Compounds containing cleavable linkers and uses thereof

Abstract

Compounds containing cleavable linkers, uses thereof, and intermediate compounds for preparing the same are provided. More specifically, the compounds containing the cleavable linkers of the present invention may contain an active agent (e.g., a drug, a toxin, a ligand, a detection probe, etc.) having a specific function or activity, an -S(=O)(=N-)- functional group that can selectively release the active agent, and a functional group that induces a chemical reaction, a physicochemical reaction, and / or a biological reaction in response to an external stimulus, and may further contain a ligand (e.g., an oligopeptide, a polypeptide, an antibody, etc.) that has binding specificity for a desired target receptor.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 788,039, filed January 3, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have emerged as a powerful class of antitumor agents with efficacy across a variety of cancers. ADCs commonly consist of three distinct features: a cell-binding or targeting moiety, a linker, and a cytotoxic drug. The linker component of an ADC is a key feature in developing targeted anticancer drugs with desirable target specificity, i.e., high activity in tumor cells but low activity in healthy cells.

[0003] Thus, there is a need for improved linkers useful in the preparation of ADCs. Summary of the Invention

[0004] A conjugate of formula (I'): (DL) n -(CB) cb (I') or a pharmaceutically acceptable salt thereof, During the ceremony, CB is a targeting moiety, cb and n are each independently an integer having a value of 1 to about 20, preferably 1 to about 10; each DL is independently a group having a structure of formula (I") or formula (I'"); [ka] each Q is independently an activator linked to L' by a heteroatom, preferably O or N; Z', in each occurrence, independently converts the structure of formula (I") or formula (I"') to (CB)cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, provided that at least one occurrence of Z' converts the structure of Formula (I") or Formula (I"') to (CB) cb provided that it is connected to each L' is independently a spacer moiety attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, preferably O or N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- promotes cleavage of the bond between L' and Q, releasing the active agent; Each X is independently -O-, -C(R b )2-, or -N(R c )-, preferably -O-, Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and positioned such that when y is 1, the N, O, or S atom is bonded to TG; When activated, TG reacts with -S(=O)(=N-)- to form (Q) q -(L') w is an inducing group that generates an N, O, or S atom capable of replacing and forming a 5- to 6-membered ring containing an intervening atom of XS(=O)(=N-)- and Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each Ra and R c are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the atom to which they are attached, However, when w is 0, q is 1.

[0005] In certain preferred embodiments, E is 0.

[0006] In some embodiments, the present invention relates to a composition (eg, a pharmaceutical composition) comprising a compound of Formula (I') and a carrier (eg, a pharmaceutically acceptable carrier).

[0007] In certain embodiments, the present invention provides conjugates of formula (I') and compositions comprising such conjugates for use, e.g., in therapy, imaging, as sensors, as molecular switches, as molecular machines, and / or as nanomachines.

[0008] In some embodiments, the present invention provides conjugates of formula (I') and pharmaceutical compositions thereof for use in methods for delivering active agents to cells, wherein the targeting moiety is selected to bind to a molecule associated with the target cell. In particular, the compounds, conjugates, and compositions of the present invention may be useful for inhibiting abnormal cell growth or treating proliferative disorders in mammals (e.g., humans), such as when the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least associated preferentially with tumor cells over healthy cells).

[0009] The conjugates of formula (I') of the present invention and pharmaceutical compositions thereof may be useful for treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), transplant rejection, lupus, myositis, infectious diseases, immunodeficiencies such as AIDS, and inflammatory diseases in mammals (e.g., humans). [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the results of an enzymatic cleavage assay of compound A-1. [Figure 2] 1 shows the results of an enzymatic cleavage assay of compound A-2. [Figure 3] 1 shows the results of an enzymatic cleavage assay of compound A-3. [Figure 4] 1 shows the results of an enzymatic cleavage assay of compound A-4. [Figure 5] 1 shows the results of an enzymatic cleavage assay of compound B-1. [Figure 6] 1 shows the results of a stability study of compound A-2 in various plasmas. [Figure 7] 1 shows the results of a stability study of compound A-2 in various plasmas. DETAILED DESCRIPTION OF THE INVENTION

[0011] In some embodiments, the present invention relates to compounds and conjugates containing cleavable linkers and their uses. Representative compounds and conjugates disclosed herein comprise an active agent (e.g., a chemical agent, a biological agent, a hormone, an oligonucleotide, a drug, a toxin, a ligand, a detection probe, etc.) having a desired function or activity, a functional group that undergoes a chemical reaction (e.g., a physicochemical reaction and / or a biological reaction) under predetermined conditions to release a nucleophilic heteroatom, and an —S(═O)(═N—)— functional group positioned proximal to the nucleophilic heteroatom, thereby reacting with the nucleophilic heteroatom in an intramolecular cyclization reaction to release the active agent. In some embodiments, the compounds and conjugates disclosed herein further comprise a targeting moiety (e.g., an oligopeptide, a polypeptide, an antibody, etc.) that has binding specificity for a desired target receptor or other molecule associated with a target cell.

[0012] definition The meaning of the term "alkyl" is understood in the art. For example, "alkyl," used alone or as part of a larger moiety such as "alkoxy," "haloalkyl," "cycloalkyl," "heterocycloalkyl," etc., may refer to a straight-chain or branched hydrocarbon that is fully saturated. Typically, straight-chain or branched alkyl groups are acyclic groups having 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms, unless otherwise specified. Examples of straight-chain and branched alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups. Alkyl groups having two possible valencies are sometimes referred to with the suffix "ene," as in alkylene. Exemplary alkylene groups include methylene, ethylene, propylene, and the like.

[0013] Moreover, the term "alkyl" (or "lower alkyl") can include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety in which substituents replace a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those of skill in the art will understand that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, substituents of substituted alkyls may include alkyl groups, amino groups, azide groups, imino groups, amido groups, phosphoryl groups (including phosphonates and phosphinates), sulfonyl groups (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as substituted and unsubstituted forms of ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0014] For example, "C" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. x ~C y The term "C" may include groups containing x to y carbons in the chain, where "x" and "y" are integers selected from 1 to about 20, and x is an integer less than y, and x and y are not the same value. For example, "C x ~C yThe term "C2-C alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups containing x to y number of carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. y -alkenyl" and "C2-C y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond respectively. When applied to heteroalkyl, "C x ~C y " indicates that the group contains x to y number of carbon and heteroatoms in the chain. When applied to carbocyclic structures such as aryl and cycloalkyl groups, "C x ~C y " indicates that the ring contains x to y carbon atoms in the ring.

[0015] The term "alkoxy" is art-recognized and may refer to, for example, an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0016] The terms "hal," "halo," and "halogen" are used interchangeably throughout and refer to fluorine or fluoro (F), chlorine or chloro (Cl), bromine or bromo (Br), or iodine or iodo (I).

[0017] The term "cycloalkyl" is art-recognized and may refer, for example, to a substituted or unsubstituted cyclic hydrocarbon that is fully saturated. Cycloalkyl includes monocyclic and bicyclic rings. Typically, monocyclic cycloalkyl groups have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms, unless otherwise specified. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, three, or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each ring shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0018] The term "aryl" has an art-recognized meaning and may refer, for example, to a substituted or unsubstituted monocyclic aromatic group in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is aromatic, and the other cyclic rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0019] The terms "heterocyclyl" and "heterocycle" have art-recognized meanings and may refer, for example, to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, in which the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. Such heterocycles also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is heterocyclic, and in which, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0020] The term "heteroaryl" has an art-recognized meaning and may refer, for example, to a substituted or unsubstituted aromatic monocyclic ring structure, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring, in which the ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is heteroaromatic, and the other cyclic rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0021] The term "substituted" refers to a moiety in which a substituent replaces a hydrogen on one or more carbon or heteroatoms of the moiety. Those of ordinary skill in the art will recognize that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permissible valences of the replacing atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds.

[0022] In some embodiments, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Substituents may include any of the substituents described herein, such as, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkyl, aralkyl, or aromatic or heteroaromatic moieties. Those of skill in the art will understand that the substituents themselves can be substituted, where appropriate. Unless expressly specified as "unsubstituted," references to chemical moieties herein are understood to include substituted variations. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variations.

[0023] The term "subject" to which administration is contemplated includes, for example, humans (i.e., male or female of any age, e.g., a pediatric subject (e.g., infant, child, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially significant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially significant birds such as chickens, ducks, geese, quail, and / or turkeys. A preferred subject is a human.

[0024] As used herein, a therapeutic agent that "prevents" a disorder or condition can refer to, for example, a compound that, in a statistical sample, reduces the incidence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0025] The term "treating" includes prophylactic and / or therapeutic treatments. The term "prophylactic or therapeutic" treatment is art-recognized and includes administration of one or more of the subject compositions to a host. A treatment is prophylactic (i.e., it protects the host against the development of the unwanted condition) if it is administered prior to the clinical manifestation of an unwanted condition (e.g., a disease or other unwanted condition in a host animal), whereas a treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing unwanted condition or its side effects) if it is administered after the manifestation of the unwanted condition.

[0026] In certain embodiments, the compounds and conjugates disclosed herein may be used alone or conjointly administered with another type of therapeutic compound or agent. As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic compounds, such that a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective in the subject simultaneously, which may involve a synergistic effect of the two compounds). For example, the different therapeutic compounds and conjugates may be administered simultaneously or sequentially, either in the same formulation or in separate formulations. In certain embodiments, the different therapeutic compounds and conjugates may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other. Thus, a subject receiving such treatment may benefit from the combined effects of the different therapeutic compounds and conjugates.

[0027] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably herein. As used herein, unless otherwise specified, "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the abnormal growth of: (1) tumor cells (tumors) that grow due to the expression of mutated tyrosine kinases or the overexpression of receptor tyrosine kinases; (2) benign and malignant cells of other proliferative disorders in which aberrant tyrosine kinase activation occurs; (3) any tumor that grows due to receptor tyrosine kinases; (4) any tumor that grows due to aberrant serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative disorders in which aberrant serine / threonine kinase activation occurs.

[0028] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell cancer), small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancer including adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, acute leukemia, and head / brain and neck cancer.

[0029] Compounds and conjugates of the present invention The present disclosure provides a conjugate of formula (I'): (DL) n -(CB) cb (I') or a pharmaceutically acceptable salt thereof, During the ceremony, CB is a targeting moiety, cb and n are each independently an integer having a value of 1 to about 20, preferably 1 to about 10; each DL is independently a group having a structure of formula (I") or formula (I'"); [ka] each Q is independently an activator linked to L' by a heteroatom, preferably O or N; Z', in each occurrence, independently converts the structure of formula (I") or formula (I"') to (CB) cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, wherein at least one occurrence of Z′ converts the structure of Formula (I″) or Formula (I′″) to (CB): cb provided that it is connected to each L' is independently a spacer moiety attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, preferably O or N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- promotes cleavage of the bond between L' and Q, releasing the active agent; Each X is independently -O-, -C(R b )2-, or -N(R c )-, preferably -O-, Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and positioned such that when y is 1, the N, O, or S atom is bonded to TG; When activated, TG reacts with -S(=O)(=N-)- to form (Q) q -(L') w is an inducing group that generates an N, O, or S atom capable of replacing and forming a 5- to 6-membered ring containing an intervening atom of XS(=O)(=N-)- and Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the atom to which they are attached, However, when w is 0, q is 1.

[0030] In certain preferred embodiments, E is 0.

[0031] Each active agent can be any suitable active agent, as described in more detail below. While many traditional conjugation methods require the presence of certain functional groups, such as amine or hydroxyl groups, to form stable bonds, the disclosure herein provides strategies for forming connections using functional groups, such as phenols and tertiary amines, to form stable bonds in the conjugates disclosed herein while still allowing for release under defined conditions that activate the triggering group.

[0032] Many suitable triggering groups are known in the art, and exemplary triggering groups and the conditions for activating them are discussed below, such as the moieties described for Y below. Some triggering groups contain an N, O, or S atom but are in a non-nucleophilic form. For example, an NO group is a triggering group that is reduced to an NH or NHOH group, which can react with -S(=O)(=N-)-, under reducing conditions, and an acetate group is a triggering group that is hydrolyzed to a hydroxyl group, which can react with -S(=O)(=N-)-, under hydrolysis conditions. Other triggering groups do not contain an N, O, or S atom but are converted to a nucleophilic N, O, or S atom upon activation. For example, a boronate group is a triggering group that is converted to a hydroxyl group, which can react with -S(=O)(=N-)-, under oxidizing conditions (e.g., with peroxide). Preferably, the triggering group is selected so that the conditions for activating it result in selective activation without cleaving or degrading other portions of the conjugate, such as the targeting moiety. Once a nucleophilic N, O, or S atom is generated, it attacks the -S(=O)(=N-)- moiety intramolecularly to form a ring, forming moiety (Q). q -(L') w It ejects an --H, where the H is attached to the heteroatom of Q or L' that was originally attached to the --S(.dbd.O)(.dbd.N--)- moiety.

[0033] In embodiments where w is 0, q is 1 and Q is directly bonded to -S(=O)(=N-)- through a heteroatom. Thus, activation of the triggering group generates a nucleophilic heteroatom that intramolecularly attacks the -S(=O)(=N-)- moiety to form a ring and eject the activator QH, where H is bonded to the heteroatom originally bonded to -S(=O)(=N-)-.

[0034] In embodiments where w is 1, L' may be selected to allow attachment of multiple occurrences of Q, and Q may be the same or different. Thus, each occurrence of Q is indirectly attached to -S(=O)(=N-)- via a spacer moiety. In such embodiments, activation of the triggering group generates a nucleophilic heteroatom, which intramolecularly attacks the -S(=O)(=N-)- moiety to form a ring, resulting in the moiety (Q). q -L'-H is ejected, where H is attached to the heteroatom in L' that was originally attached to -S(=O)(=N-)-. In such embodiments, the released heteroatom induces an intramolecular reaction to eject the active agent(s) Q (such as when Q has a tertiary amine that was attached to L' as a quaternary ammonium) or QH. For example, the heteroatom may undergo an intramolecular cyclization reaction with the ester moiety formed with the hydroxyl of QH to form a ring and eject the active agent QH. Alternatively, the heteroatom may undergo intramolecular tautomerization to eject the active agent Q or QH.

[0035] Ar can be any suitable ring, including a bicyclic or other polycyclic ring, that maintains the moieties undergoing intramolecular cyclization in close proximity to facilitate reaction after activation of the initiating group. The planar nature of aromatic and heteroaromatic rings is preferred because the rigid geometry of substituents on such rings ensures favorable positioning of reactive moieties, although other types of rings, such as cycloalkenyl or heterocycloalkenyl, can impose similar geometries. The five- or six-membered ring, and / or the number or identity of heteroatoms in the ring, and / or substituents on the other ring (e.g., electron-donating or electron-withdrawing substituents), can be selected to adjust the cyclization rate based on the resulting ring bond angle. Similarly, the more flexible steric structures of cycloalkyl and heterocyclyl rings can be useful when it is desired to slow the rate of intramolecular cyclization.

[0036] Z' can be any suitable linking group connecting Ar to one or more CB groups. Typically, the linking group should be sufficiently hydrophilic to promote aqueous solubility (solubilizing groups) and prevent aggregation of the conjugate, for example, by including moieties such as polyethylene glycol (PEG) moieties, peptide sequences, charge-carrying moieties (carboxylates, amines, nitrogen-containing rings, etc.) to balance the hydrophobic nature of any alkyl chains that may be included. Because it is often advantageous to prepare conjugates in a modular manner, Z' may also contain a linking unit, i.e., a functional group resulting from the conjugation of one reactive moiety to another. Representative linking units are discussed in more detail below (e.g., in connection with variable Z), and common linking groups include amides, triazoles, oximes, carbamates, etc. Representative Z' groups include L, as discussed in more detail below. 1’ In some embodiments, all of the DL groups attached to each CB are the same, while in other embodiments, each CB may be attached to two or more distinct DL groups. For example, some DL groups may have triggering groups that are activated under a first condition, while other DL groups may have triggering groups that are activated under a second condition, thereby allowing, for example, one active agent to be selectively released under a first condition, while a second active agent to be selectively released under a second condition.

[0037] The present disclosure also provides compounds that can serve as intermediates or reagents in the formation of the group DL in formula (I'), as described in formula (I'') or formula (I'''). Thus, in some embodiments, a compound of formula (Ia) or formula (Ia'): [ka] or a pharmaceutically acceptable salt thereof, wherein: each Q is independently an activator linked to L' through a heteroatom, preferably O or N; Z', in each occurrence, independently represents absence, a structure of formula (Ia) or a structure of formula (Ia') as (CB)cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, provided that at least one occurrence of Z' converts the structure of Formula (Ia) or Formula (Ia') to (CB): cb provided that it is connected to each L' is independently a linking group attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, preferably O or N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- facilitates cleavage of the bond between L' and Q, releasing the active agent; Each X is independently -O-, -CR a 2-, or -NR'-, preferably -O-; Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and positioned such that when y is 1, the N, O, or S atom is bonded to TG; When activated, TG reacts with -S(=O)(=N-)- to form (Q) q -(L') w is an inducing group that generates an N, O, or S atom capable of replacing and forming a 5- to 6-membered ring containing an intervening atom of XS(=O)(=N-)- and Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each R a and R care independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the carbon atom to which they are attached, However, when w is 0, q is 1.

[0038] In some embodiments, Z' is independently in each occurrence a reactive group (eg, a precursor group).

[0039] In certain preferred embodiments, E is 0.

[0040] In certain embodiments of Formulas (I'), (Ia), and (Ia'), -Y' is -(CH2) y NR''-, -(CH2) y O- or -(CH2) y S- and y is 1, the N, O, or S atom is positioned to bind to TG, R″ is hydrogen or C1-C6-alkyl, and y is an integer having a value of 0 or 1. In some such embodiments, TG is a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0041] In some embodiments of formula (I'), (Ia), and (Ia'), (L') w is an activator that links each Q to —S(═O)(═N—)—, and each Q is linked to one of the L′ groups by a heteroatom, preferably O or N, to form an —O—, —OC(O)—, —OC(O)O—, or —OC(O)NH— bond with the Q heteroatom.

[0042] In other embodiments, (Q) q -(L') w - is selected from the following: [ka] During the ceremony, each Q is independently an activator linked to L' by a heteroatom, preferably O or N; X 4 is absent or forms an —O—, —OC(O)—, —OC(O)O—, or —OC(O)NH— bond together with the heteroatom of Q; X 1 is -O- or -NR a - and X 2 is -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH-, X 3 is -OC(=O)-, w' is an integer having a value of 1, 2, 3, 4, or 5; X is -O-, -C(R b )2-, or -N(R c )-, preferably -O-, each Z″ is independently alkyl, aryl, or heteroaryl, wherein alkyl, aryl, and heteroaryl are unsubstituted or substituted with one or more substituents; R 9 and R 10 are each independently hydrogen, alkyl, aryl, or heteroaryl, where alkyl, aryl, and heteroaryl are unsubstituted or substituted, e.g., alkyl, —(CH) u NH2, -(CH2) u NR u1 R u2 , and -(CH2) u SO2R u3 and is substituted with one or more substituents selected from R u1 , R u2 , and R u3 are each independently hydrogen, alkyl, aryl, or heteroaryl; u is an integer having a value of 1 to about 10.

[0043] In some such embodiments, (Q) q -(L')w is selected from: [ka]

[0044] Furthermore, the present invention provides intermediates for preparing conjugates according to formula (Ia) or (Ia') or compounds according to formula (Ia), wherein (Q) q -(L') w (Q) q -(L') w is replaced by a leaving group such as a halogen (preferably fluorine) to allow for the bonding of

[0045] In certain such embodiments, Z' comprises a reactive group (e.g., a precursor group as discussed in more detail below with respect to Z) that can be used to attach the compound to an inducer such as CB (e.g., to prepare a compound of formula (I') as discussed in more detail above), to a solid surface (e.g., to form a bead, nanoparticle, solid support array, or sensor particle), or to any other molecule or support of interest. In certain preferred embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657153000007.tif114170In the formula, R 1 is a C1-C6 alkyl, R 21 and R 22 are each independently hydrogen or C1-C6 alkyl.

[0046] In other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657153000009.tif67166

[0047] In still other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657153000011.tif66166

[0048] In other embodiments, the compound of formula (I') is selected from: [ka]

[0049] In certain preferred embodiments, Z is a linking group having the structure of formula (F), (G), (H), (J), (K), (L), (M), or (N): [ka] During the ceremony, * is the point of attachment to CB, ** is the point of attachment to Ar, R e is alkyl, X'' is -O-, -S-, -NH-, or -CH2-; X 4 is -NHC(O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, W b1 and W b2 are each independently -C(O)NH-, -NHC(O)-, [ka] L 2 is an optionally present spacer moiety, C1-C6 alkyl, C5-C 14 It may be further substituted with one or more substituents such as aryl, and C3-C8 heteroaryl, where alkyl, aryl, and heteroaryl are, for example, C1-C 10Alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 , -(CH2) u CO2H, -(CH2) u CO2R u1 , and -(CH2) u SO2R u3 and optionally further substituted with one or more substituents selected from the group consisting of: u1 , R u2 , and R u3 are each independently hydrogen, C1 to C 15 Alkyl, C6-C 20 Aryl, or C3-C 10 heteroaryl, and u is an integer having a value of 1 to about 10; R 12 is hydrogen, C1-C8 alkyl, or an amino acid moiety such as a naturally occurring amino acid moiety; b, c, d, e, g, h, o, and qq are each independently an integer having a value from 1 to about 10; s' is an integer having a value of 1 to about 10.

[0050] In other embodiments, Z is a linking group having the structure of formula (F'), (G'), (H'), (J'), (K'), (L'), (M'), or (N'). [ka] TIFF0007657153000016.tif128151

[0051] In certain preferred embodiments, CB is selected from: [ka]

[0052] In certain preferred embodiments, (Q) q -(L') w is selected from: [ka] TIFF0007657153000019.tif217146TIFF0007657153000020.tif232162TIFF0007657153000021.tif108162

[0053] Also, a compound of formula (Ib) or formula (Ib'): [ka] or a pharmaceutically acceptable salt thereof, wherein: X is -O-, -CH2-, or -NR'-; R' is hydrogen, C1-C6-alkyl, C6-C 14 -Aryl or C2-C 20 -heteroaryl, Ar is C5 to C 20 -Aromatic ring, C2~C 20 -Heteroaromatic rings, C2-C 30 -Fused ring or C5-C 20 -Aromatic ring -C2~C 20 -heteroaromatic ring, R is Ar or -L 1’ -Z-(CB) cb , preferably -L 1’ -Z-(CB) cb is the above substituent, L 1’ C1-C further containing at least one of a peptide bond, an amino bond, an ether bond, a triazole bond, a tetrazole bond, a sugar bond, a sulfonamide bond, a phosphonate bond, a sulfo bond, or a dendrimer structure. 200 -Alkylene or C1-C 200 is alkylene, Z is CB and L 1’ is a linking unit or reactive group (e.g., a precursor group that allows for connection to CB) that connects Z', in each occurrence, independently represents absence, a structure of formula (Ib) or a structure of formula (Ib') as follows: cba linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, which connects to CB is a targeting moiety such as a ligand that has receptor binding properties; cb is an integer having a value of 0, 1, or 2; n is an integer having a value of 1, 2, 3, or 4; Y is -NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -NHOH, -NHNH2, -BR 2 R 3 , [ka] or -Y'-TG, preferably, Y is -NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -NHNH2, -BR 2 R 3 , [ka] or -Y'-TG, R 1 is a C1-C6 alkyl, r is an integer having a value of 1, 2, 3, 4, or 5; Ar 1 C6~C 20 is an arylene, R 2 and R 3 are each independently hydrogen, C1-C6-alkyl, C1-C6-alkoxy, or hydroxy, R a , R b , R c , and Rd are each independently hydrogen or C1-C6 alkyl, Y' is -(CH2) x NR''-, -(CH2) x O- or -(CH2) x S- and R" is hydrogen or C1-C6 alkyl; x is an integer having a value of 0 or 1; TG is the inducing group, Q is -Q 1 or -L'-(Q 1 ) w and L' has -O- or -NR'''- at one end and -O-, -OC(O)-, -O(CO)O-, -OC(O)NR'''-, or -OC(O)NR at the other end. 4 C7-C with CH2O- 30 - a hydrocarbon spacer, where -O-, -OC(O)-, -O(CO)O-, or -OC(O)NR''''- is a C7-C 30 The hydrocarbon spacer may further include C7 to C 30 Hydrocarbon spacers are C1-C6 alkyl, C5-C 14 and further substituted with one or more substituents such as aryl, and C3-C8 heteroaryl, where alkyl, aryl, and heteroaryl are, for example, C1-C 10 Alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 , -(CH2) u CO2H, -(CH2) u CO2R u1 , and -(CH2) u SO2R u3 and optionally further substituted with one or more substituents selected from the group consisting of: u1 , R u2 , and R u3 are each independently hydrogen, C1 to C 15 Alkyl, C6-C 20 Aryl, or C3-C 10heteroaryl, and u is an integer having a value of 1 to about 10; Q 1 is an activator containing at least one functional group of -OH, -NH-, -NR5R6, -SH, -SO2NH2, or -COOH; R 4 is hydrogen, C1-C6 alkyl, C5-C 14 -aryl, or C3-C8-heteroaryl, where alkyl, aryl, and heteroaryl are substituted or unsubstituted; R 5 and R 6 are each independently hydrogen, C1-C6-alkyl, C3-C9-cycloalkyl, or C5-C 10 -heteroaryl, wherein heteroaryl is substituted or unsubstituted; R''' and R'''' are each independently hydrogen or C1-C6-alkyl; w is an integer having a value of 1, 2, 3, 4, or 5.

[0054] In some embodiments, compounds of Formula (I) contain a functional group (e.g., Y) that can induce intramolecular cyclization upon an external stimulus. In certain embodiments, the functional group is introduced at the ortho position relative to X.

[0055] In some embodiments, R' is C1-C6-alkyl, C6-C 14 -Aryl or C2-C 20 -heteroaryl.

[0056] In some embodiments, Ar is C-C 20 -Aromatic ring, C2~C 20 -Heteroaromatic rings, C2-C 30 -Fused ring or C5-C 20 -Aromatic ring -C2~C 20-heteroaromatic ring. For example, Ar may be a benzene ring, a naphthalene ring, a pyridine ring, or a quinolone ring. Preferably, Ar is a benzene ring or a naphthalene ring. In some embodiments, the compound of Formula (Ib) or Formula (Ib') is a compound having a structure according to Formula (II) or Formula (II'): [ka] TIFF0007657153000026.tif31160 or a pharmaceutically acceptable salt thereof.

