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 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 SO functional group capable of selectively releasing the active agent, and a functional group that induces a chemical, physicochemical, and / or biological response in response to an external stimulus, and may further contain a ligand (e.g., an oligopeptide, a polypeptide, an antibody, etc.) having 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 / 788,013, 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 range of cancers. ADCs are commonly composed 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 the desired target specificity, i.e., high activity in tumor cells but low activity in healthy cells.

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

[0004] A conjugate of formula (I'): (DL) n -(CB) cb (I') or a pharma- ceutically acceptable salt thereof is provided herein: 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 the structure of formula (I″): [ka] each Q is independently an active agent linked to L' by a heteroatom, preferably O or N; Z′, independently at each occurrence, represents a group having the structure of formula (I″) as follows: cba linking group, solubilizing group, reactive group (e.g., a precursor group), a solid surface (e.g., a particle), a stabilizing group, a chelating agent, a biopolymer (e.g., an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, an antigen, a fragment of a polypeptide, or a lipibody), an active agent, or a detectable moiety, provided that at least one occurrence of Z′ transforms the structure of formula (I″) into cb Provided that you connect to Each L' is independently a heteroatom selected from O, S, and N, preferably SO through O or N. 2 The spacer portion is bound to L' and SO 2 is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q to release the active agent; Each X is independently -O-, -C(R b )(R c )- or -N(R c )-, preferably -C(R b )(R c )-and E is an integer having a value of 1, 2, or 3, preferably 1; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y' is -N(R a )-, -O-, or -S-; When TG is cut, SO 2 and (Q) q -(L') w is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently Z', hydrogen, or lower alkyl, with the proviso that R b is Z', or Rb and R c 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 embodiments, each X is positioned in an ortho or para relationship to Y' on Ar.

[0006] In certain such embodiments, at least one X and Y' are positioned in an ortho relationship to one another on Ar. In certain such embodiments, E is 2 or 3. In certain such embodiments where E is 2, both occurrences of X are positioned in an ortho relationship to Y'. In certain such embodiments, E is 1.

[0007] In other embodiments, at least one X and Y' are positioned in a para relationship to each other on Ar. In certain such embodiments, E is 1.

[0008] In certain embodiments, at least one X is positioned in an ortho or para relationship to Y'.

[0009] In certain embodiments, R attached to Ar b At least one R other than b represents Z'.

[0010] The present invention also relates to compositions (eg, pharmaceutical compositions) comprising a compound of formula (I') and a carrier (eg, a pharma- ceutically acceptable carrier).

[0011] In one aspect, 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.

[0012] In another aspect, the present invention further provides a conjugate of formula (I') and pharmaceutical compositions thereof for use in a method for delivering an active agent to a cell, 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 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).

[0013] The conjugates of formula (I') of the present invention and pharmaceutical compositions thereof may be useful in treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), transplant rejection, lupus, myositis, infectious diseases, immune deficiencies such as AIDS, and inflammatory diseases in mammals (e.g., humans). [Brief description of the drawings]

[0014] [Figure 1] 1 shows the results of a stability analysis of compound A-1. [Diagram 2] 1 shows the results of a stability analysis of compound A-2. [Diagram 3] 1 shows the results of a stability analysis of compound B-1. [Figure 4] 1 shows the results of an enzyme cleavage assay of compound A-2. [Diagram 5] 1 shows the results of an enzyme cleavage assay of compound Int-T3. [Figure 6] 1 shows the results of in vitro analysis of conjugates T-DM1, T-2-AB, and T-3-AB to NCI-N87. [Figure 7] 1 shows the results of in vitro analysis of conjugates T-DM1, T-4-AB to NCI-N87. [Figure 8] 1 shows the results of in vitro analysis of conjugates T-DM1, T-5-AB, and T-6-AB against SK-BR3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention relates to compounds and conjugates containing cleavable linkers and their uses. Exemplary 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 triggering group that undergoes a chemical reaction (e.g., a physicochemical reaction and / or a biological reaction) under predetermined conditions, and a SO 3 -linker positioned in relation to the triggering group on the aryl or heteroaryl ring such that activation of the triggering group causes the conjugate to release the active agent via 1,4-elimination, intramolecular cyclization, or 1,6-elimination. 2 In some embodiments, the compounds and conjugates disclosed herein further comprise a targeting moiety (e.g., an oligopeptide, polypeptide, antibody, etc.) that has binding specificity for a desired target receptor or other molecule associated with a target cell.

[0016] 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, a straight-chain or branched alkyl group is an acyclic group 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. 1 ~C 6 Straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups. Alkyl groups having two possible valences may be referred to with the suffix "ene", as in alkylene. Exemplary alkylene groups include methylene, ethylene, propylene, and the like.

[0017] Moreover, the term "alkyl" (or "lower alkyl") may include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety in which a substituent replaces a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, may 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, where appropriate. For example, substituents of substituted alkyls include alkyl groups, amino groups, azide groups, imino groups, amide groups, phosphoryl groups (including phosphonates and phosphinates), sulfonyl groups (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF 3 Exemplary substituted alkyls are described below. Cycloalkyl includes alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF 3 , -CN, and the like.

[0018] For example, "C" when used in conjunction with a chemical moiety 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 where x is an integer less than y, and x and y are not the same value. For example, "Cx ~C y The term "C-alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain 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. 2 ~C y -alkenyl" and "C 2 ~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. 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 number of carbon atoms in the ring.

[0019] The term "alkoxy" has an art-recognized meaning 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.

[0020] 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).

[0021] The meaning of the term "cycloalkyl" is understood in the art 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 a saturated ring, an unsaturated ring, and an aromatic ring. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0022] 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.

[0023] 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 1-4 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.

[0024] 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 1-4 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.

[0025] The term "substituted" refers to a moiety in which a substituent replaces a hydrogen on one or more carbon or heteroatoms of the moiety. One of ordinary skill in the art will appreciate 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 spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds.

[0026] 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, sulfonamide, sulfonyl, heterocyclyl, alkyl, aralkyl, or aromatic or heteroaromatic moieties. Those of skill in the art will appreciate that the substituents themselves may be substituted, where appropriate. Unless expressly specified as "unsubstituted," references to chemical moieties herein are understood to include substituted variations. For example, references to "aryl" groups or moieties implicitly include both substituted and unsubstituted variations.

[0027] 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.

[0028] 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.

[0029] 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 an undesired condition) if it is administered prior to the clinical manifestation of an undesired condition (e.g., a disease or other undesired condition of a host animal), whereas a treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing undesired condition or its side effects) if it is administered after the manifestation of an undesired condition.

[0030] In certain embodiments, the compounds and conjugates disclosed herein may be used alone or may be 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 the second compound is administered while the previously administered therapeutic compound is still effective in the body, e.g., the two compounds are effective in the subject at the same time (which may include a synergistic effect of the two compounds). For example, the different therapeutic compounds and conjugates may be administered either simultaneously or sequentially, 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 within 1 week of each other. Thus, subjects receiving such treatment may benefit from the combined effects of the different therapeutic compounds and conjugates.

[0031] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably herein. As used herein, "abnormal cell growth" refers to cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition), unless otherwise specified. This includes, for example, the abnormal growth of: (1) tumor cells (tumors) that grow due to expression of mutated tyrosine kinases or overexpression of receptor tyrosine kinases, (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs, (3) any tumor that grows due to receptor tyrosine kinases, (4) any tumor that grows due to abnormal serine / threonine kinase activation, and (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs.

[0032] 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 cancer (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.

[0033] Compounds and conjugates of the present invention The present disclosure relates to a conjugate of formula (I'): (DL) 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 of 1 to about 20, preferably 1 to about 10; each DL is independently a group having the structure of formula (I″): [ka] each Q is independently an active agent linked to L' by a heteroatom, preferably O or N; Z′, independently at each occurrence, represents a group having the structure of formula (I″) as follows: cb a linking group, solubilizing group, reactive group (e.g., a precursor group), a solid surface (e.g., a particle), a stabilizing group, a chelating agent, a biopolymer (e.g., an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, an antigenic fragment of a polypeptide, or a lipibody), an active agent, or a detectable moiety, with the proviso that at least one occurrence of Z′ converts the structure of formula (I″) to (CB): cb Provided that you connect to Each L' is independently a heteroatom selected from O, S, and N, preferably SO through O or N. 2 The spacer portion is bound to L' and SO 2 is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q to release the active agent; Each X is independently -O-, -C(R b )(R c )- or -N(R c )-, preferably -C(R b )(R c )-and E is an integer having a value of 1, 2, or 3, preferably 1; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y' is -N(R a )-, -O-, or -S-; at least one X is positioned in an ortho or para relationship to Y' on Ar; When TG is cut, SO 2 and (Q) q -(L') w is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each Rb are independently Z', hydrogen, or lower alkyl, with the proviso that R b is Z', or R b and R c 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.

[0034] In certain embodiments, each X is positioned in an ortho or para relationship to Y' on Ar.

[0035] In certain embodiments, at least one X and Y' are positioned in an ortho relationship to one another on Ar. In certain such embodiments, E is 2 or 3. In certain such embodiments where E is 2, both occurrences of X are positioned in an ortho relationship to Y'.

[0036] In other embodiments, at least one X and Y' are positioned in a para relationship to each other on Ar.

[0037] In certain embodiments, at least one X is positioned in an ortho or para relationship to Y'.

[0038] In certain embodiments, R attached to Ar b At least one R other than b represents Z'.

[0039] Each activator can be any suitable activator, as described in more detail below. While many conventional conjugation methods require having 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 release under defined conditions that activate the triggering group. Upon activation of the triggering group, the aryl / heteroaryl unit undergoes elimination (e.g., 1,6-elimination for the para-position group) to form SO 2 and releases part (Q) q -(L') w -H, where H is the SO 2 In the case of ortho-related groups, activation of the inducing group initiates a similar 1,4-elimination and / or intramolecular cyclization, thereby forming (Q) q -(L') w Examples of 1,6-elimination and 1,4-elimination and / or cyclization induced by inducing group cleavage are shown in Schemes 1 and 2.

[0040] Many suitable triggering groups are known in the art, and exemplary triggering groups and the conditions that activate them are discussed below, such as the moieties described for Y below. Some triggering groups contain N, O, or S atoms, but in a non-nucleophilic form. For example, NO 2 The group is converted to SO under reducing conditions. 2 NH that can react with 2 The acetate group is a triggering group that, under hydrolysis conditions, is hydrolyzed to a hydroxyl group capable of inducing 1,6-elimination of the self-immolative segment. Other triggering groups do not contain N, O, or S atoms, but when activated, are converted to nucleophilic N, O, or S atoms. For example, the boronate group is reduced to an SO 2The triggering group is a hydroxyl group that can be converted to a hydroxyl group that can react with the targeting moiety. Preferably, the triggering group is selected such that the conditions that activate it result in selective activation without cleaving or degrading other portions of the conjugate, such as the targeting moiety. In some embodiments, once a nucleophilic N, O, or S atom is generated, it can be converted to a hydroxyl group that can react with the targeting moiety. 2 The moiety attacks intramolecularly to form a ring, forming the moiety (Q). q -(L') w -H, where H is the SO 2 is bonded to a heteroatom of Q or L' that was originally bonded to the moiety.

[0041] In embodiments where w is 0, q is 1 and Q is joined to SO via a heteroatom. 2 Therefore, activation of the inducing group generates a nucleophilic heteroatom, which reacts with the SO 2 The moiety attacks intramolecularly to form a ring and expel the activator QH, where H is SO 2 is bonded to the heteroatom that was originally bonded to

[0042] In embodiments where w is 1, L' may be selected to permit the attachment of multiple occurrences of Q, where Q may be the same or different. Thus, each occurrence of Q is connected to SO via a spacer moiety. 2 In such embodiments, activation of the triggering group produces a nucleophilic heteroatom, which is indirectly bonded to SO 2 The moiety attacks intramolecularly to form a ring, forming the moiety (Q). q -L'-H, where H is SO 2In 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 an 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.

[0043] Ar may be any suitable ring, including bicyclic or other polycyclic rings, such that the moieties undergoing intramolecular cyclization are held in close proximity to facilitate reaction after activation of the inducing group. The planar nature of aromatic and heteroaromatic rings is preferred because the rigid geometry of the substituents on such rings ensures favorable positioning of reactive moieties, but other types of rings, such as cycloalkenyl or heterocycloalkenyl, may impose similar geometries. Five- or six-membered rings, and / or the number or identity of heteroatoms in the ring, and / or the substituents on the other ring (e.g., electron-donating or electron-withdrawing substituents) may be selected to adjust the cyclization rate based on the bond angles of the resulting ring. Similarly, the more flexible conformations of cycloalkyl and heterocyclyl rings may be useful when it is desired to slow down the rate of intramolecular cyclization.

[0044] 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 water 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. Since it is often advantageous to prepare conjugates in a modular manner, Z' may contain a linking unit, i.e., a functional group resulting from the conjugation of one reactive moiety to another reactive moiety. 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, and the like. Representative Z' groups include L, as discussed in more detail below. 1 '-Z groups. In some embodiments, all of the DL groups attached to each CB are identical, while in other embodiments, each CB may be attached to two or more distinct DL groups. For example, some DL groups may have trigger groups that are activated under a first condition, while other DL groups may have trigger groups that are activated under a second condition, such that, for example, one active agent can be selectively released under a first condition, while a second active agent can be selectively released under a second condition.

[0045] 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''). Thus, in some embodiments, a compound of formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof, each Q is independently an activator linked to L' through a heteroatom, preferably O or N; Z', independently in each occurrence, is absent, a linking group; Each L' is independently a heteroatom selected from O, S, and N, preferably SO through O or N. 2 is a linking group bonded to L' and SO 2 is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q to release the active agent; Each X is independently -O-, -C(R b )(R c )-, or -N(R c )-, preferably -C(R b )(R c )-and E is an integer having a value of 1, 2, or 3, preferably 1; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y' is -N(R a )-, -O-, or -S-; at least one X is positioned in an ortho or para relationship to Y' on Ar; When the TG is cut off, the SO 2 and (Q) q -(L') w is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is 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 Z', hydrogen, or lower alkyl, with the proviso that R b is Z, or R b and R c 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.

[0046] In some embodiments, a compound of formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof, each Q is independently an activator linked to L' through a heteroatom, preferably O or N; Z' is a reactive group (e.g., a precursor group that can be used to attach an inducer such as CB); Each L' is independently a heteroatom selected from O, S, and N, preferably SO through O or N. 2 is a linking group bonded to L' and SO 2 is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q to release the active agent; Each X is independently -O-, -C(R b )(R c )-, or -N(R c )-, preferably -C(R b )(R c )-and E is an integer having a value of 1, 2, or 3, preferably 1; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y' is -N(R a )-, -O-, or -S-; at least one X is positioned in an ortho or para relationship to Y' on Ar; When the TG is cut off, the SO 2 and (Q) q -(L') w is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is 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 Z', hydrogen, or lower alkyl, with the proviso that R b is Z, or R b and R c 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.

[0047] In certain embodiments, each X is positioned in an ortho or para relationship to Y' on Ar.

[0048] In certain such embodiments, at least one X and Y' are positioned in an ortho relationship to one another on Ar. In certain such embodiments, E is 2 or 3. In certain such embodiments where E is 2, both occurrences of X are positioned in an ortho relationship to Y'. In certain such embodiments, E is 1.

[0049] In other embodiments, at least one X and Y' are positioned in a para relationship to each other on Ar.

[0050] In certain embodiments, at least one X is positioned in an ortho or para relationship to Y'.

[0051] In certain embodiments, R attached to Ar b At least one R other than b represents Z'.

[0052] In certain embodiments of formula (I') and (Ia), -Y' is -N(R a )-, -O-, or -S-. In some such embodiments, the TG is a β-galactoside, a β-glucuronide, or a combination of a β-galactoside and a β-glucuronide.

[0053] In some embodiments of formula (I') and (Ia), (L') w For each Q, 2 and each Q is an activator linked to one of the L' groups by a heteroatom, preferably O or N, forming, with the heteroatom of Q, an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- bond.

[0054] In another embodiment, (Q) q -(L') w - is selected from the following: [ka] During the ceremony, Q is an active agent linked to L' by a heteroatom, preferably O or N; X 4 is absent or forms, together with the heteroatom of Q, an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- bond; X 1 is -O- or -NR a -, preferably -O-, 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; 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 2 ) u NH 2 , -(CH 2 ) u NR u1 R u2 , and -(CH 2 ) u SO 2 Ru3 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.

[0055] In some such embodiments, (Q) q -(L') w is selected from: [ka]

[0056] Furthermore, the present invention provides intermediates for preparing a conjugate according to formula (I') or a compound 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 bonding of

[0057] 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 supported array, or sensor particle), or to any other molecule or support of interest.

