM in MDA-MB-468, NCI-N87, and T47D cells, respectively, and were greater than 10 μM in both Calu-3 and HCC827 cells.

[0723] 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.

[0724] The invention illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies) not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" are intended to be read broadly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention to use such terms and expressions to exclude any equivalents of the features shown and described, or portions thereof, recognizing that various modifications are possible within the scope of the claimed disclosure.

[0725] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0726] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative and not limiting of 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 this disclosure pertains.

Claims

1. A conjugate of formula AI or formula A-II, or a pharmaceutically acceptable salt thereof 【Chemistry 1】 And, During the ceremony, m is an integer from 1 to 10, r is 1 or 2; Each D １ is independently a taxane, topoisomerase inhibitor, or MMAE payload, or a derivative or analog thereof; L １ and L ２ are each independently a linker, G is an optionally substituted trans-cyclooctene moiety; Each S １ are independently solubilizing groups, However, the compound is 【Chemistry 2-1】 【Chemistry 2-2】 is not a conjugate, or a pharmaceutically acceptable salt thereof.

2. The part 【Transformation 3】 is represented by formula A-IIA 【Chemistry 4】 It is of During the ceremony, q is 0, 1, or 2; m is an integer from 1 to 10, R １Ａ In each occurrence, C １－４ Alkyl, C １－４ Haloalkyl, and C １－４ independently selected from the group consisting of alkoxy; L ２ is the linker, Each S １ are independently solubilizing groups, The conjugate of claim 1.

3. The part 【Transformation 5】 is represented by formula A-IIA 【Transformation 6】 It is of During the ceremony, m is an integer from 1 to 10, R １Ａ is C １－４ Alkyl, C １－４ Haloalkyl, and C １－４ alkoxy; L ２ is the linker, Each S １ are independently solubilizing groups, 3. The conjugate of claim 1 or 2.

4. Each S １ is -NHC(NH)NH ２ , -P(O)(OH) ２ , -S(O) ２ OH, -(OCH ２ CH ２ ) ３０－８５ -OCH ３ , -N(CH ２ CH ２ C(O)OH) ２ ,or 【Transformation 7】 10. The conjugate of any preceding claim, independently selected from the group consisting of:

5. Each S １ is -NHC(NH)NH ２ , -P(O)(OH) ２ , -S(O) ２ OH, -N(CH ２ CH ２ C(O)OH) ２ ,or 【Transformation 8】 10. The conjugate of any preceding claim, independently selected from:

6. L １ is -OC(O)-, -C(O)O-, -NR １ｆ C(O)- or -C(O)NR １ｆ - and R １ｆ is hydrogen, C １－６ Alkyl, or C ０－４ Alkylene-CO ２ 10. The conjugate of any preceding claim, wherein R is H.

7. L １ is -OC(O)- ａａ or -NHC(O)- ａａ and bond aa is D １ 10. A conjugate according to any preceding claim, wherein the conjugate is linked to

8. L ２ 4. A conjugate according to any preceding claim, wherein is a linear or branched heteroalkylene linker.

9. 10. A conjugate according to any preceding claim, wherein m is 1.

10. 10. A conjugate according to any preceding claim, wherein m is 2.

11. L ２ teeth, -Y １０ -C ０－３ Alkylene-C(R １００ ) ｎ’ [((CH ２ ) ｎ’’ -Y ２０ ) ｍ’ - (CH ２ ) ｍ’’ -Y ３０ ] ｎ’－１ - and During the ceremony, 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)-, -C(O)O-, -OC(O)O-, alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene, each of the alkylene, alkenylene, alkynylene, arylene, heteroarylene, cycloalkylene, or heterocycloalkylene independently represents oxo, halo, C １－４ Alkyl, C １－４ Alkoxy, and C １－４ optionally substituted with 1 to 5 substituents independently selected from haloalkyl; Each R １００ are independently hydrogen, —C(O)OH, C １－４ Alkyl, C １－４ haloalkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl; 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'', m', and m'' are each independently 0, 1, 2, 3, 4, 5, 6, 7, or 8; n' is 2; A conjugate according to any of the preceding claims.

12. The part 【Chemistry 9】 teeth, 【Chemistry 10】 10. A conjugate according to any preceding claim, wherein:

13. D １ 10. A conjugate according to any preceding claim, wherein is paclitaxel or isotaxel, or a derivative or analogue thereof.