[0057] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (III) or formula (III'): [ka] or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, the compound is of Formula (Ib), (Ib'), (II), (II'), (III), or (III'), where R is hydrogen, halogen, aldehyde, acetal, ketal, -R * , -OR * , -SR * , -NR * R ** , -C(hull)3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, -N3, -NC, -C(O)R * , -OC(O)R * , -OS(O)R * , -S(O)2R * , -S(O)2OR * , -OS(O)OR * , -OS(O)2OR * , -S(O)NR * R ** , -S(O)NR * R ** , -S(O)R * , -OP(O)(OR * )2, -P(O)(OR * )2, -OP(OR* )2, -OP(OR * )N(R ** )2, -OP(O)(OR * )N(R ** )2, -PR * , -P(O)2, -P(O)R * , -C(O)Hal, -C(S)R * , -CO2R * , -C(S)OR * , -C(O)SR * , -C(S)SR * , -C(O)NR * R ** , -C(S)NR * R ** , -C(=NR * )NR * R ** , -NR * C(O)R ** , -NR * S(O)2OR ** , -NR * S(O)R ** , -NR * C(O)NR ** , -SS-R * , or -R * SSR ** where R * and R ** are each independently hydrogen, C1 to C 18 Alkyl, C6-C 20 Aryl, C3-C 15 Heterocycle, or C3-C 20 It is heteroaryl.

[0059] In some embodiments, the compound is of Formula (Ib), (Ib'), (II), (II'), (III), or (III'), wherein R is hydrogen or * -(L a -A1-L b -L c -Z) m -CB, where L a is a single bond or C1-C 20 - alkylene, A1 is -C(O)NR * -, -NR * C(O)-, -NR * -, -O-, -PO3-, -OPO3-, -SO-, -SO2-, -S(=O)(=N-)-, or -SO3-; L b is -(CH2CH2O) a -or-(CH2) a - and R * is hydrogen, C1 to C 18 -Alkyl, C6-C 20 -Aryl, C3-C 15 -Heterocycle, or C3-C 20 is heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C1-C 20 - alkylene, n is an integer having a value of 1 or 2; Z is CB and L c or Z can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 and dihydrogen phosphate (—OP(═O)(OH)), Z', in each occurrence, independently represents absence, a structure of formula (Ib), a structure of formula (Ib'), (II), (II'), (III), or (III') as (CB) cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, which connects to CB is a targeting moiety, such as a ligand, capable of binding to a receptor; m is an integer having a value of 0, 1, or 2.

[0060] In some embodiments, the compound is of Formula (Ib), (Ib'), (II), (II'), (III), or (III'), wherein R is hydrogen or * -L a -A1-L b -L c -Z, where L a is a single bond or C1-C 20 - alkylene, A 1 is -C(O)NR * -, -NR * C(O)-, -NR * -, -O-, -PO3-, -PO4-, -SO-, -SO2-, -S(=O)(=N-)-, or -SO3-; L b is -(CH2CH2O) a -or-(CH2) a - and R * is hydrogen, C1 to C 18 -Alkyl, C6-C 20 -Aryl, C3-C 15 -Heterocycle, or C3-C 20 -heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C1-C 20 - alkylene, n is an integer having a value of 1 or 2; Z can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 and dihydrogen phosphate (—OP(═O)(OH)), Z', in each occurrence, independently represents absence, a structure of formula (Ib), a structure of formula (Ib'), (II), (II'), (III), or (III') as (CB) cb The term "antibody" can be used to refer to a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigen, fragment of a polypeptide, or lipibody), active agent, or detectable moiety.

[0061] In some embodiments, the compound is of Formula (Ib), (Ib'), (II), (II'), (III), or (III'), wherein R is * (-L a -A1-L b -L c -Z) m -CB, where L a is a single bond or C1-C 20 - alkylene, A1 is -C(O)NR * -, -NR * C(O)-, -NR * -, -O-, -PO3-, -PO4-, -SO-, -SO2-, -S(=O)(=N-)-, or -SO3-; L b is -(CH2CH2O) a -or-(CH2) a - and R * is hydrogen, C1 to C 18 -Alkyl, C6-C 20 -Aryl, C3-C 15 -Heterocycle, or C3-C 20 -heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C1-C 20 - alkylene, n is an integer having a value of 1 or 2; Z is CB and L c is a concatenation unit that connects CB is a targeting moiety such as a ligand that has receptor binding properties; m is an integer having a value of 1 to 2.

[0062] In some embodiments, the compound is a compound of formula (Ib), (Ib'), (II), (II'), (III), or (III'), wherein L' is a C7-C alkyl group further comprising -O-, -OC(O)-, -O(CO)O-, or -OC(O)NR''''-. 30 It is a hydrocarbon spacer.

[0063] In some embodiments, the compound is of Formula (Ib), (Ib'), (II), (II'), (III), or (III'), where Q is -L'-(Q 1 ) w and [ka] During the ceremony, Q 1 -OH, -NR 5 R 6 an activator comprising at least one functional group selected from -SH, and -COOH; Q 2 is -NR 5 R 6 an activator comprising X 1 is -O- or -NR'''-, X 2 and X 4 are each independently absent or selected from —O—, —OC(O)—, —OC(O)O—, and —OC(O)NH—; X 3 is -OC(=O)-, Z″ is alkyl, aryl, or heteroaryl, wherein the alkyl, aryl, and heteroaryl are unsubstituted or substituted with one or more substituents; R 5 and R 6 is the same as defined above, R 9 and R 10 are each independently hydrogen, C1-C6 alkyl, C6-C 14 aryl, or C3-C9 heteroaryl, and R9 and R 10 The alkyl, aryl, and heteroaryl are C1 to C 10 Alkyl, -(CH2) u NH2, -(CH2) u NR u1 R u2 , and -(CH2) u SO2R u3 and R u1 , R u2 , and R u3 are each independently hydrogen, C1 to C 15 Alkyl, C6-C 20 Aryl, or C3-C 10 heteroaryl, and u is an integer having a value of 1 to about 10; R''' is hydrogen or C1-C6-alkyl, w is an integer having a value of 1, 2, 3, 4, or 5.

[0064] In certain embodiments, -L'-(Q 1 ) w teeth, [ka] is selected from.

[0065] -OH, -NR 5 R 6 Q, Q are selected from -SH and -COOH 1 , or Q 2 At least one functional group of serves as a point of attachment to L' of the active agent. This functional group may be present as part of an ester, thioester, carbonate, carbamate, amide, sulfonamide, sulfonate, sulfate, or other suitable bond, i.e., the -OH, -NR 5 R 6 The -SH, and -COOH moieties are not present by themselves while the active agent is part of the conjugate.

[0066] In some embodiments, Q 2 is -NR 5 R 6 and the activator can be attached at a quaternary amine structure, for example, -NR of the activator. 5 R 6 The moiety is capable of forming a quaternary amine bond with L'.

[0067] In some embodiments of formula (I″), (Ia), (Ib), (Ib′), (II), (II′), (III), or (III′), R 4 is a substituted alkyl, aryl, or heteroaryl. In some such embodiments, R 4 is C1~C 10 Alkyl, -(CH2) u NH2, -(CH2)u NR u1 R u2 , -(CH2) u CO2H, -(CH2) u CO2R u1 , and -(CH2) u SO2R u3 and wherein R u1 , R u2 , and R u3 are each independently hydrogen, C1 to C 15 -Alkyl, C6-C 20 -Aryl, or C3-C 10 -heteroaryl, and u is an integer having a value of 1 to about 10.

[0068] In some embodiments of formula (I″), (Ia), (Ib), (Ib′), (II), (II′), (III), or (III′), R 5 and / or R 6 is -NR 7 R 8 heteroaryl substituted with, where R 7 and R 8 are each independently hydrogen, C1-C6-alkyl, C3-C9-cycloalkyl, or C5-C 14 -aryl.

[0069] In some embodiments of formula (I″), (Ia), (Ib), (Ib′), (II), (II′)(III), or (III′), Q or -(L′) w -(Q) q is selected from: [ka] TIFF0007657153000033.tif202166TIFF0007657153000034.tif69154

[0070] In certain embodiments, provided herein are compounds of formula (Ib), (Ib'), (II), (II'), (III), or (III'), wherein: Y is -NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -NHOH, -BR 2 R 3 or -Y'-TG, preferably, Y is -NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -BR 2 R 3 or -Y'-TG, R 1 is a C1-C6 alkyl, r is an integer having a value of 1, 2, 3, 4, or 5; Ar 1 is phenylene, biphenylene, or naphthylene, R 2 and R 3 are each independently hydrogen, C1-C6-alkyl, C1-C6-alkoxy, or hydroxy, Y' is -(CH2) x NR''-, -(CH2) x O- or -(CH2) x S- and R" is hydrogen or C1-C6-alkyl, x is an integer having a value of 0 or 1; R" is hydrogen or C1-C6-alkyl, TG is a derivative group such as a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0071] In certain embodiments, the compound of formula (Ib), (Ib'), (II), (II'), (III), or (III') is selected from: [ka] TIFF0007657153000036.tif61167In formula, R 1 is a C1-C6 alkyl, R 21 and R 22 are each independently hydrogen or acetyl; R is hydrogen, * -L a -A1-L b -L c -Z, or a group having the structure of formula (F), (G), (H), (J), (K), (L), (M), or (N), [ka] L a is a single bond or C1-C 20 is alkylene, A 1 is -C(O)NH-, -NHC(O)-, -NH-, -O-, -PO3-, -PO4-, -SO-, -SO2-, -S(=O)(=N-)-, or -SO3-, L b is -(CH2CH2O) a -or-(CH2) a - and a is an integer having a value from 1 to about 20; L c is C1~C 20 is alkylene, X'' is -O-, -S-, -NH-, or -CH2-; W b1 and W b2 are each independently -C(O)NH-, -NHC(O)-, [ka] R 12 is hydrogen, C1-C8 alkyl, amino acid moiety, -(CH2) s COR 13 , or -(CH2) p NR 14 R 15 and R 13 is OH or -NH(CH2) s’ (X''CH2CH2)s’’ Z', R 14 and R 15 are each independently hydrogen or -(C(O)(CH) s’ (X''CH2CH2) s’’ Z) m -CB, X'' is -O-, -S-, -NH-, or -CH2-; R e is C1-C8 alkyl or -(L 1’ -Z) m -CB, X 4 is -NHC(O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, b, c, d, e, g, h, o, and q are each independently an integer having a value from 1 to about 10; p is an integer having a value from 1 to about 10; s and s″ are each independently an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; Z' can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 -Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10alkyl) or dihydrogen phosphate (-OP(=O)(OH)2), Z', in each occurrence, independently represents absence, a structure of formula (Ib) or a structure of formula (Ib') as follows: cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, which connects to CB is a ligand selected from: [ka] Q is selected from: [ka] TIFF0007657153000042.tif242156TIFF0007657153000043.tif162154

[0072] Active agent release As noted above, in certain embodiments, the compounds and conjugates disclosed herein can be coupled to one or more active agents (Q, Q) through an intramolecular cyclization reaction followed by a chemical reaction to activate the triggering group. 1 , Q 2 In certain embodiments, the chemical reaction is a physicochemical reaction and / or a biochemical reaction.

[0073] In some embodiments, the compounds and conjugates disclosed herein comprise a nucleophilic functional group (Y or Y') introduced on Ar at the atom adjacent to X (e.g., O). Typically, the nucleophilic functional group is masked by a triggering group (TG), as further detailed below. Upon activation, the triggering group releases the nucleophilic functional group, which reacts with a nearby -S(=O)(=N-)- moiety in an intramolecular cyclization, ultimately yielding one or more activators (Q, Q). 1 , or Q 2In some such embodiments, one or more active agents are released through a chemical, physicochemical, and / or biochemical reaction followed by an intramolecular cyclization reaction (see, e.g., Reaction Scheme 1), or the active agent(s) are released through an intramolecular cyclization reaction followed by 1,6-elimination or 1,4-elimination (see, e.g., Reaction Scheme 2).

[0074] As an example, the mechanism when Y is -Y'-TG is shown in Reaction Scheme 1. Reaction Scheme 1: [ka] Q is [ka] The mechanism for this case is shown in Reaction Scheme 2. Reaction Scheme 2: [ka]

[0075] In some embodiments, the Q 1 is an activator comprising at least one functional group selected from -OH, -NH-, -SH, and -COOH. According to these embodiments, Q, as further described herein, 1 is conjugated to a compound as described herein by a functional group selected from -OH, -NH-, -SH, and -COOH, e.g., ester, amide, thioester, carbamate, urea, oxime, hydrazone, etc. In some such embodiments, Q 2 Q 1 is used instead of Q 2 is an amine group-containing drug. In another embodiment, Q 2 is an activator capable of binding to the ammonium unit. 2 Q 2Upon release, it is capable of dissociating in its original form with the amine group, where the active agent can be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

[0076] In some embodiments, the compounds and conjugates disclosed herein are chemically and physiologically stable, and in some such embodiments, the compounds and conjugates disclosed herein reach the desired target cells with little dissociation of the active agent in the blood, thereby selectively releasing the drug.

[0077] Inducing group (TG) In some embodiments, the conjugates of the present invention comprise a triggering group (TG). TG is a group that is cleavable, preferably selectively cleavable, by a chemical reaction, such as a biological reaction. Generally, the triggering group serves to mask the nucleophilic nature of the Y or Y′ group, thereby providing stability to the compounds and conjugates disclosed herein (e.g., by preventing self-immolation or intramolecular cyclization before the conjugate reaches the target location or experiences a predetermined triggering condition). Upon activation, the triggering group releases the nucleophilic Y or Y′ group, allowing self-immolation or intramolecular cyclization to occur, as described above.

[0078] In some embodiments, the TG comprises a sequence (e.g., a peptide sequence) or moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which may be hydrolyzed by an enzyme (e.g., oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc.), and / or may comprise a moiety selected from phosphodiester, phospholipid, ester, β-galactose, β-glucose, fucose, oligosaccharide, etc.

[0079] In some embodiments, the TG comprises a reactive chemical moiety or functional group that can be cleaved under nucleophilic conditions (e.g., a silyl ether, a 2-N-acylnitrobenzenesulfonamide, an unsaturated vinyl sulfide, a sulfonamide after activation, a malondialdehyde-indole derivative, a levulinoyl ester, a hydrazone, or an acylhydrazone).

[0080] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved under basic reagent conditions (e.g., 2-cyanoethyl ester, ethylene glycolyl disuccinate, 2-sulfonylethyl ester, alkylthioester, or thiophenyl ester).

[0081] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by light irradiation (e.g., 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonates, coumarins, phosphotriesters, bis-arylhydrazones, or bimanbi-thiopropionic acid derivatives).

[0082] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by reducing agent conditions (eg, hydroxylamine, disulfide, levulinate, nitro, or 4-nitrobenzyl derivatives).

[0083] In some embodiments, the TG may contain reactive chemical moieties or functional groups that can be cleaved using acidic conditions (e.g., sugars, tert-butyl carbamate analogs, dialkyl or diaryl dialkoxysilanes, orthoesters, acetals, aconityls, hydrazones, β-thiopropionates, phosphoramidates, imines, trityl, vinyl ethers, polyketals, and alkyl 2-(diphenylphosphino)benzoate derivatives; alkyl esters, 8-hydroxyquinoline esters, and picolinic acid esters).

[0084] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved under oxidative conditions (eg, a boronate, a vicinal diol, a para-methoxybenzyl derivative, or a selenium compound).

[0085] In certain preferred embodiments, the TG comprises a sugar, which can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the triggering group is -NO2, which can be cleaved under reducing conditions. In certain preferred embodiments, the triggering group is boronate, which can be cleaved under oxidative conditions. In certain preferred embodiments, the triggering group is an ester, which can be cleaved under acidic, basic, or enzymatic conditions. In certain preferred embodiments, the triggering group is a hydrazone, which can be cleaved under nucleophilic or acidic conditions. In certain preferred embodiments, the triggering group is a hydroxylamine, which can be cleaved under reducing conditions.

[0086] sugar-derived groups In some embodiments, the compounds and conjugates disclosed herein comprise a saccharide derivative group, for example, a derivative group selected from: [ka] In the formula, each R 21 are independently hydrogen or OR 21 is selected to be a hydroxy protecting group (e.g., acetyl), and R 22is hydrogen or lower alkyl (e.g., C1-C6-alkyl). In certain embodiments, hydroxy protecting groups can be used in organic synthesis, including, but not limited to, methyl ether, methoxymethyl ether, methylthiomethyl ether, 2-methoxyethoxymethyl ether, bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether, tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, t-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-o-methyl-2-methyl-4 ... Examples of the silyl ether include, but are not limited to, 2,4,6-trimethylbenzoate, methyl carbonate, 2,2,2-trichloroethyl carbonate, 2,4,6-trichlorobenzoate, 2,4,6-trichlorobenzoate, 2,4,6-trichloroethyl carbonate ...

[0087] Protecting groups as triggering groups In some embodiments, TG is a group that can be cleaved by chemical reaction, physicochemical reaction, and / or biological reaction. In certain embodiments, TG is a protecting group. In some such embodiments, the protecting group is an amine protecting group, an alcohol protecting group, or a thiol protecting group.

[0088] Amine Protecting Groups In certain embodiments, the amine protecting group is a general protecting group that can be used in organic synthesis, including, but not limited to, m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, alkyl carbamate, 9-fluorenylmethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate (Teoc), t-butyl carbamate (Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), 8-quinolyl carbamate, 1-isopropylallyl carbamate (Ipaoc), ... acetamide, N-hydroxypiperidinyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenylmethyl carbamate, acetamide, chloroacetamide, trichloroacetamide, phenylacetamide, benzamide, N-phthalimide, N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, benzenesulfenamide, o-nitrobenzenesulfenamide, triphenylmethylsulfenamide, p-toluenesulfonamide, methanesulfonamide, and the like.

[0089] Alcohol Protecting Groups In certain embodiments, the alcohol protecting group is a general protecting group that can be used in organic synthesis, including, but not limited to, methyl ether, methoxymethyl ether (MOM ether), benzyloxymethyl ether (BOM ether), 2-(trimethylsilyl)ethoxymethyl ether (SEM ether), phenylthiomethyl ether (PTM ether), 2,2-dichloro-1,1-difluoroethyl ether, p-bromophenacyl ether, chloropropyl methyl ether, isopropyl ether, cyclohexyl ether, 4-methoxybenzyl, 2,6-dichlorobenzyl ether, 4-(dimethylaminocarbonyl)benzyl ether, 9-anthrylmethyl ether, 4-picolyl ether, methylthiomethyl ether (MTM ether), 2-methoxyethoxymethyl ether (MEM ether), bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether (THP ether), tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothio Pyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, t-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether (TMS ether), isopropyldimethylsilyl ether Silyl ether, t-butyldimethylsilyl ether (TBDMS ether), t-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivaloate ester, adamantoate ester, benzoate ester, 2,4,6-trimethylbenzoic acid (mesitoate) ester, methyl carbonate, carbonate 2,2,These include, but are not limited to, 2-trichloroethyl, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, N-phenyl carbamate, nitrate ester, 2,4-dinitrophenylsulfenic acid ester, dimethylphosphinyl ester (DMP ester), dimethylthiophosphinyl ester (MPT ester), arylmethanesulfonate, aryltoluenesulfonate, and the like.

[0090] Thiol Protecting Groups In certain embodiments, thiol protecting groups can be used in organic synthesis, including, but not limited to, S-benzyl thioether, Sp-methoxybenzyl thioether, So- or p-hydroxyl or acetoxybenzyl thioether, Sp-nitrobenzyl thioether, S-4-picolyl thioether, S-2-picolyl N-oxide thioether, S-9-anthrylmethyl thioether, S-9-fluorenylmethyl thioether, S-methoxymethyl monothioacetal, A-acetyl derivatives, S-benzoyl derivatives, S-(N-ethylcarbamate), S-(N-methoxymethylcarbamate), and the like.

[0091] linking group In some embodiments, the compounds and conjugates disclosed herein comprise a linking group that covalently connects each CB and Ar. Exemplary linking groups include, for example, C 10 ~C 100 It is a stable, non-hydrolyzable moiety, such as a linear or branched, saturated or unsaturated alkylene. In certain embodiments, the linking unit satisfies at least two, and more preferably at least three, of the following four criteria: (i) at least one -CH2- in the alkylene moiety is replaced with (i.e., replaced by) one or more heteroatoms selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) at least one heteroarylene is contained in an alkylene moiety; (iii) at least one amino acid moiety, glycoconjugate, peptide bond, or amide bond is contained in the alkylene moiety; and (iv) Alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH and -(CH2) p NH2, where s is an integer having a value of 0 to 10 and p is an integer having a value of 1 to about 10.

[0092] In certain embodiments, the linking unit comprises at least two, more preferably at least three, of the following: (i) at least one heteroatom selected from —NH—, —C(═O), —O—, —S—, and —P—; (ii) at least one heteroarylene; (iii) at least one amino acid moiety, glycobond, peptide bond, or amide bond; and (iv) Alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH and -(CH2) p NH2, where s is an integer having a value of 0 to 10 and p is an integer having a value of 1 to about 10.

[0093] In other embodiments, the linking group connecting each CB and Ar comprises a functional group produced through click chemistry.

[0094] In an alternative embodiment, the linking unit comprises a reactive functional group capable of participating in a click chemistry reaction.

[0095] Click chemistry is a reaction that can be performed under mild conditions and is highly selective for functional groups not commonly found in biomolecules (e.g., azide groups, acetylene groups, etc.). Therefore, this reaction can be carried out in the presence of conjugation triggering groups, targeting moieties, etc. Furthermore, click chemistry has high reaction specificity. For example, the click chemistry reaction between an azide group and an acetylene group proceeds selectively without interference from other functional groups present in the molecule. For example, azide-acetylene click chemistry can produce triazole moieties in high yield.

[0096] Thus, in some embodiments, the linking group connecting each CB and Ar is: [ka] and V is a single bond, -O-, -S-, or -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2- or -SO2NR 25 -, -NR 24 -S(=O)(=N-)- or -S(=O)(=N-)-NR 25 - may be R 21 ~R 25 are each independently hydrogen, (C1-C6) alkyl, (C1-C6) alkyl(C6-C 20 ) aryl, or (C1-C6) alkyl (C3-C 20 ) heteroaryl, r can be an integer having a value of 1 to about 10, p can be an integer having a value of 0 to about 10, q can be an integer having a value of 1 to about 10, and L'' can be a single bond.

[0097] In other embodiments, the linking unit connecting each CB and Ar is a linking group represented by formula (A): ** -L c -W b1 -(CH2) b -W a3 -(P 1 ) a -Y2 -W a2 -Y 1 -W a1 - * (A) During the ceremony, * is the point of attachment to CB, ** is the point of attachment to Ar, W a1 , W a2 , and W a3 are each independently -NH-, -C(=O)-, or (-CH2-) b and W b1 is an amide bond or a triazolylene, P 1 is W a3 and Y 2 is a linker connecting L c is alkylene, Y 2 is a single bond, -W a4 -(CH2) c -W b2 -(CH2) d -W a5 -, or -W a6 -(CH2) e -CR e R f -X-, R e is C1-C8 alkyl or CB-W a7 -Y3-W c1 -(CH2) f - and R f is BW a7 -Y 3 -W c1 -(CH2) f - and X is -NHC(=O)-(CH2) g -W a8 - or -C(=O)NH-(CH2) h -W a9 - and W a4 , Wa5 , W a6 , W a7 , W a8 , and W a9 are each independently -NH-, -C(=O)-, or -CH2-; W b2 is an amide bond or a triazolylene, W c1 is -NHC(=O)- or -C(=O)NH-, Y 3 is -(CH2) i -(X'CH2CH2) j -(CH2) k - and X' is -O-, -S-, -NH-, or -CH2-; CB is as defined above, b, c, d, e, f, g, h, i, and j are each independently an integer having a value from 1 to about 10; k and y are each independently an integer having a value from 0 to about 10; Y 1 is -(CH2) q -(CH2CH2X'') o -or-(CH2) q -(X''CH2CH2) o - and X'' is -O-, -S-, -NH-, or -CH2-; o and q are integers having a value of 1 to about 10.

[0098] In some embodiments, P 1 comprises at least one unit represented by formula (B) or (C), [ka] During the ceremony, R 12is hydrogen, C1-C8-alkyl, amino acid side chains such as natural amino acid side chains (e.g., H, methyl, isopropyl, isobutyl, sec-butyl, S-methylthioether, benzyl, indole, pyrrolidine, pyrroline, hydroxymethyl, tyrosyl, lysyl, imidazole, glycyl, glutamyl, carbamoylbutanoic acid, carboxamide, aspartic acid, 1-hydroxyethyl, and 2-hydroxyethyl), -(CH2) s COR 13 , or -(CH2) p NR 14 R 15 and R 13 is OH or -NH(CH2) s’ (X''CH2CH2) s’’ Z, R 14 and R 15 are each independently hydrogen or -(C(O)(CH) s’ (X''CH2CH2) s’’ Z) m -CB, X'' is -O-, -S-, -NH-, or -CH2-; Z and CB are as defined above; p is an integer having a value from 1 to about 10; s and s″ are integers having values ​​from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1.

[0099] In some embodiments of formula (B) or (C), R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R 13is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''-(CB) m and R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''-(CB) m and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH2-; Z'' is CB and R 14 Or R 15 or Z'' is a linking group that connects the remainder of the formula (I) to the remainder of the formula (I), or Z'' is a linking group that includes a reactive group.

[0100] In some such embodiments of formula (B) or (C), R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z'' and R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z'' and Z'' can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 phosphate (-O-P(=O)(OH)2), and dihydrogen phosphate (-O-P(=O)(OH)2).

[0101] In other such embodiments of formula (B) or (C): R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''CB, Z'' is CB and R 14 or R 15 isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH2-hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 and dihydrogen phosphate (-OP(=O)(OH)).

[0102] In some embodiments, Y 2 is a single bond or is selected from: [ka] During the ceremony, W b2 -C(O)NH-, -NHC(O)-, [ka] R e is C1-C8 alkyl or -(L 1’ -Z-) m CB, R f is BW b2’ -(CH2) i -(X''CH2CH2) j -NH-C(=O)-(CH2) f - and X a is -NHC(=O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, W b2 ’ is -C(O)NH- or -NHC(=O)-, c, d, e, f, g, h, i, and j are each independently an integer having a value from 1 to about 10; X'' is -O-, -S-, -NH-, or -CH2-; L 1’ , Z, m, and B are the same as defined above.

[0103] In certain embodiments, the linking units connecting each CB and Ar are connected to each other by a covalent bond (CH) b , L c , (P 1 ) a , W a1 , W a2 , W a3 , Y 1 , and Y 2is a linking group comprising a group, wherein W a1 , W a2 , and W a3 are each independently -NH-, -C(O)-, or -CH2-; W b1 is an amide bond or a triazolylene, P 1 is an amide bond, an amino acid residue, or a peptide, L c is alkylene, Y 1 is -(CH2) q -(CH2CH2X'') o -or-(CH2) q -(X''CH2CH2X'') o - and X'' is -O-, -S-, -NH-, or -CH2-; Y 2 is a single bond or a group selected from the following: [ka] TIFF0007657153000055.tif15167W b2 is an amide bond or a triazolylene, a is 0 to 10, b, c, and d are each independently an integer having a value from 1 to about 10; o and q are each independently an integer having a value of 1 to about 10.

[0104] In some embodiments, R 12 is a natural amino acid side chain. In another embodiment, R 12 is an unnatural amino acid side chain.

[0105] In some embodiments, the linking unit connecting each CB and Ar is a linking group represented by formula (A): ** -L c -W b1 -(CH2) b -W a3-(P 1 ) a -Y 2 -W a2 -Y 1 -W a1 - * (A) During the ceremony, * is the point of attachment to CB, ** is the point of attachment to Ar.