[0058] In certain embodiments, Z' comprises a reactive group (eg, a precursor group discussed in more detail below with respect to Z) that can be used to attach the compound to a targeting moiety such as CB.

[0059] In certain preferred embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657152000008.tif237162TIFF0007657152000009.tif59148In the formula, X is -O-, -C(R b )(R c )-, or -N(R c )-and R 1 is C 1 ~C 6 is alkyl, R 21 and R 22 are each independently hydrogen or C 1 ~C 6 -alkyl, R 9 and R 10 are each independently hydrogen, alkyl, aryl, or heteroaryl, where alkyl, aryl, and heteroaryl are unsubstituted or substituted, e.g., -(CH 2 ) u NH 2 , -(CH 2 ) u NR u1 R u2 , and -(CH 2 ) u SO 2 R 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.

[0060] In other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657152000011.tif170159

[0061] In other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657152000013.tif222147TIFF0007657152000014.tif98146

[0062] In still other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657152000016.tif251156TIFF0007657152000017.tif177157

[0063] In other embodiments, the compound of formula (I') is selected from: [ka] TIFF0007657152000019.tif220145

[0064] In other embodiments, the compound of formula (I') is selected from: [ka]

[0065] 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 - 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)-, [ka] L 2 is an optionally present spacer moiety, C 1 ~C 6 Alkyl, C 5 ~C 14 Aryl and C 3 ~C 8 Heteroaryl, where alkyl, aryl, and heteroaryl may be, for example, C 1 ~C 10 Alkyl, -(CH 2 ) u NH 2 , -(CH 2 ) u NR 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 u1 , R u2 , and R u3 are each independently hydrogen, C 1 ~C 15 Alkyl, C 6 ~C 20 Aryl, or C 3 ~C 10heteroaryl, and u is an integer having a value from 1 to about 10; R 12 is hydrogen, C 1 ~C 8 an amino acid moiety, such as an alkyl or naturally occurring amino acid moiety; b, c, d, e, g, h, o, and qq are each independently an integer having a value of 1 to about 10; s' is an integer having a value from 1 to about 10.

[0066] In other embodiments, Z is a linking group having the structure of formula (F'), (G'), (H'), (J'), (K'), (L'), (M'), or (N'). [ka] TIFF0007657152000024.tif129150

[0067] In certain preferred embodiments, CB is selected from: [ka]

[0068] In certain preferred embodiments, (Q) q -(L') w is selected from the following: [ka] TIFF0007657152000027.tif215149TIFF0007657152000028.tif230163TIFF0007657152000029.tif111158In the formula, * (Q) q -(L') w -of, -SO 2 - represents the attachment point to.

[0069] In some embodiments, (Q) q -(L') w is selected from the following: [ka] During the ceremony, * (Q) q -(L') w -of, -SO 2 - represents the attachment point to.

[0070] Also, the compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: Each X is independently -O-, -CH 2 - or -NR'-, E is an integer having a value of 1, 2, or 3, preferably 1; R' is hydrogen, C 1 ~C 6 -Alkyl, C 6 ~C 14 -aryl, or C 2 ~C 20 -heteroaryl, Ar is C 5 ~C 20 -aromatic ring, C 2 ~C 20 -Heteroaromatic ring, C 2 ~C 30 -Fused ring, or C 5 ~C 20 -Aromatic ring-C 2 ~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' C further comprises 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. 1 ~C 200 -Alkylene or C 1 ~C200 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 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 -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHOH, -NHNH 2 , -BR 2 R 3 , [ka] or -Y'-TG, preferably Y is -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHNH 2 , -BR 2 R 3 , [ka] or -Y'-TG, R 1 is C 1 ~C 6 is alkyl, r is an integer having a value of 1, 2, 3, 4, or 5; Ar 1 is C 6 ~C 20 is an arylene, R 2 and R 3are each independently hydrogen, C 1 ~C 6 -Alkyl, C 1 ~C 6 -alkoxy, or hydroxy; R a , R b , R c , and R d are each independently hydrogen or C 1 ~C 6 is alkyl, Y' is -(CH 2 ) x NR''-, -(CH 2 ) x O- or -(CH 2 ) x S-, R'' is hydrogen or C 1 ~C 6 is 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' is -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 CH 2 O-bearing, C 7 ~C 30 - a hydrocarbon spacer, where -O-, -OC(O)-, -O(CO)O-, or -OC(O)NR''''- is 7 ~C 30 The hydrocarbon spacer may further comprise 7 ~C 30 The hydrocarbon spacer is C 1 ~C 6 Alkyl, C 5 ~C 14 Aryl and C 3 ~C 8 and heteroaryl, where the alkyl, aryl, and heteroaryl are, for example, C1 ~C 10 Alkyl, -(CH 2 ) u NH 2 , -(CH 2 ) u NR 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 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; Q 1 -OH, -NH-, -NR 5 R 6 , -SH, -SO 2 NH 2 or -COOH, R 4 is hydrogen, C 1 ~C 6 -Alkyl, C 5 ~C 14 -aryl, or C 3 ~C 8 -heteroaryl, where alkyl, aryl, and heteroaryl are substituted or unsubstituted; R 5 and R 6 are each independently hydrogen, C 1 ~C 6 -Alkyl, C 3 ~C 9 -cycloalkyl, or C5 ~C 10 -heteroaryl, where heteroaryl is substituted or unsubstituted; R''' and R'''' are each independently hydrogen or C 1 ~C 6 -alkyl, w is an integer having a value of 1, 2, 3, 4, or 5.

[0071] In some embodiments, the compound of formula (I) comprises a functional group (e.g., Y) that can induce intramolecular cyclization by an external stimulus. In certain embodiments, the functional group is introduced at the ortho and / or para positions relative to each X.

[0072] In some embodiments, R' is C 1 ~C 6 -Alkyl, C 6 ~C 14 -aryl, or C 2 ~C 20 -heteroaryl.

[0073] In some embodiments, Ar is C 5 ~C 20 -aromatic ring, C 2 ~C 20 -Heteroaromatic ring, C 2 ~C 30 -Fused ring, or C 5 ~C 20 -Aromatic ring-C 2 ~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 (I) is a compound having a structure according to formula (II): [ka] or a pharma- ceutically acceptable salt thereof.

[0074] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (III): [ka] or a pharma- ceutically acceptable salt thereof.

[0075] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (IV): [ka] or a pharma- ceutically acceptable salt thereof.

[0076] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (V): [ka] or a pharma- ceutically acceptable salt thereof.

[0077] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (VI): [ka] or a pharma- ceutically acceptable salt thereof.

[0078] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (VII): [ka] or a pharma- ceutically acceptable salt thereof.

[0079] In other embodiments, the compound of formula (I) is a compound having a structure according to formula (VIII): [ka] or a pharma- ceutically acceptable salt thereof.

[0080] In another embodiment, the compound of formula (I) is a compound having a structure according to formula (IX). [ka]

[0081] In some embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), where R is hydrogen, halogen, aldehyde, acetal, ketal, -R * , -OR * , -SR * , -NR * R ** , -C(Hull) 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , -N 3 , -NC, -C(O)R * , -OC(O)R * , -OS(O)R * , -S(O) 2 R * , -S(O) 2 OR * , -OS(O)OR * , -OS(O) 2 OR * , -S(O)NR * R ** , -S(O) 2 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* , -CO 2 R * , -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) 2 OR ** , -NR * S(O)R ** , -NR * C(O)NR ** , -SS-R * , or -R * SSR ** where R * and R ** are each independently hydrogen, C 1 ~C 18 Alkyl, C 6 ~C 20 Aryl, C 3 ~C 15 Heterocycle, or C 3 ~C 20 It is heteroaryl.

[0082] In some embodiments, the compound is of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), where R is hydrogen or * -(L a -A 1 -L b -L c -Z) m -CB, where L a is a single bond or C 1 ~C 20 - alkylene, A 1 is -C(O)NR * -, -NR *C(O)-, -NR * -, -O-, -PO 3 -, -OPO 3 -, -SO-, -SO 2 - or -SO 3 - and L b is -(CH 2 CH 2 O) a -or-(CH 2 ) a - and R * is hydrogen, C 1 ~C 18 -Alkyl, C 6 ~C 20 -aryl, C 3 ~C 15 -heterocycle, or C 3 ~C 20 is heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C 1 ~C 20 - alkylene, n is an integer having a value of 1 or 2; Z is CB and L c or Z is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) wherein 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.

[0083] In some embodiments, the compound is of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), where R is hydrogen or * -L a -A 1 -L b -L c -Z, where L a is a single bond or C 1 ~C 20 - alkylene, A 1 is -C(O)NR * -, -NR * C(O)-, -NR * -, -O-, -PO 3 -, -PO 4 -, -SO-, -SO 2 - or -SO 3 - and L b is -(CH 2 CH 2 O) a -or-(CH 2 ) a - and R * is hydrogen, C 1 ~C 18 -Alkyl, C 6 ~C 20 -aryl, C 3 ~C 15 -heterocycle, or C 3 ~C 20-heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C 1 ~C 20 - alkylene, n is an integer having a value of 1 or 2; Z is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a precursor selected from

[0084] In some embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), where R is * (-L a -A 1 -L b -L c -Z) m -CB, where L a is a single bond or C 1~C 20 - alkylene, A 1 is -C(O)NR * -, -NR * C(O)-, -NR * -, -O-, -PO 3 -, -PO 4 -, -SO-, -SO 2 - or -SO 3 - and L b is -(CH 2 CH 2 O) a -or-(CH 2 ) a - and R * is hydrogen, C 1 ~C 18 -Alkyl, C 6 ~C 20 -aryl, C 3 ~C 15 -heterocycle, or C 3 ~C 20 -heteroaryl, a is an integer having a value from 1 to about 20; L c is a single bond or C 1 ~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.

[0085] In some embodiments, the compound is a compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), wherein L′ further comprises —O—, —OC(O)—, —O(CO)O—, or —OC(O)NR″″—. 7 ~C 30 It is a hydrocarbon spacer.

[0086] In some embodiments, the compound is a compound of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), where Q is selected from -L'-(Q 1 ) w and [ka] During the ceremony, Q 1 -OH, -NR 5 R 6 , -SH, and -COOH; Q 2 -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)-, R 5 and R 6 is the same as defined above, R 9 and R 10 are each independently hydrogen, C 1 ~C 6 Alkyl, C 6 ~C 14 Aryl, or C 3 ~C 9 Heteroaryl, R 9 and R 10 The alkyl, aryl, and heteroaryl of 1 ~C 10 Alkyl, -(CH 2 ) u NH 2 , -(CH 2 ) u NR u1 R u2 , and -(CH2 ) u SO 2 R u3 and R 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''' is hydrogen or C 1 ~C 6 -alkyl, w is an integer having a value of 1, 2, 3, 4, or 5.

[0087] In certain embodiments, -L'-(Q 1 ) w teeth, [ka] is selected from.

[0088] -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.

[0089] In some embodiments, Q 2 -NR 5 R 6and the active agent can be attached at a quaternary amine structure, e.g., the active agent can be attached at a -NR 5 R 6 The moiety is capable of forming a quaternary amine bond with L'.

[0090] In some embodiments of formula (I″), (Ia), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), R 4 is a substituted alkyl, aryl, or heteroaryl. In some such embodiments, R 4 is C 1 ~C 10 Alkyl, -(CH 2 ) u NH 2 , -(CH 2 ) u NR 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 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 of 1 to about 10.

[0091] In some embodiments of formula (I″), (Ia), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), R 5 and / or R 6 -NR 7 R 8Heteroaryl substituted with, where R 7 and R 8 are each independently hydrogen, C 1 ~C 6 -Alkyl, C 3 ~C 9 -cycloalkyl, or C 5 ~C 14 -aryl.

[0092] In some embodiments of formula (I″), (Ia), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), Q or -(L′) w -(Q) q is selected from: [ka] TIFF0007657152000047.tif208149TIFF0007657152000048.tif69157In the formula, * (Q) q -(L') w -of, -SO 2 - represents the attachment point to.

[0093] In some embodiments of formula (I″), (Ia), (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), Q or -(L′) w -(Q) q is selected from the following: [ka] During the ceremony, * (Q) q -(L') w -of, -SO 2 - represents the attachment point to.

[0094] In certain embodiments, provided herein are compounds of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), wherein: Y is -NO2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHOH, -BR 2 R 3 or -Y'-TG, preferably Y is -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -BR 2 R 3 or -Y'-TG, R 1 is C 1 ~C 6 is 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, C 1 ~C 6 -Alkyl, C 1 ~C 6 -alkoxy, or hydroxy; Y' is -(CH 2 ) x NR''-, -(CH 2 ) x O- or -(CH 2 ) x S-, R'' is hydrogen or C 1 ~C 6 -alkyl, x is an integer having a value of 0 or 1; R'' is hydrogen or C 1 ~C 6 -alkyl, TG is a derivative group such as a β-galactosides, β-glucuronides, or a combination of β-galactosides and β-glucuronides.

[0095] In certain embodiments, the compound of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX) is selected from: [ka] TIFF0007657152000051.tif239156TIFF0007657152000052.tif222153TIFF0007657152000053.tif226155T IFF0007657152000054.tif218149TIFF0007657152000055.tif232154TIFF0007657152000056.tif121153In the formula, R 1 is C 1 ~C 6 is alkyl, R 21 and R 22 are each independently hydrogen or acetyl; R is hydrogen, * -L a -A 1 -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 C 1 ~C 20 is alkylene, A 1 is -C(O)NH-, -NHC(O)-, -NH-, -O-, -PO 3 -, -PO 4 -, -SO-, -SO 2 - or -SO 3 - and L bis -(CH 2 CH 2 O) a -or-(CH 2 ) a - and a is an integer having a value from 1 to about 20; L c is C 1 ~C 20 is alkylene, X'' is -O-, -S-, -NH-, or -CH 2 - and W b1 and W b2 each independently represents -C(O)NH-, -NHC(O)-, [ka] R 12 is hydrogen, C 1 ~C 8 Alkyl, amino acid moiety, -(CH 2 ) s COR 13 , or -(CH 2 ) p NR 14 R 15 and R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z, R 14 and R 15 are each independently hydrogen or -(C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z) m -CB, X'' is -O-, -S-, -NH-, or -CH 2 - and R e is C 1 ~C 8 -alkyl or -(L 1' -Z) m -CB, X 4 is -NHC(O)-(CH 2 ) g -NH- or -C(O)NH-(CH 2 ) 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 is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 -Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 alkyl) or dihydrogen phosphate (-OP(=O)(OH) 2 ) and CB is a ligand selected from [ka] Q is selected from the following: [ka] TIFF0007657152000062.tif242149TIFF0007657152000063.tif168148In the formula, * Q,-SO 2 - represents the attachment point to.

[0096] In some embodiments, the compound of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX) is selected from: [ka] TIFF0007657152000065.tif242155TIFF0007657152000066.tif221157TIFF0007657152000067.tif220147T IFF0007657152000068.tif223146TIFF0007657152000069.tif228156TIFF0007657152000070.tif234153In the formula, R 1 is C 1 ~C 6 is alkyl, R 21 and R 22 are each independently hydrogen or acetyl; R is hydrogen, * -L a -A 1 -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 C 1 ~C 20 is alkylene, A 1is -C(O)NH-, -NHC(O)-, -NH-, -O-, -PO 3 -, -PO 4 -, -SO-, -SO 2 - or -SO 3 - and L b is -(CH 2 CH 2 O) a -or-(CH 2 ) a - and a is an integer having a value from 1 to about 20; L c is C 1 ~C 20 is alkylene, X'' is -O-, -S-, -NH-, or -CH 2 - and W b1 and W b2 each independently represents -C(O)NH-, -NHC(O)-, [ka] R 12 is hydrogen, C 1 ~C 8 Alkyl, amino acid moiety, -(CH 2 ) s COR 13 , or -(CH 2 ) p NR 14 R 15 and R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z, R 14 and R 15 are each independently hydrogen or -(C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z) m -CB, X'' is -O-, -S-, -NH-, or -CH 2 - and R e is C 1 ~C 8 -alkyl or -(L 1' -Z) m -CB, X 4 is -NHC(O)-(CH 2 ) g -NH- or -C(O)NH-(CH 2 ) 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 is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 -Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10alkyl) or dihydrogen phosphate (-OP(=O)(OH) 2 ) and CB is a ligand selected from [ka] Q is selected from the following: [ka] During the ceremony, * Q-SO 2 - represents the attachment point to.

[0097] Active agent release As mentioned 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.