14. D １ teeth, 【Chemistry 11】 10. A conjugate according to any preceding claim, wherein:

15. D １ The conjugate of any one of claims 1 to 12, wherein is exatecan, or a derivative or analogue thereof.

16. D １ teeth, 【Chemistry 12】 The conjugate of any one of claims 1 to 12 or 15, wherein

17. D １ The conjugate of any one of claims 1 to 12, wherein is MMAE, or a derivative or analogue thereof.

18. D １ teeth, 【Chemistry 13】 The conjugate of any one of claims 1 to 12 or 17, wherein

19. 10. A conjugate according to any preceding claim, wherein r is 1.

20. 10. A conjugate according to any preceding claim, wherein r is 2.

21. A conjugate of formula BI, or a pharmaceutically acceptable salt thereof 【Chemistry 14】 And, During the ceremony, G is an optionally substituted trans-cyclooctene moiety; L １ is the linker, m is 1 or 2; Each D １ independently 【Chemistry 15-1】 【Chemistry 15-2】 a payload selected from the group consisting of however, D １ but 【Chemistry 16】 then m is 2; or D １ but 【Chemistry 17】 and m is 1, G is [Chemistry 18] or 【Chemistry 19】 is, or L １ teeth, 【Chemistry 20】 or containing a disulfide, The conjugate, or a pharmaceutically acceptable salt thereof.

22. G is, 【Chemistry 21】 22. The conjugate of claim 21, selected from the group consisting of:

23. D １ teeth, 【Chemistry 22-1】 【Chemistry 22-2】 23. The conjugate of claim 21 or 22, wherein:

24. A conjugate selected from Table A-1, or a pharmaceutically acceptable salt thereof.

25. A conjugate selected from Table B-1, or a pharmaceutically acceptable salt thereof.

26. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

27. A method for treating cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of the conjugate of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 26, wherein a therapeutic support composition is administered to the patient, the therapeutic support composition comprising a biocompatible support and a compound of the formula 【Chemistry 23】 and a tetrazine-containing group of the formula: During the ceremony, 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, t is 0, 1, 2, 3, or 4; method.

28. 28. The method of claim 27, wherein the tetrazine-containing group is linked or directly attached to the antibody biocompatible support.

29. A method for treating cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of the conjugate of any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 26, and a therapeutic support composition, wherein the therapeutic support composition comprises a biocompatible support and a compound of the formula 【Chemistry 24】 and a tetrazine-containing group of the formula: During the ceremony, 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, t is 0, 1, 2, 3, or 4; method.

30. 30. The method of any one of claims 27 to 29, wherein the tetrazine-containing group is linked or directly bonded to a hyaluronic acid biocompatible support.

31. The therapeutic support composition has the formula (II) 【Chemistry 25】 and a substituted hyaluronic acid unit of In the formula, G ２ teeth, 【Chemistry 26】 and R ２２ The method of any one of claims 27 to 30, wherein is a linker of 1 to 100 linked atoms.

32. G ２ teeth, 【Chemistry 27】 32. The method of claim 31 , wherein:

33. G ２ teeth, 【Chemistry 28】 and R ２０ is hydrogen or C １－４ 33. The method of claim 32, wherein the alkyl is alkyl.

34. The method according to any one of claims 27 to 33, wherein the method is a method for treating cancer.

35. 35. The method of claim 34, 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, head / neck squamous cell carcinoma, 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, malignant extrinsic or intrinsic airway compression, or cutaneous T-cell lymphoma.

36. 36. The method of claim 34 or 35, wherein the cancer is a solid tumor.

37. 36. The method of claim 34 or 35, wherein the cancer is a soft tissue sarcoma.

38. 36. The method of claim 34 or 35, wherein the cancer is lung cancer.

39. 36. The method of claim 34 or 35, wherein the cancer is malignant extrinsic airway compression or intrinsic airway compression.

40. 35. The method of claim 34, wherein the cancer is a hematological 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.

41. The method according to any one of claims 27 to 40, wherein the method is a method for enhancing or inducing an immune response.

42. 42. The method of claim 41, wherein the immune response is an increase in one or more of leukocytes, lymphocytes, monocytes, and eosinophils.

43. 43. The method of any of claims 27-42, further comprising administering a therapeutically effective amount of an additional therapeutic agent selected from the group consisting of an anti-cancer agent or a trans-cyclooctene prodrug thereof.

44. A kit comprising the conjugate according to any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, or the therapeutic support composition according to any one of claims 27 to 33, and instructions for use thereof.

45. 45. The kit of claim 44, further comprising a therapeutic support composition as defined in any one of claims 27 to 33.

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