[0106] In some such embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s COOH, or -(CH2) p NH2, p is an integer having a value from 1 to about 10; s and s″ are each independently an integer having a value of 0 to about 10.

[0107] In some embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''-(CB) m and R14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''-(CB) m and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH2-; Z'' is CB and R 14 Or R 15 or Z'' is a linking group that connects the remainder of the formula (I) to the remainder of the formula (I), or Z'' is a linking group that includes a reactive group.

[0108] P 1 In some such embodiments, R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z'' and R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z'' and Z'' can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where Ra is C1~C 10 phosphate (-O-P(=O)(OH)2), and dihydrogen phosphate (-O-P(=O)(OH)2).

[0109] P 1 In other such embodiments, R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''CB, Z'' is CB and R 14 or R 15 isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH2-hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 and dihydrogen phosphate (-OP(=O)(OH)).

[0110] In an alternative embodiment, the linking unit connecting CB and Ar is a linking group represented by formula (F), (G), (H), (J), (K), (L), (M), or (N): [ka] During the ceremony, R e is alkyl, X 4 is -NHC(O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, e, g, and h are each independently an integer having a value from 1 to about 10; s' is an integer having a value of 1 to about 10.

[0111] In some embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''-(CB) m and R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''-(CB) m and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH2-; Z'' is CB and R 14 Or R 15or Z'' is a linking group that connects the remainder of the formula (I) to the remainder of the formula (I), or Z'' is a linking group that includes a reactive group.

[0112] In some such embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z'' and R 14 and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z'' and Z'' can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 phosphate (-O-P(=O)(OH)2), and dihydrogen phosphate (-O-P(=O)(OH)2).

[0113] In some such embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 13 is OH or -NH(CH2) s’ (X'''CH2CH2) s’’ Z''CB, R 14and R 15 are each independently hydrogen or -C(O)(CH) s’ (X'''CH2CH2) s’’ Z''CB, Z'' is CB and R 14 or R 15 isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH2-hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where R a is C1~C 10 and dihydrogen phosphate (-OP(=O)(OH)).

[0114] targeting part The compounds and conjugates of the present invention may further comprise a ligand or targeting moiety, CB. In some embodiments, the ligand or targeting moiety is any molecular recognition element that can undergo specific interaction with at least one other molecule through non-covalent bonding, such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonding, electrostatic effects, and / or electromagnetic effects. In certain embodiments, the CB is selected from a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment of an antigenic polypeptide, a repebody, and the like.

[0115] The compounds and conjugates of the invention may comprise one or more targeting moieties, i.e., the variable cb may have an integer value selected from 1, 2, 3, 4, 5, 1-10, or 1-20.

[0116] In some embodiments, the CB comprises two or more independently selected natural or unnatural amino acids conjugated by a covalent bond (e.g., a peptide bond), and the peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural or unnatural amino acids conjugated by peptide bonds. In some embodiments, the ligand includes shorter amino acid sequences (e.g., fragments of natural proteins or synthetic polypeptide fragments) as well as full-length proteins (e.g., pre-engineered proteins).

[0117] In some embodiments, the CB is selected from an antibody, hormone, drug, antibody analog (e.g., non-IgG), protein, oligopeptide, polypeptide, etc. that binds to a receptor. In certain embodiments, the CB selectively targets a drug to a specific organ, tissue, or cell. In other embodiments, the CB specifically binds to a receptor that is overexpressed in cancer cells compared to normal cells and can be classified as a monoclonal antibody (mAb) or antibody fragment, and a small molecule non-antibody. Preferably, the CB is selected from a peptide, tumor cell-specific peptide, tumor cell-specific aptamer, tumor cell-specific carbohydrate, tumor cell-specific monoclonal antibody, polyclonal antibody, and antibody fragment identified in a library screen.

[0118] Exemplary ligands or targeting moieties include carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B 12, other water-soluble vitamins (vitamin B), fat-soluble vitamins (vitamins A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), transferrin, VIP (vasoactive intestinal peptide) receptor, APRPG (Ala-Pro-Arg-Pro-Gly) peptide, TRX-20 (thioredoxin-20), integrins, nucleolin, aminopeptidase N (CD13), endoglin, vascular epithelial growth factor receptor receptor), low-density lipoprotein receptor, transferrin receptor, somatostatin receptor, bombesin, neuropeptide Y, luteinizing hormone-releasing hormone receptor, folate receptor, epidermal growth factor receptor, transforming growth factor, fibroblast growth factor receptor, asialoglycoprotein receptor, galectin-3 receptor, E-selectin receptor, hyaluronan receptor, prostate-specific membrane antigen (PSMA), cholecystokinin A receptor, cholecystokinin B receptor, discoidin domain receptor, mucin receptor, opioid receptor, plasminogen receptor, bradykinin receptor, insulin receptor, insulin-like growth factor receptor, angiotensin AT1 receptor, angiotensin AT2 receptor, granulocyte-macrophage colony-stimulating factor receptor (GM-CSF receptor), galactosamine receptor, sigma-2 receptor, delta-like 3 (DLL-3), aminopeptidase P, melanotransferrin, leptin, tetanus toxin Tet1, tetanus toxin G23, RVG (rabies virus glycoprotein) peptide, HER2 (human epidermal growth factor receptor 2), GPNMB (glycoprotein nontransferase b), Ley, CA6, CanAng, SLC44A4 (solute carrier family 44 member 4), CEACAM5 (carcinoembryonic antigen associated Intercellular adhesion molecule 5), nectin-4, carbonic anhydrase 9, TNNB2, 5T4, CD30, CD37, CD74, CD70, PMEL17, EphA2 (ephrin A2 receptor), Trop-2, SC-16, tissue factor, ENPP-3 (AGS-16), SLITRK6 (SLIT and NTRK-like family member 6), CD27, Lewis Y antigen, LIV1, GPR161 (G protein-coupled receptor 161),PBR (peripheral-type benzodiazepine receptor), MERTK (Mer receptor tyrosine kinase) receptor, CD71, LLT1 (lectin-like transcript 1 or CLED2D), interleukin-22 receptor, sigma 1 receptor, peroxisome proliferator-activated receptor, DLL3, C4.4a, cKIT, ephrin A, CTLA4 (cytotoxic T lymphocyte-associated protein 4), FGFR2b (fibroblast growth factor receptor 2b), N-acetylcholine receptor, gonadotropin-releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein Receptor type 1B (BMPR1B), E16 (LAT1, SLC7A5), STEAP1 (prostate six-transmembrane epithelial antigen), 0772P (CA125, MUC16), MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b), ETBR (endothelin receptor type B), MSG783 (RNF124), STEAP2 (prostate six-transmembrane epithelial antigen 2), TrpM4 (transient receptor potential cation 5 channel, subfamily M, member 4), CRIPTO (teratocarcinoma-derived growth factor), CD 21, CD79b, FcRH2 (IFGP4), HER2 (ErbB2), NCA (CEACM6), MDP (DPEP1), IL20R-alpha (IN20Ra), brevican (BCAN), EphB2R, ASLG659 (B7h), CD276, PSCA (prostate stem cell antigen precursor), GEDA, BAFF-R (BR3), CD22 (BL-CAM), CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, SSTR1, SSTR3, SSRI, SSTR5, SSTR6, SSTR7. TR4, ITGAV (integrin, alpha 5), ​​ITGB6 (integrin, beta 6), MET, MUC1, EGFRvIII, CD33, CD19, IL2RA (interleukin 2 receptor, alpha), AXL, BCMA, CTA (cancer testis (tetis) antigen), CD174, CLEC14A, GPR78, CD25, CD32, LGR5 (GPR49), CD133 (prominin), ASG5, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase 3), PRR4 (proline-rich protein 4),GCC (guanylate cyclase 2C), Liv-1 (SLC39A6), CD56, CanAg, TIM-1, RG-1, B7-H4, PTK7, CD138, claudins, Her3 (ErbB3), RON (MST1R), CD20, TNC (tenascin C), FAP, DKK-1, CD52, CS1 (SLAMF7), annexin A1, V-CAM, gp100, MART-1, MAGE-1 (melanoma antigen coding gene) These include, but are not limited to, MAGE-3 (melanoma-associated antigen 3), BAGE, GAGE-1, MUM-1 (multiple myeloma oncogene 1), CDK4, TRP-1 (gp75), TAG-72 (tumor-associated glycoprotein-72), gangliosides GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

[0119] target In some embodiments, the target(s) of the molecular recognition element are specifically associated with one or more particular cell types or tissue types. In some embodiments, the target is specifically associated with one or more particular disease states. In some embodiments, the target is specifically associated with one or more particular developmental stages. For example, a cell type-specific marker is typically expressed at a level at least two-fold higher in that cell type than in a reference cell population. In some embodiments, a cell type-specific marker is present at a level at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, at least ten-fold, at least 50-fold, at least 100-fold, or at least 1,000-fold higher than its average expression level in a reference population. Detection or measurement of a cell type-specific marker may enable discrimination of a cell type(s) of interest from many, most, or all other types of cells. In some embodiments, the target may include proteins, carbohydrates, lipids, and / or nucleic acids, as described herein.

[0120] In some embodiments, a substance is considered to be "targeted" if it specifically binds to a targeting moiety, such as a nucleic acid targeting moiety. In some embodiments, a targeting moiety, such as a nucleic acid targeting moiety, specifically binds to a target under stringent conditions.

[0121] In certain embodiments, the conjugates and compounds described herein comprise a targeting moiety that specifically binds to one or more targets (e.g., antigens) associated with an organ, tissue, cell, extracellular matrix component, and / or intracellular compartment. In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that specifically binds to a target associated with a particular organ or organ system. In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that specifically binds to one or more intracellular targets (e.g., organelles, intracellular proteins). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that specifically binds to a target associated with a diseased organ, tissue, cell, extracellular matrix component, and / or intracellular compartment. In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that specifically binds to a target associated with a particular cell type (e.g., endothelial cells, cancer cells, malignant cells, prostate cancer cells, etc.).

[0122] In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular tissue types (e.g., liver tissue vs. prostate tissue). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular cell types (e.g., T cells vs. B cells). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular disease states (e.g., tumor cells vs. healthy cells). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular developmental stages (e.g., stem cells vs. differentiated cells).

[0123] In some embodiments, a target may be a marker associated exclusively or primarily with one or a few cell types, one or a few diseases, and / or one or a few developmental stages. A cell type-specific marker is typically expressed at a level at least two-fold higher in that cell type than in a reference cell population, which may consist, for example, of a mixture containing approximately equal amounts of cells from multiple (e.g., 5-10 or more) different tissues or organs. In some embodiments, a cell type-specific marker is present at a level at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold, at least eight-fold, at least nine-fold, at least ten-fold, at least 50-fold, at least 100-fold, or at least 1000-fold higher than its average expression level in the reference population. Detection or measurement of a cell type-specific marker may enable one to distinguish a cell type(s) of interest from many, most, or all other types of cells.

[0124] In some embodiments, targets include proteins, carbohydrates, lipids, and / or nucleic acids. In some embodiments, targets include proteins and / or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intracellular proteins, ion channels, membrane transporter proteins, enzymes, antibodies, chimeric proteins, glycoproteins, etc. In some embodiments, targets include carbohydrates and / or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, polysaccharides), glycocalyx (i.e., the carbohydrate-rich marginal zone on the outer surface of most eukaryotic cells), etc. In some embodiments, targets include lipids and / or characteristic portions thereof, such as oils, fatty acids, glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, lipoproteins, etc. In some embodiments, targets include nucleic acids and / or characteristic portions thereof, such as DNA nucleic acids; RNA nucleic acids; modified DNA nucleic acids; modified RNA nucleic acids; nucleic acids including any combination of DNA, RNA, modified DNA, and modified RNA.

[0125] Numerous markers are known in the art. Typical markers include cell surface proteins, such as receptors. Exemplary receptors include, but are not limited to, transferrin receptors; LDL receptors; growth factor receptors such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors; cytokine receptors; cell adhesion molecules; integrins; selectins; and CD molecules. Markers can be molecules that are present exclusively or in higher amounts on malignant cells, such as tumor antigens.

[0126] nanoparticles In some embodiments, the targeting moiety comprises a particle (e.g., a targeted particle), preferably a nanoparticle, optionally conjugated to a targeting molecule that can specifically or preferably bind to a target. In some embodiments, the targeted particle itself directs the compound of the invention (e.g., by enrichment in tumor cells or tissues), and no additional targeting molecule is conjugated to the targeted particle.

[0127] As used herein, "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In some embodiments, a therapeutic agent and / or targeting molecule can be associated with a group of particles, for example, in a polymer matrix. In some embodiments, a targeting molecule can be covalently associated with the surface of the polymer matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, a therapeutic agent can be associated with the surface of the polymer matrix, encapsulated within, surrounded by, and / or dispersed throughout the polymer matrix. See, e.g., U.S. Pat. No. 8,246,968, which is incorporated herein by reference in its entirety.

[0128] Generally, nanoparticles of the present invention include any type of particle. Any particle can be used in accordance with the present invention. In some embodiments, the particle is biodegradable and biocompatible. Generally, a biocompatible substance is not toxic to cells. In some embodiments, a substance is considered biocompatible if its addition to cells results in cell death below a certain threshold. In some embodiments, a substance is considered biocompatible if its addition to cells does not induce adverse effects. Generally, a biodegradable substance is one that undergoes degradation under physiological conditions over a therapeutically significant period of time (e.g., weeks, months, or years). In some embodiments, a biodegradable substance is a substance that can be broken down by cellular mechanisms. In some embodiments, a biodegradable substance is a substance that can be broken down by chemical processes. In some embodiments, a particle is a substance that is both biocompatible and biodegradable. In some embodiments, a particle is a substance that is biocompatible but not biodegradable. In some embodiments, a particle is a substance that is biodegradable but not biocompatible.

[0129] It is often desirable to use a population of particles that are relatively uniform in size, shape, and / or composition so that each particle has similar properties. For example, at least 80%, at least 90%, or at least 95% of the particles may have a diameter or largest dimension that falls within 5%, 10%, or 20% of the average diameter or largest dimension. In some embodiments, the population of particles may be heterogeneous with respect to size, shape, and / or composition. A variety of different particles may be used in accordance with the present invention. In some embodiments, the particles are spheres or spherical bodies. In some embodiments, the particles are spheres or spherical bodies. In some embodiments, the particles are flat or plate-like. In some embodiments, the particles are cubes or rectangular prisms. In some embodiments, the particles are ellipsoids or ellipsoids. In some embodiments, the particles are cylinders, cones, or pyramids.

[0130] In some embodiments, the particle is a microparticle (e.g., a microsphere). Generally, "microparticle" refers to any particle having a diameter of less than 1000 μm. In some embodiments, the particle is a picoparticle (e.g., a picosphere). Generally, "picosphere" refers to any particle having a diameter of less than 1 nm. In some embodiments, the particle is a liposome. In some embodiments, the particle is a micelle.

[0131] Particles can be solid or hollow and can include one or more layers (e.g., nanoshells, nanorings). In some embodiments, each layer has a unique composition and unique properties relative to the other layer(s). For example, a particle may have a core / shell structure, where the core is one layer and the shell is a second layer. A particle may include multiple different layers. In some embodiments, one layer may be substantially cross-linked, while the second layer is substantially not cross-linked, etc. In some embodiments, one, a few, or all of the different layers may include one or more therapeutic or diagnostic agents to be delivered. In some embodiments, one layer includes an agent to be delivered and a second layer does not include an agent to be delivered, etc. In some embodiments, each individual layer includes a different agent or set of agents to be delivered.

[0132] In some embodiments, the particles are porous, meaning that the particles contain holes or channels, and these holes or channels are typically small compared to the size of the particles. For example, the particles may be porous silica particles, such as mesoporous silica nanoparticles, or may have a mesoporous silica coating (Lin et al., 2005, J. Am. Chem. Soc., 17:4570). The particles may have pores with diameters ranging from about 1 nm to about 50 nm, for example, about 1 to 20 nm. Approximately 10% to 95% of the volume of the particles may consist of voids within the pores or channels.

[0133] The particles may have a coating layer. The use of a biocompatible coating layer may be advantageous, for example, when the particles contain a material that is toxic to cells. Suitable coating materials include, but are not limited to, natural proteins such as bovine serum albumin (BSA), biocompatible hydrophilic polymers such as polyethylene glycol (PEG) or PEG derivatives, phospholipids (PEG), silica, lipids, polymers, carbohydrates such as dextran, other nanoparticles that can be bound to the nanoparticles of the present invention, and the like. The coating can be applied or assembled in various ways, for example, by immersion, using layer-by-layer techniques, by self-assembly, conjugation, etc. Self-assembly refers to the process of spontaneous assembly of a higher-order structure, relying on the natural attraction of the components (e.g., molecules) of the higher-order structure to each other. It typically occurs through the random movement of molecules and the formation of bonds based on size, shape, composition, or chemical properties.

[0134] Examples of polymers include polyalkylenes (e.g., polyethylene), polycarbonates (e.g., poly(1,3-dioxane-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polyhydroxy acids (e.g., poly(3-hydroxyalkanoates)), polyfumarates, polycaprolactones, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactides, polyglycolides), poly(orthoesters), polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, and polyamines. In some embodiments, polymers according to the present invention include polymers approved for human use by the U.S. Food and Drug Administration (FDA) under 21 CFR §177.2600, including, but not limited to, polyesters (e.g., polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0135] In some embodiments, the particles may be non-polymeric particles (e.g., metal particles, quantum dots, ceramic particles, inorganic materials, bone-derived materials, polymers containing bone substitutes, viral particles, etc.). In some embodiments, the therapeutic or diagnostic agent to be delivered may be associated with the surface of such non-polymeric particles. In some embodiments, the non-polymeric particles are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms). In some embodiments, the therapeutic or diagnostic agent to be delivered may be associated with the surface of the aggregate of non-polymeric components and / or encapsulated within, surrounded by, and / or dispersed throughout the aggregate of non-polymeric components.

[0136] Particles (e.g., nanoparticles, microparticles) may be prepared using any method known in the art. For example, microparticle formulations may be formed by methods such as nanoprecipitation, flow focusing fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, grinding, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other suitable methods. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconductor, conductive nanoparticles, magnetic nanoparticles, organic nanoparticles, and other nanoparticles have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843).

[0137] Methods for preparing microparticles for delivery of encapsulated drugs are described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, δ:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755).

[0138] Nucleic acid targeting moiety In some embodiments, the targeting moiety comprises a nucleic acid targeting moiety.

[0139] In general, a nucleic acid targeting moiety is any polynucleotide that binds to a component (target) associated with an organ, tissue, cell, extracellular matrix component, and / or intracellular compartment.

[0140] In some embodiments, the nucleic acid targeting moiety is an aptamer. Aptamers are typically polynucleotides that bind to specific target structures associated with specific organs, tissues, cells, extracellular matrix components, and / or intracellular compartments. Generally, the targeting function of aptamers is based on the three-dimensional structure of the aptamer. In some embodiments, the binding of an aptamer to a target is typically mediated by interactions between the two-dimensional and / or three-dimensional structures of both the aptamer and the target. In some embodiments, the binding of an aptamer to a target is not based solely on the primary sequence of the aptamer, but rather depends on the three-dimensional structure(s) of the aptamer and / or the target. In some embodiments, aptamers bind to their targets through complementary Watson-Crick base pairing interrupted by structures that disrupt base pairing (e.g., hairpin loops).

[0141] In some embodiments, the nucleic acid targeting moieties are spiegelmers (PCT Publication Nos. WO 98 / 08856, WO 02 / 100442, and WO 06 / 117217). Generally, spiegelmers are synthetic mirror-image nucleic acids capable of specifically binding to a target (i.e., a mirror-image aptamer). Spiegelmers are characterized by structural features that make them less susceptible to exonucleases and endonucleases.

[0142] Those skilled in the art will recognize that any nucleic acid targeting moiety (e.g., aptamer or spiegelmer) capable of specifically binding to a target can be used in accordance with the present invention. In some embodiments, nucleic acid targeting moieties for use in accordance with the present invention can target markers associated with a disease, disorder, and / or condition. In some embodiments, nucleic acid targeting moieties for use in accordance with the present invention can target cancer-associated targets. In some embodiments, nucleic acid targeting moieties for use in accordance with the present invention can target tumor markers. Any type of cancer and / or any tumor marker can be targeted using nucleic acid targeting moieties in accordance with the present invention. To name just a few examples, nucleic acid targeting moieties can target markers associated with prostate cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, bone cancer, esophageal cancer, liver cancer, stomach cancer, brain tumors, skin melanoma, and / or leukemia.

[0143] Nucleic acids of the present invention (nucleic acid nucleic acid targeting moieties and / or functional RNAs, such as RNAi-inducing entities, ribozymes, tRNAs, etc., described in more detail below) may be prepared according to any available technique, including, but not limited to, chemical synthesis, enzymatic synthesis, enzymatic or chemical cleavage of longer precursors, etc.

[0144] Methods for synthesizing RNA are known in the art (see, e.g., Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984, and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in molecular biology, v. 288 (Clifton, NJ.) Totowa, NJ: Humana Press, 2005).

[0145] The nucleic acids forming the nucleic acid targeting moiety may comprise naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with a hydrocarbon linker (e.g., alkylene) or polyether linker (e.g., PEG linker) inserted between one or more nucleosides, modified nucleosides with a hydrocarbon or PEG linker inserted between one or more nucleosides, or combinations thereof. In some embodiments, the nucleotides or modified nucleotides of the nucleic acid targeting moiety may be substituted with nucleotides or modified nucleotides, provided that the binding affinity and selectivity of the nucleic acid targeting moiety are not substantially reduced by the substitution (e.g., the dissociation constant of the nucleic acid targeting moiety for the target is about 1×10 -3 M), may be replaced with a hydrocarbon linker or a polyether linker.

[0146] Those skilled in the art will understand that the nucleic acids of the present invention may contain exclusively nucleotides of the type found in naturally occurring nucleic acids, or alternatively, may contain one or more nucleotide analogs or have a structure that is otherwise different from that of naturally occurring nucleic acids. U.S. Patent Nos. 6,403,779, 6,399,754, 6,225,460, 6,127,533, 6,031,086, 6,005,087, and 5,977,089, and references therein, disclose a wide variety of specific nucleotide analogs and modifications that can be used. See Crooke, S. (ed.) Antisense Drug Technology: Principles, Strategies, and Applications (1st ed.), Marcel Dekker; ISBN: 0824705661; 1st edition (2001), and references therein. For example, 2'-modifications include halo, alkoxy, and allyloxy groups. In some embodiments, the 2'-OH group is replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I. Examples of modified linkages include phosphorothioate and 5'-N-phosphoramidite linkages.

[0147] Nucleic acids containing a variety of different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages can be utilized in the present invention. The nucleic acids of the present invention can contain natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of modified nucleotides include base-modified nucleosides (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3-nitropyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, Ml-methyladenosine, pyrrolo-pyrimidine, 2-azacyt ... Amino-6-chloropurine, 3-methyladenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2'-fluororibose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, the L-enantiomeric nucleosides arabinose and hexose), modified phosphate groups (e.g., phosphorothioate linkages and 5'-N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids containing such modifications exhibit improved properties compared to nucleic acids consisting only of naturally occurring nucleotides. In some embodiments, the nucleic acid modifications described herein are utilized to reduce and / or prevent digestion by nucleases (e.g., exonucleases, endonucleases, etc.).For example, the structure of the nucleic acid may be stabilized by including nucleotide analogues at the 3' end of one or both strands to reduce digestion.

[0148] Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may be present at various positions in the nucleic acid. Those skilled in the art will understand that nucleotide analogs or other modification(s) may be located at any position(s) in the nucleic acid such that the function of the nucleic acid is not substantially affected. By way of example only, modifications may be located at any position in the nucleic acid targeting moiety such that the ability of the nucleic acid targeting moiety to specifically bind to the target is not substantially affected. The modified region may be at the 5' and / or 3' end of one or both strands. For example, modified nucleic acid targeting moieties in which approximately 1 to 5 residues at the 5' and / or 3' end of either of both strands are nucleotide analogs and / or have backbone modifications have been used. The modification may be at the 5' or 3' end. One or both nucleic acid strands may contain at least 50% unmodified nucleotides, at least 80% unmodified nucleotides, at least 90% unmodified nucleotides, or 100% unmodified nucleotides.

[0149] Nucleic acids according to the present invention may contain modifications, e.g., to sugars, nucleosides, or internucleoside linkages, such as those described in U.S. Patent Application Publication Nos. 2003 / 0175950, 2004 / 0192626, 2004 / 0092470, 2005 / 0020525, and 2005 / 0032733. The present invention encompasses the use of any nucleic acid having any one or more of the modifications described therein. For example, some terminal conjugates, e.g., lipids such as cholesterol, lithocholic acid, aluric acid, or long alkyl branched chains, have been reported to improve cellular uptake. Analogs and modifications may be tested, e.g., using any suitable assay known in the art, to select those that result in, e.g., improved delivery of therapeutic or diagnostic agents, improved specific binding of nucleic acid targeting moieties to targets, etc. In some embodiments, nucleic acids according to the present invention may contain one or more unnatural nucleoside linkages. In some embodiments, one or more internal nucleotides at the 3' end, the 5' end, or both the 3' and 5' ends of the nucleic acid targeting moiety are inverted to create a linkage, such as a 3'-3' linkage or a 5'-5' linkage.

[0150] In some embodiments, nucleic acids according to the present invention are not synthetic, but rather are naturally occurring entities that have been isolated from their natural environment.

[0151] Any method can be used to design novel nucleic acid targeting moieties (see, e.g., U.S. Patent Nos. 6,716,583, 6,465,189, 6,482,594, 6,458,543, 6,458,539, 6,376,190, 6,344,318, 6,242,246, 6,184,364, 6,001,577, 5,958,691, 5,874,218, 5,853,984, 5,843,732, 5,843,74 ... ,653, 5,817,785, 5,789,163, 5,763,177, 5,696,249, 5,660,985, 5,595,877, 5,567,588, and 5,270,163, and U.S. Patent Application Publication Nos. 2005 / 0069910, 2004 / 0072234, 2004 / 0043923, 2003 / 0087301, 2003 / 0054360, and 2002 / 0064780).

[0152] Nucleic acid targeting moieties that bind to proteins, carbohydrates, lipids, and / or nucleic acids can be designed and / or identified. In some embodiments, nucleic acid targeting moieties that bind to proteins and / or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intracellular proteins, ion channels, membrane transporter proteins, enzymes, antibodies, chimeric proteins, etc., can be designed and / or identified for use in the conjugates of the invention. In some embodiments, nucleic acid targeting moieties that bind to carbohydrates and / or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, and polysaccharides), glycocalyx (i.e., the carbohydrate-rich marginal zone on the outer surface of most eukaryotic cells), etc., can be designed and / or identified for use in the conjugates of the invention. In some embodiments, nucleic acid targeting moieties that bind to lipids and / or characteristic portions thereof, such as oils, saturated fatty acids, unsaturated fatty acids, glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, lipoproteins, etc., can be designed and / or identified for use in the conjugates of the invention. In some embodiments, nucleic acid targeting moieties that bind to nucleic acids and / or characteristic portions thereof, such as DNA nucleic acids, RNA nucleic acids, modified DNA nucleic acids, modified RNA nucleic acids, and nucleic acids comprising any combination of DNA, RNA, modified DNA, and modified RNA, can be designed and / or identified for use in the complexes of the invention.