[0098] In some embodiments, the compounds and conjugates disclosed herein have an X (e.g., -CH 2 The nucleophilic functional group (Y or Y') is introduced at the atom adjacent to the nucleophilic group (Y-) (Y-). Typically, the nucleophilic functional group is masked by a triggering group (TG), as described in further detail below. When activated, the triggering group reacts with a nearby SO in an intramolecular cyclization and / or 1,4-elimination. 2 The nucleophilic functional groups react with the moiety, ultimately yielding one or more active agents (Q, Q 1 , or Q 2 ) or their protonated forms. In some such embodiments, one or more active agents are released through an intramolecular cyclization reaction after a chemical, physicochemical, and / or biochemical reaction (see, e.g., Reaction Scheme 1), or the active agent is released through an intramolecular cyclization reaction after a 1,6-elimination or 1,4-elimination (see, e.g., Reaction Scheme 2).

[0099] As an example, the mechanism when Y is -Y'-TG is shown in Reaction Scheme 1. Reaction Scheme 1: [ka]

[0100] Q is [ka] The mechanism for this is shown in Reaction Scheme 2. Reaction Scheme 2: [ka]

[0101] The mechanism of polysubstituted construction is shown in Reaction Scheme 3. [ka]

[0102] In some embodiments, the Q 1 is an activator that includes 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, and the like. In some such embodiments, Q 2 Q 1 is used instead of Q 2 is an amine group-containing drug. 2 is an activator capable of binding to an ammonium unit. In still other embodiments, Q 2 Q 2Upon release, it is capable of dissociating in its original form bearing the amine groups, where the active agent can be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

[0103] 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.

[0104] 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. In general, 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 predefined triggering condition). When activated, the triggering group releases the nucleophilic Y or Y' group, as described above, allowing self-immolation or intramolecular cyclization to occur.

[0105] In some embodiments, the TG comprises a sequence (such as a peptide sequence, e.g., a dipeptide (Val-Cit, Val-Ala)) or moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which can be hydrolyzed by an enzyme (e.g., oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc.), and / or may comprise a moiety selected from phosphodiesters, phospholipids, esters, β-galactose, β-glucose, fructose, oligosaccharides, etc.

[0106] 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).

[0107] 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).

[0108] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by irradiation with light (e.g., 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonates, coumarins, phosphotriesters, bis-arylhydrazones, or bimanbi-thiopropionic acid derivatives).

[0109] 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).

[0110] 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).

[0111] 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 paramethoxybenzyl derivative, or a selenium compound).

[0112] In certain preferred embodiments, the TG comprises a sugar group, which can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the triggering group is -NO 2 and can be cleaved under reducing conditions. In certain preferred embodiments, the triggering group is a 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.

[0113] 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 22 is hydrogen or lower alkyl (e.g., C 1 ~C 6-alkyl). In certain embodiments, the 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-phenyl)ethyl ether, 1-phenyl-10-phenyl-2-methyl-2-phenyl ... Examples of the silyl ethers include, but are not limited to, 2,4,6-trimethylbenzoate, methyl carbonate, 2,2,2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, N-phenyl carbamate, nitrate ester, 2,4-dinitrophenyl sulfenate ester, and the like.

[0114] Protective groups as triggers 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.

[0115] 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 carbamates, 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-isopropyl aryl carbamate (Ipaoc), ... amine, N-phenylmethylcarbamate, N-hydroxypiperidinylcarbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenylmethylcarbamate, 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.

[0116] 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, triphenyl methyl ether, α-naphthyl diphenyl methyl ether, p-methoxyphenyl diphenyl methyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether (TMS ether), isopropyl dimethylsilyl 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, 2,2,Examples of the aryl esters 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), aryl methanesulfonate, aryl toluenesulfonate, and the like.

[0117] 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.

[0118] linking group In some embodiments, the compounds and conjugates disclosed herein include a linking group that connects each CB and Ar by a covalent bond. 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, more preferably at least three, of the following four criteria: (i) at least one -CH in the alkylene moiety 2 - is substituted (i.e., replaced) with 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, glycobond, peptide bond, or amide bond is contained in the alkylene moiety; and (iv) Alkylene is C 1 ~C 20 Alkyl, C 6 ~C 20 Aryl C 1 ~C 8 Alkyl, -(CH 2 ) s COOH, and -(CH 2 ) p NH 2 where s is an integer having a value from 0 to 10 and p is an integer having a value from 1 to about 10.

[0119] 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 C 1 ~C 20 Alkyl, C 6 ~C 20 Aryl C 1 ~C 8 Alkyl, -(CH 2 ) s COOH, and -(CH 2 ) p NH 2 where s is an integer having a value from 0 to 10 and p is an integer having a value from 1 to about 10.

[0120] In other embodiments, the linking group connecting each CB and Ar comprises a functional group produced through a click chemistry reaction.

[0121] In an alternative embodiment, the linking unit comprises a reactive functional group capable of participating in a click chemistry reaction.

[0122] Click chemistry is a reaction that can be carried out under mild conditions and is highly selective for functional groups not commonly found in biomolecules (e.g., azide groups, acetylene groups, etc.). Thus, the 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 azide groups and acetylene groups proceeds selectively without interference from other functional groups present in the molecule. For example, azide-acetylene click chemistry can lead to triazole moieties in high yields.

[0123] Thus, in some embodiments, the linking group connecting each CB and Ar is [ka] and V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO 2 - or -SO 2 NR 25 -, can be R 21 ~R 25 are each independently 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 can be an integer having a value from 1 to about 10, p can be an integer having a value from 0 to about 10, q can be an integer having a value from 1 to about 10, and L'' can be a single bond.

[0124] In another embodiment, the linking unit connecting each 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, ** is the point of attachment to Ar, W a1 , W a2 , and W a3 are each independently -NH-, -C(=O)-, or (-CH 2 -) b and W b1 is an amide bond or a triazolylene; P 1 is W a3 and Y 2 is an amino acid moiety, a peptide bond, or an amide bond; L c is alkylene, Y 2 is a single bond, -W a4 -(CH 2 ) c -W b2 -(CH 2 ) d -W a5 -, or -W a6 -(CH 2 ) e -CR e Rf -X-, R e is C 1 ~C 8 Alkyl or CB-W a7 -Y 3 -W c1 -(CH 2 ) f - and R f is BW a7 -Y 3 -W c1 -(CH 2 ) f - and X is -NHC(=O)-(CH 2 ) g -W a8 - or -C(=O)NH-(CH 2 ) h -W a9 - and W a4 , W a5 , W a6 , W a7 , W a8 , and W a9 are each independently -NH-, -C(=O)-, or -CH 2 - and W b2 is an amide bond or a triazolylene; W c1 is -NHC(=O)- or -C(=O)NH-, Y 3 is -(CH 2 ) i -(X'CH 2 CH 2 ) j -(CH 2 ) k - and X' is -O-, -S-, -NH-, or -CH 2 - and 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 -(CH 2 ) q -(CH 2 CH 2 X'') o -or-(CH 2 ) q -(X''CH 2 CH 2 ) o - and X'' is -O-, -S-, -NH-, or -CH 2 - and o and q are integers having a value of 1 to about 10.

[0125] In some embodiments, P 1 comprises at least one unit represented by formula (B) or (C), [ka] During the ceremony, R 12 is hydrogen, C 1 ~C 8 -alkyl, amino acid side chains such as naturally occurring amino acid side chains (e.g., H, methyl, isopropyl, isobutyl, sec-butyl, S-methyl thioether, benzyl, indole, pyrrolidine, pyrroline, hydroxymethyl, tyrosyl, lysyl, imidazole, glycyl, glutamyl, carbamoyl butanoate, carboxamide, aspartate, 1-hydroxyethyl, and 2-hydroxyethyl), -(CH 2 ) s COR 13 , or -(CH 2 ) p NR 14 R 15 and R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z, R 14 and R 15 are each independently hydrogen or -(C(O)(CH2 ) s' (X''CH 2 CH 2 ) s'' Z) m -CB, X'' is -O-, -S-, -NH-, or -CH 2 - and 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 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.

[0126] In some embodiments of formula (B) or (C), R 12 is hydrogen, alkyl, amino acid side chain, -(CH 2 ) s C(O)R 13 , or -(CH 2 ) 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(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''-(CB) m and R 14 and R 15 are each independently 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 to R 14 Or R 15 or Z'' is a linking group that includes a reactive group.

[0127] In some such embodiments of formula (B) or (C): R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', R 14 and R 15 are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', Z'' is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a reactive precursor of a linking unit selected from

[0128] In other such embodiments of formula (B) or (C): R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''CB, Z'' is CB to R 14 or R 15 Isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10-alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a linking unit formed from a precursor selected from

[0129] In some embodiments, Y 2 is a single bond or is selected from: [ka] During the ceremony, W b2 is -C(O)NH-, -NHC(O)-, [ka] R e is C 1 ~C 8 -alkyl or -(L 1' -Z-) m CB, R f is BW b2' -(CH 2 ) i -(X''CH 2 CH 2 ) j -NH-C(=O)-(CH 2 ) f - and X a is -NHC(=O)-(CH 2 ) g -NH- or -C(O)NH-(CH 2 ) 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 -CH 2 - and L 1' , Z, m, and B are as defined above.

[0130] In certain embodiments, the linking units connecting each CB and Ar are covalently connected to each other (CH 2 ) b , L c , (P 1 ) a , W a1 , W a2 , W a3 , Y 1 , and Y 2 is a linking group comprising a group, W a1 , W a2 , and W a3 are each independently -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 is -(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: [ka] 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; o and q are each independently an integer having a value of 1 to about 10.

[0131] In some embodiments, R 12 is a naturally occurring amino acid side chain. In another embodiment, R 12 is an unnatural amino acid side chain.

[0132] In some embodiments, the linking unit connecting each 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, ** is the point of attachment to Ar.

[0133] In some such embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH 2 ) s COOH, or -(CH 2 ) p NH 2 and 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.

[0134] In some embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH 2 ) s C(O)R 13 , or -(CH 2 ) 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(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''-(CB) m and R 14 and R 15 are each independently 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 to R 14 Or R 15 or Z'' is a linking group that includes a reactive group.

[0135] P 1 In some such embodiments, R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', R 14 and R 15are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', Z'' is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a reactive precursor of a linking unit selected from

[0136] P 1 In other such embodiments, R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH2 ) s'' Z''CB, Z'' is CB to R 14 or R 15 Isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a linking unit formed from a precursor selected from

[0137] 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)-(CH 2 ) g -NH- or -C(O)NH-(CH 2 )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 from 1 to about 10.

[0138] In some embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 12 is hydrogen, alkyl, amino acid side chain, -(CH 2 ) s C(O)R 13 , or -(CH 2 ) 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(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''-(CB) m and R 14 and R 15 are each independently 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 to R 14 Or R 15 or Z'' is a linking group that includes a reactive group.

[0139] In some such embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', R 14 and R 15 are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z'', Z'' is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a reactive precursor of a linking unit selected from

[0140] In some such embodiments of formula (F), (G), (H), (I), (J), (K), (L), or (M), R 13 is OH or -NH(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH 2 ) s' (X''CH 2 CH 2 ) s'' Z''CB, Z'' is CB to R 14 or R 15 Isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkynyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 -alkyl) and dihydrogen phosphate (-OP(=O)(OH) 2 ) is a linking unit formed from a precursor selected from

[0141] 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 by non-covalent binding, such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonds, electrostatic effects, and / or electromagnetic effects. In certain embodiments, the CB is selected from nanoparticles, immunoglobulins, nucleic acids, proteins, oligopeptides, polypeptides, antibodies, fragments of antigenic polypeptides, repebodies, and the like.

[0142] 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.

[0143] 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 ligands include 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).

[0144] 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 in 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, a tumor cell-specific peptide, a tumor cell-specific aptamer, a tumor cell-specific carbohydrate, a tumor cell-specific monoclonal antibody, a polyclonal antibody, and an antibody fragment identified in a library screen.

[0145] 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 benzodiazeoine 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, SSTR4, SSTR5, and SSTR6. 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, claudin, 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, gangliosides GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

[0146] 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 2-fold higher in the cell type than in a reference cell population. In some embodiments, the cell type specific marker is present at a level at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1,000-fold higher than its average expression in a reference population. Detection or measurement of a cell type specific marker may allow for the identification 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.

[0147] 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.

[0148] 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.).

[0149] 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).

[0150] In some embodiments, a target may be a marker that is exclusively or primarily associated with one or a few cell types, with one or a few diseases, and / or with one or a few developmental stages. A cell type specific marker is typically expressed at a level at least 2-fold higher in that cell type than in a reference cell population, which may consist of, for example, 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 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold higher than its average expression 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.

[0151] In some embodiments, the targets include proteins, carbohydrates, lipids, and / or nucleic acids. In some embodiments, the 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, and the like. In some embodiments, the 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), and the like. In some embodiments, the 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, and the like. In some embodiments, the 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.

[0152] Many markers are known in the art. Typical markers include cell surface proteins, such as receptors. Exemplary receptors include, but are not limited to, transferrin receptor; LDL receptor; 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.

[0153] Nanoparticles In some embodiments, the targeting moiety comprises a particle (e.g., a targeted particle), preferably a nanoparticle, optionally bound to a targeting molecule that can specifically or preferentially 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 bound to the targeted particle.

[0154] By "nanoparticle" herein is meant any particle having a diameter less than 1000 nm. In some embodiments, a therapeutic agent and / or a 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 a 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 a 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.

[0155] In general, the nanoparticles of the present invention include any type of particle. Any particle can be used according to the present invention. In some embodiments, the particles are biodegradable and biocompatible. In general, a biocompatible material is not toxic to cells. In some embodiments, a material is considered biocompatible if its addition to cells results in cell death below a certain threshold. In some embodiments, a material is considered biocompatible if its addition to cells does not induce adverse effects. Generally, a biodegradable material 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 material is a material that can be degraded by cellular mechanisms. In some embodiments, a biodegradable material is a material that can be degraded by chemical processes. In some embodiments, the particles are materials that are both biocompatible and biodegradable. In some embodiments, the particles are materials that are biocompatible but not biodegradable. In some embodiments, the particles are materials that are biodegradable but not biocompatible.

[0156] 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 maximum dimension that falls within 5%, 10%, or 20% of the average diameter or maximum dimension. In some embodiments, the population of particles may be heterogeneous in size, shape, and / or composition. A variety of different particles may be used according to 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 ovals or ellipses. In some embodiments, the particles are cylinders, cones, or pyramids.

[0157] In some embodiments, the particle is a microparticle (e.g., a microsphere). In general, "microparticle" refers to any particle having a diameter less than 1000 μm. In some embodiments, the particle is a picoparticle (e.g., picospheres). In general, "picospheres" refers to any particle having a diameter less than 1 nm. In some embodiments, the particle is a liposome. In some embodiments, the particle is a micelle.

[0158] The 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, the particles can have a core / shell structure, where the core is one layer and the shell is a second layer. The particles can include multiple different layers. In some embodiments, one layer can be substantially cross-linked, the second layer is substantially not cross-linked, etc. In some embodiments, one, a few, or all of the different layers can 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.

[0159] In some embodiments, the particles are porous, by porous we mean that the particles contain holes or channels, and these holes or channels are typically small compared to the size of the particle. For example, the particles may be porous silica particles, e.g., mesoporous silica nanoparticles, or may have a coating of mesoporous silica (Lin et al., 2005, J. Am. Chem. Soc, 17:4570). The particles may have pores ranging from about 1 nm to about 50 nm in diameter, e.g., about 1 to 20 nm in diameter. About 10% to 95% of the volume of the particle may consist of voids within the pores or channels.

[0160] 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 associated with the nanoparticles of the present invention, and the like. The coatings can be applied or assembled in various ways, for example, by immersion, using layer-by-layer techniques, by self-assembly, conjugation, and the like. Self-assembly refers to the process of spontaneous assembly of higher-order structures that depends 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.

[0161] 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 use in humans 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.

[0162] In some embodiments, the particles can be non-polymeric particles (e.g., metal particles, quantum dots, ceramic particles, inorganic materials, bone-derived materials, polymers including bone substitutes, viral particles, etc.). In some embodiments, the therapeutic or diagnostic agent to be delivered can 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 can 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.

[0163] 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, fine emulsion 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).

[0164] Methods for making 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).

[0165] Nucleic acid targeting moiety In some embodiments, the targeting moiety comprises a nucleic acid targeting moiety.

[0166] 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.

[0167] 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 subcellular compartments. In general, 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 depends on the three-dimensional structure(s) of the aptamer and / or the target. In some embodiments, aptamers bind to their targets via complementary Watson-Crick base pairing that is interrupted by a structure that disrupts the base pairing (e.g., a hairpin loop).

[0168] In some embodiments, the nucleic acid targeting moieties are spiegelmers (PCT Publication Nos. WO98 / 08856, WO02 / 100442, and WO06 / 117217). In general, spiegelmers are synthetic mirror-image nucleic acids that can specifically bind to a target (i.e., mirror-image aptamers). Spiegelmers are characterized by structural features that make them less susceptible to exonucleases and endonucleases.

[0169] One of skill in the art will recognize that any nucleic acid targeting moiety (e.g., an aptamer or spiegelmer) capable of specifically binding to a target may be used in accordance with the present invention. In some embodiments, a nucleic acid targeting moiety for use in accordance with the present invention may target a marker associated with a disease, disorder, and / or condition. In some embodiments, a nucleic acid targeting moiety for use in accordance with the present invention may target a cancer-associated target. In some embodiments, a nucleic acid targeting moiety for use in accordance with the present invention may target a tumor marker. Any type of cancer and / or any tumor marker may be targeted using a nucleic acid targeting moiety in accordance with the present invention. To name just a few examples, a nucleic acid targeting moiety may target a marker associated with prostate cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, bone cancer, esophageal cancer, liver cancer, gastric cancer, brain tumors, skin melanoma, and / or leukemia.

[0170] Nucleic acids of the invention (nucleic acid nucleic acid targeting moieties and / or functional RNAs, e.g., 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.

[0171] 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).

[0172] 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 or unsubstituted, provided that the binding affinity and selectivity of the nucleic acid targeting moiety is not substantially reduced by the substitution (e.g., the dissociation constant of the nucleic acid targeting moiety for the target is less than about 1×10 -3 M), may be replaced with a hydrocarbon linker or a polyether linker.

[0173] It will be understood by those skilled in the art that the nucleic acid according to the present invention may contain only the types of nucleotides found in naturally occurring nucleic acids, or may alternatively 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, 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 H, OR, R, halo, SH, SR, NH 2 , N.H.R., N.R. 2 or CN, where R is C 1 ~C 6is alkyl, alkenyl, or alkynyl, and halo is F, CI, Br, or I. Examples of modified linkages include phosphorothioate linkages and 5'-N-phosphoramidite linkages.

[0174] 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-azacytidine, 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 the 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.

[0175] 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. One of skill in the art will appreciate 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 a target is not substantially affected. The modified regions may be at the 5' and / or 3' ends of one or both strands. For example, modified nucleic acid targeting moieties have been used in which approximately 1-5 residues at the 5' and / or 3' ends of either of both strands are nucleotide analogs and / or have backbone modifications. The modifications may be at the 5' or 3' ends. One or both nucleic acid strands may comprise at least 50% unmodified nucleotides, at least 80% unmodified nucleotides, at least 90% unmodified nucleotides, or 100% unmodified nucleotides.

[0176] Nucleic acids according to the invention may include 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 invention encompasses the use of any nucleic acid with 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 invention may include one or more non-natural 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.

[0177] 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.

[0178] Any method can be used to design novel nucleic acid targeting moieties (see, e.g., U.S. Pat. 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, 5,843,743, and 5,843,743). (see U.S. Patent Application Publication Nos. 2005 / 0069910, 2004 / 0072234, 2004 / 0043923, 2003 / 0087301, 2003 / 0054360, and 2002 / 0064780).

[0179] Nucleic acid targeting moieties that bind to proteins, carbohydrates, lipids, and / or nucleic acids may 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., may be designed and / or identified for use in the complexes 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., may be designed and / or identified for use in the complexes 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., may be designed and / or identified for use in the complexes 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 including any combination of DNA, RNA, modified DNA, and modified RNA, may be designed and / or identified for use in the complexes of the invention.

[0180] Nucleic acid targeting moieties (e.g., aptamers or spiegelmers) may be designed and / or identified using any available method, in some embodiments, nucleic acid targeting moieties are designed and / or identified by identifying nucleic acid targeting moieties from a candidate mixture of nucleic acids.

[0181] Methods for preparing the compounds of the present invention The compounds and conjugates disclosed herein can be prepared via simple preparation methods (see, for example, Examples 1-36), which allow for convenient purification.

[0182] 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 4, 5, or 6: Reaction Scheme 4: [ka] In the formula, M is Cl or F; X, Y, Ar, R, and n are as defined above.

[0183] The invention further provides compounds as described above that are useful in these methods.

[0184] 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 pharma- ceutically acceptable salt thereof, wherein X is -O-, -C(R b )(R c )-, or -N(R c )-, preferably -C(R b )(R c )-and W is -SO 2 -G, G is a halogen (preferably fluorine), imidazole, or N-methylimidazolium; R is a substituent or -L 1' -Z, L 1' C optionally includes 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. 1 ~C 200 - alkylene, Z is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH 2 -hal), maleimides, dienes, alkenes, halides, tosylates (TsO - ), aldehydes, sulfonates (R-SO 3 - ), [ka] Phosphonic acid (-P(=O)(OH) 2 ), ketone, C 8 ~C 10 Cycloalkyl, -OH, -NHOH, -NHNH 2 , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N 3 ), amino (-NH 2 ), sulfonic acid (-SO 3 H), alkynone derivatives (-C(O)C≡CR a (where R a is C 1 ~C 10 alkyl), and dihydrogen phosphate (-OP(=O)(OH) 2 ) wherein n is an integer having a value from 1 to 4; Y is -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHNH 2 , -BR 2 R 3 , [ka] or -Y'-TG, etc. C )-dipeptides (e.g., Val-Cit, Val-Ala), -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH2 ) r -Ar 1 -NO 2 , -NHOH, -NHNH 2 , -BR 2 R 3 , [ka] or -Y'-TG, R 1 is C 1 ~C 6 is alkyl, r is an integer from 1 to 5; Ar 1 is C 6 ~C 20 - arylene, R 2 and R 3 are each independently hydrogen, C 1 ~C 6 -Alkyl, C 1 ~C 6 -alkoxy, or hydroxy; R a , R b , R c , and R d are each independently hydrogen or C 1 ~C 6 is alkyl, Y' is -(CH 2 ) x NR''-, -(CH 2 ) x O- or -(CH 2 ) x S-, R'' is hydrogen or C 1 ~C 6 is alkyl, x is an integer of 0 or 1; TG is the inducing group.

[0185] In some embodiments, the compounds and conjugates disclosed herein include an intermediate compound having a structure according to formula (V): [ka] or a pharma- ceutically acceptable salt thereof, wherein W, L 1' and Z are as defined for formula (IV); TG is a derivative group such as a β-galactosides, β-glucuronides, or a combination of β-galactosides and β-glucuronides.

[0186] In other embodiments, the compounds and conjugates disclosed herein include an intermediate compound having a structure according to formula (VI): [ka] or a pharma- ceutically acceptable salt thereof, wherein W is as defined for formula (IV); Y is N(R C )-dipeptides (e.g., Val-Cit, Val-Ala), -NO 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHOH, -NHNH 2 , -BR 2 R 3 or -O-TG, R 1 No 2 , -OC(O)(CH 2 ) r C(O)R 1 , -O(CH 2 ) r -Ar 1 -NO 2 , -NHOH, -NHNH 2 , -BR 2 R 3 , or -O-TG, etc. 1 ~C 6 -alkyl, R 1 is C 1~C 6 -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, C 1 ~C 6 -Alkyl, C 1 ~C 6 -alkoxy, or hydroxy; R a , R b , R c , and R d are each independently hydrogen or C 1 ~C 6 -alkyl, TG is a derivative group that is a β-galactosides, β-glucuronides, or a combination of β-galactosides and β-glucuronides.

[0187] Antibody-drug conjugates (ADCs) In some embodiments, CB is an antibody and Q is a drug. Thus, 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 disease, e.g., cancer, due to the ability of ADCs 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.

[0188] The ADC of the present invention comprises 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.

[0189] 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 the moiety are covalently linked.

[0190] In some embodiments, the ADC has the following formula (Formula VII): (DL) n -Ab(VII) where 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″): [ka] each Q is independently an active agent linked to L' by a heteroatom, preferably O or N; Z′, independently at each occurrence, represents a group having the structure of formula (I″) as follows: cb a linking group, solubilizing group, reactive group (e.g., a precursor group), a solid surface (e.g., a particle), a stabilizing group, a chelating agent, a biopolymer (e.g., an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, an antigen, a fragment of a polypeptide, or a lipibody), an active agent, or a detectable moiety, provided that at least one occurrence of Z′ transforms the structure of formula (I″) into cb Provided that you connect to Each L' is independently a heteroatom selected from O, S, and N, preferably SO through O or N. 2 The spacer portion is bound to L' and SO 2 is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q to release the active agent; Each X is independently -O-, -C(R b )(R c)- or -N(R c )-, preferably -C(R b )(R c )-and E is an integer having a value of 1, 2, or 3, preferably 1; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y' is -N(R a )-, -O-, or -S-; at least one X is positioned in an ortho or para relationship to Y' on Ar; When TG is cut, SO 2 and (Q) q -(L') w is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently Z', hydrogen, or lower alkyl; R b and R c 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.

[0191] In certain embodiments, each X is positioned in an ortho or para relationship to Y' on Ar.

[0192] In certain such embodiments, at least one X and Y' are positioned in an ortho relationship to one another on Ar. In certain such embodiments, E is 2 or 3. In certain such embodiments where E is 2, both occurrences of X are positioned in an ortho relationship to Y'. In certain such embodiments, E is 1.

[0193] In other embodiments, at least one X and Y' are positioned in a para relationship to each other on Ar. In certain such embodiments, E is 1.

[0194] In certain embodiments, at least one X is positioned in an ortho or para relationship to Y'.

[0195] In certain embodiments, R attached to Ar b At least one R other than b represents Z'.

[0196] In some embodiments, n has a value in the range of 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 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 equivalent to the number of drugs present in (DL). When cb is other than 1, the drug-to-antibody ratio (DAR) of the ADC is equivalent to the ratio of the number of drugs present in (DL) to the number of antibodies present in the conjugate.

[0197] Exemplary Drugs for Conjugation The ADCs of the invention provide targeted therapy, which may reduce side effects often seen with, for example, anti-cancer therapies, since one or more active agent(s) or drug(s) are delivered to specific cells.

[0198] For example, drugs include erlotinib (TARCEVA; Genentech / OSI Pharm.); bortezomib (VELCADE; MilleniumPharm.); 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); ethylenimines, methylmelamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins (e.g., brazil, tacin or blatacinone; camptothecins, including the synthetic analog topotecan; bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, or bizelesin); cryptophycins (e.g., cryptophycin 1 or cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189, and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatins;Nitrogen mustards (e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, or uracil mustard); nitrousureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimnustine ne); antibiotics (e.g., dynemicins, including calicheamicins or dynemicins A, as enediyne antibiotics, selected from calicheamicin gamma 1I and calicheamicin omega 1I); bisphosphonates (e.g., clodronate); enediyne antibiotic chromophores that are esperamicins, neocarzinostatin chromophores or related chromoproteins, aclacinomycins, actinomycins, anthramycins, azaserines, bleomycins, cactinomycins, carabimycins, carminomycins, carzinophilins, chromomas 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-adrenals (e.g., aminoglutethimide, mitotane, or trilostane); folic acid supplements (e.g., frolinic acid, acid);aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;eflornithine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;maytansinoids (e.g., maytansine or ansamitocins);trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, or anguidine);mitoguazone;mitoxantrone;mopida Mol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) 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, Ill.), 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 (registered trademark) vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids (e.g., retinoic acid); capecitabine; and pharma- ceutically acceptable salts, solvates, acids, or derivatives thereof.

[0199] Mitotic inhibitors In some embodiments, the linkers of the invention may be used to conjugate an antibody to one or more mitotic inhibitor(s) to form an ADC for the treatment of cancer. As used herein, the term "mitotic inhibitor" refers to a cytotoxic and / or therapeutic agent that prevents mitosis or cell division, a biological process that is particularly important to cancer cells. Mitotic inhibitors 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 mitotic inhibitor(s) that disrupt microtubule formation by inhibiting tubulin polymerization. In one embodiment, the mitotic inhibitor used in the ADC of the invention is Taxol® (paclitaxel), Taxotere® (docetaxel), or Ixempra® (ixabepilone). Examples of mitotic inhibitors that may be used in the ADCs disclosed herein are provided below. The auristatins mentioned above are included in the genus of mitotic inhibitors.

[0200] Auristatins 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 (US 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 mitotic inhibitors include, but are not limited to, monomethylauristatin D (MMAD or auristatin D derivatives), monomethylauristatin E (MMAE or auristatin E derivatives), monomethylauristatin 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 No. WO04 / 010957, International Patent Publication No. 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, 5,6 Nos. 3,599,902, 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.

[0201] Dolastatin The antibody may be conjugated to at least one dolastatin using the linker 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 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, the ADC of the invention comprises an antibody, a linker as described herein, and at least one dolastatin. The auristatins mentioned above are synthetic derivatives of dolastatin 10.

[0202] 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 conventional chemotherapeutic agents such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020).

[0203] 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). The 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).

[0204] Suitable maytansinoids 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 achieved in the final conjugated molecule. The structure of an exemplary maytansinoid, mertansine (DM1), is provided below: [ka]

[0205] Representative examples of maytansinoids include, but are not limited to, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine; mertansine, also referred to as 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 analogue). Other examples of maytansinoids are described in U.S. Pat. No. 8,142,784, incorporated herein by reference.

[0206] Ansamitocins are a group of maytansinoid antibiotics isolated from various bacterial sources.These compounds have strong antitumor activity.Representative examples include, but are not limited to, ansamitocin P1, ansamitocin P2, ansamitocin P3, and ansamitocin P4.

[0207] Plant alkaloids The linker of the present invention may be used to conjugate the 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.

[0208] Taxanes The antibody may be conjugated to at least one taxane using the linker of the present invention. The term "taxane" as used herein refers to a class of antineoplastic agents that have a mechanism of action on microtubules and that have a taxane ring structure and a structure that includes 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 nanoparticulate paclitaxel (ABI-007 / Abraxene®; Abraxis Bioscience).

[0209] 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.

[0210] Vinca alkaloids In one embodiment, the antibody can be conjugated to at least one vinca alkaloid using the linker of the present invention.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.The examples of vinca alkaloids that can be used in the ADC of the present invention include, but are not limited to, vindesine sulfate, vincristine, vinblastine, and vinorelbine.

[0211] Antitumor antibiotics The linker of the present invention may be used to conjugate the antibody 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 that prevents cell growth by interfering with DNA and is made from a microorganism. In many cases, antitumor antibiotics break down DNA strands or slow down or stop DNA synthesis. Examples of antitumor antibiotics that may be included in the ADCs disclosed herein include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracycline, calicheamicin, and duocarmycin, which are described in more detail below.

[0212] Actinomycin The linker 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 Streptomyces genus. 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, polosramycin, prothracarcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sivanomycin, sibiromycin, and tomaymycin. Thus, in one embodiment, D is an actinomycin, e.g., actinomycin D, or a PBD, e.g., a pyrrolobenzodiazepine (PBD) dimer.

[0213] The structure of a PBD 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]

[0214] PBDs vary in the number, type, and position of substituents in both their aromatic A-ring and pyrrolo C-ring, as well as the degree of saturation of the C-ring. In the B-ring, there is generally an imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center involved in DNA alkylation. All of the known natural products have the (S) configuration at the chiral C11α position, thereby giving them a right-handed twist when viewed from the C-ring towards 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.

[0215] Anthracyclines The linker of the present invention may be used to conjugate the antibody to at least one anthracycline. Anthracyclines are a subclass of antitumor antibiotics isolated from bacteria of the Streptomyces genus. Representative examples include, but are not limited to, daunorubicin (Cerubidine, Bedford Laboratories), doxorubicin (Adriamycin, Bedford Laboratories; also called doxorubicin hydrochloride, hydroxydaunorubicin, and Rubex), epirubicin (Ellence, Pfizer), and idarubicin (Idamycin; Pfizer Inc.). Thus, in one embodiment, D is an anthracycline, e.g., doxorubicin.

[0216] Calicheamicin The antibody may be conjugated to at least one calicheamicin using the linker of the present invention. 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 over other chemotherapeutic drugs (Damle et al. (2003) Curr Opin Pharmacol 3:386). Preparations of calicheamicin that may 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 may be used include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG, and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998), as well as 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 a calicheamicin.

[0217] Duocarmycin The antibody may be conjugated to at least one duocarmycin using the linker of the present invention. Duocarmycins are a subclass of antitumor antibiotics isolated from bacteria of the Streptomyces genus (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 carzeresin. Thus, in one embodiment, D is a duocarmycin.