[0153] Nucleic acid targeting moieties (e.g., aptamers or spiegelmers) may be designed and / or identified using any available method, hi some embodiments, nucleic acid targeting moieties are designed and / or identified by identifying nucleic acid targeting moieties from a candidate mixture of nucleic acids.

[0154] Methods for preparing the compounds of the present invention The compounds and conjugates disclosed herein can be prepared via simple preparative methods (see, for example, Examples 1-78), which allow for convenient purification.

[0155] Thus, methods for preparing the compounds of the invention are also provided herein. For example, the compounds of the invention can be prepared as shown in any one of Reaction Schemes 3, 4, 5, 6, or 7: Reaction Scheme 3: [ka] Reaction Scheme 4: [ka] Reaction Scheme 5: [ka] Reaction Scheme 6: [ka] Reaction Scheme 7: [ka] wherein X, Y, Ar, R, and n are as defined above.

[0156] Also provided herein is a method for preparing a compound, the method comprising the step of: [ka] TIFF0007657153000069.tif6148 or a pharmaceutically acceptable salt thereof, [ka] to produce a compound of formula (Iaa): [ka] or a pharmaceutically acceptable salt thereof, wherein X a is a halogen, Q is an activator linked to L' by a heteroatom, preferably O or N; Z' is absent or independently in each occurrence converts the structure of formula (IIc), sulfonyl halide, or (Iaa) to (CB) cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, fragment of an antigenic polypeptide, or lipibody), active agent, or detectable moiety, provided that at least one occurrence of Z' is selected from the group consisting of formula (IIc), sulfonyl halide, or (Iaa), and (CB) cb provided that it is connected to L' is a linking group attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, preferably O or N, and is selected such that cleavage of the bond between L' and -S(=O)(=N-)- facilitates cleavage of the bond between L' and Q, releasing the active agent; Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S-, whereby an N, O, or S atom is bonded to TG when y is 1; O and Y' are located on adjacent atoms of Ar; When activated, TG reacts with -S(=O)(=N-)- to form (Q) q -(L') w is an inducing group that generates an N, O, or S atom capable of replacing and forming a 5- to 6-membered ring containing an intervening atom of XS(=O)(=N-)- and Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; Each R a and Rc are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the carbon atom to which they are attached, However, when w is 0, q is 1.

[0157] The reactive group of the linking group can be a moiety capable of participating in 1,3-dipolar cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, non-aldol carbonyl reactions, addition to carbon-carbon multiple bonds, oxidation reactions, click reactions, or any other intermolecular coupling reactions. Preferably, the reactive group is selected to participate in selective reactions with reaction partners that are uncommon in biomolecules, such as 1,3-dipolar cycloaddition, hetero-Diels-Alder reactions, oxime / hydrazone condensations, or click reactions.

[0158] In certain preferred embodiments, the linking group may comprise an alkyne or azide (which reacts to form a triazole), an alkyne and nitrile oxide (which reacts to form an isoxazole), or a carbonyl (e.g., an aldehyde or ketone), or a hydrazine or hydroxylamine (which reacts to form an oxime or hydrazone).

[0159] Stabilizing groups (e.g., PEG) limit the clearance and metabolism of chemotherapeutic agents or markers by enzymes that may be present in blood or non-target tissues. Stabilizing groups can serve to block degradation of the chemotherapeutic agent or marker and can also provide other physical characteristics of the agent or marker (e.g., increasing the solubility of the ligand-drug conjugate or reducing the aggregation properties of the ligand-drug conjugate). Stabilizing groups can also improve the stability of the chemotherapeutic agent or marker during storage, either in formulated or unformulated form. Ideally, a stabilizing group is useful for stabilizing a therapeutic agent or marker if, when tested by storing the agent or marker in human blood at 37°C for 2 hours, the stabilizing agent serves to protect the agent or marker from degradation and results in less than 20%, preferably less than 10%, more preferably less than 5%, and even more preferably less than 2% of the agent or marker being cleaved by enzymes present in human blood under given assay conditions. The present invention also relates to conjugates containing these linkers.

[0160] Examples of suitable stabilizing groups include non-amino acids such as succinic acid, diglycolic acid, maleic acid, polyethylene glycol, pyroglutamic acid, acetic acid, naphthylcarboxylic acid, terephthalic acid, and glutaric acid derivatives, as well as non-genetically encoded amino acids or aspartic acid or glutamic acid attached to the N-terminus of the peptide through the beta-carboxy group of aspartic acid or the gamma-carboxyl group of glutamic acid.

[0161] In some embodiments, the method utilizes an intermediate compound of formula (IIa), (IIb), or (IIc) to provide a compound of formula (Iaa), wherein Ar, TG, Y′, Z′, and R a is as defined above for the conjugate of formula (I') or the compound of formula (Ia) or (Ia').

[0162] The present invention further provides compounds as described above that are useful in these methods.

[0163] intermediate compound In some embodiments, the compounds and conjugates disclosed herein include an intermediate compound having a structure according to formula (IV): [ka] or a pharmaceutically acceptable salt thereof, wherein W is hydrogen, -SiR 16 R 17 R 18 , or -S(=O)(=N-)-G, R 16 , R 17 , and R 18 are each independently C1-C6 alkyl, G is a halogen (preferably fluorine), imidazole, or N-methylimidazolium; R is a substituent or -L 1’ -Z, L 1’ is a C1-C 1-hydroxybenzoate optionally containing at least one of a peptide bond, an amino bond, an ether bond, a triazole bond, a tetrazole bond, a sugar bond, a sulfonamide bond, a phosphonate bond, a sulfo bond, or a dendrimer structure. 200 - alkylene, Z can be any of isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a (where Ra is C1~C 10 and dihydrogen phosphate (-OP(=O)(OH)), n is an integer having a value of 1 to 4, Y is -NO2, -OC(O)(CH2) r C(O)R1, -O(CH2) r -Ar1-NO2, -NHNH2, -BR2R3, [ka] or -Y'-TG, -NO2, -OC(O)(CH2) r C(O)R1, -O(CH2) r -Ar1-NO2, -NHOH, -NHNH2, -BR2R3, [ka] or -Y'-TG, R 1 is a C1-C6 alkyl, r is an integer from 1 to 5, Ar 1 C6~C 20 - arylene, R 2 and R 3 are each independently hydrogen, C1-C6-alkyl, C1-C6-alkoxy, or hydroxy, R a , R b , R c , and R d are each independently hydrogen or C1-C6 alkyl, Y' is -(CH2) x NR''-, -(CH2) x O- or -(CH2) x S- and R" is hydrogen or C1-C6 alkyl; x is an integer of 0 or 1, TG is the inducing group.

[0164] In some embodiments, the compounds and conjugates disclosed herein comprise an intermediate compound having a structure according to formula (V): [ka] or a pharmaceutically acceptable salt thereof, wherein W, L 1’ and Z are as defined for formula (IV), TG is a derivative group such as a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0165] In other embodiments, the compounds and conjugates disclosed herein include an intermediate compound having a structure according to formula (VI): [ka] or a pharmaceutically acceptable salt thereof, wherein W is as defined for formula (IV); Y is -NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -NHOH, -NHNH2, -BR 2 R 3 , or -O-TG, R 1 is NO2, -OC(O)(CH2) r C(O)R 1 , -O(CH2) r -Ar 1 -NO2, -NHOH, -NHNH2, -BR 2 R 3 or -O-TG, R 1 is C1-C6-alkyl, r is an integer from 1 to 5, Ar 1 is phenylene, biphenylene, or naphthalene, R 2 and R 3 are each independently hydrogen, C1-C6-alkyl, C1-C6-alkoxy, or hydroxy, R a , R b , R c , and R d are each independently hydrogen or C1-C6-alkyl, TG is a derivative group of β-galactoside, β-glucuronide, or a combination of β-galactoside and β-glucuronide.

[0166] Also, intermediate compounds of formula (IIa), (IIb), or (IIc): [ka] or a pharmaceutically acceptable salt thereof, wherein: G is a halogen, imidazole, or N-methylimidazolium; Each R 11 are independently C1-C6-alkyl, Ar represents a ring such as an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; When activated, TG induces the X-SO 2 and an inducing group that generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing the intervening atom of Ar; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and positioned such that when y is 1, the N, O, or S atom is bonded to TG; O and Y' are located on adjacent atoms of Ar; x and y are each independently an integer having a value of 0 or 1; Z' is absent or, independently in each occurrence, converts the structure of formula (IIa), (IIb), or (IIc) to (CB) cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, provided that at least one occurrence of Z′ converts the structure of formula (IIa), (IIb), or (IIc) to (CB), cb provided that it is connected to Each R a are independently hydrogen or alkyl; Each R b are independently hydrogen or alkyl, or The Two R's b form a 3- to 5-membered ring, such as a 3-membered ring, together with the carbon atom to which they are attached.

[0167] In some embodiments, the intermediate compound is a compound of formula (IIa), (IIb), or (IIc), wherein Ar, TG, Y′, Z′, and R a is as defined above for the conjugate of formula (I') or the compound of formula (Ia) or (Ia').

[0168] In preferred embodiments, the intermediate compound is a compound of formula (IIa), (IIb), or (IIc), where Ar is aryl (eg, phenyl or naphthyl).

[0169] In some embodiments, provided herein are intermediate compounds that are compounds of formula (IIa), (IIb), or (IIc), wherein at least one Z′ (e.g., (CB) cb Z' connected to , optionally each Z' is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halide, tosylate (TsO - ), aldehydes, sulfonates (R-SO3- ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a ), and dihydrogen phosphate (—OP(═O)(OH)2).

[0170] In other embodiments, the intermediate compound is a compound of formula (IIa), (IIb), or (IIc), where x is 0. In some such embodiments, TG is selected from the group consisting of NO, —OC(O)(CH) r C(O)R 1 , -NHNH2, -BR 2 R 3 , [ka] -NO2, -OC(O)(CH2), etc. r C(O)R 1 , -NHOH, -NHNH2, -BR 2 R 3 , [ka] where: R 1 is a C1-C6 alkyl, R 2 and R 3 are each independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, or hydroxy; R 4 , R 5 , R 6 , and R 7 are each independently hydrogen or C1-C6 alkyl, r is an integer having a value of 1, 2, 3, 4, or 5.

[0171] In alternative embodiments, the intermediate compound is a compound of formula (IIa), (IIb), or (IIc), where TG is a triggering group comprising a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0172] In certain embodiments, the intermediate compound is [ka] or a pharmaceutically acceptable salt thereof.

[0173] In certain other embodiments, the intermediate compound is [ka] or a pharmaceutically acceptable salt thereof.

[0174] Antibody-drug conjugates (ADCs) In some embodiments, CB is an antibody and Q is a drug. Accordingly, the compounds and conjugates disclosed herein may be used to conjugate an antibody to a drug moiety to form an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) may increase therapeutic efficacy in treating diseases, e.g., cancer, due to the ability of the ADC to selectively deliver one or more drug moieties(s) to target tissues, such as tumor-associated antigens. Thus, in certain embodiments, the present invention provides ADCs for therapeutic use, e.g., for the treatment of cancer.

[0175] The ADCs of the present invention comprise an antibody linked to one or more drug moieties. The specificity of the ADC is determined by the specificity of the antibody. In one embodiment, the antibody is linked to one or more cytotoxic drug(s) that are delivered to cancer cells in the body.

[0176] Examples of drugs that can be used in the ADCs of the invention are provided below. The terms "drug," "agent," and "drug moiety" are used interchangeably herein. The terms "linked" and "conjugated" are also used interchangeably herein to indicate that the antibody and moiety are covalently linked.

[0177] In some embodiments, the ADC has the following formula (Formula VII): (DL) n -Ab(VII) wherein Ab is an antibody and (DL) is a linker-drug moiety. The linker-drug moiety is made up of a linker, L, and a drug moiety, D. The drug moiety may have, for example, cytostatic, cytotoxic, or other therapeutic activity against a target cell. n is an integer having a value of 1 to about 20, preferably 1 to about 10. Preferably, DL has the structure of formula (I") or formula (I'"): [ka] each Q is independently an activator linked to L' by a heteroatom, preferably O or N; Z', in each occurrence, independently converts the structure of formula (I") or formula (I"') to (CB) cb a linking group, solubilizing group, reactive group (e.g., precursor group), solid surface (e.g., particle), stabilizing group, chelator, biopolymer (e.g., immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, or lipibody), active agent, or detectable moiety, provided that at least one occurrence of Z' converts the structure of Formula (I") or Formula (I"') to (CB) cb provided that it is connected to each L' is independently a spacer moiety attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, preferably O or N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- promotes cleavage of the bond between L' and Q, releasing the active agent; Each X is independently -O-, -C(Rb )2-, or -N(R c )-, preferably -O-, Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y' is -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and positioned such that when y is 1, the N, O, or S atom is bonded to TG; When activated, TG reacts with -S(=O)(=N-)- to form (Q) q -(L') w is an inducing group that generates the formation of an N, O, or S atom capable of replacing and forming a 5-6 membered ring containing an intervening atom of XS(=O)(=N-)- and Ar, q is an integer having a value of 1 to about 20, preferably 1 to about 10; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring together with the atom to which they are attached, However, when w is 0, q is 1.

[0178] In certain embodiments, E is 0.

[0179] In some embodiments, n has a value ranging from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is an integer having a value of 1. When cb is 1 and n is 1, the drug-to-antibody ratio (DAR) of the ADC is equal to the number of drugs present in (DL). When cb is other than 1, the drug-to-antibody ratio (DAR) of the ADC is equal to the ratio of the number of drugs present in (DL) to the number of antibodies present in the conjugate.

[0180] Exemplary Drugs for Conjugation The ADCs of the invention provide targeted therapy, whereby one or more active agent(s) or drug(s) are delivered to specific cells, which may reduce side effects often seen with, for example, anti-cancer therapies.

[0181] For example, drugs include erlotinib (TARCEVA; Genentech / OSI Pharm.); bortezomib (VELCADE; MillenniumPharm.); fulvestrant (FASLODEX; AstraZeneca); Sutent (SU11248; Pfizer); letrozole (FEMARA; Novartis); imatinib mesylate (GLEEVEC; Novartis); PTK787 / ZK 222584 (Novartis); oxaliplatin (Eloxatin; Sanofi); 5-fluorouracil (5-FU); leucovorin; rapamycin (sirolimus, RAPAMUNE; Wyeth); lapatinib (TYKERB, GSK572016; GlaxoSmithKline); lonafarnib (SCH 66336); sorafenib (BAY43-9006; Bayer Labs.); gefitinib (IRESSA; Astrazeneca); AG1478, AG1571 (SU 5271; Sugen); alkylating agents (e.g., thiotepa or CYTOXAN® cyclophosphamide); alkyl sulfonates (e.g., busulfan, improsulfan, or piposulfan); aziridines (e.g., benzodopa, carboquone, meturedopa, or uredopa); ethyleneimine, methylmelamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins (e.g., brazil, camptothecin, including the synthetic analog topotecan; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, or bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 or cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards (e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterine, prednimustine, trofosfamide, or uracil mustard); nitrousureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimustine) ne); antibiotics (e.g., dynemicin, including calicheamicin or dynemicin A, as enediyne antibiotics, selected from calicheamicin gamma 1I and calicheamicin omega 1I); bisphosphonates (e.g., clodronate); enediyne antibiotic chromophores that are esperamicin, neocarzinostatin chromophores or related chromoproteins, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromoma isin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRLIMYCIN® doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, or deoxydoxorubicin), epirubicin, esorubicin, marcelomycin, mitomycin (e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin (potf iromycin, puromycin, quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; antimetabolites (e.g., 5-fluorouracil (5-FU)); folic acid analogs (e.g., denopterin, methotrexate, pteropterin, or trimetrexate); purine analogs (e.g., fludarabine, 6-mercaptopurine, thiamiprine, or thiguanine);Pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, or floxuridine); androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane, or testolactone); anti-adrenal agents (e.g., aminoglutethimide, mitotane, or trilostane); folic acid supplements (e.g., florinic acid acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids (e.g., maytansine or ansamitocin); trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, or anguidine); mitoguazone; mitoxantrone; mopida Mol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, or anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxoids (e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumber, IL), or TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs (e.g., cisplatin or carboplatin); vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids (e.g., retinoic acid); capecitabine; and pharmaceutically acceptable salts, solvates, acids, or derivatives thereof.

[0182] Mitotic inhibitors In some embodiments, the linkers of the invention may be used to conjugate an antibody to one or more antimitotic agent(s) to form an ADC for the treatment of cancer. As used herein, the term "antimitotic agent" refers to a cytotoxic drug and / or therapeutic agent that interferes with mitosis, or cell division, a biological process particularly important to cancer cells. Antimitotic agents often affect microtubule polymerization or microtubule depolymerization, thereby disrupting microtubules and preventing cell division. Thus, in certain embodiments, an antibody is conjugated to one or more antimitotic agents that disrupt microtubule formation by inhibiting tubulin polymerization. In one embodiment, the antimitotic agent used in the ADCs of the invention is Taxol® (paclitaxel), Taxotere® (docetaxel), or Ixempra® (ixabepilone). Examples of antimitotic agents that can be used in the ADCs disclosed herein are provided below. The auristatins mentioned above are included in the genus of antimitotic agents.

[0183] Auristatin The antibody may be conjugated to at least one auristatin using the linker of the present invention. Auristatins represent a group of dolastatin analogs that have generally been shown to have anti-cancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analog of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anti-cancer drug vincristine (GR Pettit, Prog. Chem. Org. Nat. Prod, 70:1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides with four amino acids, three of which are unique to the dolastatin class of compounds. Exemplary embodiments of the auristatin subclass of antimitotic agents include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivatives), monomethyl auristatin E (MMAE or auristatin E derivatives), monomethyl auristatin F (MMAF or auristatin F derivatives), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB).The synthesis and structure of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, and 2005-0009751, International Patent Publication Nos. WO04 / 010957, WO02 / 088172, and U.S. Patent Nos. 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,664,150, and 5,665,160. 35,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, each of which is incorporated herein by reference.

[0184] Dolastatin An antibody may be conjugated to at least one dolastatin using the linkers of the invention to form an ADC. Dolastatins are short peptide compounds isolated from the Indian Ocean sea hare Dolabella auricularia (see Pettit et al., J. Am. Chem. Soc., 1976, 98, 4677). Examples of dolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. Thus, in one embodiment, an ADC of the invention comprises an antibody, a linker as described herein, and at least one dolastatin. The auristatins described above are synthetic derivatives of dolastatin 10.

[0185] Maytansinoids The linkers of the invention may be used to conjugate an antibody to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as from several species of mosses (Kupchan et al., J. Am. Chem. Soc. 94:1354-1356

[1972] ; Wani et al., J. Chem. Soc. Chem. Commun 390:

[1973] ; Powell et al., J. Nat. Prod. 46:660-666

[1983] ; Sakai et al., J. Nat. Prod. 51:845-850

[1988] ; and Suwanborirux et al., Experientia 46:117-120

[1990] ). Evidence suggests that maytansinoids inhibit mitosis by inhibiting the polymerization of the microtubule protein tubulin, thereby blocking the formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Moreover, the cytotoxic effects of maytansinoids are 1,000-fold greater than those of conventional chemotherapeutic agents such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020).

[0186] Maytansinoids include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated herein by reference). C-3 esters of maytansinol can be naturally occurring or synthetic. Moreover, both naturally occurring and synthetic C-3 maytansinol esters can be classified as C-3 esters with simple carboxylic acids or C-3 esters with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogs are described, for example, in Kupchan et al., J. Med. Chem., 21, 31-37 (1978).

[0187] Maytansinoids suitable for use in the ADCs of the invention can be isolated from natural sources, synthetically produced, or semisynthetically produced. Moreover, maytansinoids can be modified in any suitable manner, so long as sufficient cytotoxicity is maintained in the final conjugate molecule. The structure of an exemplary maytansinoid, mertansine (DM1), is provided below. [ka]

[0188] Representative examples of maytansinoids include, but are not limited to, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine; mertansine, also referred to as the drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al. (1992) Cancer Res 52:127), DM2, DM3 (N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine), DM4 (4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (a synthetic maytansinoid analog). Other examples of maytansinoids are described in U.S. Pat. No. 8,142,784, incorporated herein by reference.

[0189] Ansamitocins are a group of maytansinoid antibiotics isolated from various bacterial sources. These compounds have potent antitumor activity. Representative examples include, but are not limited to, ansamitocin P1, ansamitocin P2, ansamitocin P3, and ansamitocin P4.

[0190] plant alkaloids The linkers of the invention may be used to conjugate an antibody to at least one plant alkaloid, such as a taxane or vinca alkaloid. Plant alkaloids are chemotherapy treatments derived from and made from certain types of plants. Vinca alkaloids are made from the periwinkle plant (Catharanthus rosea), while taxanes are made from the bark of the Pacific yew tree (Taxus). Both vinca alkaloids and taxanes are also known as microtubule inhibitors and are described in more detail below.

[0191] Taxanes The linker of the present invention may be used to conjugate an antibody to at least one taxane. As used herein, the term "taxane" refers to a class of anti-neoplastic agents that possess a microtubule-acting mechanism and a structure that includes a taxane ring structure and a stereospecific side chain required for cytostatic activity. Also included within the term "taxane" are various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113, the piperazino and other derivatives described in WO 99 / 14209, the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680, the 6-thio derivatives described in WO 98 / 28288, the sulfenamide derivatives described in U.S. Pat. No. 5,821,263, and the taxol derivatives described in U.S. Pat. No. 5,415,869, each of which is incorporated herein by reference. Taxane compounds are also disclosed in U.S. Patent Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263, 5, Taxanes have been previously described in US Pat. Nos. 840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which are expressly incorporated by reference. Further examples of taxanes include, but are not limited to, docetaxel (Taxotere®; Sanofi Aventis), paclitaxel (Abraxane® or Taxol®; Abraxis Oncology), and nanoparticle paclitaxel (ABI-007 / Abraxene®; Abraxis Bioscience).

[0192] In one embodiment, the linker of the present invention may be used to conjugate the antibody to at least one docetaxel.In one embodiment, the linker of the present invention may be used to conjugate the antibody to at least one paclitaxel.

[0193] Vinca alkaloids In one embodiment, the linker of the present invention may be used to conjugate an antibody to at least one vinca alkaloid. Vinca alkaloids are a class of cell cycle-specific drugs that act by blocking the formation of microtubules by acting on tubulin, thereby inhibiting the ability of cancer cells to divide. Examples of vinca alkaloids that can be used in the ADCs of the present invention include, but are not limited to, vindesine sulfate, vincristine, vinblastine, and vinorelbine.

[0194] Antitumor antibiotics The linkers of the invention may be used to conjugate antibodies to one or more antitumor antibiotic(s) for the treatment of cancer. As used herein, the term "antitumor antibiotic" refers to an anti-neoplastic drug produced by a microorganism that prevents cell growth by interfering with DNA. Often, antitumor antibiotics degrade DNA strands or slow or stop DNA synthesis. Examples of antitumor antibiotics that can be included in the ADCs disclosed herein include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracyclines, calicheamicin, and duocarmycin, which are described in more detail below.

[0195] Actinomycin The linkers of the invention may be used to conjugate an antibody to at least one actinomycin. Actinomycins are a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces. Representative examples of actinomycins include, but are not limited to, actinomycin D (Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV, actinomycin C1], Lundbeck, Inc.), anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, porothramycin, prothracarcin B, SG2285, sibanomicin, sibiromycin, and tomaymycin. In one embodiment, D is a pyrrolobenzodiazepine (PBD). Examples of PBDs include, but are not limited to, anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, polusramycin, prothracarcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sibanomycin, sibiromycin, and tomaymycin. Thus, in one embodiment, D is an actinomycin, e.g., actinomycin D, or a PBD, e.g., a pyrrolobenzodiazepine (PBD) dimer.

[0196] The structure of PBDs can be found, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917 and 2013 / 0028919, and WO2011 / 130598 A1, each of which is incorporated by reference in its entirety. The general structure of a PBD is provided below: [ka]

[0197] PBDs vary in the number, type, and position of substituents on both their aromatic A-ring and pyrrolo C-ring, as well as the degree of saturation of the C-ring. The B-ring generally contains an imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)) at the N-C position, the electrophilic center responsible for DNA alkylation. All known natural products possess the (S) configuration at the chiral C-α position, thereby providing them with a right-handed twist when viewed from the C-ring toward the A-ring. Further examples of PBDs that can be conjugated to antibodies via the linkers disclosed herein can be found, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917 A1 and 2013 / 0028919 A1, U.S. Patent No. 7,741,319 B2, and WO2011 / 130598 A1 and WO2006 / 111759 A1, each of which is incorporated by reference in its entirety.

[0198] Anthracyclines The linkers of the invention may be used to conjugate an antibody to at least one anthracycline. Anthracyclines are a subclass of antitumor antibiotics isolated from the bacterium Streptomyces. Representative examples include, but are not limited to, daunorubicin (Cerubidine, Bedford Laboratories), doxorubicin (Adriamycin, Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxydaunorubicin, and Rubex), epirubicin (Ellence, Pfizer), and idarubicin (Idamycin; Pfizer Inc.). Thus, in one embodiment, D is an anthracycline, e.g., doxorubicin.

[0199] Calicheamicin The linker of the present invention may be used to conjugate an antibody to at least one calicheamicin. Calicheamicin is a family of enediyne antibiotics derived from the soil organism Micromonospora echinospora. Calicheamicin binds to the minor groove of DNA and induces double-stranded DNA breaks, resulting in 100-fold increased cell death compared to other chemotherapeutic agents (Damle et al. (2003) Curr Opin Pharmacol 3:386). Preparations of calicheamicin that can be used as drug conjugates according to the present invention have been described; see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296. Structural analogs of calicheamicin that can be used include, but are not limited to, γΙ, αΙ, αΙ, N-acetyl-γΙ, PSAG, and θΙ (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296). Thus, in one embodiment, D is calicheamicin.

[0200] Duocarmycin The linkers of the present invention may be used to conjugate an antibody to at least one duocarmycin. Duocarmycins are a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces (see Nagamura and Saito (1998) Chemistry of Heterocyclic Compounds, Vol. 34, No. 12). Duocarmycins bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position (Boger (1993) Pure and Appl Chem 65(6):1123, and Boger and Johnson (1995) PNAS USA 92:3642). Synthetic analogs of duocarmycin include, but are not limited to, adozelesin, bizeresin, and carzelesin. Thus, in one embodiment, D is a duocarmycin.

[0201] Other antitumor antibiotics In addition to those previously mentioned, additional antitumor antibiotics that may be used in the ADCs of the invention include bleomycin (Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).

[0202] immunomodulators In some embodiments, an antibody may be conjugated to at least one immunomodulatory agent using a linker of the invention. As used herein, the term "immunomodulatory agent" refers to an agent capable of stimulating or modifying an immune response. In one embodiment, an immunomodulatory agent is an immunostimulatory agent that enhances a subject's immune response. In some embodiments, an immunomodulatory agent is an immunosuppressant that blocks or reduces a subject's immune response. An immunomodulatory agent may regulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells) and any further differentiated cells thereof. Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (Ergamisol). Other examples of immunomodulatory agents that may be used in the ADCs of the invention include, but are not limited to, cancer vaccines, cytokines, and immunomodulatory gene therapies.