[0218] Other Antitumor Antibiotics In addition to those mentioned above, 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).

[0219] Immunomodulators In some embodiments, the antibody may be conjugated to at least one immunomodulatory agent using the linker of the invention. As used herein, the term "immunomodulatory agent" refers to an agent that can stimulate or modify an immune response. In one embodiment, the immunomodulatory agent is an immunostimuator that enhances the immune response of a subject. In some embodiments, the immunomodulatory agent is an immunosuppressant that blocks or reduces the immune response of a subject. The 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 therapy.

[0220] Cancer Vaccines The linker of the present invention may be used to conjugate an antibody to a cancer vaccine. As used herein, the term "cancer vaccine" refers to a composition (e.g., tumor antigen and cytokine) that elicits a tumor-specific immune response. The response is elicited from the subject's own immune system by administering a cancer vaccine, or in the present case, an ADC that includes an antibody and a cancer vaccine. In a preferred embodiment, 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 a particular cancer cell or present on the surface of a particular infectious agent 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) vaccines types 16 and 18 (Cervarix, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 (Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, D is a cancer vaccine that is either an immunostimulant or an immunosuppressant.

[0221] Cytokines The linker of the invention may be used to conjugate the 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 multiple animal tumor models have demonstrated that cytokines have a broad range of antitumor activity, which has been 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).

[0222] 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; Mullerian 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 alpha, beta, and gamma, 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.

[0223] Colony-stimulating factors (CSFs) The linkers of the invention may be used to conjugate the antibody to at least one colony stimulating factor (CSF). Colony stimulating factors (CSF) are growth factors that assist the bone marrow in the production of red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infections), 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 start 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.

[0224] Gene therapy The linker of the invention may be used to conjugate the 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 it concerns immunomodulatory agents, gene therapy is used to stimulate the subject's natural ability to inhibit cancer cell growth or kill cancer cells. In one embodiment, the ADC of the 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, the ADC of the invention comprises a nucleic acid encoding or otherwise enabling the production of a therapeutic protein for treating cancer. The nucleic acid encoding the therapeutic gene may be directly conjugated to the antibody, or alternatively, may be conjugated to the antibody via a carrier. Examples of carriers that may be used to deliver nucleic acids for gene therapy include, but are not limited to, viral vectors or liposomes.

[0225] Alkylating Agents The linker of the present invention may be used to conjugate the antibody to one or more alkylating agents. Alkylating agents are a class of antineoplastic compounds that bind alkyl groups to DNA. Examples of alkylating agents that can be used in the ADC of the present invention include, but are not limited to, alkyl sulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0226] 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 2 -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.).

[0227] Nitrogen Mustard The antibody may be conjugated to at least one nitrogen mustard using the linker of the present invention. 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 sodium phosphate or Estracyt®), Pfizer, Inc.), ifosfamide (Ifex®, Bristol-Myers Squibb), mechlorethamine (Mustargen®, Lundbeck Inc.), and melphalan (Alkeran® or L-Pam®, i.e., phenylalanine mustard; GlaxoSmithKline).

[0228] Nitrosoureas The antibody may be conjugated to at least one nitrosourea using the linker of the present invention. Nitrosoureas are a subclass of alkylating agents that are lipid-soluble. 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-chloro-ethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb), nimustine (also known as ACNU), and streptozocin (Zanosar®, Teva Pharmaceuticals).

[0229] Triazine and hydrazine The linker of the present invention may be used to conjugate the 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 that 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).

[0230] Other Alkylating Agents The linker of the present invention may be used to conjugate an antibody to at least one ethylenimine, 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 that are characterized by a cyclic ether with only three ring atoms.

[0231] 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 that contains an aziridine ring and appears to induce cytoxicity via 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).

[0232] Antiangiogenic Agents In some embodiments, the antibody may be conjugated to at least one antiangiogenic agent using the linker of the invention. Antiangiogenic agents inhibit the growth of new blood vessels. Antiangiogenic agents exert their effects in various 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 main proteins involved in the initiation of angiogenesis by binding to a specific receptor on the cell surface. Thus, certain antiangiogenic agents that block the interaction of VEGF with its cognate receptors block VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once a specific receptor on the cell surface is triggered, a cascade of other chemical signals is initiated to promote blood vessel growth. Thus, certain enzymes, such as some 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 yet 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 found to have anti-angiogenic properties, with numerous direct and indirect inhibitory effects.

[0233] Representative examples of antiangiogenic agents that may 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, 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 (defolimus, 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).

[0234] Antimetabolites The linker of the present invention may be used to conjugate an antibody to at least one antimetabolite. Antimetabolites are a type of chemotherapy therapeutic agent that is very similar to normal substances in cells. When a cell incorporates an antimetabolite into the cell 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 ADC of the present invention include, but are not limited to, folate antagonists (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, foxuridine, 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.

[0235] Folate antagonists The linker of the present invention may be used to conjugate the 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.).

[0236] Purine antagonists The antibody may be conjugated to at least one purine antagonist using the linker of the present invention. 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 [aka 2-CdA], Janssen Biotech, Inc.), mercaptopurine (Purinethol [aka 6-mercaptoethanol], GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation), pentostatin (Nipent [aka 2'-deoxycoformycin (DCF)]), 6-thioguanine (Lanvis [aka thioguanine], GlaxoSmithKline).

[0237] Pyrimidine antagonists The antibody may be conjugated to at least one pyrimidine antagonist using the linker of the present invention. 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, azacytidine (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).

[0238] Boron-containing drugs The antibody may be conjugated to at least one boron-containing drug using the linker of the present invention.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).

[0239] Chemical Protectants The antibody may be conjugated to at least one chemoprotectant using the linker of the present invention. Chemoprotective drugs are a class of compounds that help protect the body against the specific toxic effects of chemotherapy. Chemoprotective agents can be administered with various chemotherapy to allow cancer cells to be treated with the administered chemotherapy while simultaneously protecting healthy cells from the toxic effects of the 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 vascular leakage 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 ifocfamide.

[0240] Hormones The antibody may be conjugated to at least one hormonal agent using the linker of the invention. 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.

[0241] Antihormonal drugs The antibody may be conjugated to at least one anti-hormonal agent using the linker of the present invention. An "anti-hormonal" agent is an agent that suppresses the production and / or blocks the function of a particular endogenous hormone. In one embodiment, the anti-hormonal 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).

[0242] 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 ADC 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.).

[0243] Photoactive Therapeutic Agents The antibody may be conjugated to at least one photoactive therapeutic agent using the linker of the invention. Photoactive therapeutic agents include compounds that can be deployed to kill treated cells when the cells are exposed to electromagnetic radiation of a particular wavelength. Therapeutically significant compounds absorb electromagnetic radiation at wavelengths that penetrate the tissue. In preferred embodiments, the compounds are administered in a non-toxic form that is capable of producing photochemical effects that are toxic to cells or tissues when fully activated. In other preferred embodiments, these compounds are retained by cancerous tissues and easily cleared from normal tissues. Non-limiting examples include various chromagens and dyes.

[0244] Oligonucleotides The antibody may be conjugated to at least one oligonucleotide using the linker of the present invention. Oligonucleotides are made of short nucleic acid strands that work by interfering with the processing of genetic information. In some embodiments, oligonucleotides for use in ADCs are unmodified single-stranded and / or double-stranded DNA or RNA molecules, while in other embodiments, these therapeutic oligonucleotides are chemically modified single-stranded 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 a cell. Some of the important oligonucleotide technologies include antisense oligonucleotides (including RNA interference (RNAi)), aptamers, CpG oligonucleotides, and ribozymes.

[0245] Antisense oligonucleotides The antibody may be conjugated to at least one antisense oligonucleotide using the linker of the present invention. 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, and about 18 to about 25 nucleotides.

[0246] There are several mechanisms that can be used to inhibit the function of RNA once the oligonucleotide binds to the target RNA (Crooke ST. (1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterized antisense mechanisms result 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 expression of target genes by non-catalytic mechanisms such as modulation of splicing or translation arrest can also be potent and selective modulators of gene function.

[0247] Another RNase-dependent antisense mechanism that has recently received 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 the introduction of relatively longer double-stranded RNA (dsRNA), while in preferred embodiments, the RNAi effect is achieved by the introduction of shorter double-stranded RNA, such as small interfering RNA (siRNA) and / or microRNA (miRNA). In yet another embodiment, RNAi can also be achieved by the introduction of a plasmid that produces dsRNA complementary to a target gene. In each of the foregoing embodiments, the double-stranded RNA is designed to interfere with gene expression of a specific target sequence in a cell. In general, this mechanism involves converting dsRNA into short RNA, which directs ribonuclease to homologous mRNA target (summarized in Ruvkun, Science 2294:797 (2001)), which then 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 envisioned for therapeutic use (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 leukemic 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 allows the use of antisense oligonucleotides in ADCs for the treatment of breast cancer.In other embodiments, the present invention provides compositions and methods for initiating antisense oligonucleotide therapy, where 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 acid (LNA)), deoxyribonucleotides, and any combination thereof. The RNA of the present invention only needs to be sufficiently similar to natural RNA that it has the ability to mediate antisense oligonucleotide-based regulation as described herein.

[0248] Aptamers The antibody may be conjugated to at least one aptamer using the linker of the present invention. An aptamer is a nucleic acid molecule that is selected from a random pool based on its ability to bind to other molecules. Like antibodies, aptamers can bind to target molecules with extraordinary affinity and specificity. In many embodiments, aptamers exhibit complex, sequence-dependent three-dimensional shapes, which allow them to interact with target proteins and produce 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 promise as targeted molecular therapy.

[0249] CpG oligonucleotides The linker of the present invention may be used to conjugate the 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 oligonucleotides containing this CpG motif can be utilized to initiate antitumor immune responses.

[0250] Ribozymes The antibody may be conjugated to at least one ribozyme using the linker of the present invention. 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.

[0251] 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 that are characterized by an unstable nucleus that can undergo radioactive decay.The success of radionuclide therapy is based on sufficient concentration of 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 travel. In a preferred embodiment, the therapeutic agent is a radionuclide selected from the group consisting of 111In, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 64Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, and 211Pb. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111 1, Sb-119, 1-125, Ho-161, Os-189m, and Ir-192. The decay energies of useful nuclides that emit beta particles 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 radionuclides that emit alpha particles 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 11C, 13N, 150, 75Br, 198Au, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, 169Yb, and the like.

[0252] Radiosensitizers The antibody may be conjugated to at least one radiosensitizer using the linker of the present invention. The term "radiosensitizer" as used herein is defined as a molecule, preferably a low molecular weight molecule, that is administered to an animal in a therapeutically effective amount to increase the sensitivity of cells of the radiosensitizer 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 may be combined with a radiolabeled antibody or ADC. The addition of a radiosensitizer may result in enhanced efficacy when 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.

[0253] 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, the following: 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 their therapeutically effective analogs and derivatives. Alternatively, the radiosensitizer may be activated using photodynamic therapy (PDT). Representative examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, photofrin (r), benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and their therapeutically active analogs and derivatives.

[0254] Topoisomerase inhibitors The antibody may be conjugated to at least one topoisomerase inhibitor using the linker of the present invention. 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 during normal cell cycle, followed by cleavage and recombination. 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.

[0255] Tyrosine Kinase Inhibitors The linker of the present invention may be used to conjugate the antibody to at least one tyrosine kinase inhibitor. Tyrosine kinase is an intracellular enzyme that functions to attach phosphate group to the amino acid tyrosine. By preventing protein tyrosine kinase from functioning, 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.

[0256] 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 proteins, amatoxins, crotin, curcin, dianthin proteins, diptheria toxins (e.g., diptheria A chain and nonbinding active fragments of diptheria 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 toxoid, 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), adrenal cortex 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.).

[0257] It should be noted that the aforementioned groups of drug moieties that may be used in the ADCs of the invention are not exclusive, in that an example of a particular drug may be found in more than one category; e.g., ansamitocins are both mitotic inhibitors and antitumor antibiotics.

[0258] All stereoisomers of the drug moieties listed above, ie, any combination of R and S configurations at the chiral carbon of D, are contemplated for the compounds of the present invention.

[0259] 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, e.g., 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, densitometer, flow cell analysis, ELISA, or direct analysis of the cyclic peptide or the subsequently 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.

[0260] 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 to alter 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 an interaction with an antigen or ligand or increasing binding affinity, (iv) a material capable of affecting electromobility or cell invasiveness through physical parameters such as charge, hydrophobicity, or (v) a material capable of modulating ligand affinity, antigen-antibody binding, or ion complex formation.

[0261] In certain embodiments, FRET technology can be used to distinguish intact molecules from those that have been exposed to conditions that activate the triggering group, for example by attaching a donor chromophore to the central Ar ring and an acceptor chromophore as Q.

[0262] 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).

[0263] 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 target cell of interest. The antigen is not required, but in some embodiments, allows for the ADC bound to it to be internalized. The target cell of interest 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 target cell of interest, 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 compared 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.

[0264] 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 usually heterotetrameric glycoproteins of about 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.

[0265] 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.

[0266] 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, including polyclonal, monoclonal, 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.

[0267] Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens generally have multiple binding sites, also called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an 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 that contain an antigen-binding site or a portion thereof that immunospecifically binds to an antigen of a desired target, such targets 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 aspect, the immunoglobulins are of human, murine, or rabbit origin.

[0268] 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')2, 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 arrangement that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In most cases, six CDRs confer binding specificity to the antibody for a target. 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. A "single-chain Fv" or "scFv" antibody fragment contains 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).

[0269] 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.

[0270] Both light and heavy chain variable domains have complementarity determining regions (CDRs), also known as hypervariable regions. The more highly conserved parts of the variable domains are called frameworks (FRs). As is known in the art, the amino acid positions / boundaries that define the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, in that these positions may be considered to be within the hypervariable region under one set of criteria, while being considered to be outside the hypervariable region under a different set of criteria. One or more of these positions may also be found in an extended hypervariable region. The CDRs in each chain are held together in close proximity by the FR regions and, 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.

[0271] 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 (including, for example, any eukaryotic, prokaryotic, or phage clone), and 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 as well as antibody fragments (such as, for example, 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 the circulatory system of animals, and may have 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 wide 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.

[0272] In most cases, antibodies are composed only of genetically 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.

[0273] In certain embodiments, the antibody of the ADC described herein is a chimeric antibody. The term "chimeric" antibody as used herein 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, for example, 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 entirety.

[0274] 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. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions 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 contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., 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; and 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 U.S. Pat. No. 5,565,332, all of which are incorporated herein by reference in their entireties.

[0275] In certain embodiments, the antibody of the ADC described herein is a human antibody. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. As used herein, "human antibody" includes antibodies having amino acid sequences of human immunoglobulins, and also includes antibodies isolated from human immunoglobulin libraries, or antibodies isolated from animals transgenic with one or more human immunoglobulins and that do not express endogenous immunoglobulins. Human antibodies can be produced by a variety of 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. Pat. 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. In addition, 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 techniques similar to those described above. Fully human antibodies that recognize a selected epitope can be generated using a technique referred to as "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).

[0276] In certain embodiments, the antibody of the ADC described herein is a primatized antibody. The term "primatized antibody" refers to an antibody that comprises a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated herein by reference in their entirety.

[0277] 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.

[0278] In certain embodiments, the antibody of the ADC described herein is a derivatized antibody. For example, but not limited to, a derivatized antibody is typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cellular ligand or other protein, and the like. 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, and the like. Additionally, a derivative can contain one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0279] In certain embodiments, the antibodies of the ADCs described herein have sequences that are modified to alter at least one biological effector function mediated by the constant region, as compared to the corresponding wild-type sequence. For example, in some embodiments, the antibodies can be modified to reduce at least one biological effector function mediated by the constant region, as compared to an unmodified antibody, for example, to reduce binding to Fc receptors (FcRs). Binding to FcRs can be reduced by mutating the immunoglobulin constant region segment of the antibody in specific regions required for interaction with FcRs (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491, and Lund et al., 1991, J. Immunol. 147:2657-2662).

[0280] 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, as compared to an 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.

[0281] 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 (TAA). In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have attempted to identify transmembrane or otherwise tumor-associated polypeptides that are differentially expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cell(s). In many cases, 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.

[0282] 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 according to the National Center for Biotechnology Information (NCBI) Nucleic Acid and Protein Sequence Identification Conventions, alternative names, Genbank accession numbers, and primary reference(s). Nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases, such as GenBank.

[0283] [Table 1] TIFF0007657152000106.tif253157TIFF0007657152000107.tif254157TIFF0007657152000108.tif157157

[0284] 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 mertansine (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 vogatoxin (bogatox (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 (Epitumomab 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), rolvo Tutumab Mertansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23 (IgE receptor)), Mapatumumab (TRAIL-R1), Margetuximab (ch4DS), Matuzumab (EGFR), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumomab Passudotoxin (Moxetumomab pasudotox (CD22), Nacolomab tafenatox (C2-42 antigen), Naptumomab estafenatox (5T4), Narutuzumab (RON), Natalizumab (integrin α4), Necitumumab (EGFR), Nesvacumab (angiopoietin 2), Nimotuzumab (EGFR), Nivolumab (IgG4), Ocaratuzumab (CD20), Ofatumumab (CD20), Olaratumab (PDGF-Rα), Onartuzumab (human scatter factor receptor kinase), Ontuxizumab (TEM1), Oportuzumab monat monato (EpCAM), Olefovomab (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 moleukin (EpCAM), Ublituximab (MS4A), Urelumab (4-1BB), Vandetanib (VEGF), Vanticutumab (Frizzled receptor), Volociximab (integrin α5β1), Vorsetuzumab-mafodotin mafodotin (CD70), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

[0285] How to generate antibodies The antibodies of the ADCs can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody recombinantly, a host cell is 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 cell and, optionally, secreted into the medium in which the host cell is 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 the host cell. This is, for example, as described 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.

[0286] 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, 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 can be synthesized using conventional mutagenesis techniques and used as a template for mutagenesis to generate an antibody as described herein. Alternatively, a DNA fragment encoding the antibody can be directly synthesized.

[0287] Once the 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 DNA fragment encoding the CH is operatively linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. The term "operatively linked" as used in this context 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.

[0288] 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 such 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 the transcriptional and translational control sequences in 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 and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

[0289] 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 variable region sequence of the antibody. 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 may 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).

[0290] 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 appreciated by those of ordinary skill in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on such factors 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 sequences thereof, 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.

[0291] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (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, typically the selectable marker gene confers resistance to drugs, such as G418, puromycin, blasticidin, hygromycin, or methotrexate, on the host cell 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 a host cell 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, and the like.

[0292] Antibodies can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the antibody is carried out in eukaryotic cells, e.g., mammalian host cells, to obtain optimal secretion of a properly folded, immunologically active antibody. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including DHFR-CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as 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 a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is 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.

[0293] In some embodiments, the antibody of the ADC may be a bifunctional antibody. Such an antibody, in which one heavy and one light chain is specific for one antigen and the other heavy and light chains are specific for a second antigen, may be produced by crosslinking the antibody to a second antibody by standard chemical crosslinking methods. Bifunctional antibodies may also be made by expressing a nucleic acid engineered to encode the bifunctional antibody.

[0294] 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, such as 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 a nucleic acid engineered to encode the bispecific antibody.

[0295] 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)).

[0296] 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.

[0297] 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 by any other standard technique for the purification of proteins.

[0298] 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).

[0299] Imaging Compounds and Sensors In certain embodiments, provided herein are uses of the disclosed compounds in imaging compositions and as sensors.

[0300] 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 a specific material (e.g., cancer cells, viruses, various chemical substances, etc.) with bio-receptors (portions designed to be capable of adsorbing to and reacting with biological materials such as DNA, RNA, antibodies, enzyme proteins, cells, biological membranes, and hormone receptors) having selective specificity, and performing measurements using signal transducers (devices that convert the reaction between a specific material and a biological receptor into an electrical signal using various methods), and can be used in the medical industry, environmental industry, processing industry, military (chemical warfare), research, food, etc. (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).

[0301] Chemical sensors rapidly and accurately monitor specific materials in many fields such as clinical diagnosis, medical research, chemical material measurement, and environmental measurement by using electrical properties such as electricity, resistance, and potential difference, and optical properties such as color and fluorescence. 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), dust / soot sensors (suspended dust, dust, soot, turbidity), etc. (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;2013).

[0302] A molecular switch is a molecule that can be reversibly switched between two or more stable states. The molecule can be switched between states in response to environmental stimuli such as changes in the chemical environment (such as pH), light irradiation (e.g., light of a particular wavelength), temperature, electric current, the microenvironment, or the presence of a ligand. In some cases, the switching between states can depend on a combination of stimuli. The oldest form of synthetic molecular switch is the pH indicator, which displays distinct colors as a function of pH. Synthetic molecular switches may be applied in molecular computers or responsive drug delivery systems. Molecular switches are also important in biology, since many biological functions, such as allosteric regulation and vision, are based on them.

[0303] Such biosensors, chemical sensors, and molecular switches may further comprise additional photoreactive moieties such as rhodamine, phenol red, orange azo dyes, papa red, non-sulfonated cyanines, sulfonated cyanines, chemiluminescent fluoride sensors (1,2-dioxetane derivatives), and D2A dyes (NIR fluorescent dyes). Alternatively, the photoreactive moieties may be selected from compounds having functional groups and structures similar to: [ka] During the ceremony, R 100 is H or C 1 ~C 6 -alkyl, R 101 is H or SO 3 H and R 102 is C 1 ~C 6 -Alkyl, or -(CH 2 ) z COOH, z is an integer from 3 to 8; R 103 and R 104 are each independently H or C 1 ~C 6 is alkyl, R 105 and R 106are each independently hydrogen, COOH, or SO 3 It's H.

[0304] 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).

[0305] Treatment method targeted therapy The targeting moiety of the conjugate may be recognised by cells, thereby providing a so-called targeted therapy.

[0306] In some embodiments, the conjugate comprises an active agent for use in targeted therapy for the treatment of autoimmune disease. In some such embodiments, the active agent is selected from cyclosporine, cyclosporine A, mycophenolate mofetil, sirolimus, tacrolimus, enercept, prednisone, azathioprine, methotrexate cyclophosphamide, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlororambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab, and the like.

[0307] In some embodiments, the compounds comprise 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, clacillin, cyclosporine, cyclosporine), cyclosporine, ... Rithromycin, erythromycin, lincomycin, clindamycin), tetracyclines (demeclocycline, 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 (idoxuradine, vidarabine, aciclovir, rifampicin, rifampicin, spectinomycin ... lovir, famcicyclovir, pencicyclovir, valacyclovir, gancicyclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir, antisense oligonucleotides, immunoglobulins, interferons, HIV infection treatments (tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine,The medicament is selected from the group consisting of nevirapine, delaviridine, saquinavir, ritonavir, indinavir, nelfinavir, and the like.

[0308] In some embodiments, the compounds and conjugates disclosed herein comprise an active agent Q for use in a method for delivery of an active agent to a cell for the treatment of a tumor, where the targeting moiety is selected to bind to a 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).

[0309] In some such embodiments, the active agent is selected from cytotoxic or immunomodulatory agents, anti-cancer agents, antitubulin agents, cytotoxic agents, etc. Preferably, cytotoxic or immunomodulatory agents include antitubulin agents, auristatins, DNA minor groove binders, DNA transcription inhibitors, alkylating agents, anthracyclines, antibiotics, folate antagonists, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposide, fluorindated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinol, pore forming compounds, purine antimetabolites, puromycins, radiosensitizers, rapamycin, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc., and anti-cancer agents include methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydrochloride, hydrazine ... These include cyclophosphamide, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, proocarbazine, 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 drugs 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 , 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), duocarmycins , 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, eleutherobin, mitoxantrone, and the like.

[0310] Cell proliferation and apoptosis The compounds and conjugates disclosed herein may be used in methods for inducing apoptosis in a cell.

[0311] 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 Sjogren'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 sufficient amount of the compounds, conjugates, or compositions described herein to provide a therapeutic benefit. The identity of the antibody of the administered compound, conjugate, or composition will depend on the disease being treated, and 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 particular disease being treated. In certain cases, the compounds and compositions disclosed herein may treat or improve the disease itself or symptoms of the disease when administered as a monotherapy. In other cases, the compounds and compositions disclosed herein may be part of an overall treatment regimen that includes other agents that, together with the inhibitors or compounds and compositions disclosed herein, treat or improve the disease or symptoms of the disease being treated. Agents useful for treating or improving 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.

[0312] Although an absolute cure is always desirable in any therapeutic regimen, achieving a cure is not required to provide a therapeutic benefit. A therapeutic benefit may include halting or slowing the progression of a disease, regressing a disease without a cure, and / or improving 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.

[0313] One particular class of disease that involves dysregulation of apoptosis and is a significant health burden worldwide is cancer. In a specific embodiment, 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 with 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 of treating a disease associated with dysregulation of intrinsic apoptosis, comprising administering to a subject having a disease associated with dysregulation of 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 of treating cancer, comprising administering to a subject having 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.

[0314] 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.

[0315] The compounds and conjugates disclosed herein may be administered as monotherapy to provide therapeutic benefit or may be administered to supplement or in conjunction with other chemotherapeutic agents and / or radiotherapy. The chemotherapeutic agents for which the compounds and compositions disclosed herein may be utilized as supplemental therapy may 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.

[0316] The compounds and conjugates disclosed herein, which may not be effective as monotherapy in treating cancer, may be administered to supplement or together with other chemotherapeutic agents or radiotherapy to provide therapeutic benefit. One embodiment relates to a method of administering the compounds or compositions 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 to supplement 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 started chemotherapy and / or radiotherapy or have not yet shown signs of resistance, or in patients who have begun to show signs of resistance.

[0317] 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, for example comprising the disclosed compound and a pharmaceutically acceptable carrier.

[0318] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for administration to humans, particularly for invasive routes of administration (i.e., routes such as injection or implantation that bypass transport or diffusion through 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, injections, and the like. The compositions can also be present in transdermal delivery systems, for example, skin patches. The compositions can also be present in liquids suitable for topical administration, such as ointments or creams.

[0319] A pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound, such as the 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 choice of a pharma- ceutical acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The pharmaceutical composition preparation may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be, for example, a liposome or other polymer matrix that may have the compound of the present invention incorporated therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to prepare and administer.

[0320] The phrase "pharmacologically 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 undue toxicity, irritation, allergic response, or other problem or complication, within the scope of prudent medical judgment, commensurate with a reasonable benefit / risk ratio.

[0321] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically 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 pharma-ceutically 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; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, 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 buffers, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0322] 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, pastes for application to the tongue, such as in aqueous or non-aqueous solutions or suspensions); absorption through the oral mucosa (e.g., sublingually); anally, rectally, or intravaginally (e.g., as a pessary, cream, or foam); parenterally (e.g., as a sterile solution or suspension, including intramuscularly, intravenously, subcutaneously, or intrathecally); intranasally; 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.

[0323] 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, 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 one hundred percent, this amount will range from about 1 percent to about ninety-nine 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.

[0324] 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 carriers 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.

[0325] 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 powders, powders, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (with an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compound, conjugate, or composition may also be administered as a bolus, electuary, or paste.

[0326] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically 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) disintegrants, 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, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of 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 such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0327] 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 dispersants. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0328] Tablets and other solid dosage forms of the pharmaceutical composition, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example, with hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various ratios 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 any other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents, and may belong to compositions that release the active ingredient(s) only, or preferentially, in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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 a salicylate, which are solid at room temperature, but liquid at body temperature, and therefore will melt in the rectum or vaginal cavity and release the active compounds.

[0333] Formulations of the pharmaceutical composition for administration to the buccal cavity may be presented as a mouthwash, or as an oral spray, or as an oral ointment.

[0334] 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.

[0335] 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.

[0336] 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 pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0337] 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.

[0338] 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.

[0339] Transdermal patches have the added 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 dispersing the compound in a polymer matrix or gel.

[0340] Ophthalmic formulations, eye ointments, powders, solutions, and the like are also contemplated 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.

[0341] The phrases "parenteral administration" and "administered parenterally" as used herein mean 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.

[0342] Pharmaceutical compositions suitable for parenteral administration contain one or more active compounds in combination with one or more pharma- ceutically 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.

[0343] 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.

[0344] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersing agents. Prevention of microbial action may be ensured by incorporating various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. In addition, prolonged absorption of the injectable dosage form may be achieved by incorporating agents that delay absorption, such as aluminum monostearate and gelatin.

[0345] In some cases, it is desirable to slow down the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or amorphous materials with low water solubility.The absorption rate of drugs then depends on their dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drugs can be achieved by dissolving or suspending the drug in an oil vehicle.

[0346] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer 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 are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0347] When used in the methods of the present invention, the active compound may be administered by itself or in combination with a pharma- ceutically 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.

[0348] In some embodiments of the present invention, the compounds of the present invention are co-administered with one or more additional compounds / agents.

[0349] In certain such embodiments, the co-administration is simultaneous. In certain such embodiments, the compound of the present invention is combined with one or more additional compounds. In certain other such embodiments, the compound of the present invention is administered separately but simultaneously with one or more additional compounds. In certain such embodiments, the co-administration is sequential, with the compound of the present invention being administered minutes or hours before or after the administration of one or more additional compounds.

[0350] The method of introducing the compound of the present invention can also be enabled by rechargeable or biodegradable devices.In order to obtain controlled delivery of drugs, including proteinaceous biological agents, various slow release polymeric devices have been developed and tested in vivo in recent years.Various biocompatible polymers, including hydrogels, including both biodegradable and non-degradable polymers, can be used to form implants to obtain sustained release of compounds at specific target sites.

[0351] 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.

[0352]

[0000] 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, used, the route of administration, the time of administration, the excretion rate of the particular compound(s) being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) being used, the age, sex, weight, medical condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0353] 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 begin 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 varies 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, another type of therapeutic agent being administered together with the compound of the present invention. A higher total dose may be delivered by multiple administrations of the agent. 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).

[0354] In general, 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 on the factors mentioned above.

[0355] If desired, the effective daily dose of the active compound or conjugate can be administered as one, two, three, four, five, six or more sub-doses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage form.In certain embodiments of the present invention, the active compound can be administered two or three times a day.In preferred embodiments, the active compound will be administered once a day.

[0356] The patient undergoing this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as horses, cows, pigs, and sheep, as well as poultry and pets in general.

[0357] 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 the second compound or conjugate is administered while the previously administered therapeutic compound or conjugate is still effective in the body (e.g., the two compounds or conjugates are effective in the patient at the same time, which may include a synergistic effect of the two compounds or conjugates). For example, the different therapeutic compounds or conjugates may be administered either simultaneously or sequentially, in the same formulation or in separate formulations. In certain embodiments, the 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, an individual undergoing such treatment may benefit from the combined effect of the different therapeutic compounds or conjugates.

[0358] The present invention includes the use of pharma- ceutically acceptable salts of the compounds or conjugates disclosed herein.In certain embodiments, the 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, the contemplated salts of the present invention include, but are not limited to, L-arginine salts, benenthamine 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.

[0359] 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 the crystallization solvent adventitious to such solvent.

[0360] 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.

[0361] Examples of pharma- ceutically 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, butylated hydroxyanisole (BHA), butylated hydroxytoluene (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.

[0362] Having now generally described the invention, it will be more readily understood with 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 be limiting of the invention. EXAMPLES

[0363] 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

[0364] [Example 1] Preparation of Int-TG [ka]

[0365] 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 with N 2Dissolution was allowed to occur under atmospheric pressure. The mixture was allowed to warm to room temperature. After stirring at room temperature for 4 h, the mixture was concentrated under reduced pressure, then EA (1000 mL) and saturated sodium bicarbonate (1000 mL) were added. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., 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, CDCl 3 ) 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).

[0366] [Example 2] Preparation of compound FA-Int [ka]

[0367] 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.

[0368] [Example 3] Preparation of compound IntC-L-1 [ka]

[0369] 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 Boc 2 O (14.7 g, 67.47 mmol) 2 The mixture was stirred at room temperature overnight and then cooled to room temperature. 2The reaction was quenched with O (500 mL) and brine (150 mL x 3). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. After concentration, compound IntCl-L-1a was used directly in the next reaction without purification (13.01 g, 78%). 1 H NMR (400 MHz, CDCl 3 ) 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).

[0370] 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), PyBOP (15.72 g, 30.20 mmol) and DIPEA (10.52 mL, 60.39 mmol) were added at 0 °C and N 2 The mixture was stirred at room temperature for 2 h. EA (20 mL x 6), H 2 O (20 mL), and brine (200 mL) were added to the mixture. The organic layer was extracted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., 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, CDCl 3 ) 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 (s,4H),3.44-3.43(m,2H),3.38-3.21(m,2H),2.30-2.20(m,3H),2.04-2.00(m,1H),1.76(s,1H),1.44(s,9H).EI-MS m / z:526(M + ).

[0371] Preparation of compound IntCl-L-1c To a solution of compound IntCl-L-1b (3 g, 5.71 mmol) in MeOH (25 mL), Pd / C (900 mg) was added at room temperature and cooled to 50° C. 2 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, CDCl 3 ) 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 + ).