[0203] Cancer vaccines The linkers of the present invention may be used to conjugate antibodies to cancer vaccines. As used herein, the term "cancer vaccine" refers to a composition (e.g., tumor antigens and cytokines) that elicits a tumor-specific immune response. The response is elicited from a subject's own immune system by administering a cancer vaccine, or in the case of the present invention, an ADC comprising the antibody and cancer vaccine. In preferred embodiments, the immune response results in the eradication of tumor cells (e.g., primary or metastatic tumor cells) in the body. The use of cancer vaccines generally involves the administration of a specific antigen or group of antigens, for example, present on the surface of specific cancer cells or present on the surface of a specific infectious pathogen shown to promote cancer formation. In some embodiments, the use of cancer vaccines is for prophylactic purposes, while in other embodiments, the use is for therapeutic purposes. Non-limiting examples of cancer vaccines that may be used in the ADCs disclosed herein include recombinant bivalent human papillomavirus (HPV) types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, D is a cancer vaccine that is either an immunostimulatory agent or an immunosuppressant.

[0204] cytokines The linker of the invention may be used to conjugate an antibody to at least one cytokine. The term "cytokine" generally refers to a protein released by one cell population and acting on another cell as an intercellular mediator. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and enhance the recognition of tumor cells by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856). Studies in numerous animal tumor models have demonstrated that cytokines have broad antitumor activity, which has translated into several cytokine-based approaches for cancer therapy (Lee and Margoli, supra). Recently, several cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have entered clinical trials for patients with advanced cancer (Lee and Margoli, supra).

[0205] Examples of cytokines that can be used in the ADCs of the invention include parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); factor); fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF; platelet growth factors; transforming growth factors (TGF); insulin-like growth factors-I and -II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon α, β, and γ, colony-stimulating factors (CSF); granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and other polypeptide factors including LIF and Kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. Thus, in one embodiment, D is a cytokine.

[0206] Colony-stimulating factors (CSFs) The linkers of the invention may be used to conjugate an antibody to at least one colony-stimulating factor (CSF). Colony-stimulating factors (CSFs) are growth factors that assist the bone marrow in producing red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), colony-stimulating factors may be introduced to help support white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used following bone marrow transplantation to help the new bone marrow begin producing white blood cells. Representative examples of CSFs that may be used in the ADCs disclosed herein include, but are not limited to, erythropoietin (Epoetin), filgrastim (Neopogen (also known as granulocyte colony-stimulating factor (G-CSF); Amgen, Inc.), sargramostim (Leukine (granulocyte-macrophage colony-stimulating factor and GM-CSF); Genzyme Corporation), promegapoietin, and oprelvekin (recombinant IL-11; Pfizer, Inc.). Thus, in one embodiment, D is a CSF.

[0207] Gene therapy The linkers of the present invention may be used to conjugate an antibody to at least one nucleic acid for gene therapy (directly or indirectly via a carrier). Gene therapy generally refers to the introduction of genetic material into cells, which is designed to treat a disease. When referring to immunomodulatory agents, gene therapy is used to stimulate a subject's natural ability to inhibit cancer cell growth or kill cancer cells. In one embodiment, an ADC of the present invention comprises a nucleic acid encoding a functional therapeutic gene used to replace a mutated or otherwise dysfunctional (e.g., truncated) gene associated with cancer. In other embodiments, an ADC of the present invention comprises a nucleic acid encoding a therapeutic protein for treating cancer or otherwise enabling the production of a therapeutic protein. The nucleic acid encoding the therapeutic gene can be directly conjugated to the antibody, or alternatively, can be conjugated to the antibody via a carrier. Examples of carriers that can be used to deliver nucleic acids for gene therapy include, but are not limited to, viral vectors or liposomes.

[0208] Alkylating agents The linkers of the invention may be used to conjugate antibodies to one or more alkylating agent(s). Alkylating agents are a class of anti-neoplastic compounds that attach alkyl groups to DNA. Examples of alkylating agents that can be used in the ADCs of the invention include, but are not limited to, alkyl sulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0209] Alkyl sulfonate The linkers of the invention may be used to conjugate an antibody to at least one alkyl sulfonate. An alkyl sulfonate has the general formula: R-SO-OR 1 wherein R and R 1is typically an alkyl or aryl group. A representative example of an alkyl sulfonate is busulfan (Myleran®, GlaxoSmithKline; Busulfex IV®, PDL BioPharma, Inc.).

[0210] Nitrogen Mustard The linkers of the invention may be used to conjugate an antibody to at least one nitrogen mustard. Representative examples of this subclass of anti-cancer compounds include, but are not limited to, chlorambucil (Leukeran®, GlaxoSmithKline), cyclophosphamide (Cytoxan®, Bristol-Myers Squibb; Neosar, Pfizer, Inc.), estramustine (estramustine phosphate sodium or Estracyt®), Pfizer, Inc.), ifosfamide (Ifex®, Bristol-Myers Squibb), mechlorethamine (Mustargen®, Lundbeck Inc.), and melphalan (Alkeran® or L-Pam®, i.e., phenylalanine mustard; GlaxoSmithKline).

[0211] Nitrosourea The linkers of the present invention may be used to conjugate an antibody to at least one nitrosourea. Nitrosoureas are a subclass of lipid-soluble alkylating agents. Representative examples include, but are not limited to, carmustine (BCNU [also known as BiCNU, N,N-bis(2-chloroethyl)-N-nitrosourea, or 1,3-bis(2-chloroethyl)-1-nitrosourea], Bristol-Myers Squibb), fotemustine (also known as Muphoran®), lomustine (CCNU or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb), nimustine (also known as ACNU), and streptozocin (Zanosar®, Teva Pharmaceuticals).

[0212] Triazine and hydrazine The linkers of the present invention may be used to conjugate an antibody to at least one triazine or hydrazine. Triazines and hydrazines are subclasses of nitrogen-containing alkylating agents. In some embodiments, these compounds may spontaneously decompose or be metabolized to produce alkyldiazonium intermediates, which facilitate the transfer of alkyl groups to nucleic acids, peptides, and / or polypeptides, thereby causing mutagenic, carcinogenic, or cytotoxic effects. Representative examples include, but are not limited to, dacarbazine (DTIC-Dome, Bayer Healthcare Pharmaceuticals Inc.), procarbazine (Mutalane®, Sigma-Tau Pharmaceuticals, Inc.), and temozolomide (Temodar®, Schering Plough).

[0213] Other alkylating agents The linkers of the present invention may be used to conjugate antibodies to at least one ethyleneimine, methylamine derivative, or epoxide. Ethylenimines are a subclass of alkylating agents that typically contain at least one aziridine ring. Epoxides represent a subclass of alkylating agents characterized by a cyclic ether with only three ring atoms.

[0214] Representative examples of ethylenimines include, but are not limited to, thiopeta (Thioplex, Amgen), diaziquone (also known as aziridinylbenzoquinone (AZQ)), and mitomycin C. Mitomycin C is a natural product containing an aziridine ring and is thought to induce cytoxicity through DNA cross-linking (Dorr RT, et al. Cancer Res. 1985;45:3510; Kennedy KA, et al. Cancer Res. 1985;45:3541). Representative examples of methylamine derivatives and their analogs include, but are not limited to, altretamine (Hexalen, MGI Pharma, Inc.) (also known as hexamethylamine and hexastat). Representative examples of epoxides of this class of anticancer compounds include, but are not limited to, dianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) is chemically related to aziridine and generally facilitates the transfer of alkyl groups by a similar mechanism as described above. Dibromodulcitol is hydrolyzed to dianhydrogalactitol and is therefore a prodrug for epoxides (Sellei C, et al. Cancer Chemother Rep. 1969;53:377).

[0215] Antiangiogenic agents In some embodiments, the antibody may be conjugated to at least one anti-angiogenic agent using the linker of the invention. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in a variety of ways. In some embodiments, these agents interfere with the ability of growth factors to reach their targets. For example, vascular endothelial growth factor (VEGF) is one of the major proteins involved in the initiation of angiogenesis by binding to specific receptors on the cell surface. Thus, certain anti-angiogenic agents that block the interaction of VEGF with its cognate receptor prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once specific receptors on the cell surface are triggered, a cascade of other chemical signals is initiated to promote blood vessel growth. Thus, certain enzymes, such as several tyrosine kinases, known to promote intracellular signaling cascades that contribute to cell proliferation, are targets for cancer therapy. In other embodiments, these agents interfere with intercellular signaling cascades. In still other embodiments, these agents disable specific targets that activate and promote cell growth or directly interfere with the growth of vascular cells. Over 300 substances have been discovered to have anti-angiogenic properties, with numerous direct and indirect inhibitory effects.

[0216] Representative examples of antiangiogenic agents that can be used in the ADCs of the invention include angiostatin, ABX EGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux), ZD1839 (Iressa), OSI-774, erlotinib (tarceva), angiostatin, arrestin, endostatin, BAY 12-9566 and in combination with fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and paclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, interleukin-12, interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimastat, prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, in combination with paclitaxel, in combination with gemcitabine and cisplatin, and in combination with irinotecan and cisplatin and in combination with radiation, tecogalan, temozolomide and PEG-interferon alpha 2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (Afinitor, Novartis) Pharmaceutical Corporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.)), nilotinib (Tasigna, Novartis Pharmaceuticals Corporation), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), and phosphoinositide 3-kinase (PI3K) inhibitors.

[0217] Antimetabolites The linkers of the invention may be used to conjugate an antibody to at least one antimetabolite. Antimetabolites are a class of chemotherapy therapeutic agents that closely resemble normal substances within cells. When cells incorporate antimetabolites into cellular metabolism, the result is negative for the cell, for example, the cell cannot divide. Antimetabolites are classified according to the substance they interfere with. Examples of antimetabolites that can be used in the ADCs of the invention include, but are not limited to, folate antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, and pentostatin), as described in more detail below.

[0218] folate antagonists The linkers of the invention may be used to conjugate an antibody to at least one antifolate. Antifolates are a subclass of antimetabolites that are structurally similar to folic acid. Representative examples include, but are not limited to, methotrexate, 4-amino-folic acid (also known as aminopterin and 4-aminopteroic acid), lometrexol (LMTX), pemetrexed (Alimpta, Eli Lilly and Company), and trimetrexate (Neutrexin, Ben Venue Laboratories, Inc.).

[0219] Purine antagonists The linker of the present invention may be used to conjugate an antibody to at least one purine antagonist. Purine analogs are a subclass of antimetabolites that are structurally similar to the group of compounds known as purines. Representative examples of purine antagonists include, but are not limited to, azathioprine (Azasan, Salix; Imuran, GlaxoSmithKline), cladribine (Leustatin [also known as 2-CdA], Janssen Biotech, Inc.), mercaptopurine (Purinethol [also known as 6-mercaptoethanol], GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation), pentostatin (Nipent [also known as 2'-deoxycoformycin (DCF)]), and 6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

[0220] Pyrimidine antagonists The linker of the present invention may be used to conjugate an antibody to at least one pyrimidine antagonist. Pyrimidine antagonists are a subclass of antimetabolites that are structurally similar to the group of compounds known as purines. Representative examples of pyrimidine antagonists include, but are not limited to, azacitidine (Vidaza, Celgene Corporation), capecitabine (Xeloda, Roche Laboratories), cytarabine (also known as cytosine arabinoside and arabinosyl cytosine, Bedford Laboratories), decitabine (Dacogen, Eisai Pharmaceuticals), 5-fluorouracil (Adrucil, Teva Pharmaceuticals; Efudex, Valeant Pharmaceuticals, Inc.), 5-fluoro-2'-deoxyuridine 5'-phosphate (FdUMP), 5-fluorouridine triphosphate, and gemcitabine (Gemzar, Eli Lilly and Company).

[0221] Boron-containing drugs The linker of the present invention may be used to conjugate an antibody to at least one boron-containing drug. Boron-containing drugs are a class of cancer therapeutic compounds that interfere with cell proliferation. Representative examples of boron-containing drugs include, but are not limited to, borophycin and bortezomib (Velcade, Millenium Pharmaceuticals).

[0222] Chemical protectants The linker of the present invention may be used to conjugate an antibody to at least one chemoprotective agent. Chemoprotective drugs are a class of compounds that help protect the body against the toxic effects of chemotherapy. Chemoprotective agents can be administered with various chemotherapy regimens to protect healthy cells from the toxic effects of chemotherapy while simultaneously allowing cancer cells to be treated with the administered chemotherapy. Representative chemoprotective agents include, but are not limited to, amifostine (Ethyol, Medimmune, Inc.), which is used to reduce nephrotoxicity associated with cumulative doses of cisplatin; dexrazoxane (Totect, Apricus Pharma; Zinecard), which is used to treat extravasation caused by the administration of anthracyclines and heart-related complications caused by the administration of the antitumor antibiotic doxorubicin; and mesna (Mesnex, Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitis during chemotherapy treatment with ifosfamide.

[0223] Hormones The linkers of the invention may be used to conjugate an antibody to at least one hormonal agent. Hormonal agents (including synthetic hormones) are compounds that interfere with the production or activity of endogenously produced hormones of the endocrine system. In some embodiments, these compounds interfere with cell growth or produce cytotoxic effects. Non-limiting examples include androgens, estrogens, medroxyprogesterone acetate (Provera, Pfizer, Inc.), and progestins.

[0224] Antihormonal drugs The linkers of the invention may be used to conjugate an antibody to at least one antihormonal agent. An "antihormonal" agent is an agent that suppresses the production and / or blocks the function of certain endogenous hormones. In one embodiment, the antihormonal agent interferes with the activity of a hormone selected from the group including androgen, estrogen, progesterone, and gonadotropin-releasing hormone, thereby interfering with the growth of various cancer cells. Representative examples of antihormonal agents include aminoglutethimide, anastrozole (Arimidex, AstraZeneca Pharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals), cyproterone acetate (Cyprostat, Bayer PLC), degarelix (Firmagon, Ferring Pharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide (Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZeneca Pharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals), letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide (Prostap), lupron, medroxyprogesterone acetate (Provera, Pfizer Inc.), and megestrol acetate (Megace, Bristol-Myers Squibb). Company), tamoxifen (Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl, Ferring).

[0225] Corticosteroids The linker of the present invention may be used to conjugate an antibody to at least one corticosteroid. Corticosteroids can be used in the ADCs of the present invention to reduce inflammation. Examples of corticosteroids include, but are not limited to, glucocorticoids such as prednisone (Deltasone, Pharmacia & Upjohn Company, a division of Pfizer, Inc.).

[0226] Photoactive Therapeutic Agents The linkers of the invention may be used to conjugate the antibody to at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be deployed to kill treated cells when the cells are exposed to electromagnetic radiation of a specific wavelength. Therapeutically significant compounds absorb electromagnetic radiation at wavelengths that are transparent to tissue. In preferred embodiments, the compounds are administered in a non-toxic form that, upon sufficient activation, is capable of producing photochemical effects that are toxic to cells or tissues. In other preferred embodiments, these compounds are retained by cancerous tissue and easily cleared from normal tissue. Non-limiting examples include various chromagens and dyes.

[0227] Oligonucleotides The linker of the present invention may be used to conjugate an antibody to at least one oligonucleotide. Oligonucleotides are made of short nucleic acid strands that act by interfering with the processing of genetic information. In some embodiments, oligonucleotides for use in ADCs are unmodified single- and / or double-stranded DNA or RNA molecules, while in other embodiments, these therapeutic oligonucleotides are chemically modified single- and / or double-stranded DNA or RNA molecules. In one embodiment, the oligonucleotides used in ADCs are relatively short (19-25 nucleotides) and hybridize to unique nucleic acid sequences in the total pool of nucleic acid targets present in cells. Some important oligonucleotide technologies include antisense oligonucleotides (including RNA interference (RNAi)), aptamers, CpG oligonucleotides, and ribozymes.

[0228] antisense oligonucleotides The linker of the present invention may be used to conjugate an antibody to at least one antisense oligonucleotide. The antisense oligonucleotide is designed to bind to RNA by Watson-Crick hybridization. In some embodiments, the antisense oligonucleotide is complementary to nucleotides encoding a region, domain, portion, or segment of the conjugated antibody. In some embodiments, the antisense oligonucleotide comprises about 5 to about 100 nucleotides, about 10 to about 50 nucleotides, about 12 to about 35 nucleotides, or about 18 to about 25 nucleotides.

[0229] There are several mechanisms that can be used to inhibit RNA function once the oligonucleotide binds to the target RNA (Crooke ST. (1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterized antisense mechanism results in cleavage of the targeted RNA by endogenous cellular nucleases such as RNase H or nucleases associated with the RNA interference machinery. However, oligonucleotides that inhibit target gene expression through non-catalytic mechanisms such as modulation of splicing or translation arrest can also be potent and selective modulators of gene function.

[0230] Another RNase-dependent antisense mechanism that has recently attracted much attention is RNAi (Fire et al. (1998). Nature, 391, 806-811; Zamore PD. (2002). Science, 296, 1265-1269). RNA interference (RNAi) is a post-transcriptional process in which double-stranded RNA inhibits gene expression in a sequence-specific manner. In some embodiments, the RNAi effect is achieved by introducing relatively longer double-stranded RNA (dsRNA), while in preferred embodiments, the RNAi effect is achieved by introducing shorter double-stranded RNA, such as small interfering RNA (siRNA) and / or microRNA (miRNA). In yet another embodiment, RNAi can also be achieved by introducing a plasmid that produces dsRNA complementary to a target gene. In each of the aforementioned embodiments, the double-stranded RNA is designed to interfere with gene expression of a specific target sequence in a cell. Generally, this mechanism involves converting dsRNA into short RNA, which directs ribonuclease to homologous mRNA target (summarized in Ruvkun, Science 2294:797(2001)), and then ribonuclease degrades corresponding endogenous mRNA, thereby resulting in the regulation of gene expression.Notably, dsRNA has been reported to have antiproliferative properties, which also allows it to be used for treatment (Aubel et al., Proc.Natl.Acad.Sci.,USA 88:906(1991)). For example, synthetic dsRNA has been shown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA, 62:357-361(1969)), is active in treating leukemia in mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128(1969)), and inhibits chemically induced tumor development in mouse skin (Gelboin et al., Science 167:205-207(1970)). Thus, in a preferred embodiment, the present invention enables the use of antisense oligonucleotides in ADCs for the treatment of breast cancer.In another embodiment, the present invention provides compositions and methods for initiating antisense oligonucleotide therapy, wherein the dsRNA interferes with the expression of EGFR by target cells at the mRNA level. As used above, dsRNA refers to naturally occurring RNA, partially purified RNA, recombinantly produced RNA, synthetic RNA, and modified RNA that differs from naturally occurring RNA by incorporating non-standard nucleotides, non-nucleotide materials, nucleotide analogs (e.g., locked nucleic acids (LNA)), deoxyribonucleotides, and any combination thereof. The RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate antisense oligonucleotide-based modulation as described herein.

[0231] Aptamers The linker of the present invention can be used to conjugate an antibody to at least one aptamer. Aptamers are nucleic acid molecules selected from random pools based on their ability to bind to other molecules. Like antibodies, aptamers can bind to target molecules with exceptional affinity and specificity. In many embodiments, aptamers exhibit complex, sequence-dependent three-dimensional shapes, which allow them to interact with target proteins to form tightly bound complexes similar to antibody-antigen interactions, thereby interfering with the function of the protein. This particular ability of aptamers to tightly and specifically bind to their target proteins is the basis for their promising potential as targeted molecular therapy.

[0232] CpG oligonucleotides The linker of the present invention can be used to conjugate an antibody to at least one CpG oligonucleotide.It is known that bacterial and viral DNA are strong activators of both innate and specific immunity in humans.These immunological properties have been associated with the unmethylated CpG dinucleotide motifs found in bacterial DNA.Due to the fact that these motifs are rare in humans, the human immune system has evolved the ability to recognize these motifs as early indicators of infection and subsequently initiate immune responses.Therefore, the oligonucleotide containing this CpG motif can be used to initiate anti-tumor immune responses.

[0233] Ribozymes The linkers of the present invention may be used to conjugate an antibody to at least one ribozyme. Ribozymes are catalytic RNA molecules ranging from about 40 to 155 nucleotides in length. The ability of ribozymes to recognize and cleave specific RNA molecules makes them promising candidates for therapeutic drugs. A representative example is angiozyme.

[0234] Radionuclide drugs (radioisotopes) The antibody may be conjugated to at least one radionuclide drug using the linker of the present invention.Radionuclide drugs include drugs characterized by an unstable nucleus that can undergo radioactive decay.The success of radionuclide therapy is based on the sufficient concentration of the radionuclide and its long-term retention by cancer cells.Other factors to be considered include the half-life of the radionuclide, the energy of the emitted particle, and the maximum range that the emitted particle can move. In preferred embodiments, the therapeutic agent is a radionuclide selected from the group consisting of In, Lu, Bi, Bi, At, Cu, Cu, Cu, Y, I, I, P, P, Sc, Ag, Ga, Pr, Sm, Tb, Dy, Ho, Re, Re, Re, Pb, Ra, Ac, Fe, Se, As, Sr, Mo, Rh, Pd, Pr, Pm, Er, Ir, Au, Au, and Pb. Radionuclides that substantially decay with Auger-emitting particles are also preferred. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, and Ir-192. The decay energies of useful beta-particle-emitting nuclides are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm-255. The decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV.Additional potentially useful radioisotopes include C, N, Tl, Br, Au, Ru, Ru, Ru, Hg, Hg, Te, Te, Tm, Tm, Tm, Pt, Pd, Rh, Pr, Pr, Tb, Ho, Au, Co, Co, Cr, Fe, Se, Tl, Ac, Br, Yb, and the like.

[0235] Radiosensitizers The linker of the present invention may be used to conjugate an antibody to at least one radiosensitizer. As used herein, the term "radiosensitizer" is defined as a molecule, preferably a low-molecular-weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of target cells to electromagnetic radiation and / or to promote the treatment of diseases treatable with electromagnetic radiation. A radiosensitizer is an agent that increases the sensitivity of cancer cells to radiation therapy while typically having a much lower effect on normal cells. Thus, a radiosensitizer can be used in combination with a radiolabeled antibody or ADC. The addition of a radiosensitizer can result in enhanced efficacy compared to treatment with a radiolabeled antibody or antibody fragment alone. Radiosensitizers are described in D.M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxanes, and cisplatin.

[0236] The radiosensitizer may be activated by the electromagnetic radiation of X-rays. Representative examples of radiosensitizers activated by X-rays include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives thereof. Alternatively, the radiosensitizer may be activated using photodynamic therapy (PDT). Representative examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and therapeutically effective analogs and derivatives thereof.

[0237] Topoisomerase inhibitors The linker of the present invention may be used to conjugate an antibody to at least one topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control changes in DNA structure by catalyzing the phosphodiester backbone of DNA strands, followed by cleavage and recombination, during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecin and its derivatives irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, and teniposide.

[0238] tyrosine kinase inhibitors The linker of the present invention can be used to conjugate an antibody to at least one tyrosine kinase inhibitor. Tyrosine kinase is an intracellular enzyme that functions to attach a phosphate group to the amino acid tyrosine. By preventing the function of protein tyrosine kinase, tumor growth can be inhibited. Examples of tyrosine kinases that can be used in the ADC of the present invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, and vandetanib.

[0239] Other drugs Examples of other agents that can be used in the ADCs of the invention include abrin (e.g., abrin A chain), alpha toxin, Aleurites fordii protein, amatoxin, crotin, curcin, dianthin protein, diptheria toxin (e.g., diphtheria A chain and non-binding active fragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogellin, modeccin A chain, momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (e.g., exotoxin A chain (from Pseudomonas aeruginosa)), restrictocin, ricin A chain, ribonuclease (Rnase), sapaonaria officinalis inhibitors, saporin, alpha-sarcin, Staphylococcal enterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g., PS-341 [bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck & Company, Inc.)), belinostat, entinostat, mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (e.g., geldanamycin and its many analogs), adrenocortical suppressants, and trichothecenes (see, e.g., WO 93 / 21232). Other medications also include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), and tretinoin (Renova, Valeant Pharmaceuticals Inc.).

[0240] It should be noted that the aforementioned groups of drug moieties that can be used in the ADCs of the invention are not exclusive, in that a particular drug example can be found in more than one category; for example, ansamitocins are both antimitotic agents and antitumor antibiotics.

[0241] All stereoisomers of the drug moiety described above are contemplated for the compounds of the present invention, ie, any combination of R and S configurations at the chiral carbon of D.

[0242] A "detectable moiety" or "marker" refers to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, radioactive, or chemical means. For example, useful labels include: 32 P, 35 Detectable moieties include S, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin-streptavidin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with sequences complementary to the target. Detectable moieties often generate a measurable signal, such as a radioactive, color, or fluorescent signal, that can be used to quantify the amount of detectable moiety bound in a sample. Quantification of the signal may be accomplished, for example, by scintillation counting, densitometry, flow cell analysis, ELISA, or direct analysis of the cyclic peptide or subsequent digested peptides by mass spectrometry (one or more peptides may be assayed). Those skilled in the art are familiar with the techniques and detection means for labeling compounds of interest. These techniques and methods are conventional and well known in the art.

[0243] A detection probe refers to (i) a material capable of providing a detectable signal, (ii) a material capable of interacting with a first probe or a second probe and altering the detectable signal provided by the first probe or the second probe, such as by fluorescence resonance energy transfer (FRET), (iii) a material capable of stabilizing the interaction with an antigen or a ligand or increasing the binding affinity, (iv) a material capable of influencing electromobility or cell invasiveness through physical parameters such as charge or hydrophobicity, or (v) a material capable of adjusting ligand affinity, antigen-antibody binding, or ion complex formation.

[0244] In certain embodiments, FRET technology can be used to distinguish intact molecules from molecules that have been exposed to conditions that activate the triggering group, for example, by attaching a donor chromophore as the central Ar ring and an acceptor chromophore as Q.

[0245] In some embodiments, provided herein is the use of the disclosed compounds as imaging agents (e.g., fluorophores or chelators), such as fluoresceins, rhodamines, lanthanide fluorophores, and derivatives thereof. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelating agents include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N''-tetraacetic acid (BAPTA).

[0246] antibody The antibody of the ADC may be any antibody that typically, but not necessarily, specifically binds to an antigen expressed on the surface of a desired target cell. The antigen is not required, but in some embodiments, the bound ADC can be internalized within the cell. The desired target cell may include a cell in which induction of apoptosis is desired. The target antigen may be any protein, glycoprotein, polysaccharide, lipoprotein, etc., expressed on the desired target cell, but typically will be a protein that is either uniquely expressed on the target cell and not expressed on normal or healthy cells, or that is overexpressed on the target cell relative to normal or healthy cells, such that the ADC selectively targets the specific cell of interest, e.g., tumor cell. As will be appreciated by those skilled in the art, the specific antigen, and therefore the antibody, selected will depend on the identity of the desired target cell of interest. In a specific embodiment, the antibody of the ADC is an antibody suitable for administration to humans.