[0372] 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), HBTU (2.36 g, 6.23 mmol) and DIPEA (1.36 mL, 7.78 mmol) were added at 0 °C and N 2 The mixture was stirred at room temperature for 2.5 h and then diluted with EA (100 mL x 7) and H 2 O (100 mL) was added. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., 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-d 6) 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 + ).

[0373] 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 h. The mixture was concentrated under reduced pressure and used directly in the next step without further purification (4.08 g, crude). EI-MS m / z: 682 (M + ).

[0374] [Example 4] Preparation of compound IntC-L [ka]

[0375] 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) at 0 °C under N 2 The mixture was stirred at room temperature overnight. EA (80 mL x 4) and saturated citric acid (60 mL) were added to the mixture, and the organic layer was washed with NaHCO 3 (120 mL), brine (100 mL), and anhydrous Na 2 SO 4The mixture was dried at 40° C., 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 + ).

[0376] Preparation of compound K-2 0°C, N 2 To a solution of compound K-1 (3.29 g, 9.66 mmol) in MeOH (15 mL) under atmospheric pressure, 2 LiOH H dissolved in O (15 mL) 2 O (2.03 g, 48.32 mmol) was added.

[0377] The mixture was stirred at 0° C. for 30 min and warmed to room temperature for 2 h. The mixture was acidified with saturated aqueous citric acid and EA (40 mL×2) was added to the mixture. The organic layer was extracted with H 2 20 mL) and brine (30 mL). 2 SO 4 The mixture was dried at 40° C., 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 + ).

[0378] Preparation of Compound K To a solution of compound K-2 (3.69 g, 11.31 mmol) in DMF (20 mL), NHS (1.69 mg, 14.7 mmol) and EDCI (2.93 g, 15.26 mmol) were added 2 The mixture was stirred at room temperature overnight and concentrated. The residue, compound K, was used directly in the next step without further purification (4.79 g, crude). EI-MS m / z: 446(M + +Na).

[0379] 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), DIPEA (5.21 mL, 29.93 mmol) was added with N 2 The mixture was stirred at room temperature overnight. 2 O (70 mL) and brine (60 mL) were added to the mixture, and the mixture was extracted with EA (70 mL×7). The organic layer was then diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., 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+).

[0380] 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 H 2 LiOH H dissolved in O (10 mL) 2 O (356 mg, 8.48 mmol) at 0 °C, 2 The mixture was added under atmospheric pressure. The mixture was stirred at 0° C. for 30 min and warmed to room temperature for 3 h. The mixture was acidified with 2M HCl and concentrated under reduced pressure. The residue, compound IntC-L-3, was used directly in the next step without further purification (996 mg, crude). EI-MS m / z: 880(M + ).

[0381] Preparation of compound IntC-L To a solution of compound IntC-L-3 (996 mg, 1.13 mmol) in DCM (30 mL), TFA (8 mL) was added at 0 °C and N 2 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 + ).

[0382] [Example 5] Preparation of compound MPS-D1 [ka]

[0383] Preparation of compound MPS-D1a A solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 mL) was added with 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 N 2 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-d 6 ) 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).

[0384] 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 obtain compound MPS-D1b (5.56 g, 90%). 1 H NMR (400 MHz, CDCl 3 ) 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).

[0385] 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 and N 2 After stirring at room temperature for 14 h, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL x 3). The organic layer was washed with brine (200 mL) and anhydrous Na 2 SO 4 The mixture was dried over rt, filtered and concentrated under reduced pressure to give compound MPS-D1 (5.29 g, 86%). 1 H NMR (400 MHz, CDCl 3 ) 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 + ).

[0386] [Example 6] Preparation of compound MPS-D2 [ka]

[0387] 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 Ag 2 O (4.92 g, 19.48 mmol) was added to N 2 The mixture was added under atmospheric pressure. 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, CDCl 3) 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).

[0388] Preparation of compound L-1b To a solution of compound L-1a (5.98 g, 13.7 mmol) in DMF (30 mL), NaN 3 (1.34 g, 20.55 mmol) 2 The mixture was added under atmospheric pressure. The mixture was stirred at 110° C. for 1 hour 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, CDCl 3 ) δ 3.72-3.60(m,22H),3.39(t,J=4.8 Hz,2H),2.78(br,1H).

[0389] Preparation of compound L-1c A solution of compound L-1b (2 g, 6.51 mmol) in acetone (56 mL) was diluted with Jones reagent (5 mL) at -5 °C and heated with N 2 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 x 2) and water (5 mL). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., 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, CDCl 3 ) δ 4.15(s,2H),3.76-3.67(m,18H),3.40(t,J=4.8 Hz,2H).

[0390] Preparation of compound L-1d To a solution of compound L-1c (500 mg, 1.56 mmol) in DCM (10 mL), t-BuOH (305 μL, 3.11 mmol), DIC (292.5 μL, 1.87 mmol), and DMAP (19 mg, 0.16 mmol) were added under N 2 The mixture was stirred at room temperature for 4 h and diluted with DCM (30 mL x 2). The organic layer was washed with water (5 mL) and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1d (278.5 mg, 47%). 1 H NMR (400 MHz, CDCl 3 ) δ 4.01(s,2H),3.70-3.66(m,18H),3.38(t,J=4.8 Hz,2H),1.47(s,9H).

[0391] Preparation of compound L-1e A solution of compound L-1d (278 mg, 0.74 mmol) in EtOH (5 mL) was added with Pd / C (236 mg, 0.11 mmol) and 4 M HCl (in 1,4-dioxane) under N 2 The mixture was added under atmosphere. The mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite® to remove Pd / C and concentrated to give compound L-1e (255.3 mg, 89.2%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.32(s,1H),3.98(s,2H),3.55-3.40(m,18H),3.86(t,J=5.6 Hz,2H),2.70-2.64(m,2H),1.42(s,9H).

[0392] Preparation of compound MPS-D2 To a solution of compound L-1e (255.3 mg, 0.66 mmol) and compound MPS-D1 (240.6 mg, 0.72 mmol) in DMF (6 mL) was added HBTU (300 mg, 0.79 mmol) and DIPEA (229.3 μL, 1.32 mmol) under nitrogen atmosphere. The mixture was stirred at room temperature for 2 h and diluted with EA (20 mL × 2) and water (5 mL). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D2 (306 mg, 71%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.95(s,4H),7.82(d,J=8.0 Hz,2H),7.38(d,J=8.0 Hz,2H),7.33-7.30(m,1H),3.98(s,2H),3.68-3.63(m,18H),3.55-3.53(m,2H),3.49-3.47(m,2H),2.95(s,1H),2.88(s,1H),2.46(s,3H) 1.46(s,9H).EI-MS m / z:666(M + +1).

[0393] [Example 7] Preparation of compound MPS-D4 [ka]

[0394] Preparation of compound MPS-D3 To a solution of compound MPS-D2 (120 mg, 0.18 mmol) in DCM (8 mL) was added TFA (4 mL) at 0° C. The reaction was cooled to 5° C. 2The mixture was allowed to warm to room temperature under atmosphere for 2 hours. After the reaction was completed, the mixture was concentrated under reduced pressure three times by using toluene as a co-solvent, thereby removing TFA. The mixture was then redissolved in DMF, and NHS (31 mg, 0.27 mmol) and EDCI (52 mg, 0.27 mmol) were added thereto. The mixture was stirred at room temperature overnight. After the reaction was completed, compound MPS-D3 was used directly in the next step without further purification (127 mg, crude). EI-MS m / z: 707(M + ).

[0395] Preparation of compound MPS-D4 To a solution of compound IntC-L (60 mg, 0.08 mmol) and compound MPS-D3 (82 mg, 0.12 mmol) in DMF (6 mL), DIPEA (112 μL, 0.64 mmol) was added and N 2 The mixture was stirred for 30 min, dissolved in DMSO (3 mL), and purified by HPLC to produce compound MPS-D4 (77 mg, 73%). EI-MS m / z: 1373(M + ).

[0396] [Example 8] Preparation of compound MPS-D5 [ka]

[0397] To a solution of compound MPS-D1 (500 mg, 1.50 mmol) in DMF (8 mL), propargylamine (106 μL, 1.65 mmol) was added at room temperature with N 2 The reaction was cooled to 0° C. and PyBop (1.17 g, 2.26 mmol) and DIPEA (524 μL, 3.01 mmol) were added to it. The mixture was stirred at room temperature for 2 h and diluted with EA (30 mL×2) and distilled water (20 mL). The organic layer was extracted, washed with brine (50 mL) and diluted with anhydrous Na 2 SO 4The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D5 (510 mg, 92%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.11(t,J=5.2 Hz,1H),7.98-7.89(m,4H),7.79(d,J=8.0 Hz,2H),7.43(d,J=8.4 Hz,2H),4.05-4.03(m,2H),3.60(t,J=7.6 Hz,2H),3.39(t,J=7.2 Hz,2H),3.12(s,1H),2.38(s,3H).

[0398] [Example 9] Preparation of compound A-15-1 [ka]

[0399] Preparation of Compound A-15-1a To a solution of z-valine (1.01 g, 3.81 mmol) and N-methylaniline (412 μL, 3.81 mmol) in DCM (15 mL), DCC (1.18 g, 5.71 mmol) and DMAP (92 mg, 0.76 mmol) were added at room temperature with N 2 Add under atmospheric pressure, followed by stirring at room temperature for 3 hours. The mixture was filtered through Celite® and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound A-15-1a (1.05 g, 78%). EI-MS m / z: 584(M + ).

[0400] Preparation of Compound A-15-1b Compound A-15-1a (1.05 g, 2.96 mmol) was dissolved in MeOH (15 mL) under a nitrogen atmosphere, and Pd / C (378 mg, 0.18 mmol) was added. The mixture was stirred at room temperature for 2 h and then heated at 50° C. for 2 h. 2 After stirring under reduced pressure, the mixture was filtered through Celite and washed with MeOH (30 mL). The filtrate was concentrated to give compound A-15-1b (560 mg, 86.0%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.45-7.41(m,2H),7.38-7.36(m,1H),7.19(d,J=7.6 Hz,1H),3.32(s,3H),2.88(d,J=6.0 Hz,1H),2.33(s,3H),1.73(q,J=6.8 Hz,1H),0.86(d,J=6.8 Hz,3H),0.80(d,J=6.8 Hz,3H).

[0401] Preparation of Compound A-15-1 To a solution of compound A-15-1b (220 mg, 0.99 mmol) in DMF (8 mL), 37% formaldehyde (223 μL, 2.99 mmol) and AcOH (1.14 mL, 19.8 mmol) were added under N 2 After stirring at room temperature for 5 min, NaCNBH 3 (125 mg, 1.98 mmol) was added. The mixture was stirred at room temperature for 2 h and saturated NaHCO 3 The reaction was quenched with EA (15 mL×2). To this mixture was added EA (20 mL×2) and brine (20 mL). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound A-15-1 (189 mg, 81%). EI-MS m / z: 235(M + ).

[0402] [Example 10] Preparation of compound POS-D1 [ka]

[0403] Preparation of compound POS-D1a To a solution of ethyl 4-hydrobenzoate (20 g, 120.35 mmol) in EtOH (60 mL), NH 2 NH 2 H 2 O (88 mL, 1805.4 mmol) 2The mixture was added under atmospheric pressure and stirred overnight at reflux. After the reaction was completed, the mixture was cooled to room temperature and concentrated under reduced pressure, followed by trituration with EtOH, thereby obtaining compound POS-D1a (17.539 g, 96%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.50(s,1H),7.68(d,J=8.4 Hz,2H),6.78(d,J=8.8 Hz,2H),4.37(s,2H).EI-MS m / z:431(M + ).

[0404] Preparation of compound POS-D1b To a solution of compound POS-D1a (17.54 g, 115.28 mmol) in EtOH (200 mL) and DMF (100 mL), CS 2 (45 mL, 749.32 mmol) and KOH (6.5 g, 115.28 mmol) were dissolved in N 2 After stirring at 85° C. for 18 h, the mixture was added with EA (500 mL) and H 2 2H2O (500 mL) was added, which was then acidified with 1M HCl. The organic layer was 2 2H2O (500 mL), and brine (500 mL). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was subjected to ether / hexane trituration to give compound POS-D1b (20.7 g, 93%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.44(s,1H),7.72(d,J=8.4 Hz,2H),6.94(d,J=8.0 Hz,2H).EI-MS m / z:195(M + ).

[0405] Preparation of compound POS-D1c To a solution of compound POS-D1b (5 g, 25.75 mmol) in THF (100 mL), Et 3N (4.3 mL, 30.9 mmol) and MeI (1.76 mL, 28.33 mmol) were added dropwise at 0° C. After stirring at 0° C. for 10 min, the mixture was warmed to room temperature. The mixture was then stirred for 2 h and diluted with EA (100 mL×2). The organic layer was extracted with H 2 2H2O (100 mL), and brine (100 mL). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was subjected to ether trituration to obtain compound POS-D1c (5.15 g, 96%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.80(d,J=8.4 Hz,2H),6.94(d,J=8.4 Hz,2H),2.74(s,3H).EI-MS m / z:209(M + ).

[0406] Preparation of compound POS-D1d To a solution of compound POS-D1c (3.2 g, 15.37 mmol) in EtOH (150 mL), 70% m-CPBA (11.4 g, 46.11 mmol) was added at 0 °C and N 2 After stirring at room temperature for 5 hours, additional 70% m-CPBA (11.4 g, 46.11 mmol) was added. The mixture was then stirred at room temperature overnight and heated under H 2 O (500 mL), saturated NaHCO 3 (300 mL) and diluted with EA (500 mL×2). The organic layer was washed with brine (300 mL) and anhydrous Na 2 SO 4 The mixture was dried over 100 mL of ethyl acetate, filtered, and concentrated under reduced pressure. The residue was subjected to trituration with HX / EA=1:1 (100 mL) to obtain compound POS-D1d (3.2 mg, 89%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.95(d,J=8.8 Hz,2H),7.01(d,J=8.8 Hz,2H),3.69(4s,3H).EI-MS m / z:241(M + ).

[0407] Preparation of compound POS-D1 To a solution of tetraethylene glycol (17.3 ml, 0.10 mol) in THF (50 mL) was added NaH (2.6 g, 0.065 mmol) dropwise at 0° C. The mixture was stirred at 0° C. for 1 h, and then propargyl bromide (5.95 g, 0.05 mol) was added. The mixture was stirred at room temperature overnight, quenched with ice / water and diluted with EA (100 mL×2). The organic layer was extracted with H 2 2H2O (100 mL), and brine (100 mL). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. Trituration of the residue with ether afforded 3,6,9,12-tetraoxapentadec-14-yn-1-ol (5.87 g, 51%). 1 H NMR (400 MHz, CDCl 3 ) δ 4.21(s,2H),3.73-3.66(m,14H),3.59-3.61(m,2H),2.60(s,1H),2.42(t,J=2.4 Hz,1H)).

[0408] 3,6,9,12-Tetraoxapentadec-14-yn-1-ol (660 mg, 2.84 mmol) and compound D-4-5 (310 mg, 1.29 mmol) were dissolved in THF (8 mL) and DMF (0.8 mL), and PPh 3 (667 mg, 2.58 mmol) was added to it. The mixture was cooled to 0° C. 2.2 M DEAD (1.17 mL, 2.58 mmol) was added to it, and the mixture was stirred at 0° C. for 3 h. After the reaction was completed, EA (15 mL×2) and distilled water (15 mL) were added, and the organic layer was extracted and washed with brine (20 mL). The obtained organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried over rt, filtered and concentrated under reduced pressure to give compound POS-D1 (205 mg, 30%). EI-MS m / z: 455(M + ).

[0409] [Example 11] Preparation of compound Int-TG13 [ka]

[0410] To a solution of the compound (1R,2S)-(-)-norephedrine (1 g, 6.61 mmol) in DCM (5 mL), TEA (0.92 mL, 6.61 mmol) and acetyl chloride (0.47 mL, 6.61 mmol) were added at 0 °C and N 2 After stirring at room temperature for 2 h, the mixture was diluted with H 2 The reaction was quenched with O (7 mL). The organic layer was extracted with DCM (2 × 8 mL) and anhydrous MgSO 4 The mixture was dried at 40° C., filtered and concentrated. The residue was purified by column chromatography to give compound Int-TG13 (987 mg, 78%). 1 H-NMR (400 MHz, CDCl 3 ) δ 7.28 (m, 5H), 5.70 (m, 1H), 4.86 (s, 1H), 4.34-4.30 (m, 1H), 3.65 (m, 1H), 2.00(s, 3H), 1.01 (d, J = 7.2 Hz, 3H)

[0411] [Example 12] Preparation of compound IntB-Q3-1 [ka]

[0412] A solution of PNU-1529682 (52 mg, 0.081 mmol) in MeOH (5 mL) / distilled water (3 mL) was diluted with NaIO 4 (18 mg, 0.081 mmol) was added at room temperature. After stirring for 2 h, the mixture was concentrated under reduced pressure to produce crude compound IntB-Q3-1 (51 mg, 99%). EI-MS m / z: 628 (M +1 ).