[0247] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end.

[0248] References to "VH" refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to "VL" refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

[0249] The term "antibody" herein is used in the broadest sense to refer to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, murine antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. The term "scFv" refers to a single-chain Fv antibody in which the variable domains of the heavy and light chains from a conventional antibody have been joined to form a single chain.

[0250] Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, a single antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing an antigen-binding site or portion thereof that immunospecifically binds to a desired target antigen, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. However, in one embodiment, the immunoglobulins are of human, murine, or rabbit origin.

[0251] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments. An "Fv" fragment is the smallest antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target-binding site on the surface of the VH-VL dimer. In many cases, six CDRs confer target-binding specificity to an antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to a target. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. A "single-domain antibody" is composed of a single VH or VL domain that exhibits sufficient affinity for the target. In a specific embodiment, a single-domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0252] Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are generated by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art.

[0253] Both the light chain variable domain and the heavy chain variable domain have complementarity-determining regions (CDRs), also known as hypervariable regions. The more highly conserved portions of variable domains are called framework regions (FRs). As is known in the art, the amino acid positions / boundaries defining the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions, in that these positions can be considered within a hypervariable region under one set of criteria, while being considered outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in an extended hypervariable region. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)). As used herein, numbering of amino acid residues in immunoglobulins is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0254] In certain embodiments, the antibodies of the ADCs of the present disclosure are monoclonal antibodies. The term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone (e.g., including any eukaryotic, prokaryotic, or phage clone), not the method by which it is produced. Preferably, the monoclonal antibodies of the present disclosure are present in a homogenous or substantially homogenous population. Monoclonal antibodies include both intact molecules and antibody fragments (e.g., Fab and F(ab')2 fragments) that are capable of specifically binding to a protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from an animal's circulatory system, and may exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., 1983, J. Nucl. Med 24:316). Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Antibodies of the present disclosure include chimeric, primatized, humanized, or human antibodies.

[0255] In most cases, antibodies are composed only of gene-encoded amino acids, although in some embodiments, non-encoded amino acids may be incorporated at specific positions. Examples of non-encoded amino acids that can be incorporated into antibodies for use in controlling stoichiometry and binding positions, as well as methods for making such modified antibodies, are discussed in Tian et al., 2014, Proc Nat'l Acad Sci USA 111(5):1766-1771 and Axup et al., 2012, Proc Nat'l Acad Sci USA 109(40):16101-16106, the entire contents of which are incorporated herein by reference.

[0256] In certain embodiments, the antibodies of the ADCs described herein are chimeric antibodies. As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entireties.

[0257] In certain embodiments, the antibodies of the ADCs described herein are humanized antibodies. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, and 5,693,762; U.S. Pat. No. 6,180,370 to Queen et al.; EP 239400; PCT Publication No. WO 91 / 09967; U.S. Pat. No. 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, as well as US Pat. No. 5,565,332, all of which are incorporated herein by reference in their entireties.

[0258] In certain embodiments, the antibodies of the ADCs described herein are human antibodies. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. As used herein, "human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, and also include antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins and that does not express endogenous immunoglobulins. Human antibodies can be produced by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887, 4,716,111, 6,114,598, 6,207,418, 6,235,883, 7,227,002, 8,809,151, and U.S. Published Application No. 2013 / 189218, the contents of which are incorporated by reference in their entireties. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, 5,939,598, 7,723,270, 8,809,051, and U.S. Published Application No. 2013 / 117871, which are incorporated by reference in their entireties. Additionally, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, NY) may be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection."In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[0259] In certain embodiments, the antibody of the ADCs described herein is a primatized antibody. The term "primatized antibody" refers to an antibody comprising a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated herein by reference in their entireties.

[0260] In certain embodiments, the antibodies of the ADCs described herein are bispecific or dual variable domain antibodies (DVDs). Bispecific and DVD antibodies are monoclonal, often human or humanized, antibodies that have binding specificities for at least two different antigens. DVDs are described, for example, in U.S. Patent No. 7,612,181, the disclosure of which is incorporated herein by reference.

[0261] In certain embodiments, the antibodies of the ADCs described herein are derivatized antibodies. For example, but not limited to, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, derivatives can contain one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0262] In certain embodiments, the antibodies of the ADCs described herein have sequences that have been modified to alter at least one biological effector function mediated by the constant region, relative to the corresponding wild-type sequence. For example, in some embodiments, the antibodies may be modified to reduce at least one biological effector function mediated by the constant region, e.g., to reduce binding to Fc receptors (FcR), relative to an unmodified antibody. Binding to FcR may be reduced by mutating the immunoglobulin constant region segment of the antibody in specific regions required for interaction with FcR (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491, and Lund et al., 1991, J. Immunol. 147:2657-2662).

[0263] In certain embodiments, the antibodies of the ADCs described herein are modified to acquire or improve at least one constant region-mediated biological effector function, e.g., to enhance interaction with FcγR, relative to the unmodified antibody (see, e.g., US2006 / 0134709). For example, antibodies having constant regions that bind to FcγRIIA, FcγRIIB, and / or FcγRIIIA with higher affinity than the corresponding wild-type constant regions can be produced according to the methods described herein.

[0264] In certain specific embodiments, the antibodies of the ADCs described herein are antibodies that bind to tumor cells, such as antibodies against cell surface receptors or tumor-associated antigens (TAAs). In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are differentially expressed on the surface of one or more particular types of cancer cells compared to one or more normal, non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. Such cell surface receptors and tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art.

[0265] Exemplary Cell Surface Receptors and TAAs Examples of cell surface receptors and TAAs that can be targeted by the antibodies of the ADCs described herein include, but are not limited to, the various receptors and TAAs listed below in Table 1. For convenience, information about these antigens, all of which are known in the art, is listed below, including their names, alternative names, GenBank accession numbers, and primary reference(s) in accordance with the National Center for Biotechnology Information (NCBI) Nucleic Acid and Protein Sequence Identification Conventions. The nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases such as GenBank.

[0266] [Table 1] TIFF0007657153000088.tif252159TIFF0007657153000089.tif255159TIFF0007657153000090.tif150160

[0267] Exemplary Antibodies Exemplary antibodies of interest for use in the ADCs of the disclosure include 3F8 (GD2), Abagovomab (CA-125 (mimetic)), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), ALD518 (IL-6), alemtuzumab (CD52), altumomab pentetate (CEA), amatuximab (mesothelin), anatumomab mafenatox (Anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), besilesomab (CEA-related antigen), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30 (TNFRSF8)), cantuzumab mertansine (mucin CanAg), cantuzumab vedotin (Cantuzumab ravtansine) (MUC1), capromab pendetide (prostate cancer cells), carlumab (MCP-1), catumaxomab (EpCAM, CD3), CC49 (Tag-72), cBR96-DOX ADC (Lewis Y antigen), cetuximab (EGFR), sitatuzumab bogatoxin (Citatuzumabbogatox (EpCAM), cixutumumab (IGF-1 receptor), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-E2), dacetuzumab (CD40), darotuzumab (insulin-like growth factor 1 receptor), deratumumab (CD38 (cyclic ADP-ribose hydrolase)), demcizumab (DLL4), denosumab (RANKL), detumomab (B-cell lymphoma) tumor cells), Drozitumab (DR5), Dusigitumab (ILGF2), Eclomeximab (D3 ganglioside), Eculizumab (C5), Edrecolomab (EpCAM), Elotuzumab (SLAMF7), Elsilimomab (IL-6), Enavatuzumab (TWEAK receptor), Enotikumab (DLL4), Ensituximab (5AC), Epitumomab-situxetan cituxetan (episialin), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etancizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (HGF), figitumumab (IGF-1 receptor), framvotumab (TYRP1 (glycoprotein 75)), fresolimumab (TGF-1), galiximab ( CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovomab (CA-125), IMAB362 (CLDN18.2), imgatuzumab (EGFR), indatuximab ravtansineravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30 (TNFRSF8)), labetuzumab (CEA), lambrolizumab (PDCD1), lexatumumab (TRAIL-R2), lintuzumab (CD33), rolbo Tuzumab Mertansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23 (IgE receptor)), Mapatumumab (TRAIL-R1), Margetuximab (ch4DS), Matuzumab (EGFR), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumomab Pasudotoxin (Moxetumomab) pasudotox (CD22), nacolomab tafenatox (C2-42 antigen), naptumomab estafenatox (5T4), narutumab (RON), natalizumab (integrin α4), necitumumab (EGFR), nesvacumab (angiopoietin 2), nimotuzumab (EGFR), nivolumab (IgG4), ocaratuzumab (CD20), ofatumumab (CD20), olaratuzumab (PDGF-Rα), onartuzumab (human scatter factor receptor kinase), ontuximab (TEM1), oportuzumab monato (EpCAM), oregovomab (CA-125), otlertuzumab (CD37), panitumumab (EGFR), pancomab (tumor-specific glycosylation of MUC1), parsatuzumab (EGFL7), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pidilizumab (PD-1), pinatuzumab vedotinvedotin (CD22), Pritumumab (Vimentin), Racotumomab (N-Glycolylneuraminic Acid), Radretumab (Fibronectin Extra Domain B), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Lobatumumab (IGF-1 Receptor), Samalizumab (CD200), Satumomab Pendetide (TAG-72), Seriban ERBB3, Sibrotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), Siltuximab (IL-6), Solitomab (EpCAM), Sonepcizumab (Sphingosine-1-phosphate), Tabalumab (BAFF), Tacatuzumab tetraxetan (alpha-fetoprotein), Taplitumomab paptox paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), TGN1412 (CD28), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tobetumab (CD40a), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab-cell molleukin (EpCAM), ublituximab (MS4A), urelumab (4-1BB), vandetanib (VEGF), vanticutumab (Frizzled receptor), volociximab (integrin α5β1), borsetuzumab-mafodotin mafodotin (CD70), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

[0268] How to generate antibodies The antibodies of the ADCs can be prepared by recombinantly expressing immunoglobulin light and heavy chain genes in host cells. For example, to express an antibody recombinantly, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, thereby allowing the light and heavy chains to be expressed in the host cells and, optionally, secreted into the medium in which the host cells are cultured, from which the antibody can be recovered. Standard recombinant DNA techniques are used to obtain antibody heavy and light chain genes, incorporate these genes into a recombinant expression vector, and introduce the vector into host cells. This is described, for example, in Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook, Fritsch, and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Patent No. 4,816,397.

[0269] In one embodiment, Fc variant antibodies are similar to their wild-type counterparts except for changes in their Fc domain. To generate nucleic acids encoding such Fc variant antibodies, conventional mutagenesis techniques can be used to synthesize a DNA fragment encoding the Fc domain or a portion of the Fc domain (referred to as the "wild-type Fc domain") of a wild-type antibody, which can then be used as a template for mutagenesis to generate antibodies as described herein. Alternatively, the DNA fragment encoding the antibody can be synthesized directly.

[0270] Once DNA fragments encoding the wild-type Fc domain are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the constant region genes into full-length antibody chain genes. In these manipulations, the CH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. As used in this context, the term "operatively linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0271] To express an Fc variant antibody, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector so that these genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The variant antibody light chain gene (gne) and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0272] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction enzyme sites are present). Prior to insertion of the variant Fc domain sequence, the expression vector can already carry the antibody variable region sequence. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0273] In addition to the antibody chain genes, the recombinant expression vector carries regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be apparent to those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the desired expression level of protein, etc. Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and their sequences, see, e.g., U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al., and U.S. Patent No. 4,968,615 by Schaffner et al.

[0274] In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all to Axel et al.). For example, the selectable marker gene typically confers resistance to drugs, such as G418, puromycin, blasticidin, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use with methotrexate selection / amplification in DHFR-host cells) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0275] Antibodies can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, antibody expression is carried out in eukaryotic cells, such as mammalian host cells, to obtain optimal secretion of properly folded, immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO) cells (e.g., including DHFR-CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, such as those described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules.

[0276] In some embodiments, the antibody of the ADC may be a bifunctional antibody. Such an antibody, in which one heavy chain and one light chain are specific for one antigen and the other heavy and light chains are specific for a second antigen, may be produced by cross-linking the antibody to a second antibody by standard chemical cross-linking methods. Bifunctional antibodies may also be made by expressing nucleic acids engineered to encode the bifunctional antibody.

[0277] In certain embodiments, bispecific antibodies, i.e., antibodies that use the same binding site to bind one antigen and a second, unrelated antigen, can be produced by mutating amino acid residues in the light and / or heavy chain CDRs. Exemplary second antigens include proinflammatory cytokines (e.g., lymphotoxin, interferon-γ, or interleukin-1). Bispecific antibodies can be produced, for example, by mutating amino acid residues in the vicinity of the antigen-binding site (see, e.g., Bostrom et al., 2009, Science 323:1610-1614). Bifunctional antibodies can be made by expressing nucleic acids engineered to encode the bispecific antibody.

[0278] Antibodies can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Antibodies can also be produced using cell-free platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals)).

[0279] Methods for recombinant expression of Fc fusion proteins are described in Flanagan et al., Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols.

[0280] Once an antibody has been produced by recombinant expression, it may be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography for the antigen, particularly after selection with Protein A or Protein G, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins.

[0281] Once isolated, the antibodies can be further purified, if desired, by, for example, high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds., Elsevier, 1980)) or by gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0282] Imaging Compounds and Sensors In certain embodiments, provided herein are uses of the disclosed compounds in imaging compositions and as sensors.

[0283] The sensor can be a biosensor, a chemical sensor, or a molecular switch. Biosensors are capable of identifying the presence or amount of a specific material by reacting it with bioreceptors (portions designed to be able to adsorb to and react with biological materials such as DNA, RNA, antibodies, enzyme proteins, cells, biological membranes, and hormone receptors) having selective specificity, and then measuring it using a signal transducer (a device that converts the reaction between a specific material and a biological receptor into an electrical signal using various methods). Biosensors can be used in the medical industry, environmental industry, processing industry, military (chemical warfare), research, food, and the like (e.g., Biosensors and Bioelectronics, 2016, 32-45; Pol. J. Environ. Stud. 2015, 19-25; Analytica Chimica Acta 568 (2006) 200-210; Biosensors and Bioelectronics 2017, 217-231; ACS Appl. Mater. Interfaces 2015,7,20190-20199, Journal of Coastal Life Medicine 2016;4(3):200-202, Artificial Cells, Blood Substitutes, and Biotechnology,39:281-288, Journal of Controlled Release 159(2012)154-163).

[0284] Chemical sensors use electrical properties such as electricity, resistance, and potential difference, and optical properties such as color and fluorescence to quickly and accurately monitor specific materials in many fields, such as clinical diagnosis, medical research, chemical material measurement, and environmental measurement. These include gas sensors (hydrogen, oxygen, carbon monoxide), ion sensors (cations, anions, gas-sensitive ions), constituent sensors (gas phase, liquid phase, luminescent constituents), humidity sensors (relative humidity, absolute humidity, condensation), and dust / soot sensors (airborne dust, dust, soot, turbidity) (e.g., Chem. Soc. Rev., 2015, 44, 3358; Journal of the Korean Chemical Society, 2010, 451-459; Chem. Sci., 2015, 6, 1150-1158; KR 10-1549347; J. Phys. Chem. B 2016, 120, 7053-7061; ACS Appl. Mater. Interfaces 2015, 7, 704-712; J. Am. Chem. Soc. 2011, 133, 10960-10965; J. Am. Chem. Soc. 2012, 134, 20412-20420; Org. Lett. 2014, 16, 1680-1683; J. Org. Chem. 2013, 78, 702-705; J. Org. Chem. 2015, 80, 12129-12136; ACS Macro Lett. 2014, 3, 1191-1195; New J. Chem., 2012, 36, 386-393; Chem. Commun., 2010, 46, 6575-6577; see 2013).

[0285] A molecular switch is a molecule that can be reversibly switched between two or more stable states. The molecule can switch between states in response to environmental stimuli, such as changes in the chemical environment (e.g., pH), light irradiation (e.g., light of a particular wavelength), temperature, electrical current, the microenvironment, or the presence of a ligand. In some cases, switching between states can depend on a combination of stimuli. The earliest form of synthetic molecular switch is the pH indicator, which displays distinct colors as a function of pH. Synthetic molecular switches may also be applied in molecular computers or reactive drug delivery systems. Molecular switches are also important in biology, as many biological functions, such as allosteric regulation and vision, are based on them.

[0286] Such biosensors, chemical sensors, and molecular switches may further comprise additional photoreactive moieties such as rhodamine, phenol red, orange azo dye, papa red, non-sulfonated cyanine, sulfonated cyanine, chemiluminescent fluoride sensors (1,2-dioxetane derivatives), and D2A dye (NIR fluorescent dye). Alternatively, the photoreactive moiety may be selected from compounds having functional groups and structures similar to: [ka] During the ceremony, R 100 is H or C1-C6-alkyl, R 101 is H or SO3H, and R 102 is C1-C6 alkyl, or -(CH2) z COOH, z is an integer from 3 to 8, R 103 and R 104 are each independently H or C1-C6 alkyl, R 105 and R 106 are each independently hydrogen, COOH, or SO3H.

[0287] Additional photoreactive moieties are known in the art (see, e.g., Org. Lett. 2014, 16, 1680-1683; J. Am. Chem. Soc. 2011, 133, 10960-10965; Dye Lasers, 3rd Ed. (Springer-Verlag, Berlin, 1990); J. Am. Chem. Soc. 2012, 134, 20412-20420).

[0288] Treatment method targeted therapy The targeting moiety of the conjugate may be recognized by cells, thereby providing so-called targeted therapy.

[0289] In some embodiments, the conjugate comprises an active agent for use in targeted therapy for the treatment of autoimmune diseases, hi some such embodiments, the active agent is selected from cyclosporine, cyclosporine A, mycophenolate mofetil, sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate cyclophosphamide, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab, and the like.

[0290] In some embodiments, the compound comprises an active agent Q for use in targeted therapy for the treatment of infectious diseases. In some such embodiments, Q is selected from the group consisting of beta-lactams (penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, becampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin), aminoglycosides (amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin), macrolides (azithromycin, clamicin, thiamin monophosphate, thiamin monophosphate), thiamin monophosphate, ... Rithromycin, erythromycin, lincomycin, clindamycin), tetracyclines (domeclocycline, doxycycline, minocycline, tetracycline), quinolones (cinoxacin, nalidixic acid), fluoroquinolones (ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxicin), polypeptides (bacitracin Antibiotics (trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampicin, spectinomycin, nitrofurantoin), general antivirals (idoxuridine, vidarabine, aspirin, rifampicin, rifampicin, spectinomycin, nitrofurantoin), sulfonamides (sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, sulfacetamide), other antibiotics (trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampicin, spectinomycin, nitrofurantoin), general antivirals (idoxuridine, vidarabine, aspirin, rifampicin, spectinomycin, nitrofurantoin), lovir, famcicyclovir, pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir, antisense oligonucleotides, immunoglobulins, interferons, HIV infection treatments (tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine,It is selected from nevirapine, delaviridine, saquinavir, ritonavir, indinavir, nelfinavir, etc.

[0291] In some embodiments, the compounds and conjugates disclosed herein comprise an active agent Q for use in a method for delivering an active agent to a cell for the treatment of a tumor, wherein the targeting moiety is selected to bind to the target cell (i.e., a cancer cell). In particular, the compounds, conjugates, and compositions of the invention may be useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), such as when the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least associated preferentially with tumor cells over healthy cells).

[0292] In some such embodiments, the active agent is selected from cytotoxic or immunomodulatory agents, anti-cancer agents, anti-tubulin agents, cytotoxic agents, etc. Preferably, cytotoxic or immunomodulatory agents include anti-tubulin agents, auristatins, DNA minor groove binders, DNA transcription inhibitors, alkylating agents, anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposide, fluorindated pyrimidines, ionophores, lexitropsin, maytansinoids, nitrosoureas, platinol, pore-forming compounds, purine antimetabolites, puromycin, radiosensitizers, rapamycin, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc., and anti-cancer agents include methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydrochloride, hydroxybenzoates ... Antitubulin agents include thrombin, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, and the like. Antitubulin agents include taxanes (e.g., paclitaxel, docetaxel), T67, vinca alkaloids (vinca alkyloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), baccatin derivatives, taxane derivatives, epothiolone (e.g., epothilone A, epothilone B), nocodazole, colchicine, colcemid, estramustine, cryptophycins, cemadotin, maytansinoids, combrestatin, discodermolide, erytherobin, auristatin derivatives (AFP, MMAF, MMAE), and the like; cytotoxic agents include androgens, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin,Busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (actinomycin), daunorubicin, dacarbazine (decarbazine), D M1, DM4, docetaxel, doxorubicin, etoposide, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate methicone, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbidine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16, VM-26; DNA minor groove binders (e.g., enediynes, lexitropsin, CBI compounds), duocarmycin , taxanes (e.g., paclitaxel, docetaxel), puromycin, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A, epothilone B, estramustine, cryptophycin, cemadotin, maytansinoids, discodermolide, eluterobin, mitoxantrone, and the like.

[0293] Cell proliferation and apoptosis The compounds and conjugates disclosed herein may be used in methods for inducing apoptosis in cells.

[0294] Dysregulation of apoptosis has been implicated in a variety of diseases, including, for example, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma, or Crohn's disease), hyperproliferative disorders (e.g., breast cancer, lung cancer), viral infections (e.g., herpes, papilloma, or HIV), and other conditions such as osteoarthritis and atherosclerosis. The compounds, conjugates, and compositions described herein may be used to treat or ameliorate any of these diseases. Such treatment generally involves administering to a subject suffering from the disease a compound, conjugate, or composition described herein in an amount sufficient to provide a therapeutic benefit. The identity of the antibody of the administered compound, conjugate, or composition will depend on the disease being treated; thus, the antibody should bind to a cell surface antigen expressed in a cell type whose inhibition would be beneficial. The therapeutic benefit achieved will also depend on the specific disease being treated. In certain cases, the compounds and compositions disclosed herein, when administered as a monotherapy, may treat or ameliorate the disease itself or the symptoms of the disease. In other cases, the compounds and compositions disclosed herein may be part of an overall treatment regimen that includes other agents that, in conjunction with the inhibitor or compounds and compositions disclosed herein, treat or ameliorate the disease or symptoms of the disease being treated. Agents useful for treating or ameliorating a particular disease that can be administered to supplement or in conjunction with the compounds and compositions disclosed herein will be apparent to those skilled in the art.

[0295] Although an absolute cure is always desirable in any therapeutic regimen, achieving a cure is not required to provide a therapeutic benefit. Therapeutic benefit may include halting or slowing the progression of the disease, regressing the disease without a cure, and / or ameliorating or slowing the progression of disease symptoms. Prolonged survival and / or improved quality of life compared to the statistical mean may also be considered a therapeutic benefit.

[0296] One particular class of disease that involves dysregulation of apoptosis and is a significant global health burden is cancer. In specific embodiments, the compounds and compositions disclosed herein can be used to treat cancer. The cancer can be, for example, a solid tumor or a hematological tumor. Cancers that can be treated with the compounds and compositions disclosed herein include, but are not limited to, bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer, small cell lung cancer, and splenic cancer. The compounds and compositions disclosed herein may be particularly beneficial in the treatment of cancer, as they can be used to specifically target tumor cells using antibodies, thereby potentially avoiding or ameliorating undesirable side effects and / or toxicity that may be associated with systemic administration of unconjugated inhibitors. One embodiment relates to a method for treating a disease associated with dysregulated intrinsic apoptosis, comprising administering to a subject with a disease associated with dysregulated apoptosis an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand of the compounds and compositions disclosed herein binds to a cell surface receptor on cells in which intrinsic apoptosis is dysregulated. One embodiment relates to a method for treating cancer, comprising administering to a subject with cancer an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand is capable of binding to a cell surface receptor or tumor-associated antigen expressed on the surface of cancer cells.

[0297] In the context of tumorigenic cancers, therapeutic benefit, in addition to including the effects discussed above, may also specifically include halting or slowing the progression of tumor growth, regressing tumor growth, eradicating one or more tumors, and / or increasing patient survival compared to the statistical mean for the type and stage of cancer being treated. In one embodiment, the cancer being treated is a tumorigenic cancer.

[0298] The compounds and conjugates disclosed herein can be administered as monotherapy to provide therapeutic benefit, or can be administered adjunctively or in conjunction with other chemotherapeutic agents and / or radiotherapy. The chemotherapeutic agents for which the compounds and compositions disclosed herein can be utilized as adjunctive therapy can be targeted (e.g., ADCs, protein kinase inhibitors, etc.) or non-targeted (e.g., non-specific cytotoxic agents such as radionucleotides, alkylating agents, and intercalating agents). Non-targeted chemotherapeutic agents with which the compounds and compositions disclosed herein may be administered adjunctively include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbidine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asperaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

[0299] The compounds and conjugates disclosed herein, which may not be effective in treating cancer as monotherapy, can be administered adjunctively or together with other chemotherapeutic agents or radiotherapy to provide a therapeutic benefit. One embodiment relates to a method of administering a compound or composition disclosed herein in an amount effective to sensitize tumor cells to standard chemotherapy and / or radiotherapy. Thus, in the context of cancer treatment, "therapeutic benefit" includes administering the compounds and compositions disclosed herein adjunctively or together with chemotherapeutic agents and / or radiotherapy as a means of sensitizing tumors to chemotherapy and / or radiotherapy, either in patients who have not yet begun chemotherapy and / or radiotherapy or who have not yet shown signs of resistance, or in patients who have begun to show signs of resistance.

[0300] Pharmaceutical Compositions and Administration Thereof The compounds and conjugates disclosed herein can be used to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a disclosed compound and a pharmaceutically acceptable carrier.

[0301] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for administration to humans, particularly for invasive administration routes (i.e., routes such as injection or implantation that bypass transport or diffusion across epithelial barriers), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical compositions can be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophiles for reconstitution, powders, liquids, syrups, suppositories, injectables, etc. The compositions can also be present in transdermal delivery systems, e.g., skin patches. The compositions can also be present in liquid formulations suitable for topical administration, such as ointments or creams.

[0302] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that act, for example, to stabilize, increase the solubility, or increase the absorption of a compound such as a compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The pharmaceutical composition preparation can be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be, for example, a liposome or other polymer matrix that can have a compound of the present invention incorporated therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0303] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0304] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; and (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and corn oil. (10) oils such as propylene glycol, (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0305] Pharmaceutical compositions (preparations) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., drenches, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, or pastes for application to the tongue, such as in aqueous or non-aqueous solutions or suspensions); absorbed through the oral mucosa (e.g., sublingually); anally, rectally, or vaginally (e.g., as a pessary, cream, or foam); parenterally (e.g., as a sterile solution or suspension, including intramuscularly, intravenously, subcutaneously, or intrathecally); nasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for such may be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein.

[0306] The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0307] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0308] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), lyophilized tablets, powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (with an inert base such as gelatin and glycerin, or sucrose and acacia), and / or mouthwashes, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compounds, conjugates, or compositions may also be administered as a bolus, electuary, or paste.

[0309] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin. (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0310] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0311] Tablets and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art of pharmaceutical formulation. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a type that releases the active ingredient(s) only, or preferentially, in a certain portion of the digestive tract, optionally in a delayed manner. Examples of implantable compositions that may be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0312] The liquid dosage form useful for oral administration includes pharmaceutically acceptable emulsion, lyophilized agent for reconstitution, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form may contain inert diluent commonly used in the art, such as water or other solvent, solubilizer and emulsifier, such as cyclodextrin and its derivatives, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and mixtures thereof.

[0313] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0314] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar and tragacanth, and mixtures thereof.

[0315] Formulations of the pharmaceutical compositions for rectal, intravaginal, or urethral administration may be presented as suppositories, which can be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and will melt in the rectum or vaginal cavity to release the active compounds.

[0316] Formulations of the pharmaceutical composition for administration to the mouth may be presented as a mouthwash, or a mouth spray, or a mouth ointment.

[0317] Alternatively or additionally, the compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device, which may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0318] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as are known in the art to be appropriate.

[0319] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0320] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0321] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0322] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0323] Ophthalmic formulations, eye ointments, powders, solutions, and the like, are also contemplated as being within the scope of the present invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, the liquid ophthalmic formulation has properties similar to those of tears, aqueous humor, or vitreous humor, or is compatible with such fluids.

[0324] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0325] Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable isotonic aqueous or non-aqueous sterile solutions, dispersions, suspensions, or emulsions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use.

[0326] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0327] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action may be ensured by incorporating various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable dosage form can be achieved by incorporating agents that delay absorption, such as aluminum monostearate and gelatin.

[0328] In some cases, in order to prolong the effect of a drug, it is desirable to slow down the absorption of the drug from subcutaneous injection or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, the absorption delay of parenterally administered drugs can be achieved by dissolving or suspending the drug in an oil vehicle.

[0329] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug-to-polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0330] When used in the methods of the present invention, the active compound may be administered by itself or in combination with a pharmaceutically acceptable carrier, for example, as a pharmaceutical composition containing 0.1 to about 99.5% (more preferably about 0.5 to about 90.0%) of the active ingredient.

[0331] In some embodiments of the present invention, the compounds of the present invention are co-administered with one or more additional compounds / agents.

[0332] In certain such embodiments, the co-administration is simultaneous. In certain such embodiments, a compound of the invention is combined with one or more additional compounds. In certain other such embodiments, a compound of the invention is administered separately but simultaneously with one or more additional compounds. In certain such embodiments, the co-administration is sequential, with a compound of the invention being administered minutes or hours before or after the administration of one or more additional compounds.

[0333] The method of introducing the compounds of the present invention can also be enabled by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years to achieve controlled delivery of drugs, including proteinaceous biologics. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants to achieve sustained release of compounds at specific target sites.

[0334] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0335] The selected dosage level will depend on a variety of factors, including the activity of the particular compound, conjugate, or combination of compounds and / or conjugates, or esters, salts, or amides thereof, employed, the route of administration, the time of administration, the excretion rate of the particular compound(s) employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, medical condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0336] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start dosing the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, other therapeutic agents being administered together with the compound of the present invention. A higher total dose may be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0337] Generally, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0338] If desired, the effective daily dose of the active compound or conjugate can be optionally administered as 1, 2, 3, 4, 5, 6 or more sub-doses, which are administered separately at appropriate intervals throughout the day in unit dosage form.In certain embodiments of the present invention, the active compound can be administered twice or three times a day.In preferred embodiments, the active compound will be administered once a day.

[0339] Patients receiving this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as horses, cattle, pigs, and sheep, as well as poultry and pets in general.

[0340] In certain embodiments, the compounds or conjugates disclosed herein may be used alone or co-administered with another type of therapeutic agent. As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds or conjugates, such that a second compound or conjugate is administered while a previously administered therapeutic compound or conjugate is still effective in the body (e.g., two compounds or conjugates are effective in a patient simultaneously, which may include a synergistic effect of the two compounds or conjugates). For example, different therapeutic compounds or conjugates may be administered simultaneously or sequentially in the same formulation or in separate formulations. In certain embodiments, different therapeutic compounds or conjugates may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, or longer of each other. Thus, individuals receiving such treatment can benefit from the combined effects of different therapeutic compounds or conjugates.

[0341] The present invention includes the use of pharmaceutically acceptable salts of the compounds or conjugates disclosed herein. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl salts, dialkyl salts, trialkyl salts, or tetra-alkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine salts, benethamine salts, benzathine salts, betaine salts, calcium hydroxide salts, choline salts, deanol salts, diethanolamine salts, diethylamine salts, 2-(diethylamino)ethanol salts, ethanolamine salts, ethylenediamine salts, N-methylglucamine salts, hydrabamine salts, 1H-imidazole salts, lithium salts, L-lysine salts, magnesium salts, 4-(2-hydroxyethyl)morpholine salts, piperazine salts, potassium salts, 1-(2-hydroxyethyl)pyrrolidine salts, sodium salts, triethanolamine salts, tromethamine salts, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

[0342] Pharmaceutically acceptable acid addition salts may also exist as various solvates, for example with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in the preparation or crystallization solvent or from a crystallization solvent adventitious to such solvent.

[0343] Wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0344] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium disulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0345] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention. [Example]

[0346] Synthesis protocol Abbreviation AcO: acetyl AcOH: acetic acid EA: Ethyl acetate DCM: dichloromethane m-CPBA: meta-chloroperbenzoic acid TBDMSOTf: tert-butyldimethylsilyl triflate TBDMS: tert-butyldimethylsilyl DMF: dimethylformamide EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HOBt: 1-hydroxybenzotriazole hydrate ACN: acetonitrile TBDMS-Cl: tert-butyldimethylsilyl chloride DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene THF: tetrahydrofuran DCC: N,N'-dicyclohexylcarbodiimide DMAP: 4-dimethylaminopyridine NHS: N-hydroxysuccinimide DIPEA: Diisopropylethylamine TEA: Triethylamine DEAD: Diethyl azodicarboxylate Boc: tert-butyloxycarbonyl LAH: Lithium aluminum hydride CDI: 1,1'-carbonyldiimidazole BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine TPSCl: Triphenylchlorosilane tfa: trifluoroacetyl PyBop: Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate HBTU: N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate TFA: Trifluoroacetic acid DIC: N,N'-diisopropylcarbodiimide DMPA: 2,2-dimethoxy-2-phenylacetophenone TBAF: tetra-n-butylammonium fluoride AgOTf: silver trifluoromethanesulfonate (BimC4A)3: tripotassium 5,5',5''-[2,2',2''-nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)]tripentanoate hydrate

[0347] [Example 1] Preparation of Int-TG [ka]

[0348] β-D-Galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in 33% HBr in AcOH (20 mL) at 0 °C under a N atmosphere. The mixture was allowed to warm to room temperature. After stirring at room temperature for 4 h, the mixture was concentrated under reduced pressure, followed by the addition of EA (1000 mL) and saturated sodium bicarbonate (1000 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG (5.2 g, 99%). 1 H NMR(400 MHz,CDCl3) δ 6.70(d,J=4.0 Hz,1H),5.52(d,J=2.4 Hz,1H),5.41(dd,J=7.6,2.8 Hz,1H),5.05(dd,J=6.4,4.0 Hz,1H),4.49(t,J=6.4 Hz,1H),4.22-4.09(m,2H),2.16-2.01(m,12H).

[0349] [Example 2] Preparation of compound Int-TG1 [ka]

[0350] Preparation of compound Int-TG1-1 To a solution of salicylaldehyde (Aldrich, CAS 90-02-8, 148 mg, 1.22 mmol) and compound Int-TG (0.5 g, 1.22 mmol) in acetonitrile (10 mL), dried molecular sieves (2.5 g) and AgO (845 mg, 3.65 mmol) were added under a N atmosphere. After stirring at room temperature for 1 h, distilled water (50 mL) and EA (50 mL × 2) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1-1 (441 mg, 81%). 1H NMR(400 MHz,CDCl3) δ 10.37(s,1H),7.88(t,J=7.6 Hz,1H),7.21(t,J=7.4 Hz,1H),7.14(t,J=8.4 Hz,1H),5.62(m,1H),5.48(m,1H),5.16(d,J=7.2 Hz,2H),4.27-4.23(m,1H),4.18-4.09(m,2H)m 2.21(s,3H),2.07(s,6H),2.03(s,3H).

[0351] Preparation of compound Int-TG1-2 To a solution of compound Int-TG1-1 (260 mg, 0.575 mmol) in DCM (3 mL) was added m-CPBA (283 mg, 1.149 mmol) at 0 °C under a N atmosphere. After 5 h, the mixture was concentrated under reduced pressure. EA (50 mL × 2) and aqueous sodium bicarbonate solution (30 mL) were added. The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound Int-TG1-2 (270 mg, quantitative). Compound Int-TG1-2 was used directly in the next reaction without purification. 1 H NMR(400 MHz,CDCl3) δ 8.18(s,1H),7.90(d,J=8.0 Hz,1H),7.64(d,J=8.0 Hz,1H),7.46(t,J=7.6 Hz,1H),7.15(d,J=8.4 Hz,1H),5.51(m,2H),5.11(d,J=8.8 Hz,1H),5.04(d,J=8.0 Hz,1H),4.24(m,1H),4.16(m,1H),4.08(m,1H),2.18(s,3H),2.09(s,3H),2.07(s,3H),2.02(s,3H).EI-MS m / z:491(M + +Na).

[0352] Preparation of Compound Int-TG1-3 To a solution of compound Int-TG1-2 in CHCl (3 mL) was added hydrazine hydrate (21 μL, 0.427 mmol) at 0° C. under a N atmosphere. After stirring at 0° C. for 0.5 h, EA (30 mL×2) and 1 M aqueous HCl (10 mL) were added. The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound Int-TG1-3 (161 mg, 86%). 1 H NMR(400 MHz,CDCl3) δ 7.03(t,J=8.0 Hz,1H),6.98-6.95(m,2H),6.83(t,J=7.6 Hz,1H),6.02(s,1H),5.47(d,J=3.2 Hz,2H),5.13(dd,J=10.8,2.8 Hz,1H),4.93(d,J=7.6 Hz,1H),4.26(m,1H),4.19-4.09(m,2H),4.06(m,1H),2.21(s,3H),2.13(s,3H),2.08(s,3H),2.03(s,3H).EI-MS m / z:463(M + +Na).

[0353] Preparation of compound Int-TG1 To a solution of compound Int-TG1-3 (161 mg, 0.366 mmol) in DCM (3 mL) was added EtN (102 μL, 0.732 mmol) and TBDMS-OTf (126 μL, 0.549 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2 h. Then DCM (30 mL × 2) and 1 M aqueous HCl (10 mL) were added. The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1 (147 mg, 91%). 1H NMR(400 MHz,CDCl3) δ 7.02(d,J=7.6 Hz,1H),6.95-6.84(m,3H),5.48-5.43(m,2H),5.15(d,J=8.0 Hz,1H),5.10(d,J=10.4 Hz,1H),4.21-4.11(m,2H),4.03-3.99(m,1H),2.19(s,3H),2.04(s,3H) ,2.02(s,3H),2.00(s,3H),0.99(s,9H),0.20(s,3H),0.16(s,3H).EI-MS m / z:555(M + ).

[0354] [Example 3] Preparation of compound Int-TG2 [ka]

[0355] Preparation of compound Int-TG2-1 To a solution of 3-formyl-4-hydroxybenzoic acid (3 g, 18.06 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS 134179-38-7, 5.98 g, 23.48 mmol) in DMF (20 mL) was added EDCI (5.19 g, 27.09 mmol), HOBt (4.15 g, 27.09 mmol), and EtN (10.1 mL, 72.24 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature overnight under a N atmosphere. The reaction was quenched with EA (60 mL × 2) and citric acid (60 mL). The organic layer was extracted with aqueous sodium bicarbonate (80 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2-1 (2.56 g, 39%). 1H NMR(400 MHz,CDCl3) δ 11.26(s,1H),9.96(s,1H),8.16(s,1H),7.98-7.96(d,J=8.4 Hz,1H),7.04-7.02(d,J=9.2 Hz,1H),6.91(s,1H),3.68-3.61(m,14H),3.37-3.34(m,2H),EI-MS m / z:367(M + ).

[0356] Preparation of compound Int-TG2-2 To a solution of compound Int-TG2-1 (1.41 g, 3.85 mmol) and compound Int-TG (1.74 g, 4.24 mmol) in anhydrous ACN (20 mL) was added molecular sieves (8 g) and AgO (2.68 g, 11.55 mmol) at room temperature under a N atmosphere. The mixture was stirred at room temperature for 3 hours and then filtered through Celite®. The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2-2 (1.88 g, 70%). 1 H NMR(400 MHz,CDCl3) δ 10.35(s,1H),8.20-8.17(m,2H),7.26(s,1H),7.20-7.18(d,J=9.2 Hz,1H),6.96(s,1H),5.63-5.58(m,1H),5.50-5.49(m,1H),5.23-5.21(m,1H),5.18-5.14(m,1H),4.24- 4.14(m,3H),3.69-3.64(m,14H),3.37-3.35(m,2H),2.21(s,3H),2.08-2.07(m,6H),2.03(s,3H).EI-MS m / z:697(M + ).

[0357] Preparation of compound Int-TG2-3 To a solution of compound Int-TG2-2 (1.69 g, 2.42 mmol) in DCM (15 mL) was added m-CPBA (2.4 g, 9.70 mmol) at 0 °C under a N atmosphere. After stirring at 0 °C for 7 h, the mixture was quenched by the addition of saturated sodium bicarbonate (40 mL × 2). The mixture was separated, and the organic layer was washed with brine, dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2-3 (1.25 g, 76%). 1 H NMR(400 MHz,CDCl3) δ 7.36-7.33(m,2H),7.01-6.99(d,J=8.4 Hz,1H),6.71(m,1H),6.06(s,1H),5.49-5.44(m,2H),5.15-5.12(m,1H),4.99-4.97(d,J=8.0 Hz,1H),4.24-4.09(m,3H),3.69-3.63(m,14H),3.37-3.34(m,2H),2.20(s,3H),2.13(s,3H),2.12(s,3H),2.03(s,3H),EI-MS m / z:685(M + ).

[0358] Preparation of compound Int-TG2 To a solution of compound Int-TG2-3 (750 mg, 1.09 mmol) in DCM (10 mL) was added TBDMS-OTf (504 μL, 2.19 mmol) and EtN (458 μL, 3.29 mmol) at 0° C. under a N atmosphere. The mixture was stirred at room temperature overnight and then quenched by the addition of citric acid (20 mL). The organic layer was washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2 (799 mg, 91%). 1H NMR(400 MHz,CDCl3) δ 7.35(d,J=2.4 Hz,1H),7.30(dd,J=8.4,2.0 Hz,1H),7.02(d,J=8.0 Hz,1H),6.65(t,J=5.2 Hz,1H),5.49-5.44(m,2H),5.20(d,J=7.6 Hz,1H),5.12(dd,J=10.0,3.6 Hz,1H),4.20-4.11(m,2H),4.06-4.03(m,1H),3.69-3.62(m,15H),3.37(t,J=5.2 Hz,2H),2.19(s,3H),2.05(s,3H),2.02(s,3H),2.01(s,3H),1.01(s,9H),0.22(s,3H),0.18(s,3H).EI-MS m / z:799(M + ).

[0359] [Example 4] Preparation of compound Int-TG3 [ka]

[0360] Preparation of compound Int-TG3-1 To a solution of salicylaldehyde (Aldrich, 200 mg, 1.64 mmol) and compound Bg-Br (813 mg, 1.64 mmol) in acetonitrile (12 mL) was added dried molecular sieves (1.0 g) and AgO (1.42 g, 4.92 mmol) at room temperature. The mixture was stirred overnight, and distilled water (50 mL) and EA (50 mL × 2) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG-3-1 (218 mg, 30%). 1H NMR(400 MHz,CDCl3) δ 10.35(s,1H),7.86(dd,J=6.0,1.6 Hz,1H),7.56(td,J=7.6,1.6 Hz,1H),7.20(t,J=7.6 Hz,1H),7.13(d,J=8.8 Hz,1H),5.39-5.31(m,3H),5.27-5.25(m,1H),5.24-4.19(m,1H),3.75(s,3H),2.07-2.04(m,9H).EI-MS m / z:461(M + +Na).

[0361] Preparation of compound Int-TG3-2 To a solution of compound Int-TG3-1 (217.6 mg, 0.50 mmol) in DCM (10 mL) was added m-CPBA (367.1 mg, 1.50 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature overnight, and 40 mL of DCM was added. The organic layer was washed with saturated sodium bicarbonate (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure.

[0362] The residue was dissolved in CHCl (5 ml). Hydrazine (36.2 μL, 0.74 mmol) was added. After 30 minutes, DCM (20 mL × 2) and water (10 mL) were added. The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG1-3-2 (195 mg, 92%). 1 H NMR(400 MHz,CDCl3) δ 7.09(t,J=8.0 Hz,1H),7.00-6.95(m,2H),6.83(td,J=6.8,1.6 Hz,1H),5.38-5.24(m,4H),4.19-4.13(m,1H),3.75(s,3H),2.06-2.02(m,9H).EI-MS m / z:449(M + +Na).

[0363] Preparation of compound Int-TG3 To a solution of compound Int-TG3-2 (194 mg, 0.46 mmol) in DCM (5 mL) was added EtN (190.8 μL, 1.37 mmol) and TBDMS-OTf (209.8 μL, 0.91 mmol) at 0 °C under a N atmosphere. After stirring at 0 °C for 1 h, DCM (30 mL × 2) and water (10 mL) were added. The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG3 (188.6 mg, 76%). 1 H NMR(400 MHz,CDCl3) δ 7.00(dd,J=6.0,1.6 Hz,1H),6.94-6.88(m,2H),6.84(dd,J=6.0,2.0 Hz,1H),5.38-5.21(m,5H),3.72(s,3H),2.03(d,J=6.8 Hz,9H),0.98(s,9H),0.18(s,3H),0.15(s,3H).EI-MS m / z:563(M + +Na).

[0364] [Example 5] Preparation of compound Int-TG4 [ka]

[0365] To a solution of 2-nitrophenol (500 mg, 3.59 mmol) in anhydrous pyridine (20 mL) was added TBDMS-Cl (650 mg, 4.31 mmol) at room temperature under a N atmosphere. The mixture was stirred at room temperature overnight, and DCM (30 mL × 2) and water (20 mL) were added. The resulting organic layer was washed with 2N aqueous HCl, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG4 (410 mg, 94%). 1 H NMR(400 MHz,CDCl3) δ 7.80(dd,J=6.8,0.8 Hz,1H),7.43(t,J=7.2 Hz,1H),7.04-6.97(m,2H),1.01(s,9H),0.26(s,6H).

[0366] [Example 6] Preparation of compound Int-TG6 [ka]

[0367] Preparation of compound Int-TG6-1 To a solution of 1,2-dihydroxybenzene (1.0 g, 9.08 mmol) in DMF (15 mL) was added TBDMS-Cl (1.64 g, 10.88 mmol) and imidazole (1.24 g, 18.21 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2 h. EA (30 mL × 2) and distilled water (20 mL) were added. The resulting organic layer was washed with brine, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG6-1 (1.27 g, 64%). 1 H NMR(400 MHz,CDCl3) δ 6.94(d,J=8.0 Hz,1H),6.89-6.82(m,2H),6.76(t,J=7.6 Hz,1H),5.48(s,1H),1.02(s,9H),0.28(s,6H).

[0368] Preparation of compound Int-TG6 To a solution of Int-TG6-1 (300 mg, 1.34 mmol) and levulinic acid (310.5 mg, 2.67 mmol) in 1,4-dioxane (12 mL), DCC (551.7 mg, 2.67 mmol) and DMAP (13.07 mg, 0.11 mmol) were added. The mixture was stirred at room temperature for 3 hours. Distilled water (50 mL) and EA (50 mL × 2) were added, and the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG6 (380.1 mg, 88%). 1H NMR(400 MHz,CDCl3) δ 7.09(td,J=6.0,1.6 Hz,1H),7.03(dd,J=6.4,1.6 Hz,1H) 6.95-6.88(m,2H),2.84(s,4H),2.22(s,3H),0.98(s,9H),0.20(s,6H).

[0369] [Example 7] Preparation of compound Int-TG7 [ka]

[0370] Compound Int-TG1 (80 mg, 0.14 mmol) was dissolved in anhydrous methanol (2 mL), and K2CO3 (99.7 mg, 0.72 mmol) was added thereto at 0 °C. The mixture was stirred at 0 °C for 1 hour. The residue was diluted with EA (10 mL × 2), and the organic layer was washed with 1N aqueous HCl (2 mL) and water (10 mL). The obtained organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to preparative TLC to give compound Int-TG7 (17.4 mg, 31%). 1 H NMR(400 MHz,CDCl3) δ 7.12(d,J=8.0 Hz,1H),6.95-6.85(m,3H),4.74(d,J=7.2 Hz,1H),4.05(d,J=3.2 Hz,1H),3.97(dd,J=6.0,5.6 Hz,1H),3.90-3.85(m,2H),3.68(dd,J=6.8,2.8 Hz,1H),3.63(t,J=5.6 Hz,1H),3.48(s,1H),1.03(s,9H),0.20(d,J=12.0 Hz,6H).EI-MS m / z:409(M + +Na).

[0371] [Example 8] Preparation of compound Int-TG8 [ka]

[0372] Preparation of compound Int-TG8-1 To a solution of catechol (500 mg, 0.454 mmol) and 2-nitrobenzyl bromide (333.5 mg, 1.54 mmol) in acetone (30 mL) was added K2CO3 (401.6 mg, 2.91 mmol). The mixture was refluxed for 15 h. After cooling to room temperature, the mixture was concentrated under reduced pressure, and it was diluted with EA (50 mL × 2) and 1N aqueous NaOH solution (20 mL). The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG8-1 (300.3 mg, 79%). 1 H NMR(400 MHz,CDCl3) δ 8.19(dd,J=6.8,1.2 Hz,1H),7.75(d,J=7.6 Hz,1H),7.69(td,J=7.2,1.2 Hz,1H),7.53(td,J=6.8,1.6 Hz,1H),6.99(dd,J=7.2,1.2 Hz,1H),6.93(td,J=5.2,2.4 Hz,1H),6.85-6.79(m,2H),5.64(s,1H),5.58(s,2H).

[0373] Preparation of compound Int-TG8 To a solution of compound Int-TG8-1 (300 mg, 1.22 mmol) in DCM (5 mL) was added EtN (342 μL, 2.50 mmol) and TBDMS-OTf (421.8 μL, 1.83 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 3 h, and then diluted with DCM (30 mL × 2) and 2 N aqueous HCl (10 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG8 (410 mg, 94%). 1 H NMR(400 MHz,CDCl3) δ 8.19(dd,J=6.8,1.2 Hz,1H),8.01(dd,J=8.0,0.8 Hz,1H),7.67(td,J=6.4,1.2 Hz,1H),7.48(t,J=7.6 Hz,1H),6.92-6.87(m,4H),5.49(s,2H),1.01(s,9H),0.18(s,6H).

[0374] [Example 9] Preparation of compound Int-TG9 [ka]

[0375] Preparation of compound Int-TG9-1 To a solution of 2,3-dihydroxynaphthalene (930 mg, 5.83 mmol) and compound Int-TG (1.0 g, 2.43 mmol) in acetone (10 mL) was added NaOH (230 mg, 5.75 mmol) at room temperature under nitrogen. The mixture was stirred at room temperature overnight and concentrated under reduced pressure. The mixture was diluted with distilled water (20 mL) and EA (30 mL × 2), and the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG9-1 (560 mg, 47%). 1 H NMR(400 MHz,CDCl3) δ 7.67(t,J=9.2Hz,2H),7.39-7.29(m,4H),6.07(s,1H),5.53-5.50(m,2H),5.18(dd,J=7.2,3.6 Hz,1H),5.10(d,J=7.6 Hz,1H),4.31-4.11(m,3H),2.21(s,3H),2.12(d,J=8.8 Hz,6H),2.05(s,3H).

[0376] Preparation of compound Int-TG9 To a solution of compound Int-TG9-1 (200 mg, 0.41 mmol) in DCM (7 mL) was added TBDMS-OTf (0.12 mL, 0.53 mmol) and EtN (0.11 mL, 0.82 mmol) at 0 °C under a N atmosphere. After stirring overnight at room temperature, the mixture was diluted with DCM (30 mL × 2) and extracted with distilled water (10 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG9 (230 mg, 96%). 1H NMR(400 MHz,CDCl3) δ 7.67-7.63(m,2H),7.36-7.33(m,3H),7.20(s,1H),5.52(dd,J=8.4,2.0 Hz,1H),5.47(d,J=3.2 Hz,1H),5.31(d,J=8.0 Hz,1H),5.15(dd,J=6.8,3.6 Hz,1H),4.23-4.13(m,3H),2.20(s,3H),2.05(s,3H) 2.02(d,J=3.6 Hz,6H),1.03(s,9H),0.26(s,3H),0.22(s,3H).

[0377] [Example 10] Preparation of compound Int-TG10 [ka]

[0378] Preparation of compound Int-TG10-1 To a solution of 3-(4-hydroxyphenyl)propionic acid (500 mg, 3.01 mmol) in CHCl (10 mL) was added 4 M NaOH (7.5 mL, 30 mmol), and the resulting mixture was refluxed for 6 h. After the reaction was completed, the mixture was acidified with 4 M HCl and concentrated to remove CHCl. ​​EA (30 mL × 3), HO (20 mL), and brine (20 mL) were added for extraction, and the resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-1 (408 mg, product:SM=4:6 by HPLC). 1 H NMR(400 MHz,CDCl3) δ 10.90(s,1H),9.87(s,1H),7.41-7.38(m,2H),6.94(d,J=9.6 Hz,1H),2.96(t,J =7.4 Hz,2H),2.69(t,J =7.4 Hz,2H).

[0379] Preparation of compound Int-TG10-2 To a solution of compound Int-TG10-1 (408 mg, 2.1 mmol) in DMF (10 mL), NHS (363 mg, 3.15 mmol) and EDCI (604 mg, 3.15 mmol) were added, and the resulting mixture was stirred at room temperature overnight. 11-Azido-3,6,9-trioxaundecan-1-amine (636 mg, 2.5 mmol) and DIPEA (3.66 mL, 21 mmol) dissolved in DMF (3 mL) were added to the mixture, and the resulting mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was acidified with 4 M HCl, diluted with EA (30 mL × 5), and washed with HO (30 mL) and brine (30 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-2 (288 mg, 50% pure by HPLC). EI-MS m / z: 395(M + ).

[0380] Preparation of compound Int-TG10-3 To a solution of compound Int-TG10-2 (288 mg, 0.73 mmol) and compound Int-TG (303 mg, 0.74 mmol) in ACN (10 mL) was added molecular sieves (1.5 g) under N atmosphere. After stirring at room temperature for 10 minutes, AgO (508 mg, 2.19 mmol) was added thereto. The mixture was stirred at room temperature for 3 hours, diluted with H2O (5 mL), and then filtered through Celite®. The filtrate was washed with EA (20 mL × 2), H2O (20 mL), and brine (20 mL). The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-3 (195 mg). EI-MS m / z: 725(M + ).