[0413] Preparation of compound IntB-Q3 To a solution of compound IntB-Q3-1 (51 mg, 0.081 mmol) in dry DCM (5 mL) was added 2-(dimethylamino)ethylamine (6.1 μl, 0.089 mmol), TEA (34 μl, 0.243 mmol), and TBTU (52 mg, 0.162 mmol) at room temperature. After stirring for 1 h, the mixture was diluted with DCM (2×8 mL). The organic layer was extracted with H 2 Wash with 2 mL of anhydrous NaCl. 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography to produce compound IntB-Q3 (38 mg, 67%). EI-MS m / z: 698(M +1 ).

[0414] [Example 13] Preparation of compound L-2 [ka]

[0415] Compound L-2 was synthesized by a synthetic route similar to that described in Journal of Polymer Science, Part A: Polymer Chemistry, 2012, 50(19), 3986-3995.

[0416] Preparation of compound L-2a Yield: 30% 1 H NMR (400 MHz, CDCl 3 )δ 7.80(d,J=8.4 Hz,2H),7.34(d,J=8.4 Hz,2H),4.16(t,J=4.8 Hz,2H),3.74-3.58(m,14H),2.45(s,3H).

[0417] Preparation of compound L-2b Yield: 68% 1 H NMR (400 MHz, CDCl 3 )δ 3.74-3.61(m,14H),3.40(t,J=4.8 Hz,2H),2.45(t,J=6.0 Hz,1H).

[0418] Preparation of Compound L-2c Yield 63% 1 H NMR(400 MHz, CDCl 3 ) δ 4.21 (d, J = 2.4 Hz, 2H), 3.72 - 3.67 (m, 14H), 3.39 (t, J = 5.2 Hz, 2H), 2.43 (t, J = 2.4 Hz, 1H).

[0419] Preparation of Compound L-2 Yield 76% 1 H NMR(400 MHz, CDCl 3 ) δ 4.20 (d, J = 2.4 Hz, 2H), 3.71 - 3.61 (m, 12H), 3.51 (t, J = 4.8 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H), 2.43 (t, J = 2.4 Hz, 1H).

[0420] [Example 14] Preparation of Compound L-3

Chemical Structure

[0421] Compound L-3 was synthesized by a synthetic route similar to that described in Journal of Organic Chemistry, 2002, 67, 5032 - 5035.

[0422] Preparation of Compound L-3a Yield 92% 1 H NMR(400 MHz, CDCl 3 ) δ 10.08 (s, 1H), 7.97 (q, J = 8.8 Hz, 8.8 Hz, 4H), 7.16 (brs, 1H), 4.14 (d, J = 2.4 Hz, 2H), 3.70 - 3.62 (m, 16H), 2.41 (t, J = 2.4 Hz, 1H). EI-MS m / z: 384 (M +1 )

[0423] Preparation of Compound L-3b Yield 69% 1 H NMR (400 MHz, CDCl 3 ) δ 7.82 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 6.88 (brs, 1H), 5.47 (brs, 1H), 4.14 (d, J = 2.4 Hz, 2H), 3.70 - 3.63 (m, 16H), 2.42 (brs, 1H), 2.19 (s, 3H). EI-MS m / z: 404 (M +1 )

[0424] Preparation of Compound L-3 Yield 81% 1 H NMR (400 MHz, CDCl 3 ) δ 8.19 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 7.6 Hz, 2H), 7.07 (brs, 1H), 4.16 (s, 2H), 3.70 - 3.49 (m, 16H), 2.42 (brs, 1H), 2.19 (s, 3H). EI-MS m / z: 402 (M +1 )

[0425] [Example 15] Preparation of Compound L-4

Chemical Structure

[0426] Compound L-4 was synthesized by a synthetic route similar to that described in Journal of Medicinal Chemistry, 52(19), 5816 - 5825; 2009.

[0427] Preparation of Compound L-4a Yield 55% 1 H NMR (400 MHz, CDCl 3 ) δ 4.21 (d, J = 2.0 Hz, 2H), 3.72 - 3.60 (m, 24H), 2.79 (brs, 1H), 2.43 (t, J = 2.4 Hz, 1H).

[0428] Preparation of Compound L-4 EI-MS m / z: 400 (M+1 )

[0429] [Example 16] Preparation of compound MPS-D6 [ka]

[0430] Compound L-5 was synthesized via a synthetic route similar to the syntheses described in Examples 44 and 45.

[0431] Preparation of compound MPS-D6a Yield: 91% 1H NMR (400 MHz, CDCl 3 )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),3.70-3.61(m,20H),3.39(t,J=4.8 Hz,2H),2.45(s,3H).EI-MS m / z:462(M +1 )

[0432] Preparation of compound MPS-D6b Yield 93%; EI-MS m / z:610(M +1 )

[0433] Preparation of compound MPS-D6c Yield 54%.EI-MS m / z:584(M +1 )

[0434] Preparation of compound MPS-D6 Yield 72%; EI-MS m / z:899(M +1 )

[0435] [Example 17] Preparation of compound MPS-D7 [ka]

[0436] Compound MPS-D7 was synthesized via a synthetic route similar to that described in Example 28. Yield 80%; EI-MS m / z:546(M +1 ) 1 H NMR (400 MHz, CDCl 3 )δ 8.11-7.94(m,4H),7.83(d,J=7.6 Hz,2H),7.44(brs,1H),7.38(d,J=8.0 Hz,2H),4.15(s,2H),3.69-3.65(m,14H),3.58-3.48(m,4H),2.80(s,1H),2.46(s,3H).

[0437] [Example 18] Preparation of compound IntB-Q10 [ka]

[0438] Preparation of compound IntB-Q10-1 Compound IntB-Q10-1 was synthesized by a synthetic route similar to that described in Mol.Pharmaceutics 2015,12,1813-1835.

[0439] Preparation of compound IntB-Q10-2 Compound IntB-Q10-2 was synthesized by a synthetic route similar to that described in Angew. Chem. Int. Ed. 2010, 49, 7336-7339 and International Patent Application Publication No. WO2015 / 110935A1, which are incorporated herein by reference in their entireties.

[0440] Preparation of compound IntB-Q10-3 A solution of compound IntB-Q10-1 (80 mg, 0.239 mmol) and compound IntB-Q10-2 (118 mg, 0.239 mmol) in DCM (10 mL) was added to a flask containing molecular sieves and BF 3 ·OEt 2 (14.8 μL, 0.12 mmol) at 0 °C, N 2Add under atmospheric pressure. After stirring for 2 hours, the mixture was filtered through Celite®, washed with DCM (50 mL) and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound IntB-Q10-3 (105 mg, 66%) as a white foam. 1 H NMR (400 MHz, CDCl 3 )δ 8.12(d,J=8.0 Hz,1H),7.89(brs,1H),7.63(d,J=8.0 Hz,1H),7.50(m,1H),7.35(m,1H),5.70(m,1H),5.51(s,1H),5.33(m,1 H),5.20(m,1H),4.23(m,3H),4.11(m,2H),3.93(m,2H),3.42(t,J=10.8 Hz,1H),2.18(s,3H),2.08(s,3H),2.04(s,3H),2.00(s,3H),1.55(s,9H).EI-MS m / z:564.4(M +1 ).

[0441] Preparation of compound IntB-Q10-4 Compound IntB-Q10-3 (100 mg, 0.15 mmol) was dissolved in DCM (2 mL) and then cooled to 0° C. with 4N HCl in 1,4-dioxane (1 mL) at 0° C. 2 The solution was added under atmospheric pressure. After stirring for 4 h, the reaction mixture was concentrated under reduced pressure.

[0442] The reaction mixture was incubated at room temperature for 4 h with N 2 Compound IntB-Q10-4 was used directly in the next step without further purification (90 mg, 99%).

[0443] EI-MS m / z: 564.2(M +1 ).

[0444] Preparation of compound IntB-Q10 A solution of compound IntB-Q10-4 (90 mg, 0.149 mmol) in THF (5 mL) was diluted with glutaric anhydride (18.8 μL, 0.164 mmol), Et 3N (52 μL, 0.373 mmol) and 4-DMAP (2 mg, 0.015 mmol) were dissolved in N 2 The reaction mixture was stirred at room temperature for 2 hours and purified by preparative HPLC to give compound IntB-Q10 (30 mg, 30%) as a white solid. EI-MS m / z: 678.3(M +1 ).

[0445] [Example 19] Preparation of compound IntB-Q13 [ka]

[0446] Compound IntB-Q13 was synthesized by a synthetic route similar to that described in Mol.Pharmaceutics 2015,12,1813-1835.

[0447] [Example 20] Preparation of compound IntB-Q14 [ka]

[0448] Compound IntB-Q14 was synthesized by a synthetic route similar to that described in International Patent Application Publication No. WO2015 / 038426A1, the contents of which are incorporated herein by reference in their entirety.

[0449] [Example 21] Preparation of compound IntB-Q15 [ka]

[0450] Preparation of compound IntB-Q15-1 To a solution of compound IntB-Q10-1 (55 mg, 0.016 mmol) in DCM (2 mL), acetyl chloride (26.8 μL, 0.032 mmol) and pyridine (30 μL, 0.032 mmol) were added at 0 °C with N 2After stirring for 30 min, the reaction was allowed to warm to room temperature and further stirred for 1 h. The mixture was diluted with EA (20 mL) and diluted with H 2 The mixture was washed with 0 (10 mL). Compound IntB-Q15-1 (50 mg, 80%) was isolated as a pale yellow foam. EI-MS m / z: 398.2(M +1 +Na). 1 H NMR (400 MHz, CDCl 3 )δ 8.02(brs,1H)7.79(d,J=8.0 Hz,1H),7.72(t,J=8.8 Hz,1H),7.51(m,1H),7.38(m,1H),4.16(m,1H),4.04(m,1H),3.92(m,1H),3.71(m,1H),3.36(m,1H),2.27,(s,3H),1.54(s,9H).

[0451] Preparation of compound IntB-Q15 Compound IntB-Q15 was synthesized via a ...

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 the structure of formula (I″); 【Chemistry 1】 each Q is independently an activator linked to L' by a heteroatom; Z′, independently at each occurrence, represents a group having the structure of formula (I″) as follows: 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 (I'') to (CB), ｃｂ Provided that you connect to Each L' is independently selected from O, S, and N, and is joined to SO ２ and L′ and SO ２ is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q, releasing the active agent; Each X is independently -O-, -C(R ｂ ) (R ｃ )- or -N(R ｃ ) - and E is an integer having a value of 1, 2, or 3; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y′ is —N(R ａ )-, -O-, or -S-; at least one X is positioned in a para relationship to Y′ on Ar; When TG is cut, SO ２ And (Q) ｑ -(L') ｗ is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20; each w and x is independently an integer having a value of 0 or 1; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently Z′, hydrogen, or lower alkyl, with the proviso that R ｂ is Z'; or R ｂ and R ｃ 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. X is -C(R ｂ ) (R ｃ 2. The conjugate of claim 1, wherein

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

4. The conjugate of claim 1 , wherein E is 1.

5. 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 4, which is a linear or branched, saturated or unsaturated alkylene moiety.

6. 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 2】 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 5, wherein o and q are each independently an integer having a value from 1 to about 10.

7. 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 6 , wherein is the point of attachment to Ar.

8. P １ But, 【Chemistry 3】 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.

9. TG is selected from the following: 【Chemistry 4】 During the ceremony, Each R ２１ are independently hydrogen or acetyl; R ２２ The conjugate of any one of claims 1 to 8, wherein is hydrogen or lower alkyl.

10. TG is -NO ２ , -C(O)-(CH ２ ) ２ The conjugate of claim 1 , wherein the alkyl group is selected from C(O)-alkyl and nitrobenzyl.

11. The conjugate of any one of claims 1 to 10, 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.

12. (Q) ｑ -(L') ｗ - is selected from the following: 【Chemistry 5】 During the ceremony, X １ is -O- or -NR ａ - and R ａ is hydrogen or C １ ~C ６ is alkyl, 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)-, 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; The conjugate according to any one of claims 1 to 10.

13. (Q) ｑ -(L') ｗ - is selected from the following: 【Chemistry 6】 【change】 【change】 【change】 During the ceremony, ＊ But, (Q) ｑ -(L') ｗ , -SO ２ represents a point of attachment to -, and (Q) ｑ -(L') ｗ is selected from the following: 【Chemistry 7】 During the ceremony, ＊ But, (Q) ｑ -(L') ｗ , -SO ２ The conjugate of claim 12, wherein said at least one bond is a bond to said at least one aryl group.

14. The conjugate according to any one of claims 1 to 13, 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, and the antibody is 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.

15. 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 15. The conjugate of claim 14, 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.

16. A compound of formula (Ia), 【Chemistry 8】 or a pharma- ceutically acceptable salt thereof, each Q is independently an activator linked to L' by a heteroatom; Z', independently in each occurrence, is absent, and the structure of formula (Ia) is (CB) according to claim 1 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) to (CB), ｃｂ Provided that you connect to Each L' is linked to SO through a heteroatom selected from O, S, and N. ２ is a linking group bonded to L′ and SO ２ is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q, releasing the active agent; Each X is independently -O-, -C(R ｂ ) (R ｃ )- or -N(R ｃ ) - and E is an integer having a value of 1, 2, or 3; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y′ is —N(R ａ )-, -O-, or -S-; at least one X is positioned in a para relationship to Y′ on Ar; When TG is cut, SO ２ And (Q) ｑ -(L') ｗ is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20; each w and x is independently an integer having a value of 0 or 1; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently Z′, hydrogen, or lower alkyl, with the proviso that R ｂ is Z'; or R ｂ and R ｃ 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.

17. A compound of formula (Ia), 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof, each Q is independently an activator linked to L' via a heteroatom; Z' is a reactive group; Each L' is independently selected from O, S, and N, and is joined to SO ２ is a linking group bonded to L′ and SO ２ is selected such that cleavage of the bond between L′ and Q promotes cleavage of the bond between L′ and Q, releasing the active agent; Each X is independently -O-, -C(R ｂ ) (R ｃ )- or -N(R ｃ ) - and E is an integer having a value of 1, 2, or 3; Ar is a 6-membered aryl or 6-membered heteroaryl ring; Y′ is —N(R ａ )-, -O-, or -S-; at least one X is positioned in a para relationship to Y′ on Ar; When TG is cut, SO ２ And (Q) ｑ -(L') ｗ is a triggering group that generates an N, O, or S atom capable of initiating the release of each q is independently an integer having a value from 1 to about 20; each w and x is independently an integer having a value of 0 or 1; Each R ａ and R ｃ are independently hydrogen or lower alkyl; Each R ｂ are independently Z′, hydrogen, or lower alkyl, with the proviso that R ｂ is Z'; or R ｂ and R ｃ 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.

18. 20. A method for preparing a conjugate comprising reacting a compound of claim 17 with a targeting moiety, 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.

19. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15 and a pharma- ceutically acceptable carrier or excipient.

20. An imaging composition comprising the conjugate of any one of claims 1 to 15.

21. A sensor compound comprising the conjugate according to any one of claims 1 to 15.

22. A composition comprising a conjugate according to any one of claims 1 to 15 for use in detection, said use comprising contacting a material with the composition.

23. A molecular switch, molecular machine, or nanomachine comprising the conjugate of any one of claims 1 to 15.

24. 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 23; (2) an activating agent that activates the inducing group.

25. 16. Use of a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 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.

26. 26. The use of claim 25, 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 associated preferentially with tumor cells over healthy cells).

27. 27. The use according to claim 25 or 26, wherein the disease or condition is an autoimmune disease, an infectious disease, or a tumor.

28. Use of a pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 in the manufacture of a medicament for treating a proliferative disease.

29. 29. The use of claim 28, 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.

30. 30. The use of claim 29, wherein the proliferative disease is a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

31. 16. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 15 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.

32. 32. The pharmaceutical composition of claim 31 , 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).

33. 33. The pharmaceutical composition of claim 31 or 32, wherein the disease or condition is an autoimmune disease, an infectious disease, or a tumor.

34. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 for treating a proliferative disease.

35. 35. The pharmaceutical composition of claim 34, 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.

36. 36. The pharmaceutical composition of claim 35, wherein the proliferative disease is a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancies.

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