[0381] Preparation of compound Int-TG10-4 To a solution of compound Int-TG10-3 (195 mg, 0.27 mmol) in DCM (5 mL) was added 70% m-CPBA (133 mg, 0.54 mmol) at 0 °C under a N atmosphere. The mixture was stirred at 0 °C for 3 hours. Then, 70% m-CPBA (66 mg, 0.27 mmol) was further added thereto, and the mixture was stirred at 0 °C overnight. The reaction was quenched with saturated NaHCO (20 mL × 3) and diluted with DCM (20 mL). The organic layer was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. Compound Int-TG10-4 (199 mg, crude) was used directly in the next reaction without purification. EI-MS m / z: 741(M + ).

[0382] Preparation of compound Int-TG10-5 To a solution of compound Int-TG10-4 (199 mg, 0.27 mmol) in CHCl (4 mL) was added NHNH·H0 (133 mg, 0.54 mmol) at 0 °C under a N atmosphere. After stirring at room temperature for 30 min, the mixture was quenched with EA (20 mL) and saturated citric acid (20 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-5 (146 mg). EI-MS m / z: 713(M + ).

[0383] Preparation of compound Int-TG10 To a solution of compound Int-TG10-5 (146 mg, 0.21 mmol) in DMF (2 mL) was added TEA (133 μL, 0.62 mmol) and TBDMS-OTf (94 μL, 0.41 mmol) at 0 °C under a N atmosphere. The mixture was stirred at 0 °C for 20 min and continued to stir at room temperature for 3 h. The mixture was extracted with EA (20 mL), saturated citric acid (20 mL), and brine (30 mL). The resulting organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography followed by preparative TLC to give compound Int-TG10 (32 mg, 19%). EI-MS m / z: 827(M + ).

[0384] Example 11 Preparation of Compound FA-Int [ka]

[0385] The compound FA-Int was obtained by methods similar to those described in US Patent Application Publication No. 2007 / 0276018, which is incorporated herein by reference in its entirety.

[0386] [Example 12] Preparation of compound IntC-L-1 [ka]

[0387] Preparation of compound IntCl-L-1a To a solution of 2,2-(ethylenedioxy)bis(ethylamine) (50 g, 337.4 mmol) in DCM (300 mL) was added BocO (14.7 g, 67.47 mmol) dissolved in DCM (200 mL) under N atmosphere. The mixture was stirred at room temperature overnight and quenched with HO (500 mL) and brine (150 mL × 3). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. After concentration, compound IntCl-L-1a (13.01 g, 78%) was used directly in the next reaction without purification. 1 H NMR(400 MHz,CDCl3) δ 5.20(s,1H),3.62-3.62(m,4H),3.55-3.51(m,4H),3.35-3.25(m,2H),2.90-2.87(m,2H),1.45(s,9H).

[0388] Preparation of compound IntCl-L-1b To a solution of compound IntCl-L-1a (6 g, 24.16 mmol) and zL-Glu-OMe (5.94 g, 20.13 mmol) in DMF (30 mL) was added PyBOP (15.72 g, 30.20 mmol) and DIPEA (10.52 mL, 60.39 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2 h. EA (20 mL × 6), HO (20 mL), and brine (200 mL) were added to the mixture. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntCl-L-1b (10.6 g, quantitative). 1 H NMR(400 MHz,CDCl3) δ 7.40-7.28(m,5H),6.32(s,1H),5.80(s,1H),5.11(s,2H),5.02(s,1H),4.36(s,1H),3.74(s,3H),3.60(s,4H),3.54 EI-MS m / z:526(M + ).

[0389] Preparation of compound IntCl-L-1c To a solution of compound IntCl-L-1b (3 g, 5.71 mmol) in MeOH (25 mL) was added Pd / C (900 mg) at room temperature under H. The mixture was stirred for 3 hours, filtered through Celite®, and then concentrated under reduced pressure. Compound IntCl-L-1c was used directly in the next step without further purification (2.23 g, crude). 1 H NMR(400 MHz,CDCl3) δ 6.56(s,1H),5.20(s,1H),3.73(s,3H),3.61(s,4H),3.57-3.55(m,4H),3.53-3.50(m,1H),3. 48-3.44(m,4H),2.40-2.32(m,2H),2.18-2.10(m,1H),1.88-1.81(m,1H),1.44(s,9H).EI-MS m / z:392(M + ).

[0390] Preparation of compound IntCl-L-1d To a solution of compound IntCl-L-1c (2.23 g, 5.71 mmol) and compound FA-Int (2.12 g, 5.19 mmol) in DMF (15 mL) was added HBTU (2.36 g, 6.23 mmol) and DIPEA (1.36 mL, 7.78 mmol) at 0 °C under a N atmosphere. The mixture was stirred at room temperature for 2.5 h, and EA (100 mL × 7) and HO (100 mL) were added. The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntCl-L-1d (4.06 g, quantitative). 1 H NMR(400Hz,DMSO-d6) δ 8.89(d,J =7.6 Hz,1H),8.63(s,1H),7.90(d,J =8 Hz,2H),7.64(d,J =8.4 Hz,2H),6.76-6.75(m,1H),5.12(s,1H),4.41-4.36(m,1H),3.64(s,3H),3.4 7(s,4H),3.39-3.35(m,4H),3.20-3.12(m,2H),3.07-3.02(m,2H),2.23(t,J =7.4 Hz,2H),2.09-2.06(m,1H),1.96-1.91(m,1H),1.36(s,9H).EI-MS m / z:782(M + ).

[0391] Preparation of compound IntCl-L-1 To a solution of compound IntCl-L-1d (4.68 g, 5.99 mmol) in DCM (50 mL) was added TFA (10 mL) dropwise at 0° C. The reaction was allowed to warm to room temperature and stirred for 3 hours. The mixture was concentrated under reduced pressure and used directly in the next step without further purification (4.08 g, crude).

[0392] [Example 13] Preparation of compound IntC-L [ka]

[0393] Preparation of Compound K-1 To a solution of L-Lys(Boc)-OMe (3 g, 10.11 mmol) and 4-pentynoic acid (992 mg, 10.11 mmol) in DMF (30 mL) was added PyBop (7.89 g, 15.16 mmol) followed by DIPEA (5.26 mL, 30.32 mmol) in one portion at 0 °C under a N atmosphere. The mixture was stirred at room temperature overnight. EA (80 mL × 4) and saturated citric acid (60 mL) were added to the mixture, and the organic layer was washed with NaHCO (120 mL), brine (100 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound K-1 (3.29 g, 95%). EI-MS m / z: 341(M + ).

[0394] Preparation of Compound K-2 To a solution of compound K-1 (3.29 g, 9.66 mmol) in MeOH (15 mL) at 0 °C under N atmosphere was added LiOH·H2O (2.03 g, 48.32 mmol) dissolved in H2O (15 mL).

[0395] The mixture was stirred at 0 °C for 30 minutes and warmed to room temperature for 2 hours. The mixture was acidified with saturated aqueous citric acid, and EA (40 mL × 2) was added to the mixture. The organic layer was washed with H O (30 mL) and brine (30 mL), dried over anhydrous Na SO , filtered, and concentrated under reduced pressure. Compound K-2 was used directly in the next step without further purification (3.15 g, crude). EI-MS m / z: 327(M + ).

[0396] Preparation of Compound K To a solution of compound K-2 (3.69 g, 11.31 mmol) in DMF (20 mL) was added NHS (1.69 mg, 14.7 mmol) and EDCI (2.93 g, 15.26 mmol) under a N atmosphere. The mixture was stirred at room temperature overnight and concentrated. The residue, compound K (4.79 g, crude), was used directly in the next step without further purification. EI-MS m / z: 446(M + +Na).

[0397] Preparation of compound IntC-L-2 To a solution of compound IntC-L-1 (4.08 g, 5.99 mmol) and compound K (4.79 g, 11.31 mmol) in DMF (25 mL) was added DIPEA (5.21 mL, 29.93 mmol) under N atmosphere. The mixture was stirred at room temperature overnight. HO (70 mL) and brine (60 mL) were added to the mixture, which was then extracted with EA (70 mL × 7). The organic layer was then dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntC-L-2 (1.12 g, 19%). EI-MS m / z: 991 (M+).

[0398] Preparation of compound IntC-L-3 To a solution of compound IntA-L-2 (1.12 g, 1.13 mmol) in MeOH (27 mL) was added LiOH·HO (356 mg, 8.48 mmol) dissolved in HO (10 mL) at 0 °C under a N atmosphere. The mixture was stirred at 0 °C for 30 min and warmed to room temperature for 3 h. The mixture was acidified with 2 M HCl and concentrated under reduced pressure. The residue, compound IntC-L-3 (996 mg, crude), was used directly in the next step without further purification. EI-MS m / z: 880(M + ).

[0399] Preparation of compound IntC-L To a solution of compound IntC-L-3 (996 mg, 1.13 mmol) in DCM (30 mL) was added TFA (8 mL) at 0 °C under a N atmosphere. After stirring at 0 °C for 1 h, the mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (5 mL) and purified by preparative HPLC to produce compound IntC-L (409 mg, 32%). EI-MS m / z: 780(M + ).

[0400] [Example 14] Preparation of compound MPS-D1 [ka]

[0401] Preparation of compound MPS-D1a To a solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 mL) was added piperidine hydrochloride (6.66 g, 54.82 mmol), paraformaldehyde (4.95 g, 164.5 mmol), and concentrated HCl (0.6 mL) at room temperature under a N atmosphere. The mixture was stirred at 100 °C for 16 hours and cooled to room temperature. Acetone (90 mL) was added dropwise to the mixture. The mixture was stirred at 0 °C for 1 hour. The solid was filtered and washed with diethyl ether (30 mL × 2) to obtain compound MPS-D1a (6.11 g, 38%). 1 H NMR(400 MHz,DMSO-d6) δ 8.08(s,4H),5.73(s,1H),3.65(t,J=7.2 Hz,2H),3.35(t,J=7.2 Hz,2H),3.31(m,6H),1.74(s,4H).

[0402] Preparation of compound MPS-D1b To a solution of MPS-D1a (6.11 g, 20.52 mmol) in EtOH (40 mL) and MeOH (26 mL) was added 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 mL, 3.08 mmol) at room temperature. The mixture was stirred at 100 °C for 16 h, then cooled to 0 °C and further stirred for 1 h. The solid was filtered and washed with ether (30 mL × 2) to give compound MPS-D1b (5.56 g, 90%). 1 H NMR(400 MHz,CDCl3) δ 8.04-7.99(m,4H),7.27(d,J=8.4 Hz,2H),7.15(d,J=7.6 Hz,2H),3.39-3.36(m,2H),3.25-3.21(m,2H),2.27(s,3H).

[0403] Preparation of compound MPS-D1 To a solution of MPS-D1b (5.56 g, 18.51 mmol) in MeOH (90 mL) and distilled water (90 mL) was added oxone (25.03 g, 40.72 mmol) at 0 °C under a N atmosphere. After stirring at room temperature for 14 h, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL × 3). The organic layer was washed with brine (200 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound MPS-D1 (5.29 g, 86%). 1 H NMR(400 MHz,CDCl3) δ 8.04-7.99(m,4H),7.81(d,J=8.4 Hz,2H),7.46(d,J=8.4 Hz,2H),3.63(t,J=7.2 Hz,2H),3.41(t,J=7.2 Hz,2H),2.44(s,3H).EI-MS m / z:333(M + ).

[0404] [Example 15] Preparation of compound MPS-D2 [ka]

[0405] Preparation of Compound L-1a To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was added KI (294 mg, 1.77 mmol) and AgO (4.92 g, 19.48 mmol) under a N atmosphere. The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was filtered through Celite® and washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1a (5.98 g, 73%). 1 H NMR(400 MHz,CDCl3) δ 7.80(d,J=8.4 Hz,2H),7.35(d,J=8.4 Hz,2H),4.16(t,J=4.8 Hz,2H),3.71-3.58(m,22H),2.88(br,1H),2.45(s,3H).

[0406] Preparation of Compound L-1b To a solution of compound L-1a (5.98 g, 13.7 mmol) in DMF (30 mL) was added NaN (1.34 g, 20.55 mmol) under a N atmosphere. The mixture was stirred at 110 °C for 1 h and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1b (4.1 g, 97%). 1 H NMR(400 MHz,CDCl3) δ 3.72-3.60(m,22H),3.39(t,J=4.8 Hz,2H),2.78(br,1H).

[0407] Preparation of compound L-1c To a solution of compound L-1b (2 g, 6.51 mmol) in acetone (56 mL) was slowly added dropwise Jones reagent (5 mL) at -5 °C under a N atmosphere. The mixture was stirred at room temperature for 2 hours, filtered through Celite®, and the filtrate was concentrated under reduced pressure. The filtrate was diluted with DCM (20 mL × 2) and water (5 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1c (1.85 g, 89%). 1 H NMR(400 MHz,CDCl3) δ 4.15(s,2H),3.76-3.67(m,18H),3.40(t,J...

Claims

1. A conjugate of formula (I'): (D-L) ｎ (CB) ｃｂ (I') or a pharma- ceutically acceptable salt thereof, During the ceremony, CB is a targeting moiety; cb and n are each independently an integer having a value from 1 to about 20; each D-L is independently a group having a structure of formula (I") or formula (I"'); 【Chemistry 1】 Each Q is independently selected from L′ or S(O) by a heteroatom. ２ an activator linked to Z′, independently at each occurrence, represents a group having a structure of formula (I″) or formula (I′″) represented by (CB): ｃｂ a linking group, solubilizing group, reactive group, solid surface, stabilizing group, chelator, biopolymer, active agent, or detectable moiety that connects the structure of formula (I") or formula (I") to (CB), ｃｂ Provided that you connect to each L' is independently a spacer moiety attached to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- promotes cleavage of the bond between L' and Q, releasing said active agent; Each X is independently -O-, -C(R ｂ ) ２ -, or -N(R ｃ ) - and Ar represents aryl or heteroaryl; Y' is -(CR ｂ ２ ) ｙ N (R ａ ) -, - (CR ｂ ２ ) ｙ O- or -(CR ｂ ２ ) ｙ S- and positioned such that when y is 1, said N, O or S atom is bonded to TG; When TG is activated, it reacts with the -S(=O)(=N-)- to form (Q) ｑ -(L') ｗ is an inducing group which generates an N, O, or S atom capable of replacing and forming a 5-6 membered ring containing X-S(=O)(=N-)- and an intervening atom of Ar; (Y′) ｘ TG is in the ortho position relative to X in formula (I'') and in the ortho position relative to N(=S(=O)) in formula (I'''); q is an integer having a value from 1 to about 20; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently hydrogen or lower alkyl; or Two R's ｂ form a 3- to 5-membered ring together with the atoms to which they are attached, With the proviso that when w is 0, q is 1, or a pharma- ceutically acceptable salt thereof.

2. The conjugate of claim 1, wherein X is -O-.

3. 2. The conjugate of claim 1, wherein Ar is phenyl, pyridyl or naphthyl.

4. At least one Z′ is (i) at least one heteroatom selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) at least one heteroarylene; (iii) at least one amino acid moiety, glycobond, peptide bond, or amide bond, and (iv) C １ ~C ２０ Alkyl, C ６ ~C ２０ Aryl C １ ~C ８ Alkyl, -(CH ２ ) ｓ COOH, and -(CH ２ ) ｐ N.H. ２ (wherein s is an integer having a value from 0 to 10 and p is an integer having a value from 1 to about 10), １０ ~C １００ The conjugate of any one of claims 1 to 3, which is a linear or branched, saturated or unsaturated alkylene moiety.

5. The conjugate of any one of claims 1 to 4, wherein at least one Z' comprises a functional group that can be produced through a click chemistry reaction.

6. At least one Z′ comprises: 【Chemistry 2】 During the ceremony, Each V is independently a single bond, —O—, —S—, or —NR ２１ --, --C(O)NR ２２ --, --NR ２３ C(O)-, -NR ２４ SO ２ -, -SO ２ N.R. ２５ --, --NR ２４ -S(=O)(=N-)- or -S(=O)(=N-)-NR ２５ - and R ２１ , R ２２ , R ２３ , R ２４ , and R ２５ each independently represents hydrogen, (C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkyl(C ６ ~C ２０ ) aryl, or (C １ ~C ６ ) alkyl(C ３ ~C ２０ ) heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; The conjugate of any one of claims 1 to 4, wherein L'' is a single bond.

7. The Z' connecting CB and Ar is connected to each other in a straight chain by a covalent bond (CH ２ ) ｂ , L ｃ , (P １ ) ａ , W ａ１ , W ａ２ , W ａ３ , W ｂ１ , Y １ , and Y ２ is a linking group comprising a group, W ａ１ , W ａ２ , and W ａ３ each independently represents -NH-, -C(O)-, or -CH ２ - and W ｂ１ is an amide bond or a triazolylene; P １ is an amide bond, an amino acid residue, or a peptide; L ｃ is alkylene, Y １ But -(CH ２ ) ｑ - (CH ２ CH ２ X'') ｏ - or - (CH ２ ) ｑ -(X''CH ２ CH ２ X'') ｏ - and X″ is —O—, —S—, —NH—, or —CH ２ - and Y ２ is a single bond or a group selected from the following: 【Chemistry 3】 W ｂ２ is an amide bond or a triazolylene; a is 0 to 10; b, c, and d are each independently an integer having a value from 1 to about 10; The conjugate of any one of claims 1 to 4, wherein o and q are each independently an integer having a value from 1 to about 10.

8. The Z′ connecting CB and Ar is a linking group represented by formula (A), ＊＊ -L ｃ -W ｂ１ -(CH ２ ) ｂ -W ａ３ -(P １ ) ａ -Y ２ -W ａ２ -Y １ -W ａ１ - ＊ (A) During the ceremony, ＊ is the point of attachment to CB, ＊＊ The conjugate of claim 7, wherein is the point of attachment to Ar.

9. P １ But, 【Chemistry 4】 During the ceremony, R １２ is hydrogen, alkyl, amino acid side chain, -(CH ２ ) ｓ C(O)R １３ , or -(CH ２ ) ｐ N.R. １４ R １５ and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R １３ is OH or -NH(CH ２ ) ｓ’ (X''CH ２ CH ２ ) ｓ’’ Z''-(CB) ｍ and R １４ and R １５ each independently represents hydrogen or -C(O)(CH ２ ) ｓ’ (X''CH ２ CH ２ ) ｓ’’ Z''-(CB) ｍ and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH ２ - and Z'' is CB R １４ Or R １５ or Z″ is a linking group that comprises a reactive group.

10. at least one Z' is a linking group of formula (F'), (G'), (H'), (J'), (K'), (L'), (M'), or (N'); 【Chemistry 5】 【change】 During the ceremony, L ２ is unsubstituted or has one or more C １ ~C ６ Alkyl, C ５ ~C １４ Aryl, and C ３ ~C ８ a heteroaryl substituted spacer moiety, where the alkyl, aryl, and heteroaryl are unsubstituted or C １ ~C １０ Alkyl, -(CH ２ ) ｕ N.H. ２ , -(CH ２ ) ｕ N.R. ｕ１ R ｕ２ , -(CH ２ ) ｕ CO ２ H, -(CH ２ ) ｕ CO ２ R ｕ１ , and -(CH ２ ) ｕ SO ２ R ｕ３ wherein R is substituted with one or more substituents selected from the group consisting of ｕ１ , R ｕ２ , and R ｕ３ are each independently hydrogen, C １ ~C １５ Alkyl, C ６ ~C ２０ Aryl, or C ３ ~C １０ heteroaryl, and u is an integer having a value from 1 to about 10; R ｅ is alkyl, X″ is —O—, —S—, —NH—, or —CH ２ - and X ４ is -NHC(O)-(CH ２ ) ｇ -NH- or -C(O)NH-(CH ２ ) ｈ -NH-, W ｂ１ and W ｂ２ each independently represents -C(O)NH-, -NHC(O)-, 【Chemistry 6】 R １２ is hydrogen, alkyl, amino acid side chain, -(CH ２ ) ｓ C(O)R １３ Or - (CH ２ ) ｐ N.R. １４ R １５ and R １３ is OH or -NH(CH ２ ) ｓ’ (X''CH ２ CH ２ ) ｓ’’ Z''-(CB) ｍ and R １４ and R １５ each independently represents hydrogen or -C(O)(CH ２ ) ｓ’ (X''CH ２ CH ２ ) ｓ’’ Z''-(CB) ｍ and s and s″ are each independently an integer having a value from 0 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH ２ - and Z'' is CB R １４ Or R １５ or Z″ is a linking group that includes a reactive group; a, b, c, d, e, g, h, o, p, and qq are each independently an integer having a value from 1 to about 10; The conjugate of any one of claims 7 to 9, wherein s' is an integer having a value from 1 to about 10.

11. 11. The conjugate of any one of claims 1 to 10, wherein TG is a reactive chemical moiety or functional group that can be cleaved by nucleophilic reagent conditions, basic reagent conditions, light irradiation, reducing agent conditions, acidic conditions, enzymatic conditions, or oxidative conditions.

12. TG is selected from the following: 【Chemistry 7】 During the ceremony, Each R ２１ are independently hydrogen or acetyl; R ２２ The conjugate of any one of claims 1 to 11, wherein is hydrogen or lower alkyl.

13. The conjugate of any one of claims 1 to 12, wherein Q is a chemical agent, a biological agent, a hormone, an oligonucleotide, a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

14. (Q) ｑ -(L') ｗ - is selected from the following: 【Chemistry 8】 During the ceremony, X １ is -O- or -NR ａ - and X ２ is —O—, —OC(O)—, —OC(O)O—, or —OC(O)NH—, X ４ is absent or selected from —O—, —OC(O)—, —OC(O)O—, and —OC(O)NH—; X ３ is -OC(=O)-, Z″ is alkyl, aryl, or heteroaryl, where the alkyl, aryl, and heteroaryl are unsubstituted or substituted with one or more substituents; w′ is an integer having a value of 1, 2, 3, 4, or 5; R ９ and R １０ is each independently hydrogen, alkyl, aryl, or heteroaryl, wherein alkyl, aryl, and heteroaryl are unsubstituted or substituted with one or more substituents.

15. (Q) ｑ -(L') ｗ - is selected from the following: 【Chemistry 9】 【change】 【change】 【change】 During the ceremony, ＊ But, (Q) ｑ -(L') ｗ The conjugate of claim 14, wherein:

16. The conjugate of any one of claims 1 to 15, wherein the targeting moiety is a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment of an antigenic polypeptide, or a lipibody.

17. 17. The conjugate of claim 16, wherein the targeting moiety is an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) variant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant of an antibody, and other modified immunoglobulin molecules comprising an antigen recognition site.

18. The antibody is selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, and tositumomab-I. １３１ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, cetrizumab pegol, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI-Ig, second generation anti-CD20, ACZ-885, tocilizumab, atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab (CP-675 206), ticilimumab, MDX-010, IDEC-114, inotuzumab ozogamicin, HuMax 17. The conjugate of claim 16, selected from EGFR, Aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, Catumaxomab, IGN101, MT-201, Pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, Motavizumab, Efangumab, Aurograv, Raxibacumab, 3rd generation anti-CD20, LY2469298, and Veltuzumab.

19. A compound of formula (Ia) or formula (Ia'), 【Chemistry 10】 or a pharma- ceutically acceptable salt thereof, Each Q is independently selected from L′ or S(O) by a heteroatom. ２ an activator linked to Z′, independently at each occurrence, is absent, represents a structure of formula (Ia) or of formula (Ia′) represented by (CB): ｃｂ a linking group, solubilizing group, reactive group, solid surface, stabilizing group, chelating agent, biopolymer, active agent, or detectable moiety that connects the structure of formula (Ia) or formula (Ia') to (CB), ｃｂ Provided that you connect to each L' is a linking group bonded to -S(=O)(=N-)- through a heteroatom selected from O, S, and N, selected such that cleavage of the bond between L' and -S(=O)(=N-)- facilitates cleavage of the bond between L' and Q, releasing said active agent; Each X is independently -O-, -CR ａ ２ -, or -NRc-, Ar represents aryl or heteroaryl; Y' is -(CR ｂ ２ ) ｙ N (R ａ ) -, - (CR ｂ ２ ) ｙ O- or -(CR ｂ ２ ) ｙ S- and positioned such that when y is 1, said N, O or S atom is bonded to TG; When TG is activated, it reacts with the -S(=O)(=N-)- to form (Q) ｑ -(L') ｗ is an inducing group which generates an N, O, or S atom capable of replacing and forming a 5-6 membered ring with the intervening atom of X-S(=O)(=N-)- and Ar, (Y′) ｘ TG is in the ortho position relative to X in formula (Ia) and in the ortho position relative to N(=S(=O)) in formula (Ia'); q is an integer having a value from 1 to about 20; w, x, and y are each independently an integer having a value of 0 or 1; E is an integer having a value of 0, 1, or 2; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently hydrogen or lower alkyl, or Two R's ｂ form a 3- to 5-membered ring together with the carbon atom to which they are attached, With the proviso that when w is 0, q is 1, or a pharma- ceutically acceptable salt thereof.

20. 20. A method for preparing a conjugate comprising reacting a compound of claim 19 with a targeting moiety.

21. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 18 and a pharma- ceutically acceptable carrier or excipient.

22. A composition comprising a conjugate according to any one of claims 1 to 18 for imaging.

23. A composition comprising a conjugate according to any one of claims 1 to 18 for detection.

24. A composition comprising a conjugate according to any one of claims 1 to 18 for use in detection, said use comprising contacting a material with the composition.

25. A molecular switch, molecular machine, or nanomachine comprising the conjugate of any one of claims 1 to 18.

26. A method for transferring a portion of a molecular device, comprising the steps of: (1) A molecular switch, a molecular machine, or a nanomachine according to claim 25; (2) an activating agent that activates the inducing group.

27. 20. Use of a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 18 in the manufacture of a medicament for treating a disease or condition associated with a target cell, wherein the targeting moiety is selected to bind to a molecule associated with the target cell.

28. 28. The use of claim 27, wherein the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least preferentially associated with tumor cells over healthy cells).

29. 29. The use according to claim 27 or 28, wherein the disease or condition is an autoimmune disease, an infectious disease, or a tumor.

30. Use of a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 18 in the manufacture of a medicament for treating a proliferative disease.

31. 31. The use of claim 30, wherein the proliferative disease is selected from an autoimmune disorder, a chronic inflammatory condition, a hyperproliferative disorder, a viral infection, osteoarthritis, atherosclerosis, and a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

32. 32. The use of claim 31 , wherein the proliferative disease is a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

33. 20. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 18 for treating a disease or condition associated with a target cell, wherein the targeting moiety is selected to bind to a molecule associated with the target cell.

34. 34. The pharmaceutical composition of claim 33, wherein the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least preferentially associated with tumor cells over healthy cells).

35. 35. The pharmaceutical composition of claim 33 or 34, wherein the disease or condition is an autoimmune disease, an infectious disease, or a tumor.

36. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 18 for treating a proliferative disease.

37. 37. The pharmaceutical composition of claim 36, wherein the proliferative disease is selected from an autoimmune disorder, a chronic inflammatory condition, a hyperproliferative disorder, a viral infection, osteoarthritis, atherosclerosis, and a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

38. 38. The pharmaceutical composition of claim 37, wherein the proliferative disease is a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

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